Heparan Sulfate Proteoglycans

Heparan sulfate proteoglycans are found at the cell surface and in the extracellular matrix, where they interact with a plethora of ligands. Over the last decade, new insights have emerged regarding the mechanism and biological significance of these interactions. Here, we discuss changing views on the specificity of protein–heparan sulfate binding and the activity of HSPGs as receptors and coreceptors. Although few in number, heparan sulfate proteoglycans have profound effects at the cellular, tissue, and organismal level.Heparan sulfate proteoglycans (HSPGs) are glycoproteins, with the common characteristic of containing one or more covalently attached heparan sulfate (HS) chains, a type of glycosaminoglycan (GAG) (Esko et al. 2009). Cells elaborate a relatively small set of HSPGs (∼17) that fall into three groups according to their location: membrane HSPGs, such as syndecans and glycosylphosphatidylinositol-anchored proteoglycans (glypicans), the secreted extracellular matrix HSPGs (agrin, perlecan, type XVIII collagen), and the secretory vesicle proteoglycan, serglycin (Esko et al. 1985), which allowed functional studies in the context of a cell culture model (Zhang et al. 2006). A decade later, the first HSPG mutants in a model organism (Drosophila melanogaster) were identified (Rogalski et al. 1993; Nakato et al. 1995; Häcker et al. 1997; Bellaiche et al. 1998; Lin et al. 1999), which was followed by identification of mutants in nematodes, tree frogs, zebrafish, and mice (​and3).3). HS is evolutionarily ancient and its composition has remained relatively constant from Hydra to humans (Yamada et al. 2007; Lawrence et al. 2008).

Table 1.

Heparan sulfate proteoglycans

Immunological Mechanisms Mediating Hantavirus Persistence in Rodent Reservoirs

Hantaviruses, similar to several emerging zoonotic viruses, persistently infect their natural reservoir hosts, without causing overt signs of disease. Spillover to incidental human hosts results in morbidity and mortality mediated by excessive proinflammatory and cellular immune responses. The mechanisms mediating the persistence of hantaviruses and the absence of clinical symptoms in rodent reservoirs are only starting to be uncovered. Recent studies indicate that during hantavirus infection, proinflammatory and antiviral responses are reduced and regulatory responses are elevated at sites of increased virus replication in rodents. The recent discovery of structural and non-structural proteins that suppress type I interferon responses in humans suggests that immune responses in rodent hosts could be mediated directly by the virus. Alternatively, several host factors, including sex steroids, glucocorticoids, and genetic factors, are reported to alter host susceptibility and may contribute to persistence of hantaviruses in rodents. Humans and reservoir hosts differ in infection outcomes and in immune responses to hantavirus infection; thus, understanding the mechanisms mediating viral persistence and the absence of disease in rodents may provide insight into the prevention and treatment of disease in humans. Consideration of the coevolutionary mechanisms mediating hantaviral persistence and rodent host survival is providing insight into the mechanisms by which zoonotic viruses have remained in the environment for millions of years and continue to be transmitted to humans.Hantaviruses are negative sense, enveloped RNA viruses (family: Bunyaviridae) that are comprised of three RNA segments, designated small (S), medium (M), and large (L), which encode the viral nucleocapsid (N), envelope glycoproteins (G_N and G_C), and an RNA polymerase (Pol), respectively. More than 50 hantaviruses have been found worldwide [1]. Each hantavirus appears to have coevolved with a specific rodent or insectivore host as similar phylogenetic trees are produced from virus and host mitochondrial gene sequences [2]. Spillover to humans causes hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS), depending on the virus [3]–[5]. Although symptoms vary, a common feature of both HFRS and HCPS is increased permeability of the vasculature and mononuclear infiltration [4]. Pathogenesis of HRFS and HCPS in humans is hypothesized to be mediated by excessive proinflammatory and CD8+ T cell responses (​).

Table 1

Summary of Immune Responses in Humans during Hantavirus Infection.

Stress-induced flowering

Many plant species can be induced to flower by responding to stress factors. The short-day plants Pharbitis nil and Perilla frutescens var. crispa flower under long days in response to the stress of poor nutrition or low-intensity light. Grafting experiments using two varieties of P. nil revealed that a transmissible flowering stimulus is involved in stress-induced flowering. The P. nil and P. frutescens plants that were induced to flower by stress reached anthesis, fruited and produced seeds. These seeds germinated, and the progeny of the stressed plants developed normally. Phenylalanine ammonialyase inhibitors inhibited this stress-induced flowering, and the inhibition was overcome by salicylic acid (SA), suggesting that there is an involvement of SA in stress-induced flowering. PnFT2, a P. nil ortholog of the flowering gene FLOWERING LOCUS T (FT) of Arabidopsis thaliana, was expressed when the P. nil plants were induced to flower under poor-nutrition stress conditions, but expression of PnFT1, another ortholog of FT, was not induced, suggesting that PnFT2 is involved in stress-induced flowering.Key words: flowering, stress, phenylalanine ammonia-lyase, salicylic acid, FLOWERING LOCUS T, Pharbitis nil, Perilla frutescensFlowering in many plant species is regulated by environmental factors, such as night-length in photoperiodic flowering and temperature in vernalization. On the other hand, a short-day (SD) plant such as Pharbitis nil (synonym Ipomoea nil) can be induced to flower under long days (LD) when grown under poor-nutrition, low-temperature or high-intensity light conditions.^1–9 The flowering induced by these conditions is accompanied by an increase in phenylalanine ammonia-lyase (PAL) activity.¹⁰ Taken together, these facts suggest that the flowering induced by these conditions might be regulated by a common mechanism. Poor nutrition, low temperature and high-intensity light can be regarded as stress factors, and PAL activity increases under these stress conditions.¹¹ Accordingly, we assumed that such LD flowering in P. nil might be induced by stress. Non-photoperiodic flowering has also been sporadically reported in several plant species other than P. nil, and a review of these studies suggested that most of the factors responsible for flowering could be regarded as stress. Some examples of these factors are summarized in 12^–14

Table 1

Some cases of stress-induced flowering

Cell Biology of Mitotic Recombination

Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination.Homologous recombination (HR) is defined as the homology-directed exchange of genetic information between two DNA molecules (Fig. 1). Mitotic recombination is often initiated by single-stranded DNA (ssDNA), which can arise by several avenues (Mehta and Haber 2014). They include the processing of DNA double-strand breaks by 5′ to 3′ resection, during replication of damaged DNA, or during excision repair (Symington 2014). The ssDNA is bound by replication protein A (RPA) to control its accessibility to the Rad51 recombinase (Sung 1994, 1997a; Sugiyama et al. 1997; Morrical 2014). The barrier to Rad51-catalyzed recombination imposed by RPA can be overcome by a number of mediators, such as BRCA2 and Rad52, which serve to replace RPA with Rad51 on ssDNA, and the Rad51 paralogs Rad55-Rad57 (RAD51B-RAD51C-XRCC2-XRCC3) and the Psy3-Csm2-Shu1-Shu2 complex (SHU) (RAD51D-XRCC2-SWS1), which stabilize Rad51 filaments on ssDNA (see Sung 1997b; Sigurdsson et al. 2001; Martin et al. 2006; Bernstein et al. 2011; Liu et al. 2011; Qing et al. 2011; Amunugama et al. 2013; Zelensky et al. 2014). The Rad51 nucleoprotein filament catalyzes the invasion into a homologous duplex to produce a displacement loop (D-loop) (Fig. 1). At this stage, additional antirecombination functions are exerted by Srs2 (FBH1, PARI), which dissociates Rad51 filaments from ssDNA, and Mph1 (FANCM), which disassembles D-loops (see Daley et al. 2014). Upon Rad51-catalyzed strand invasion, the ATP-dependent DNA translocase Rad54 enables the invading 3′ end to be extended by DNA polymerases to copy genetic information from the intact duplex (Li and Heyer 2009). Ligation of the products often leads to joint molecules (JMs), such as single- or double-Holliday junctions (s/dHJs) or hemicatenanes (HCs), which must be processed to allow separation of the sister chromatids during mitosis. JMs can be dissolved by the Sgs1-Top3-Rmi1 complex (STR) (BTR, BLM-TOP3α-RMI1-RMI2) (see Bizard and Hickson 2014) or resolved by structure-selective nucleases, such as Mus81-Mms4 (MUS81-EME1), Slx1-Slx4, and Yen1 (GEN1) (see Wyatt and West 2014). Mitotic cells favor recombination events that lead to noncrossover events likely to avoid potentially detrimental consequences of loss of heterozygosity and translocations.Open in a separate windowFigure 1.Primary pathways for homology-dependent double-strand break (DSB) repair. Recombinational repair of a DSB is initiated by 5′ to 3′ resection of the DNA end(s). The resulting 3′ single-stranded end(s) invade an intact homologous duplex (in red) to prime DNA synthesis. For DSBs that are repaired by the classical double-strand break repair (DSBR) model, the displaced strand from the donor duplex pairs with the 3′ single-stranded DNA (ssDNA) tail at the other side of the break, which primes a second round of DNA synthesis. After ligation of the newly synthesized DNA to the resected 5′ strands, a double-Holliday junction (dHJ) intermediate is generated. The dHJ can be either dissolved by branch migration (indicated by arrows) into a hemicatenane (HC) leading to noncrossover (NCO) products or resolved by endonucleolytic cleavage (indicated by triangles) to produce NCO (positions 1, 2, 3, and 4) or CO (positions 1, 2, 5, and 6) products. Alternatively to the double-strand break repair (DSBR) pathway, the invading strand is often displaced after limited synthesis and the nascent complementary strand anneals with the 3′ single-stranded tail of the other end of the DSB. After fill-in synthesis and ligation, this pathway generates NCO products and is referred to as synthesis-dependent strand annealing (SDSA).

Table 1.

Evolutionary conservation of homologous recombination proteins between Saccharomyces cerevisiae and Homo sapiens

Comparative Analysis of Myxococcus Predation on Soil Bacteria

Predator-prey relationships among prokaryotes have received little attention but are likely to be important determinants of the composition, structure, and dynamics of microbial communities. Many species of the soil-dwelling myxobacteria are predators of other microbes, but their predation range is poorly characterized. To better understand the predatory capabilities of myxobacteria in nature, we analyzed the predation performance of numerous Myxococcus isolates across 12 diverse species of bacteria. All predator isolates could utilize most potential prey species to effectively fuel colony expansion, although one species hindered predator swarming relative to a control treatment with no growth substrate. Predator strains varied significantly in their relative performance across prey types, but most variation in predatory performance was determined by prey type, with Gram-negative prey species supporting more Myxococcus growth than Gram-positive species. There was evidence for specialized predator performance in some predator-prey combinations. Such specialization may reduce resource competition among sympatric strains in natural habitats. The broad prey range of the Myxococcus genus coupled with its ubiquity in the soil suggests that myxobacteria are likely to have very important ecological and evolutionary effects on many species of soil prokaryotes.Predation plays a major role in shaping both the ecology and evolution of biological communities. The population and evolutionary dynamics of predators and their prey are often tightly coupled and can greatly influence the dynamics of other organisms as well (1). Predation has been invoked as a major cause of diversity in ecosystems (11, 12). For example, predators may mediate coexistence between superior and inferior competitors (2, 13), and differential trajectories of predator-prey coevolution can lead to divergence between separate populations (70).Predation has been investigated extensively in higher organisms but relatively little among prokaryotes. Predation between prokaryotes is one of the most ancient forms of predation (27), and it has been proposed that this process may have been the origin of eukaryotic cells (16). Prokaryotes are key players in primary biomass production (44) and global nutrient cycling (22), and predation of some prokaryotes by others is likely to significantly affect these processes. Most studies of predatory prokaryotes have focused on Bdellovibrionaceae species (e.g., see references 51, 55, and 67). These small deltaproteobacteria prey on other Gram-negative cells, using flagella to swim rapidly until they collide with a prey cell. After collision, the predator cells then enter the periplasmic space of the prey cell, consume the host cell from within, elongate, and divide into new cells that are released upon host cell lysis (41). Although often described as predatory, the Bdellovibrionaceae may also be considered to be parasitic, as they typically depend (apart from host-independent strains that have been observed [60]) on the infection and death of their host for their reproduction (47).In this study, we examined predation among the myxobacteria, which are also deltaproteobacteria but constitute a monophyletic clade divergent from the Bdellovibrionaceae (17). Myxobacteria are found in most terrestrial soils and in many aquatic environments as well (17, 53, 74). Many myxobacteria, including the model species Myxococcus xanthus, exhibit several complex social traits, including fruiting body formation and spore formation (14, 18, 34, 62, 71), cooperative swarming with two motility systems (64, 87), and group (or “wolf pack”) predation on both bacteria and fungi (4, 5, 8, 9, 15, 50). Using representatives of the genus Myxococcus, we tested for both intra- and interspecific variation in myxobacterial predatory performance across a broad range of prey types. Moreover, we examined whether prey vary substantially in the degree to which they support predatory growth by the myxobacteria and whether patterns of variation in predator performance are constant or variable across prey environments. The latter outcome may reflect adaptive specialization and help to maintain diversity in natural populations (57, 59).Although closely related to the Bdellovibrionaceae (both are deltaproteobacteria), myxobacteria employ a highly divergent mode of predation. Myxobacteria use gliding motility (64) to search the soil matrix for prey and produce a wide range of antibiotics and lytic compounds that kill and decompose prey cells and break down complex polymers, thereby releasing substrates for growth (66). Myxobacterial predation is cooperative both in its “searching” component (6, 31, 82; for details on cooperative swarming, see reference 64) and in its “handling” component (10, 29, 31, 32), in which secreted enzymes turn prey cells into consumable growth substrates (56, 83). There is evidence that M. xanthus employs chemotaxis-like genes in its attack on prey cells (5) and that predation is stimulated by close contact with prey cells (48).Recent studies have revealed great genetic and phenotypic diversity within natural populations of M. xanthus, on both global (79) and local (down to centimeter) scales (78). Phenotypic diversity includes variation in social compatibility (24, 81), the density and nutrient thresholds triggering development (33, 38), developmental timing (38), motility rates and patterns (80), and secondary metabolite production (40). Although natural populations are spatially structured and both genetic diversity and population differentiation decrease with spatial scale (79), substantial genetic diversity is present even among centimeter-scale isolates (78). No study has yet systematically investigated quantitative natural variation in myxobacterial predation phenotypes across a large number of predator genotypes.Given the previous discovery of large variation in all examined phenotypes, even among genetically extremely similar strains, we anticipated extensive predatory variation as well. Using a phylogenetically broad range of prey, we compared and contrasted the predatory performance of 16 natural M. xanthus isolates, sampled from global to local scales, as well as the commonly studied laboratory reference strain DK1622 and representatives of three additional Myxococcus species: M. flavescens (86), M. macrosporus (42), and M. virescens (63) (Table ​(Table1).1). In particular, we measured myxobacterial swarm expansion rates on prey lawns spread on buffered agar (31, 50) and on control plates with no nutrients or with prehydrolyzed growth substrate.

TABLE 1.

List of myxobacteria used, with geographical origin

Visualization of Ca2+ Signaling During Embryonic Skeletal Muscle Formation in Vertebrates

Dynamic changes in cytosolic and nuclear Ca²⁺ concentration are reported to play a critical regulatory role in different aspects of skeletal muscle development and differentiation. Here we review our current knowledge of the spatial dynamics of Ca²⁺ signals generated during muscle development in mouse, rat, and Xenopus myocytes in culture, in the exposed myotome of dissected Xenopus embryos, and in intact normally developing zebrafish. It is becoming clear that subcellular domains, either membrane-bound or otherwise, may have their own Ca²⁺ signaling signatures. Thus, to understand the roles played by myogenic Ca²⁺ signaling, we must consider: (1) the triggers and targets within these signaling domains; (2) interdomain signaling, and (3) how these Ca²⁺ signals integrate with other signaling networks involved in myogenesis. Imaging techniques that are currently available to provide direct visualization of these Ca²⁺ signals are also described.The recognition of Ca²⁺ as a key regulator of muscle contraction dates back to Sydney Ringer''s seminal observations in the latter part of the 19th Century (Ringer 1883; Ringer 1886; Ringer and Buxton 1887; see reviews by Martonosi 2000; Szent-Györgyi 2004). More recently, evidence is steadily accumulating to support the proposition that Ca²⁺ also plays a necessary and essential role in regulating embryonic muscle development and differentiation (Flucher and Andrews 1993; Ferrari et al. 1996; Lorenzon et al. 1997; Ferrari and Spitzer 1998, 1999; Wu et al. 2000; Powell et al. 2001; Jaimovich and Carrasco 2002; Li et al. 2004; Brennan et al. 2005; Harris et al. 2005; Campbell et al. 2006; Terry et al. 2006; Fujita et al. 2007; and see reviews by Berchtold et al. 2000; Ferrari et al. 2006; Al-Shanti and Stewart 2009). What is currently lacking, however, is extensive direct visualization of the spatial dynamics of the Ca²⁺ signals generated by developing and differentiating muscle cells. This is especially so concerning in situ studies. The object of this article, therefore, is to review and report the current state of our understanding concerning the spatial nature of Ca²⁺ signaling during embryonic muscle development, especially from an in vivo perspective, and to suggest possible directions for future research. The focus of our article is embryonic skeletal muscle development because of this being an area of significant current interest. Several of the basic observations reported, however, may also be common to cardiac muscle development and in some cases to smooth muscle development. What the recent development of reliable imaging techniques has most certainly done, is to add an extra dimension of complexity to understanding the roles played by Ca²⁺ signaling in skeletal muscle development. For example, it is clear that membrane-bound subcellular compartments, such as the nucleus (Jaimovich and Carrasco 2002), may have endogenous Ca²⁺ signaling activities, as do specific cytoplasmic domains, such as the subsarcolemmal space (Campbell et al. 2006). How these Ca²⁺ signals interact with specific down-stream targets within their particular domain, and how they might serve to communicate information among domains, will most certainly be one of the future challenges in elucidating the Ca²⁺-mediated regulation of muscle development.Any methodology used to study the properties of biological molecules and how they interact during development should ideally provide spatial information, because researchers increasingly need to integrate data about the interactions that underlie a biological process (such as differentiation) with information regarding the precise location within cells or an embryo where these interactions take place. Current Ca²⁺ imaging techniques are beginning to provide us with this spatial information, and are thus opening up exciting new avenues of investigation in our quest to understand the signaling pathways that regulate muscle development (

Immunomodulation by Mesenchymal Stem Cells in Veterinary Species

Mesenchymal stem cells (MSC) are adult-derived multipotent stem cells that have been derived from almost every tissue. They are classically defined as spindle-shaped, plastic-adherent cells capable of adipogenic, chondrogenic, and osteogenic differentiation. This capacity for trilineage differentiation has been the foundation for research into the use of MSC to regenerate damaged tissues. Recent studies have shown that MSC interact with cells of the immune system and modulate their function. Although many of the details underlying the mechanisms by which MSC modulate the immune system have been defined for human and rodent (mouse and rat) MSC, much less is known about MSC from other veterinary species. This knowledge gap is particularly important because the clinical use of MSC in veterinary medicine is increasing and far exceeds the use of MSC in human medicine. It is crucial to determine how MSC modulate the immune system for each animal species as well as for MSC derived from any given tissue source. A comparative approach provides a unique translational opportunity to bring novel cell-based therapies to the veterinary market as well as enhance the utility of animal models for human disorders. The current review covers what is currently known about MSC and their immunomodulatory functions in veterinary species, excluding laboratory rodents.Abbreviations: AT, adipose tissue; BM, Bone marrow; CB, umbilical cord blood; CT, umbilical cord tissue; DC, dendritic cell; IDO, indoleamine 2;3-dioxygenase; MSC, mesenchymal stem cells; PGE₂, prostaglandin E2; VEGF, vascular endothelial growth factorMesenchymal stem cells (MSC, alternatively known as mesenchymal stromal cells) were first reported in the literature in 1968.³⁹ MSC are thought to be of pericyte origin (cells that line the vasculature)^21,22 and typically are isolated from highly vascular tissues. In humans and mice, MSC have been isolated from fat, placental tissues (placenta, Wharton jelly, umbilical cord, umbilical cord blood), hair follicles, tendon, synovial membrane, periodontal ligament, and every major organ (brain, spleen, liver, kidney, lung, bone marrow, muscle, thymus, pancreas, skin).^23,121 For most current clinical applications, MSC are isolated from adipose tissue (AT), bone marrow (BM), umbilical cord blood (CB), and umbilical cord tissue (CT; 11,87,99 Clinical trials in human medicine focus on the use of MSC both for their antiinflammatory properties (graft-versus-host disease, irritable bowel syndrome) and their ability to aid in tissue and bone regeneration in combination with growth factors and bone scaffolds (clinicaltrials.gov).¹³¹ For tissue regeneration, the abilities of MSC to differentiate and to secrete mediators and interact with cells of the immune system likely contribute to tissue healing (Figure 1). The current review will not address the specific use of MSC for orthopedic applications and tissue regeneration, although the topic is covered widely in current literature for both human and veterinary medicine.^57,62,90

Table 1.

Tissues from which MSC have been isolated

Archaeology of Eukaryotic DNA Replication

Recent advances in the characterization of the archaeal DNA replication system together with comparative genomic analysis have led to the identification of several previously uncharacterized archaeal proteins involved in replication and currently reveal a nearly complete correspondence between the components of the archaeal and eukaryotic replication machineries. It can be inferred that the archaeal ancestor of eukaryotes and even the last common ancestor of all extant archaea possessed replication machineries that were comparable in complexity to the eukaryotic replication system. The eukaryotic replication system encompasses multiple paralogs of ancestral components such that heteromeric complexes in eukaryotes replace archaeal homomeric complexes, apparently along with subfunctionalization of the eukaryotic complex subunits. In the archaea, parallel, lineage-specific duplications of many genes encoding replication machinery components are detectable as well; most of these archaeal paralogs remain to be functionally characterized. The archaeal replication system shows remarkable plasticity whereby even some essential components such as DNA polymerase and single-stranded DNA-binding protein are displaced by unrelated proteins with analogous activities in some lineages.Double-stranded DNA is the molecule that carries genetic information in all cellular life-forms; thus, replication of this genetic material is a fundamental physiological process that requires high accuracy and efficiency (Kornberg and Baker 2005). The general mechanism and principles of DNA replication are common in all three domains of life—archaea, bacteria, and eukaryotes—and include recognition of defined origins, melting DNA with the aid of dedicated helicases, RNA priming by the dedicated primase, recruitment of DNA polymerases and processivity factors, replication fork formation, and simultaneous replication of leading and lagging strands, the latter via Okazaki fragments (Kornberg and Baker 2005; Barry and Bell 2006; Hamdan and Richardson 2009; Hamdan and van Oijen 2010). Thus, it was a major surprise when it became clear that the protein machineries responsible for this complex process are drastically different, especially in bacteria compared with archaea and eukarya. The core components of the bacterial replication systems, such as DNA polymerase, primase, and replication helicase, are unrelated or only distantly related to their counterparts in the archaeal/eukaryotic replication apparatus (Edgell 1997; Leipe et al. 1999).The existence of two distinct molecular machines for genome replication has raised obvious questions on the nature of the replication system in the last universal common ancestor (LUCA) of all extant cellular life-forms, and three groups of hypotheses have been proposed (Leipe et al. 1999; Forterre 2002; Koonin 2005, 2006, 2009; Glansdorff 2008; McGeoch and Bell 2008): (1) The replication systems in Bacteria and in the archaeo–eukaryotic lineage originated independently from an RNA-genome LUCA or from a noncellular ancestral state that encompassed a mix of genetic elements with diverse replication strategies and molecular machineries. (2) The LUCA was a typical cellular life-form that possessed either the archaeal or the bacterial replication apparatus in which several key components have been replaced in the other major cellular lineage. (3) The LUCA was a complex cellular life-form that possessed both replication systems, so that the differentiation of the bacterial and the archaeo–eukaryotic replication machineries occurred as a result of genome streamlining in both lines of descent that was accompanied by differential loss of components. With regard to the possible substitution of replication systems, a plausible mechanism could be replicon takeover (Forterre 2006; McGeoch and Bell 2008). Under the replicon takeover hypothesis, mobile elements introduce into cells a new replication system or its components, which can displace the original replication system through one or several instances of integration of the given element into the host genome accompanied by inactivation of the host replication genes and/or origins of replication. This scenario is compatible with the experimental results showing that DNA replication DNA in Escherichia coli with an inactivated DnaAgene or origin of replication can be rescued by the replication apparatus of R1 or F1 plasmids integrated into the bacterial chromosome (Bernander et al. 1991; Koppes 1992). Furthermore, genome analysis suggests frequent replicon fusion in archaea and bacteria (McGeoch and Bell 2008); in particular, such events are implied by the observation that in archaeal genomes, genes encoding multiple paralogs of the replication helicase MCM and origins of replication are associated with mobile elements (Robinson and Bell 2007; Krupovic et al. 2010). Replicon fusion also is a plausible path from a single origin of replication that is typical of bacteria to multiple origins present in archaea and eukaryotes. However, all the evidence in support of frequent replicon fusion and the plausibility of replicon takeover notwithstanding, there is no evidence of displacement of the bacterial replication apparatus with the archaeal version introduced by mobile elements, or vice versa, displacement of the archaeal machinery with the bacterial version, despite the rapid accumulation of diverse bacterial and archaeal genome sequences. Thus, the displacement scenarios of DNA replication machinery evolution are so far not supported by comparative genomic data.Regardless of the nature of the DNA replication system (if any) in the LUCA and the underlying causes of the archaeo–bacterial dichotomy of replication machineries, the similarity between the archaeal and eukaryotic replication systems is striking (Leipe et al. 1999; Bell and Dutta 2002; Bohlke et al. 2002; Kelman and White 2005; Barry and Bell 2006). Thus, the archaeal replication system appears to be an ancestral version of the eukaryotic system and hence a good model for functional and structural studies aimed at gaining mechanistic insights into eukaryotic replication.

Table 1.

The relationship between archaeal and eukaryotic replication systems

Mouse Models of Osteoarthritis: A Summary of Models and Outcomes Assessment

Osteoarthritis (OA) is a multidimensional health problem and a common chronic disease. It has a substantial impact on patient quality of life and is a common cause of pain and mobility issues in older adults. The functional limitations, lack of curative treatments, and cost to society all demonstrate the need for translational and clinical research. The use of OA models in mice is important for achieving a better understanding of the disease. Models with clinical relevance are needed to achieve 2 main goals: to assess the impact of the OA disease (pain and function) and to study the efficacy of potential treatments. However, few OA models include practical strategies for functional assessment of the mice. OA signs in mice incorporate complex interrelations between pain and dysfunction. The current review provides a comprehensive compilation of mouse models of OA and animal evaluations that include static and dynamic clinical assessment of the mice, merging evaluation of pain and function by using automatic and noninvasive techniques. These new techniques allow simultaneous recording of spontaneous activity from thousands of home cages and also monitor environment conditions. Technologies such as videography and computational approaches can also be used to improve pain assessment in rodents but these new tools must first be validated experimentally. An example of a new tool is the digital ventilated cage, which is an automated home-cage monitor that records spontaneous activity in the cages.

Osteoarthritis (OA) is a multidimensional health problem and a common chronic disease.³⁶ Functional limitations, the absence of curative treatments, and the considerable cost to society result in a substantial impact on quality of life.⁷⁶ Historically, OA has been described as whole joint and whole peri-articular diseases and as a systemic comorbidity.^9,111 OA consists of a disruption of articular joint cartilage homeostasis leading to a catabolic pathway characterized by chondrocyte degeneration and destruction of the extracellular matrix (ECM). Low-grade chronic systemic inflammation is also actively involved in the process.^42,92 In clinical practice, mechanical pain, often accompanied by a functional decline, is the main reason for consultations. Recommendations to patients provide guidance for OA management.^{22, 33,49,86} Evidence-based consensus has led to a variety of pharmacologic and nonpharmacologic modalities that are intended to guide health care providers in managing symptomatic patients. Animal-based research is of tremendous importance for the study of early diagnosis and treatment, which are crucial to prevent the disease progression and provide better care to patients.The purpose of animal-based OA research is 2-fold: to assess the impact of the OA disease (pain and function) and to study the efficacy of a potential treatment.^18,67 OA model species include large animals such as the horse, goat, sheep, and dog, whose size and anatomy are expected to better reflect human joint conditions. However, small animals such as guinea pig, rabbit, mouse, and rat represent 77% of the species used.^1,87 In recent years, mice have become the most commonly used model for studying OA. Mice have several advantageous characteristics: a short development and life span, easy and low-cost breeding and maintenance, easy handling, small joints that allow histologic analysis of the whole joint,³² and the availability of genetically modified lines.¹⁰⁸ Standardized housing, genetically defined strains and SPF animals reduce the genetic and interindividual acquired variability. Mice are considered the best vertebrate model in terms of monitoring and controlling environmental conditions.^7,14,15,87 Mouse skeletal maturation is reached at 10 wk, which theoretically constitutes the minimal age at which mice should be entered into an OA study.^64,87,102 However, many studies violate this limit by testing mice at 8 wk of age.Available models for OA include the following (32,111 physical activity and exercise induced OA; noninvasive mechanical loading (repetitive mild loading and single-impact injury); and surgically induced (meniscectomy models or anterior cruciate ligament transection). The specific model used would be based on the goal of the study.⁷ For example, OA pathophysiology, OA progression, and OA therapies studies could use spontaneous, genetic, surgical, or noninvasive models. In addition, pain studies could use chemical models. Lastly, post-traumatic studies would use surgical or noninvasive models; the most frequently used method is currently destabilization of the medial meniscus,³² which involves transection of the medial meniscotibial ligament, thereby destabilizing the joint and causing instability-driven OA. An important caveat for mouse models is that the mouse and human knee differ in terms of joint size, joint biomechanics, and histologic characteristics (layers, cellularity),^32,64 and joint differences could confound clinical translation.¹⁰ Table 1. Mouse models of osteoarthritis.

Natural Infection of Burkholderia pseudomallei in an Imported Pigtail Macaque (Macaca nemestrina) and Management of the Exposed Colony

Identification of the select agent Burkholderia pseudomallei in macaques imported into the United States is rare. A purpose-bred, 4.5-y-old pigtail macaque (Macaca nemestrina) imported from Southeast Asia was received from a commercial vendor at our facility in March 2012. After the initial acclimation period of 5 to 7 d, physical examination of the macaque revealed a subcutaneous abscess that surrounded the right stifle joint. The wound was treated and resolved over 3 mo. In August 2012, 2 mo after the stifle joint wound resolved, the macaque exhibited neurologic clinical signs. Postmortem microbiologic analysis revealed that the macaque was infected with B. pseudomallei. This case report describes the clinical evaluation of a B. pseudomallei-infected macaque, management and care of the potentially exposed colony of animals, and protocols established for the animal care staff that worked with the infected macaque and potentially exposed colony. This article also provides relevant information on addressing matters related to regulatory issues and risk management of potentially exposed animals and animal care staff.Abbreviations: CDC, Centers for Disease Control and Prevention; IHA, indirect hemagglutination assay; PEP, postexposure prophylacticBurkholderia pseudomallei, formerly known as Pseudomonas pseudomallei, is a gram-negative, aerobic, bipolar, motile, rod-shaped bacterium. B. pseudomallei infections (melioidosis) can be severe and even fatal in both humans and animals. This environmental saprophyte is endemic to Southeast Asia and northern Australia, but it has also been found in other tropical and subtropical areas of the world.^7,22,32,42 The bacterium is usually found in soil and water in endemic areas and is transmitted to humans and animals primarily through percutaneous inoculation, ingestion, or inhalation of a contaminated source.^{8, 22,28,32,42} Human-to-human, animal-to-animal, and animal-to-human spread are rare.^8,32 In December 2012, the National Select Agent Registry designated B. pseudomallei as a Tier 1 overlap select agent.³⁹ Organisms classified as Tier 1 agents present the highest risk of deliberate misuse, with the most significant potential for mass casualties or devastating effects to the economy, critical infrastructure, or public confidence. Select agents with this status have the potential to pose a severe threat to human and animal health or safety or the ability to be used as a biologic weapon.³⁹Melioidosis in humans can be challenging to diagnose and treat because the organism can remain latent for years and is resistant to many antibiotics.^12,37,41 B. pseudomallei can survive in phagocytic cells, a phenomenon that may be associated with latent infections.^19,38 The incubation period in naturally infected animals ranges from 1 d to many years, but symptoms typically appear 2 to 4 wk after exposure.^13,17,35,38 Disease generally presents in 1 of 2 forms: localized infection or septicemia.²² Multiple methods are used to diagnose melioidosis, including immunofluorescence, serology, and PCR analysis, but isolation of the bacteria from blood, urine, sputum, throat swabs, abscesses, skin, or tissue lesions remains the ‘gold standard.’^9,22,40,42 The prognosis varies based on presentation, time to diagnosis, initiation of appropriate antimicrobial treatment, and underlying comorbidities.^7,28,42 Currently, there is no licensed vaccine to prevent melioidosis.There are several published reports of naturally occurring melioidosis in a variety of nonhuman primates (NHP; 2,10,13,17,25,30,31,35 The first reported case of melioidosis in monkeys was recorded in 1932, and the first published case in a macaque species was in 1966.³⁰ In the United States, there have only been 7 documented cases of NHP with B. pseudomallei infection.^2,13,17 All of these cases occurred prior to the classification of B. pseudomallei as a select agent. Clinical signs in NHP range from subclinical or subacute illness to acute septicemia, localized infection, and chronic infection. NHP with melioidosis can be asymptomatic or exhibit clinical signs such as anorexia, wasting, purulent drainage, subcutaneous abscesses, and other soft tissue lesions. Lymphadenitis, lameness, osteomyelitis, paralysis and other CNS signs have also been reported.^{2,7,10,22,28,32} In comparison, human''s clinical signs range from abscesses, skin ulceration, fever, headache, joint pain, and muscle tenderness to abdominal pain, anorexia, respiratory distress, seizures, and septicemia.^7,9,21,22

Table 1.

Summary of reported cases of naturally occurring Burkholderia pseudomalleiinfections in nonhuman primates

Functional and structural differences between skinned and intact muscle preparations

Myofilaments and their associated proteins, which together constitute the sarcomeres, provide the molecular-level basis for contractile function in all muscle types. In intact muscle, sarcomere-level contraction is strongly coupled to other cellular subsystems, in particular the sarcolemmal membrane. Skinned muscle preparations (where the sarcolemma has been removed or permeabilized) are an experimental system designed to probe contractile mechanisms independently of the sarcolemma. Over the last few decades, experiments performed using permeabilized preparations have been invaluable for clarifying the understanding of contractile mechanisms in both skeletal and cardiac muscle. Today, the technique is increasingly harnessed for preclinical and/or pharmacological studies that seek to understand how interventions will impact intact muscle contraction. In this context, intrinsic functional and structural differences between skinned and intact muscle pose a major interpretational challenge. This review first surveys measurements that highlight these differences in terms of the sarcomere structure, passive and active tension generation, and calcium dependence. We then highlight the main practical challenges and caveats faced by experimentalists seeking to emulate the physiological conditions of intact muscle. Gaining an awareness of these complexities is essential for putting experiments in due perspective.

IntroductionIn striated muscle, force is generated by sarcomeres located within myocytes (Bers, 2001, 2002). The sarcomere is located within the selectively permeable cell membrane, which supports intracellular ionic homeostasis. Within this highly regulated space, sarcomere force generation is activated by dynamic changes in cytosolic Ca²⁺. The sarcomeric protein troponin C (TnC) binds to Ca²⁺, which prompts the formation of myosin cross-bridges between the sarcomere thick (myosin) and thin (actin) filaments. These myofilaments are arranged in a regular lattice oriented along the muscle fiber direction and form the main structural basis of myocyte contraction. The contraction process is regulated by many other intracellular molecules and ions, in particular Mg²⁺ and H⁺, as well as by cellular and sarcomeric morphologies.To identify the ionic and molecular mechanisms that regulate the sarcomere, it is necessary to control the chemical environment it is exposed to. The biochemistry of the sarcomere proteins can be studied using in vitro biochemistry assays. However, these fail to account for the regular structure of the sarcomere, which is important for both biochemistry and function. Alternatively, the sarcomeres can be accessed by skinning the muscle, i.e., removing the sarcolemma membrane (or making it permeable to compounds and ions), while preserving sarcomere functionality (Curtin et al., 2015). Exposing the sarcomeres to tailored ionic conditions provides a means to observe and control molecular behavior in a setting that more closely resembles native structures. After skinning, the sarcomere system is effectively isolated from the other cellular subsystems (except in some skeletal muscle experiments that remove the sarcolemma while preserving intracellular organelles and structures; Donaldson, 1985; Fill and Best, 1988; Posterino et al., 2000). This facilitates the study of contraction and its regulation separately from the sarcolemma. The central assumption of skinned muscle experiments is that the response of the sarcomeres to changes in the natural cytosol can be reproduced artificially and controllably through analogous changes in the bathing solution.In skinning protocols (typically used with skeletal muscle) where the SR is preserved, applying caffeine liberates the intracellular Ca²⁺ reserves to stimulate contraction (Donaldson, 1985). In cases where the T tubules are preserved in the skinning process, ionic substitution in the bathing solution may induce T-tubule membrane depolarization and hence Ca²⁺ release from the SR (Fill and Best, 1988). An alternative approach to releasing SR calcium is by electric-field stimulation, with the electric field applied transversely relative to the fiber direction (Posterino et al., 2000).The principal readouts of skinned-muscle experiments are contraction kinetics, adenosine triphosphatase (ATPase) activity, and generated force. Their value therefore rests on the premise that the structural integrity of the sarcomeres is preserved. Under this condition, skinned muscle may be viewed as an intermediary experimental system, straddling intact muscle and in vitro molecular experiments.Skinned preparations allow the probing of muscle behavior beyond the current reach of experiments on intact systems. In experiments where contraction is elicited by controlling the bath [Ca²⁺], the influence of “cytosolic” conditions on Ca²⁺ sensitivity, in the steady-state, is typically presented in terms of Hill-type force-[Ca²⁺] relationships, or “F-pCa,” where pCa ≡ − log₁₀[Ca²⁺]/(mol/liter). Other intracellular molecular structures that fulfill structural and mechanical roles (e.g., titin [Cazorla et al., 2001; Fukuda and Granzier, 2005; Fukuda et al., 2005; Li et al., 2016; Tonino et al., 2017] or the cytoskeleton [Roos and Brady, 1989]) can also be investigated. The controlled progression of the system from one equilibrium state to another has helped to reveal, for example, hysteresis in F-pCa, which may potentially fulfill a physiological role but would be difficult to identify in the dynamic natural system (Bers, 2001; Harrison et al., 1988). Dynamic mechanical experiments also yield insight into myofilament kinetics (Breithaupt et al., 2019; Palmer et al., 2020; Stelzer et al., 2006; Terui et al., 2010). In some (mechanical) skinning methods that preserve the T tubules, further details of the excitation–contraction coupling become experimentally accessible (Fill and Best, 1988; Posterino et al., 2000). The ability to perform protein-exchange manipulations (e.g., cardiac versus skeletal TnC; Babu et al., 1988; Gulati and Babu, 1989), to include fluorescent proteins (e.g., troponin; Brenner et al., 1999), and to perform time-resolved dynamics measurements through the flash photolysis of caged compounds (ATP [Goldman et al., 1982, 1984], inorganic phosphate [Araujo and Walker, 1996; Dantzig et al., 1992; Millar and Homsher, 1990; Tesi et al., 2000], and Ca²⁺ chelators [Luo et al., 2002; Wahr et al., 1998]) provide additional handles for probing molecular mechanisms. Overall, much of our understanding of striated muscle generally and cytosolic conditions (temperature, pH, etc.) is derived from skinned-muscle experiments (Bers, 2001).Historically, skinning has been performed in a wide array of animal species and striated muscle systems, ranging from single cells to multicellular fibers of cardiac, skeletal, and smooth muscle. Various skinning techniques have been proposed. In “mechanical” skinning, the sarcolemma is effectively peeled off (entirely or partially; Cassens et al., 1986; Endo, 1977; Trube, 1978) by microdissection (Azimi et al., 2020; Donaldson, 1985; Fabiato, 1985b; Fabiato and Fabiato, 1975, 1977, 1978a, 1978b; Fill and Best, 1988; Godt, 1974; Godt and Maughan, 1977; Jewell, 1977; Lamb and Stephenson, 2018; Matsubara and Elliott, 1972; Moisescu, 1976; Rebbeck et al., 2020), while preserving the structural integrity and function of the T tubules and the SR (Lamb and Stephenson, 1990; Posterino et al., 2000; Stephenson, 1981). However, the technique is difficult and no longer used routinely. In contrast, “chemical” skinning involves dissolving or permeabilizing the membrane by applying a chemical agent. The most common agent is Triton X-100 (Solaro et al., 1971), but alternatives include Brij (Hibberd and Jewell, 1982), lubrol (Scheld et al., 1989), glycerol, and saponin (Edes et al., 1995; Endo and Iino, 1980; Gwathmey and Hajjar, 1990; Launikonis and Stephenson, 1997; Patel et al., 2001). Chemical skinning is particularly appropriate for multicellular tissue preparations. Controlling the precise protocol and chemical agent reportedly allows the selective dissolution of the sarcolemma membrane while leaving intracellular organelles (mitochondria and SR) intact. Nonetheless, treatment with (typically 1%) Triton X-100 frees the myofibrils of contamination by mitochondrial, sarcolemmal, and SR membranes while preserving ATPase activity and sensitivity to Ca²⁺ (Solaro et al., 1971). This straightforwardness makes Triton X-100 demembranation the predominantly used technique today. Other reported skinning approaches use propionate (Reuben et al., 1971) or the Ca²⁺ chelators EGTA or EDTA (Thomas, 1960; Winegard, 1971; Miller, 1979), but the uncertainty in the underlying mechanisms has undermined the reliability of these methods (Miller, 1979). For completeness, we also mention a less used “freeze drying” approach that arguably preserves the protein content of the fibers better than chemical skinning (De Beer et al., 1992; Schiereck et al., 1993; Stienen et al., 1983).Although, for many years, skinned muscle experiments have served as an invaluable method for investigating fundamental physiology, they are increasingly inspiring more ambitious practical applications. At a practical level, live human cells are inevitably a highly scarce resource, with facilities for collecting, storing, and measuring samples often being displaced both geographically and temporally. These issues are more realistically resolved with skinned cells, which can be preserved frozen for several months (Mosqueira et al., 2019). The development of new sarcomere drugs, including omecamtiv mecarbil and mavacamten, demonstrate that the sarcomere is a viable drug target (Tsukamoto, 2019). Similarly, Ca²⁺-sensitizing drugs (which act by increasing either the sensitivity to [Ca²⁺] or the magnitude of the generated force) such as levosimendan (Edes et al., 1995), pimobendan (Fitton and Brogden, 1994; Scheld et al., 1989), sulmazole (Solaro and Rüegg, 1982), isomazole (Lues et al., 1988), and EMD-57033 (Gross et al., 1993; Lee and Allen, 1997) have all been assessed using measurements on skinned fibers. Identifying further novel sarcomere modulator compounds requires large high-throughput screening, which is unrealistic using intact muscle.There is also a growing appetite for exploiting the quantitative value of skinned muscle experiments for more direct clinical applications, such as guiding patient-specific therapies. Much of this ambition relies on the integrative power of computational models to simulate human heart mechanics based on individual patients’ data, linking sub-cellular mechanisms with systemic behavior (Niederer et al., 2019a, 2019b). Building upon basic understanding of muscle behavior, recent developments in biomedical engineering extrapolate physiological processes at the cellular and tissue levels to predict global whole-heart function. As this field continues to grow in maturity, and as model predictions allow more meaningful comparisons with clinical data, efforts are increasingly focusing on quantitatively elucidating the interdependence between cellular behavior, tissue properties, and the anatomy. The quantitative accuracy of the subsystems at all these levels therefore becomes paramount.In both of these evolving applications, the relevance and value of skinned-muscle experiments hinges on their ability to reliably emulate the intact system (Land et al., 2017; Margara et al., 2021; Mijailovich et al., 2021). Skinned-muscle experiments conducted over the past decades confirm the fidelity, in many respects, of these preparations as valid experimental models. However, they also highlight caveats and significant interpretational challenges. Gaining an awareness of these issues is becoming all the more essential to avoid misinterpretations that may have practical consequences. This review therefore aims to highlight these challenges, to help users of skinned-based measurements put them in an appropriate perspective.The present review is structured as follows. We first compare measurements of the principal physiological properties of skinned and intact muscle, highlighting similarities and discrepancies. We focus primarily on chemical skinning, and in particular Triton X-100 (the predominantly used chemical agent). We then describe practical challenges involved in conducting experiments, insofar as they impact on measurement outcomes. We conclude with a summary of recommendations and main caveats.Comparing skinned and intact muscleSkinned muscle experiments aim to reveal and controllably reproduce features of the physiological function of sarcomeres. However, notable discrepancies arise between skinned- and intact-muscle measurements of basic muscle properties that govern overall muscle function. To establish these differences rigorously at the single-cell level encounters significant methodological challenges. Although it might seem obvious that this would require doing measurements systematically on both preparation types in tandem, many early experiments were done predominantly on skinned rather than on intact cells (King et al., 2011). This stems largely from the specific challenges of noninjurious cell attachment and performing small-force measurement on intact single cells (Brady, 1991). More recently, technical developments (e.g., involving the use of flexible carbon fibers to hold the cells at opposite ends; Iribe et al., 2007; Le Guennec et al., 1990; Yasuda et al., 2001) have made these measurements more practicable. Despite these advances, however, only a fraction of studies in the literature have systematically made direct comparisons between skinned and intact systems taken from the same species under optimally similar conditions (see the selection listed in ReferenceSystemIntactSkinning method[Mg²⁺] (mM)Ionic strength (mM)pH Reuben et al. (1971) CrayfishEGTA-3007.0 Winegard (1971) Frog cardiacEDTA1-6.5–7.0 Matsubara and Elliott (1972) Frog skeletalXDissection1-7.0 Godt (1974) Frog skeletalDissection51507.3 Wood et al. (1975) Human skeletalEGTA2–4-7.0 Moisescu (1976) Frog skeletalDissection11507.1 Godt and Maughan (1977) Frog skeletalXDissection31507.0 Best et al. (1977) Rat cardiacHomogenization0.05, 11507.0 Trube (1978) Mouse cardiacDissection (partial)41327.0 Gordon (1978) Rabbit smoothTriton X-1001.0–6.91307.0 Stienen et al. (1983) Frog skeletalFreeze drying1.11607.0Fabiato and Fabiato (1975, 1978a, 1978b)Rat cardiacDissection0.321607.0 Fabiato and Fabiato (1978a) Frog skeletalDissection0.321607.0 Fabiato (1981) Rat cardiacXEGTA11607.1 Fabiato (1981) Rabbit cardiacXEGTA11607.1 Fabiato (1985b) Canine cardiacDissection31707.1 Hibberd and Jewell (1982) Rat cardiacBrij-580.32007.0Solaro et al. (1971, 1976); Solaro and Rüegg (1982)Canine cardiacTriton X-100Var1007.0 Donaldson (1985) Rabbit skeletalDissection11507.0 Kentish et al. (1986) Rat cardiacXTriton X-10032007.0 Fill and Best (1988) Frog skeletalDissection11507.0 Lues et al. (1988) Various cardiacTriton X-100-1406.7 Roos and Brady (1989) Rat cardiacXTriton X-100-1607.1 Scheld et al. (1989) Human cardiacLubrol PX-1406.7 Harrison and Bers (1989) Rabbit cardiacTriton X-1002.2-7.0 Lamb and Stephenson (1990) Toad skeletalDissection1-7.10 Gwathmey and Hajjar (1990) Human cardiacXSaponin31607.1 Sweitzer and Moss (1990) Rat cardia, rabbit skeletalTriton X-10011807.0 Millar and Homsher (1990) Rabbit skeletalEGTA12007.1 De Beer et al. (1992) Rabbit skeletalFreeze drying--- Gross et al. (1993) Guinea pig cardiacTriton X-100--7.4 Gao et al. (1994) Rat cardiacXTriton X-1001.2-7.0 Wolff et al. (1995a) Canine cardiacTriton X-10011807.0 Edes et al. (1995) Guinea pig cardiacSaponin-1607.4 Araujo and Walker (1996) Rat cardiacTriton X-1001180- Allen et al. (2000) Rat cardiacTriton X-1001–81507.0 Posterino et al. (2000) Rat skeletalDissection1-7.1 Irving et al. (2000) Rat trabeculaeXTriton X-100-2007.35 Patel et al. (2001) Mouse cardiacSaponin + Triton X-100-1807.0 Konhilas et al. (2002) Rat trabeculaeTriton X-1001180- Luo et al. (2002) Rabbit skeletalTriton X-10011807.0 Fukuda et al. (2003) Bovine cardiacTriton X-10011807.0 Prado et al. (2005) Rabbit skeletalXTriton X-100-1807.0 Fukuda et al. (2005) Bovine and rat cardiacTriton X-10011807.0 Stelzer et al. (2006) Mouse cardiacSaponin + Triton X-10011807.0 Terui et al. (2010) Pig cardiacTriton X-10011807.0 Gillis and Klaiman (2011) Fish cardiacTriton X-10011707.0 Curtin et al. (2015) Rabbit skeletalXTriton X-10022007.1 Li et al. (2016) Rabbit skeletalTriton X-100-1807.0 Land et al. (2017) Human cardiacTriton X-10012007.1 Stehle (2017) Guinea pig cardiacTriton X-100-1707.0 Breithaupt et al. (2019) Rat cardiacGlycerol + Triton X-10012007.0 Giles et al. (2019) Mouse cardiacSaponin + Triton X-10011807.0 Azimi et al. (2020) Rat skeletalDissection1-7.1 Rebbeck et al. (2020) Human and rat skeletalDissection1-7.4 Palmer et al. (2020) Mouse cardiacTriton X-10012007.0Open in a separate windowA mark (X) in the Intact column indicates studies that directly compared measurements on both intact and skinned muscle (either performed within the same study or by considering previously published results). Var, variable.Sarcomere structureThe geometrical configuration and separation of the myofilaments regulate their interaction in the native system and hence their ability to generate tension. Under normal physiological conditions, the filament lattice structure is influenced by a complex balance of opposing forces, which include (Millman, 1998) electrostatic interactions between both thick and thin filaments (with charge being affected by pH and screened by the surrounding ionic strength), van der Waals forces, and entropic thermal forces, as well as Donnan osmotic force (whereby water enters the filament lattice to dilute counterions surrounding the charged filaments; Ilani, 2015). It is therefore unsurprising that this balance becomes disrupted upon removal of the sarcolemma.Muscle skinning broadly conserves the sarcomere assembly, but, as illustrated below, detailed quantitative features are altered at different scales. Microscopy and synchrotron x-ray measurements on skinned muscle report a modest increase in sarcomere length (∼3%), accompanied by a greater lateral expansion (up to twofold, depending on conditions), compared with intact cells. This is apparent in both skeletal (Matsubara and Elliott, 1972) and cardiac muscle (Irving et al., 2000; Roos and Brady, 1989). In both skinned and intact preparations, longitudinal stretching decreases the myofilament lattice spacing monotonically. This occurs more slowly in the skinned system, especially at large sarcomere lengths (Fig. 1; Irving et al., 2000). Despite their similar overall behavior, different physical effects are likely to operate in the two systems. The volume of intact cells is approximately conserved (Yagi et al., 2004), and therefore, stretching the cell decreases its cross-sectional area. As the sarcomere number remains constant, this increases the sarcomere density and hence stress generation (force per unit cross-sectional area). The constant-volume constraint is removed in skinned systems (Godt and Maughan, 1977; Irving et al., 2000; Matsubara and Elliott, 1972), which allows the structure to respond more visibly to other forces.Open in a separate windowFigure 1.Average myofilament spacing as a function of the sarcomere length in intact and relaxed skinned rat trabeculae, measured by x-ray diffraction. Adapted from Irving et al. (2000).The expansion of the myofilament spacing in skinned preparations can be reversed by increasing the osmotic pressure of the solution using dextran (Cazorla et al., 2001; Konhilas et al., 2002). However, this compressive effect does not by itself return the myofilaments fully to their intact physiological state (Konhilas et al., 2002). Recent x-ray diffraction experiments have identified an alteration of the detailed molecular structure of the thick filaments below physiological temperatures (Caremani et al., 2019, 2021). Although this effect is overlooked in many experiments, it may significantly affect cross-bridge kinetics.Skinning may also impact sarcomere morphology on larger scales. While measuring the effect of skinning on the sarcomere length in rat heart trabeculae using laser diffraction, Kentish et al. (1986) observed an increase in the diffraction intensity and a decrease in the dispersion of the first-order diffraction. Although this effect might result from the loss of intracellular scatterers (mitochondria, cytosolic proteins, etc.) upon skinning, the authors hypothesize that the skinning process might effectively enhance the homogenization of the sarcomere environment of the skinned tissue, relative to the intact one, where individual cells may display spontaneous and uncoordinated contractions. Nonetheless, the relative homogeneity of the skinned tissue degrades rapidly after successive contractions, possibly due to a loss of integrity of the cellular structure and content, in both cardiac (Kentish et al., 1986) and skeletal muscle (Fabiato and Fabiato, 1978b). This reflects a degree of irreproducibility inherent to skinned systems.Sarcomere structure strongly regulates contractile properties. Changes in both sarcomere length and interfilament spacing affect cross-bridge cycling and influence the regulation and amount of tension generated by skinned sarcomeres. Recent evidence also suggests that skinning may perturb myofilament interactions via steric effects due to myosin head orientations (Caremani et al., 2019, 2021; Konhilas et al., 2002). These effects, discussed further below, highlight the complexity in the disruption of the sarcomere function caused by skinning, relative to intact muscle, and the challenge in rationalizing their discrepancies based on fundamental physics principles. Ultimately, the extent to which skinning modifies sarcomere functionality bears critically on the interpretation of skinned muscle experiments.Passive mechanical compliancePassive mechanical properties of cardiac muscle strongly govern diastolic behavior. In intact tissue, these may have contributions originating in the cells themselves and the extracellular matrix (mostly comprising collagen). Passive tension and sarcomere length vary nonlinearly in both intact and skinned rat ventricular trabeculae preparations (Fig. 2; Kentish et al., 1986). However, in the skinned case, this length dependence is weaker, and the extension range is greater, indicating the presence of additional parallel elastic elements in the intact tissue, potentially associated with the sarcolemma or extracellular structures.Open in a separate windowFigure 2.Passive stress increasing with sarcomere length in skinned and intact rat ventricular trabeculae. The skinned results indicate enhanced mechanical compliance. Adapted from Kentish et al. (1986). Fig. 2 is reprinted with permission from Circulation Research.The qualitative similarity in the passive force-length relations in intact and skinned muscle makes the attribution of their quantitative differences challenging. The direct contribution of the sarcolemma itself, although plausible in principle, is expected to be weak, given its high compliance. However, it is more likely to contribute indirectly, given that the cell volume remains approximately constant upon stretching (Yagi et al., 2004). This effect may also be exacerbated by the Coulombic repulsion of the negatively charged myofilaments that, when confined within a fixed volume, would enhance resistance to lateral cellular compression (Kentish et al., 1986). Skinning may also cause the loss of intracellular components that contribute to the passive mechanics, e.g., a nonfilamentous stroma, comprising vesicular elements that dissolve in the skinning process (Kentish et al., 1986). Similarly, the loss of tubulin dimers from the cytoplasm may interfere with the viscoelastic behavior and resistance to cell shortening of the microtubule cytoskeleton (White, 2011).Structural differences can also explain discrepancies between skinned and intact muscle properties. Variations in the ionic strength acting on skinned myocytes have identified a mechanical contribution from the intracellular cytoskeleton (Roos and Brady, 1989). Similarly, titin contributes to the passive stiffness in isolated myofibrils and skinned single fibers, separately from the extracellular (mostly collagen) contribution (Cazorla et al., 2001; Fukuda and Granzier, 2005; Fukuda et al., 2005; Herzog, 2018; Powers et al., 2017). Within the isolated sarcomeric system, the stiffness varies inversely with the titin molecular size (Mijailovich et al., 2019; Prado et al., 2005), but this correlation disappears in intact fiber bundles, where extracellular contributions (e.g., from collagen) may dominate (Brower et al., 2006; Chung and Granzier, 2011; Fomovsky et al., 2010).Although the above observations highlight the limitations of using skinned preparations as a model for investigating passive mechanics in intact tissue, there may be indirect implications for contractile function. The distribution of force between passive and active mechanisms affects contraction, e.g., via force-dependent Ca²⁺ sensitivity (Cazorla et al., 2001; Fukuda and Granzier, 2005; Fukuda et al., 2005; Martyn and Gordon, 2001; Mijailovich et al., 2019; Sweitzer and Moss, 1990). In particular, passively elastic titin influences active contraction via the release of troponin I (TnI) from actin, as a result of the redistribution of mechanical load and strain on both the thick and thin filaments (Mijailovich et al., 2019). It may also determine the sarcomere length for a given afterload or the shortest sarcomere length in isotonic contractions.Calcium dependence of tension generationSkinned preparations are often used to measure the Ca²⁺ dependence of force development under equilibrium conditions. Measured F-pCa relations (e.g., Fig. 3) are conventionally characterized by their maximum saturating value, the location of the half-maximum point (the “sensitivity,” pCa₅₀), and the Hill coefficient n (quantifying the rate of rise and taken as a measure of cooperativity). To assess their validity, analogous F-pCa relations may also be generated in intact muscle by controlling the intracellular [Ca²⁺] homeostasis via tetanization, i.e., high-frequency activation (Fig. 3). Reported F-pCa relationships vary significantly according to the muscle type and preparations (Fabiato, 1981; Fukuda et al., 2003; Hibberd and Jewell, 1982; Kentish et al., 1986). This is problematic insofar as measurements in skinned systems aim to reproduce the “authentic” behavior in the intact system. The most intuitive mechanism involves an increased Ca²⁺-troponin binding affinity (Allen and Kentish, 1985; Kentish et al., 1986; Stephenson and Wendt, 1984), but more complex contributions also originate in the thick-filament structure upon stretching (Zhang et al., 2017).Open in a separate windowFigure 3.Comparing the force-calcium relationship in intact and skinned muscle. (a) Intact (ferret, 30°C; Yue et al., 1986) versus skinned (rabbit, 29°C; Harrison and Bers, 1989) muscle. (b) Pooled measurements derived from intact (solid symbols, pCa₅₀ ≈ 6.21, n ≈ 4.9) and skinned (open symbols, 6.04, 3.8) preparations of the same rat ventricular myocytes. max, maximum. From Gao et al. (1994). Fig. 3 is reprinted with permission from Circulation Research.Both pCa₅₀ and n are significantly enhanced in the intact case (in ferret) relative to skinned tissue (rabbit), substantially exceeding typical species-dependent variability observed in skinned muscle (Fig. 3 a; Bers, 2001). A similar qualitative conclusion was drawn from comparisons of intact and skinned preparations of the same rat ventricular myocytes (Fig. 3 b; Gao et al., 1994). These discrepancies are particularly significant when comparing the measured sensitivity values (pCa₅₀ = 5.52; Land et al., 2017) with physiological systolic [Ca²⁺] levels in the heart (0.6 µM ≃ pCa 6.22; Coppini et al., 2013; Land et al., 2017). Thus, the skinned muscle measurements are clearly incompatible with observed physiological behavior in intact myocytes and hence at the organ scale. Although the dominant underlying biophysical reason for these differences is uncertain, the detailed experimental conditions are fundamentally important (Bers, 2001). A rigorous quantitative comparison is therefore challenging.Skinning may affect the F-pCa relation via the sarcomere structure. An increase in the myofilament spacing plausibly reduces the rate of myosin cross-bridge formation and hence the amount of force generated for a given [Ca²⁺]. This would translate into a reduction in pCa₅₀, induced by muscle shortening, as observed in both skinned and (more weakly) intact preparations (Komukai and Kurihara, 1997). This mechanism may arguably contribute to the Frank–Starling mechanism in muscle, whereby the strength of contraction increases with stretch. However, this intuitive explanation has been shown to be insufficient in accounting for the complete effect on calcium sensitivity (Irving and Craig, 2019; de Tombe et al., 2010). It is also contradicted by experiments in which comparable myofilament spacings were achieved either via dextran-based osmotic compression or by sarcomere stretching (Konhilas et al., 2002). These discrepancies suggest that the filament spacing may not be the dominant contributor to pCa₅₀. However, this conclusion assumes the functional equivalence of the two scenarios. This may not be the case, as skinning may perturb other intracellular structures (e.g., titin or thin-filament regulatory proteins; Komukai and Kurihara, 1997). Experiments on mouse skinned cardiomyocytes have suggested that titin regulates filament spacing (Cazorla et al., 2001). Osmotic pressure may also impact the cross-bridge structural configuration on smaller molecular scales (Caremani et al., 2021; Konhilas et al., 2002).The sensitivity of the myofilaments to their chemical environment adds a further layer of complexity to skinned experiments. As discussed further below, F-pCa curves depend on the ionic strength, [Mg²⁺], and pH, all of which are routinely specified in skinned-experiment protocols. Skeletal muscle measurements have shown that increasing the temperature of the bathing solution increases the [Ca²⁺] required to activate skinned muscle as well as the maximal generated force (Godt and Lindley, 1982). Similarly, decreasing [Mg²⁺] lowers the activation [Ca²⁺] (Godt and Lindley, 1982). However, the native cell features other regulators that are lost during skinning and are not typically included in experiments. Sensitizers like taurine, carnosine-like compounds, and myosin light-chain kinase modestly increase the Ca²⁺ sensitivity (Gao et al., 1994). β-Adrenergic stimulation of intact muscle activates PKA, which in turn affects sarcomere dynamics by phosphorylating TnI and myosin-binding protein C (Gillis and Klaiman, 2011; Kentish et al., 2001; Patel et al., 2001). TnI phosphorylation decreases its binding affinity for Ca²⁺ (de Tombe and Stienen, 1995; Patel et al., 2001; Zhang et al., 1995), while that of myosin-binding protein C induces a movement of the myosin heads that accelerates force development.Despite their appealing relative simplicity, inconsistencies between skinned and intact muscle suggest fundamental alterations to muscle function by the skinning process. Following the rapid length release and restretch of skinned rat trabeculae, force redevelopment is Ca²⁺-dependent (Wolff et al., 1995b), unlike the rate of force redevelopment after a rapid-length release of intact ferret trabeculae (Hancock et al., 1993). This discrepancy is arguably explained by the relative dominance of thin- or thick-filament kinetics, respectively (Hunter et al., 1998).Taken together, these results illustrate the challenge of objectively determining the physiological Ca²⁺ dependence of muscle tension, in large part owing to the considerable technical challenge of replicating the native conditions of the myofilament system in vitro.Force-length relationThe sarcomere length dependence of force generation that underlies the Frank–Starling mechanism is a fundamental property of muscle behavior. Contributing mechanisms include the variation in myofilament overlap as the sarcomere is stretched, the apparent increase in the binding of Ca²⁺ to TnC with increasing length (Hibberd and Jewell, 1982; Kobirumaki-Shimozawa et al., 2014), and the modulation of the thick- (Fukuda et al., 2001; Zhang et al., 2017) and thin-filament structures (Zhang et al., 2017). The passive mechanical properties of titin (which vary according to the isoform) affect the variation in the lattice spacing under tension, and hence the length dependence of the actomyosin interaction (Fukuda et al., 2003). Recent evidence shows that the strain on titin, effectively acting as a force sensor, contributes to the Frank–Starling effect by influencing the structure of both the thin and thick filaments that are different from Ca²⁺-induced changes (Ait-Mou et al., 2016).Length-dependent tension, manifested in the F-pCa relationship, is qualitatively similar in intact and skinned preparations (Fig. 4). In the intact case, active tension was measured as the difference between the maximum tension in transiently stimulated muscle and the resting (unstimulated) tension at the same sarcomere lengths. The process was repeated at different [Ca²⁺] values in the bathing solution, so as to modulate the intracellular calcium. Comparing Fig. 4, a and b, for sufficiently low [Ca²⁺] below the level for full activation, the skinned- and unskinned-tissue measurements show a qualitatively similar transition from a concave to a convex dependence as [Ca²⁺] is increased. The results suggest that, whereas the unskinned system sustains no active tension for sarcomere lengths below ∼1.6 µm, the skinned preparation allows tension generation in this regimen, albeit at unphysiologically large [Ca²⁺]. However, the ability to measure (potentially heterogeneous) sarcomere lengths accurately in this regimen is questionable.Open in a separate windowFigure 4.Active force generation in intact and skinned rat ventricular trabeculae as a function of sarcomere length, for different bath [Ca²⁺]. From Kentish et al. (1986). Fig. 4 reprinted with permission from Circulation Research.For sufficiently low [Ca²⁺], the basic contraction mechanisms are thus preserved after skinning, at least qualitatively, suggesting that the general features of the force-length relationship are inherent myofibril properties. However, this conclusion assumes that (1) the chemical environments of the myofilaments are largely similar (any experimentally defined environment can only approximate the real cytosol), and (2) myofilament properties are not appreciably modified by the skinning process. The latter condition may be affected by the reported swelling of the myofilament lattice (Godt and Maughan, 1977; Irving et al., 2000; Konhilas et al., 2002; Matsubara and Elliott, 1972) or by any damage to the filaments occurring during the skinning process. Both of these effects should reduce the gradient of the tension relative to stretch.Significant variations in measurements may originate from structural causes at different levels. The above results, derived from trabeculae, show a steeper length dependence for short sarcomere lengths, compared with those of Fabiato and Fabiato (1975) on (mechanically) skinned maximally activated single ventricular myocytes (Kentish et al., 1986). This discrepancy might be ascribed either to the conservation of intercellular connections and extracellular connective tissue that might be lost in the skinned single myocytes, or to differences in the myofilament spacing in the multicellular tissue preparation. Some more subtle effects, such as the temperature-dependent alteration of the internal thick-filament structure in demembrenated muscle, observed recently (Caremani et al., 2019, 2021), seldom receive due consideration.Length-dependent F-pCa measurements show the sensitivity of muscle activation by calcium increasing with length, as marked by an increase in pCa₅₀ (Fig. 5). The maximum generated force at saturating [Ca²⁺] also increases. However, the Hill coefficient (n ≈ 7) does not vary significantly. A small but statistically significant increase in n was previously reported (Kentish et al., 1986), albeit based on sparser data, and was explained by invoking several mechanisms, e.g., interactions between adjacent tropomyosin molecules or alterations to the number of possible cross-bridges. Nonetheless, significant discrepancies even in the absolute values of n reported in other studies are also highlighted, potentially related to experimental conditions and the choice of skinning protocol.Open in a separate windowFigure 5.Dependence of the calcium sensitivity on sarcomere length. (a) Hill-type F-pCa for sarcomere lengths (SLs) = 1.85, 1.95, 2.05, 2.15, and 2.25 µm. Forces are normalized to the maximum force measured at SL = 2.05 µm. The data do not show a change in the Hill coefficient. (b) Increase in the Ca²⁺ sensitivity (decreasing [Ca²⁺] at half-maximum) with increasing SL, measured from the position of the inflection point in the fitted Hill curves from panel a. Adapted from Dobesh et al. (2002).The force-length relation in striated muscle underpins its central physiological role. Whereas the appeal of skinned muscle experiments for characterizing force generation is highlighted by numerous experiments, rationalizing quantitative differences remains notoriously challenging. In large part, this stems from the highly multifarious influence of the skinning process on the intracellular system and on details of the preparation protocol.Practical challenges: performing skinned muscle experimentsThe previous section illustrated the ability of skinned muscle preparations to reproduce intact muscle behavior while highlighting significant quantitative differences between the two systems. Clarifying the sources of these differences is crucial when developing practical applications that seek to exploit skinned muscle as a reductionist model for native-state muscle. One important hurdle is to correctly replicate the chemical and physiological intracellular environment, in particular with regard to [Mg²⁺], [ATP], pH, and the ionic strength. By tuning the experimental parameters to match the physiological conditions, the consistency between skinned and intact systems can be significantly improved (Gao et al., 1994; Mijailovich et al., 2021). Over decades, systematic efforts have sought to achieve this through detailed computations of the chemical equilibria of the bathing solutions (Fabiato, 1985a; Fabiato and Fabiato, 1975, 1977; Godt and Maughan, 1977; Moisescu, 1976). In practice, experimental protocols vary, sometimes idiosyncratically, between laboratories.This section outlines some of the elements of experimental protocols for skinned muscle that pose particular challenges insofar as they may significantly impact measurement outcomes.Bathing solution composition

ATP

After skinning, mitochondrial function is compromised, and hence, myocytes can no longer produce ATP (Rüegg, 2012). In multicellular tissue experiments, even a plentiful supply of ATP in the bathing solution may diffuse too slowly to maintain a homogeneous concentration throughout the fiber network (Godt, 1974). However, the inherent ATPase activity of muscle contraction implies a consumption of ATP supplies over the time of experiments. ATP-regenerating systems include creatine phosphate (typically 10–15 mM; Godt, 1974; Lamb and Stephenson, 2018). Nonetheless, in multicellular tissue, the rapid hydrolysis of ATP within the contractile system may yet produce an ATP concentration gradient between the interior and exterior of the network that inaccurately reflects the native state. This problem is arguably less serious in cardiac than skeletal myocytes (typical cardiac cell diameters are ∼13−20 µm, and lengths are ∼60−120 µm [Campbell et al., 1987, 1989; Liu et al., 1991], whereas skeletal muscle fiber diameters range from several microns to thousands of microns [Jimenez et al., 2013], with lengths sometimes reaching centimeters). However, the problem may yet arise in trabeculae.The physiological role of ATP in a given experiment, in addition to its participation in cross-bridge cycling, depends on the muscle preparation. In skeletal muscle experiments that preserve intracellular membrane structures (Endo and Iino, 1980; Launikonis and Stephenson, 1997), ATP governs calcium pumping into the SR (Godt, 1974; Lamb and Stephenson, 2018). This function is of course nonexistent in preparations where the SR has been dissolved. Alongside its role as energetic fuel, ATP also maintains the extensibility of the muscle by allowing myosin to dissociate from actin (Best et al., 1977; Weber and Murray, 1973).The decrease in maximum force with increasing [ATP] (in its physiological form MgATP; Fig. 6 b) is intuitively explained by the reduction in the number of formed cross-bridges (since ATP binding is associated with the release of rigor myosin; Best et al., 1977). An accompanying decrease in pCa₅₀ and an increase in the Hill coefficient (Fig. 6 a; Best et al., 1977) are both complicated by their Mg²⁺ dependence. These observations have been explained in terms of the effective cooperativity between neighboring cross-bridges in altering the inhibitory properties of troponin, which would arguably increase cross-bridge activation at a given [Ca²⁺] (Best, 1983; Best et al., 1977; Weber and Murray, 1973). However, this scenario is difficult to reconcile with analogous studies in skeletal muscle that report a qualitatively similar behavior for pCa₅₀ but with little [MgATP] dependence on maximum tension (Godt, 1974).Open in a separate windowFigure 6.Dependence of the force–calcium relationship on MgATP in the rat heart. (a) Decrease in Ca²⁺ sensitivity (increase in [Ca²⁺] at half-maximum) as [MgATP] increases from 30 to 100 µM ([Mg²⁺] = 50 µM). (b) Decrease in the maximum tension with increasing [MgATP]. Adapted from Best et al. (1977).

Mg²⁺

Mg²⁺, the second most abundant cation in muscle cells after K⁺, regulates the Ca²⁺ sensitivity of myofilament activity via its binding affinity to troponin (Alpert et al., 1979; Bers, 2001; Best, 1983; Best et al., 1977; Rayani et al., 2018; Tikunova and Davis, 2004). The Ca²⁺-specific low-affinity binding site (site II) at the N-terminal end of cardiac TnC serves as the principal initiator of contraction in the presence of Ca²⁺ (Bers, 2001). However, the structure of TnC is also controlled by binding sites III and IV, located at the C-terminal end, which competitively bind either Ca²⁺ (with high affinity) or Mg²⁺ (low affinity; Rayani et al., 2018; Tikunova and Davis, 2004). According to some cardiac muscle experiments, more Ca²⁺ is required to achieve a given degree of activation as [Mg²⁺] increases in the millimolar range (Best, 1983; Tikunova and Davis, 2004), consistent with competitive binding of these ions on TnC. However, this interpretation is contested by other cardiac experiments claiming negligible impact to the Ca²⁺ sensitivity under even an order-of-magnitude change in Mg²⁺ (Allen et al., 2000). The precise effect of Mg²⁺, while being potentially artifactual in some cases, may also vary with the dominant mechanism of action in the specific muscle system considered.Historically, setting the physiologically correct [Mg²⁺] has been challenging. Its determination requires the consideration of multiple binding equilibria and is naturally prone to uncertainty (Lamb and Stephenson, 2018). Given its relative abundance, cytosolic Mg²⁺ was initially assumed to merely ensure the balance for anionic charge, but its regulatory role was recognized subsequently. Various techniques have measured [Mg²⁺] (using spectrophotometry, Mg²⁺-sensitive electrodes, dye-based measurements, etc.). However, these measurements carry significant uncertainties, particularly given the difficulty of discerning free cytosolic Mg²⁺ from the total cellular magnesium (up to 20 times greater, contained in MgATP or cellular compartments) or interference from other ions (Romani and Scarpa, 1992). Many measurements report [Mg²⁺] as being consistently 0.4–0.8 mM but reaching up to 3.5 mM in some cases (Romani and Scarpa, 1992). In the intact rat heart specifically, values of 0.72 mM (from epifluorescence; Gao et al., 1994) or 0.85 mM (¹⁹F-NMR; Murphy et al., 1989) have been measured. [Mg²⁺] in excess of several millimolars are used in some studies but are known to be above the physiological level (Bers, 2001; Hunter et al., 1998).

pH

Intracellular pH in intact muscle regulates all the stages of tension generation, including the handling of Ca²⁺ by sarcolemmal electrophysiology, its delivery to the myofilaments, and the response of the filaments to the Ca²⁺ signal (Orchard and Kentish, 1990). This versatility makes it difficult to establish the relative significance of pH on sarcomere function specifically.In skinned muscle, a decrease in pH decreases pCa₅₀. The results in Fig. 7 show a 0.1% drop in pH producing a 0.1% drop in pCa₅₀ (Bers, 2001; Orchard and Kentish, 1990). The precise mechanism for this effect remains uncertain but may involve competition of H⁺ with Ca²⁺ for binding to TnC, interactions within the troponin complex, or the shielding of the net effective negative charge of the TnC binding site (Orchard and Kentish, 1990). Although a decrease in calcium sensitivity was also confirmed qualitatively in tetanized intact cardiac muscle (Marban and Kusuoka, 1987), the results differ quantitatively.Open in a separate windowFigure 7.Dependence of pH on the force-calcium relationship in guinea pig trabeculae. Adapted from Orchard and Kentish (1990).The observed decrease in maximal force resulting from decreasing pH in skinned muscle may be due to a direct impact on the efficiency of the coupling of ATP hydrolysis to cross-bridge force generation (Fig. 7; Orchard and Kentish, 1990). ATPase activity is affected by pH in intact muscle, albeit more weakly (Blanchard and Solaro, 1984; Kentish and Nayler, 1979; Orchard and Kentish, 1990). However, it is uncertain whether the same dominant mechanisms are relevant in the intact and skinned cases.The suitability of skinned muscle experiments for reliably investigating pH dependence is thus questionable. Bathing solutions for skinned muscle are typically designed with a high pH-buffering capacity (e.g., with 90 mM HEPES) to maintain a stable pH ∼7 (see Lamb and Stephenson, 2018).

Ionic strength

Ionic strength impacts inversely on the maximum force generated by skinned muscle (Fig. 8; Kentish, 1984). In practice, it can be controlled experimentally, in both cardiac and skeletal experiments, for example by varying KCl in the bathing soution (Kentish, 1984; Solaro et al., 1976). Reported ionic strength values range between 150 and 200 mM (Fig. 8). The inhibition of tension appears to be associated with Ca²⁺ binding, as this ionic strength dependence is [Ca²⁺] dependent only in the presence of MgATP (in skeletal muscle; Solaro et al., 1976). However, the precise ionic strength in intact muscle is uncertain (Gao et al., 1994), as reflected in the lack of consensus in the literature (see Open in a separate windowFigure 8.Dependence of generated tension on osmolarity. The osmolarity Γ/2 was controlled by varying (a) the Cl⁻ salt (filled circles: KCl; open circles: NaCl; diamonds: TMACl; triangles: choline Cl) or (b) K⁺ salt concentrations (filled circles: KCl, filled squares: K propionate; open square: K Mes), for pCa = 3.8. The consistency between the results suggests that the tension depends predominantly on the ionic strength rather than on the size of specific ions. From Kentish (1984). Fig. 8 reprinted with permission from Journal of Physiology.

Conclusion

The above considerations of ATP, Mg²⁺, pH, and ionic strength highlight the sensitivity of skinned muscle measurements to the precise solution composition. Establishing the correct recipe is made all the more challenging given that the impact on measured force generation varies between muscle systems and species. As argued above, although differences between measurements often appear to be quantitative, this does not exclude the possibility of qualitative differences in the dominant mechanisms of action. This fundamental ambiguity introduces considerable complication in translating results meaningfully to the intact system.TemperaturePhysiological function emerges from the balance of multiple temperature-dependent processes. Although measurements should thus ideally always be done at physiological temperature, lower temperatures are often used in practice due to the impaired stability of the sarcomere structure in skinned preparations at higher temperatures. This can have significant consequences on contraction, given the highly variable temperature sensitivities of different subcellular mechanisms (Rall and Woledge, 1990).There is widespread agreement that cooling reduces the maximum generated force in a wide range of muscle types and preparations (Fig. 9; Fabiato, 1985b; Godt and Lindley, 1982; Harrison and Bers, 1989; Stephenson and Williams, 1985; Sweitzer and Moss, 1990). This result has been argued to result more from a change in the force exerted by cross-bridges than from the number of cross-bridges formed (Sweitzer and Moss, 1990). In contrast, the temperature dependence of calcium sensitivity is less consistent. Skinned muscle displays either an increase (Brandt and Hibberd, 1976; Harrison and Bers, 1989; Orentlicher et al., 1977; Sweitzer and Moss, 1990) or a decrease in pCa₅₀ (Fabiato, 1985b; Godt and Lindley, 1982; Stephenson and Williams, 1985) with increasing temperature. However, the former result may be an artifact associated with heterogeneous shortening of sarcomeres at higher temperatures (Sweitzer and Moss, 1990).Open in a separate windowFigure 9.Temperature dependence of the F-pCa relationship in skinned trabeculae from the rabbit ventricle, showing an increase in both the maximum tension C_max and the sensitivity pCa₅₀ (pCa at half-maximum) with increasing temperature. Adapted from Harrison and Bers (1989).More recent work has revealed further complications in the regulatory role of temperature in muscle. In particular, temperature influences structural thick-filament regulation in both cardiac and skeletal muscle (Caremani et al., 2019, 2021; Park-Holohan et al., 2021). Reducing the temperature disrupts the orderly configuration of the myosin lever arms along the thick filaments, making them less available for force generation and causing an almost threefold decrease in total tissue force.The above experimental results highlight the multifaceted complexity of temperature dependence that arises from the interdependence of multiple molecular processes. Skinned preparations constitute only a subsystem within the overall muscle system, and there is therefore no guarantee that the kinetic balance within the reduced system is physiologically accurate.Sarcomere heterogeneityFor conceptual convenience, muscle tissue is often represented as a homogeneous assembly of identical sarcomeres acting in synchrony. This picture is simplistic in reality. Aspects of muscle dynamics, even under isometric conditions, derive specifically from the heterogeneous behavior at the sarcomere level. For example, within a myofibril, tension relaxation proceeds with the onset of rapid lengthening (“give”), initially in a single weak sarcomere, that then propagates to other sarcomeres along the myofibril (Edman and Flitney, 1982; Poggesi et al., 2005; Stehle, 2017). This effect accounts for the [P_i]-dependent asymmetry in the force kinetics that is observed in contraction-relaxation cycles when [Ca²⁺] is stepped up and down (Poggesi et al., 2005). It also suggests that relaxation kinetics is governed not only by the rate-limiting steps of the cross-bridge cycle of a generic myosin molecule but also by collective effects at a higher structural level.This effect arguably escapes notice in skinned-fiber experiments that exploit the flash photolysis of caged compounds to time-resolve the details of cross-bridge–cycle kinetics (e.g., the photorelease of inorganic phosphate P_i modulates cross-bridge kinetics; Araujo and Walker, 1996; Dantzig et al., 1992; Millar and Homsher, 1990; Tesi et al., 2000). These experiments suffer from important practical limitations. In particular, the relatively modest (unidirectional) changes in [P_i] achievable by photorelease fail to disrupt the chemomechanical equilibrium of the sarcomeres sufficiently to generate heterogeneous give. Under these near-equilibrium conditions, observed changes in force are more likely to reflect rate-limiting single-cross-bridge kinetics than transients in sarcomere heterogeneity. This obstacle was bypassed in experiments done on isolated myofibrils, which, in contrast, allow sufficiently large jumps in [P_i] (in both directions) to be imposed by rapid solution change (Poggesi et al., 2005; Stehle, 2017). By monitoring the progression of tension decay in conjunction with the lengths of individual sarcomeres, these experiments highlight the role of sarcomere dynamics in accounting for tension relaxation. Compared with skinned-tissue experiments, they also provide better consistency with the relaxation kinetics (k_TR) observed in mechanically induced force redevelopment (Stehle, 2017).Practical considerationsThe preceding discussion has highlighted the value of skinned muscle in emulating the essential features of intact muscle contraction in vivo. On the other hand, we have also described how discrepancies between intact and skinned muscle properties are sufficiently significant as to mar the prospect of considering skinned preparations as unambiguous surrogates. The underlying causes are complex, and it is often difficult to distinguish between experimental artifacts and manifestations of genuine physiological differences. This complexity is further compounded by species- or system-dependent specificities (e.g., cardiac versus skeletal muscle). Consequently, in practice, experimental protocols often evolve organically within laboratory communities, based on direct observations and acquired practical knowhow. Interestingly, a recent meta-analysis of published measurements of specific force in skinned human skeletal muscle noted a greater consistency in the results obtained within research groups (defined in terms of commonalities in authorship) than between them (Kalakoutis et al., 2021). This observation could be interpreted as revealing a genealogy of sorts in the evolution of protocols that is at odds with rigorous and objective development, thereby possibly mitigating the appeal of the experiments altogether.Tempting as it may be to imagine a universally applicable method, we feel it would be counterproductive to seek to disentangle and confront the rationales of individual protocols, with the risk of dogmatically promoting one valid method among several. The very idea of a unique universal recipe, valid for all experiments, is indeed highly questionable. As a more fruitful approach, we instead present the following themes as set of general guiding principles for encouraging good experimental practice.Monitoring sarcomeric dynamicsGiven the importance of sarcomere length and interfilament dynamics in force generation, we recommend that mechanical force measurements be accompanied by the simultaneous measurement of striation patterns. This would include the mean sarcomere length and, ideally, an index of heterogeneity and/or stability. We recognize that these measurements may be particularly challenging in cardiac trabeculae.Fixing the pHEnsuring the constancy of pH is paramount for ensuring consistency in measurements. This is achieved by applying a suitable buffer, in many cases imidazole.Saturation with ATPA useful simplification of the experimental system is to ensure that the cross-bridge cycling kinetics is not rate-limited by ATP. In most cases, this can be achieved by using solutions with at least 4 mM free ATP.Careful control of [Ca²⁺]The importance of correctly determining the concentration of free Ca²⁺ cannot be sufficiently emphasized. Some laboratories use pCa solutions based on recipes that originate with Fabiato and Fabiato (1979) or Godt and Lindley (1982). Those wishing to make new recipes can consider using the MaxChelator software suite (Bers et al., 2010; Patton et al., 2004), which can provide appropriate stoichiometric concentrations of Ca²⁺, Mg²⁺, EGTA, and ATP for use in experimental solutions. A useful recipe for producing buffers with varying [Ca²⁺] is to prepare “low” and “high” reference buffers (e.g., with pCa = 9.0 and 4.5) and to mix them in appropriate proportions.Choice of temperatureGiven the importance of temperature as a determinant of muscle kinetics, it stands to reason that experiments should be done at physiological temperatures. However, a practical drawback is its destabilization of the sarcomere structure. Skeletal fibers have historically been measured at lower temperatures (sometimes even near above freezing) to ensure that preparations last the experiment duration. Many experiments on both skeletal and cardiac muscle can be done at 15°C. However, it is worth noting that rodent myocardium is more fragile than human (where room temperature or even 37°C is possible), possibly owing to differences in metabolic and ATPase rates. As a general recommendation, we would encourage experimentalists to choose temperatures that are nearest to physiological conditions where the preparation is stable. It is, however, perhaps even more important to only compare experimental results obtained at the same temperature.ConclusionThe aim of this review was to survey the benefits of skinned muscle measurements for characterizing cardiac muscle physiology, while highlighting intrinsic challenges for both the conduct and the interpretation of measurements. These features are summarized in Strengths• Direct access to the sarcomere system• Separation of cellular subsystems (e.g., sarcomeres versus sarcolemma)• Ability to use fluorescent probes and other analytic tools• Convenience of controllably performing different standardized experiments (e.g., isometric/isotonic contractions)• Ability to perform protein exchange experiments that preserve overall functionality (e.g., troponin; Babu et al., 1988; Brenner et al., 1999; Gulati and Babu, 1989); and to probe time-resolve sarcomere dynamics by photolysis of caged compounds (ATP [Goldman et al., 1982, 1984], inorganic phosphate [Araujo and Walker, 1996; Dantzig et al., 1992; Millar and Homsher, 1990; Tesi et al., 2000], and Ca²⁺ chelators [Luo et al., 2002; Wahr et al., 1998])• Simpler handling and storage logistics (samples can be thawed and analyzed after prior freezing) Weaknesses • Challenge of reproducing the native physiological environment• Variations in results between laboratories• Instability and sensitivity to temperature• Challenges of [Ca²⁺] calibration• Structural changes caused by skinning (e.g., altered sarcomere morphology, loss of cellular heterogeneity), impacting functional behaviorOpen in a separate windowThe potential pitfalls of mischaracterizing sarcomere behavior, based on skinned muscle measurements, are particularly exposed when considering the broader physiological context, where different cardiac subsystems operate simultaneously (Mosqueira et al., 2019; Niederer et al., 2019b). Pharmacological research increasingly exploits skinned muscle experiments to assess targeted drug action on sarcomeres (Dou et al., 2007; Edes et al., 1995; Fitton and Brogden, 1994; Hara et al., 1999; Kobayashi et al., 1991; Lamont and Miller, 1992; Lee and Allen, 1997; Lues et al., 1988; Scheld et al., 1989; Solaro and Rüegg, 1982; Sudo et al., 2001; Tadano et al., 2010). However, drug impact is notoriously multifaceted, and side effects, unseen in the isolated sarcomeres, may readily and unpredictably overwhelm intended effects (Lee and Allen, 1997; Lues et al., 1993). These side effects notwithstanding, the extrapolation of skinned-muscle measurements to the native cellular state and to systemic cardiac function encounters significant interpretational hurdles, as illustrated above.Skinned muscle measurements carry intrinsic uncertainty, as experiments performed using different animal models, temperatures, and protocols occasionally produce contradictory characterizations. Approximate quantitative accuracy is obviously highly problematic in the perspective of developing customized clinical care. This requirement is particularly important given the modular nature of models and the need to combine interacting subsystems on different length scales (Niederer et al., 2019a, 2019b). In practice, the interfacing of such modules normally requires ad hoc empirical alterations to model parameters, often relying on the modeler’s judgment (Hunter et al., 1998; Land et al., 2017). These choices are naturally often speculative.Despite these difficulties, it would be wrong to misrepresent the true potential of skinned-muscle experiments. Just as animal models are essential for investigating human physiology, skinned muscle provides an experimental setting with unique benefits. Biophysical modeling helps to formalize the conceptual basis for interpreting experimental data in terms of specific mechanisms (for example, an observed variation in pCa₅₀ may result from changes to troponin binding kinetics or cross-bridge formation). Global sensitivity analyses allow a ranking of the relative importance of individual model parameters, thus providing a handle for guiding judgment in how to use measurement-derived parameters (Longobardi et al., 2020). In this perspective, the benefit of models is in providing a framework for formulating and testing hypotheses, rather than delivering fixed and absolute representations of the muscle system.The appeal of skinned muscle preparations is best appreciated by seeing them not as a direct emulation of real muscle, but rather as one further element in the physiologist’s experimental armory. This issue is well illustrated by Irving and Craig (2019) with reference to a loosening of the thick-filament structure induced by cardiac myosin-binding protein C phosphorylation. This effect was manifested as a structural change in skinned cardiac muscle but may be eclipsed in the compact and crowded conditions of intact muscle. In such circumstances, attempting to reconcile the experiments, even qualitatively, may seem futile. Yet the skinned-muscle effect may well be the telltale indicator of a genuine regulatory mechanism that would otherwise remain invisible and unmeasurable in the intact system. Rather than seeking a literal mirroring of these skinned and intact experiments at any cost, additional physiological insight might potentially be gained by further pursuing the experiments, and comparing their quantitative results in parallel, in other cell types or under different experimental conditions. Ultimately, the integration of experimental findings remains a continual process involving a balance of pragmaticism and biophysically guided scientific judgment.