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1.
Stephane Sarrazin William C. Lamanna Jeffrey D. Esko 《Cold Spring Harbor perspectives in biology》2011,3(7)
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).
Open in a separate windowHS, heparan sulfate; CS, chondroitin sulfate; PG, proteoglycan.aThe variation in core mass is because of species differences.bThe number of chains is based on the number of putative attachment sites for chain initiation as well as data from the literature; the actual number of chains varies by method, tissue, and species.
Open in a separate window
Open in a separate windowFigure 1 shows in pictorial form many of the systems in which HSPGs participate.
Table 1.
Heparan sulfate proteoglycansClass | Proteoglycan | Core mass (kDa)a | Chain type (number)b | Tissue | Human disease |
---|---|---|---|---|---|
Membrane-bound | Syndecan-1–syndecan-4 | 31–45 | HS (2–3) in Sdc2 and Sdc4; HS/CS (3–4 HS/1-2 CS) in Sdc1 and Sdc3 | Epithelial cells, fibroblasts | |
Glypican-1–glypican-6 | 57–69 | HS (1–3) | Epithelial cells, fibroblasts | Simpson–Golabi–Behmel syndrome (overgrowth) (GPC3) (Pilia et al. 1996); omodysplasia (skeletal dysplasia) (GPC6) (Campos-Xavier et al. 2009) | |
Betaglycan (part-time PG) | 110 | HS/CS (1–2) | Fibroblasts | ||
Neuropilin-1 (part-time PG) | 130 | HS or CS (1) | Endothelial cells | ||
CD44v3 | 37 | HS (1) | Lymphocytes | ||
Secretory vesicles | Serglycin | 10–19 | Heparin/CS (10–15) | Mast cells, hematopoietic cells | |
Extracellular matrix | Perlecan | 400 | HS (1–4) | Basement membranes | Schwartz–Jampel syndrome (skeletal dysplasia) (Nicole 2000; Arikawa-Hirasawa et al. 2001) |
Agrin | 212 | HS (2–3) | Basement membranes | ||
Collagen XVIII | 150 | HS (1–3) | Epithelial cells, basement membranes | Knobloch syndrome type I (Sertie et al. 2000) |
Table 2.
Mutants altered in HSPG core proteinsGene | Proteoglycan | Phenotype (references) |
---|---|---|
Sdc1 | Syndecan-1 | Null allele: viable; increase in inflammation-mediated corneal angiogenesis (Gotte et al. 2002, 2005); corneal epithelial cells migrate more slowly, show reduced localization of α9 integrin during wound closure and fail to increase in proliferation after wounding (Stepp et al. 2002); enhanced leukocyte-endothelial interaction in the retina (Gotte et al. 2002, 2005); increase in medial and intimal smooth muscle cell replication and neointimal lesion after injury (Fukai et al. 2009); reduced cardiac fibrosis and dysfunction during angiotensin II–induced hypertension (Schellings et al. 2010); not required for follicle initiation and development (Richardson et al. 2009); accumulates plasma triglycerides, and shows prolonged circulation of injected human VLDL and intestinally derived chylomicrons (Stanford et al. 2009); juvenile mice resistant to carcinogen-induced tumorigenesis (McDermott et al. 2007); increased basal protein leakage and more susceptible to protein loss induced by combinations of IFN-γ, TNF-α, and increased venous pressure (Bode et al. 2008); exacerbates anti-GBM nephritis shifting Th1/Th2 balance toward a Th2 response (Rops et al. 2007); no role in hepatocyte infection by Plasmodium yoelii sporozoites (Bhanot 2002); normal larval development of Trichinella spiralis, but modestly reduced Th2 responses during infection (Beiting et al. 2006); less susceptible to Pseudomonas aeruginosa infection (Haynes et al. 2005); reduced P. aeruginosa infection rate and virulence (Park et al. 2001); protected from Staphylococcus aureus beta-toxin-induced lung injury (Hayashida et al. 2009a); exaggerated airway hyperresponsiveness, eosinophilia, and lung IL-4 responses to allergens (Xu et al. 2005); exaggerated CXC chemokines, neutrophilic inflammation, organ damage, and lethality in LPS endotoxemia (Hayashida et al. 2009b); prolonged recruitment of inflammatory cells in dextran sodium sulfate (DSS)-induced colitis and delayed type hypersensitivity (Masouleh et al. 2009; Floer et al. 2010). |
Sdc2 | Syndecan-2 | No mutants reported. Sdc2 antisense impairs angiogenesis in human microvascular endothelial cells (Noguer et al. 2009); morpholinos inhibit cell migration and fibrillogenesis during embryogenesis in zebrafish (Arrington and Yost 2009). |
Sdc3 | Syndecan-3 | Null allele: viable; altered feeding behavior (Strader et al. 2004); no phenotype in synovial endothelial cells (Patterson et al. 2005); enhanced long-term potentiation (LTP) in area CA1 (brain) and impaired performance in tasks assessing hippocampal function (Kaksonen et al. 2002); more sensitive to inhibition of food intake by the melanocortin agonist MTII (Reizes et al. 2003); perturbs laminar structure of the cerebral cortex as a result of impaired radial migration, and neural migration in the rostral migratory stream is impaired (Hienola et al. 2006); novel form of muscular dystrophy characterized by impaired locomotion, fibrosis, and hyperplasia of myonuclei and satellite cells (Cornelison et al. 2004). |
Sdc4 | Syndecan-4 | Null allele: viable; enhanced fibrin deposition in degenerating fetal vessels in the placental labyrinth (Ishiguro et al. 2000); delayed angiogenesis in wound granulation tissue (Echtermeyer et al. 2001); defective subcellular localization of mTOR Complex2 and Akt activation in endothelial cells, affecting endothelial cell size, NOS, and arterial blood pressure (Partovian et al. 2008); decreased macrophage uptake of phospholipase A2-modified LDL (Boyanovsky et al. 2009); mesangial expansion, enhanced matrix collagens I and IV, fibronectin and focal segmental glomerulosclerosis in males, and induction of Sdc2 in glomeruli (Cevikbas et al. 2008); more susceptible to hepatic injury, and thrombin-cleaved form of osteopontin is significantly elevated after concanavalin-A injection (Kon et al. 2008); less damage in osteoarthritic cartilage in a surgically induced model of osteoarthritis (Echtermeyer et al. 2009); explanted satellite cells fail to reconstitute damaged muscle and are deficient in activation, proliferation, MyoD expression, myotube fusion, and differentiation (Cornelison et al. 2004); vibrissae are shorter and have a smaller diameter because of suboptimal response to fibroblast growth factors (Iwabuchi and Goetinck 2006); lower phosphorylation levels of focal adhesion kinase (Wilcox-Adelman et al. 2002); random migration of fibroblasts as a result of high delocalized Rac1 activity (Bass et al. 2007); defective RGD-independent cell attachment to transglutaminase-fibronectin matrices (Telci et al. 2008); impaired suppression of production of IL-1β by TGF-α (Ishiguro et al. 2002); decreased neutrophil recruitment and increased myofibroblast recruitment and interstitial fibrosis after bleomycin-treatment, no inhibition of fibrosis with recombinant CXCL10 protein (Jiang et al. 2010); hypersensitivity to LPS because of decreased TGF-β suppression of IL-1 production in monocytes and neutrophils (Ishiguro et al. 2001). |
Gpc1 | Glypican-1 | Null allele: viable; reduced brain size (Jen et al. 2009). Athymic mutant mice show decreased tumor angiogenesis and metastasis (Aikawa et al. 2008). |
Gpc2 | Glypican-2 | No mutants reported. |
Gpc3 | Glypican-3 | Null allele: viable; resembles Simpson–Golabi–Behmel overgrowth syndrome, including somatic overgrowth, renal dysplasia, accessory spleens, polydactyly, and placentomegaly (Cano-Gauci et al. 1999; Chiao et al. 2002); defects in cardiac and coronary vascular development (Ng et al. 2009); alterations in Wnt signaling, in vivo inhibition of the noncanonical Wnt/JNK signaling, activation of canonical Wnt/β-catenin signaling (Song et al. 2005); increased Hedgehog signaling (Capurro et al. 2008); abnormal rates of proliferation and apoptosis in cortical and medullary collecting duct cells (Grisaru et al. 2001); delay in endochondral ossification, impairment in the development of the myelomonocytic lineage (Viviano et al. 2005). |
Gpc4 | Glypican-4 | Zebrafish knypek controls cell polarity during convergent extension (Topczewski et al. 2001); craniofacial skeletal defects in adult fish (LeClair et al. 2009). |
Gpc5 | Glypican-5 | No mutants reported. |
Gpc6 | Glypican-6 | Impaired endochondral ossification and omodysplasia (Campos-Xavier et al. 2009). |
Tgfbr3 | Betaglycan | Null allele: embryonic lethal; heart and liver defects (Stenvers et al. 2003); defect in seminiferous cord formation in E12.5–13.5 embryos (Sarraj et al. 2010). |
Hspg2 | Perlecan | Null allele: embryonic lethal (E10–12); developmental angiogenesis altered in zebrafish (Zoeller et al. 2009); high incidence of malformations of the cardiac outflow tract, lack of well-defined spiral endocardial ridges (Costell et al. 2002); lower amounts of collagen IV and laminins in embryonic hearts, reduced function in infarcted hearts from heterozygous mice (Sasse et al. 2008); absence of acetylcholinesterase at the neuromuscular junctions (Arikawa-Hirasawa et al. 2002); cephalic and skeletal abnormalities (Arikawa-Hirasawa et al. 1999); cerebral ectopias, exencephaly (Girós et al. 2007); increased cross-sectional area of myosin heavy chain type IIb fibers in the tibialis anterior muscle (Xu et al. 2010b); diminished osteocyte canalicular pericellular area (Thompson et al. 2011). |
Exon 3 deletion (Hspg23/3) viable: proteinuria after protein loading (Morita et al. 2005); monocyte/macrophage influx impaired in Hspg23/3Col18a1−/– mice in a model of renal ischemia/reperfusion (Celie et al. 2007). | ||
Secreted as CSPG in some tissues (Danielson et al. 1992; Govindraj et al. 2002; Vogl-Willis and Edwards 2004; West et al. 2006), but relationship of CSPG isoform to phenotypes not established. | ||
Prg1 | Serglycin | Null allele: viable; secretory granule defects in mast cells (Abrink et al. 2004); dense core formation is defective in mast cell granules (Henningsson et al. 2006); defective secretory granule maturation and granzyme B storage in cytotoxic T cells (Grujic et al. 2005); no effect on macrophages (Zernichow et al. 2006); platelets and megakaryocytes contain unusual scroll-like membranous inclusions (Woulfe et al. 2008); enlargement of multiple lymphoid organs, decrease in the proportion of CD4+ cells, more pronounced airway inflammatory response in older mice (Wernersson et al. 2009); increased virulence of Klebsiella pneumoniae (Niemann et al. 2007); defective regulation of antiviral CD8+ T-cell responses (Grujic et al. 2008). |
Agrn | Agrin | Null allele: embryonic lethal; reduced number, size, and density of postsynaptic acetylcholine receptor aggregates in muscles; abnormal intramuscular nerve branching and presynaptic differentiation (Gautam et al. 1996,1999); smaller brains (Serpinskaya et al. 1999); abnormal development of interneuronal synapses (Gingras et al. 2007); increased resistance to excitotoxic injury (Hilgenberg et al. 2002); reduced number of cortical presynaptic and postsynaptic specializations (Ksiazek et al. 2007). |
Floxed allele: Inactivation in podocytes does not affect glomerular charge selectivity or glomerular architecture (Harvey et al. 2007). | ||
Col18a1 | Collagen XVIII | Null allele: viable; increased microvascular growth (Li and Olsen 2004); increased angiogenesis associated with atherosclerotic plaques (Moulton et al. 2004); delayed regression of blood vessels in the vitreous along the surface of the retina after birth and lack of or abnormal outgrowth of retinal vessels (Fukai et al. 2002); larger choroidal neovascularization lesions and increased vascular leakage (Marneros et al. 2007); accelerated healing and vascularization rate of excisional dorsal skin wounds (Seppinen et al. 2008); anomalous anastomoses of vasculature; disruption of the posterior iris pigment epithelial cell layer with release of melanin granules, severe thickening of the stromal iris basement membrane zone (Marneros and Olsen 2003); increase in the amount of retinal astrocytes (Hurskainen et al. 2005); more severe glomerular and tubulointerstitial injury in induced anti-GBM glomerulonephritis (Hamano et al. 2010); monocyte/macrophage influx impaired in Hspg23/3Col18a1−/– mice in a model of renal ischemia/reperfusion (Celie et al. 2007); mild chylomicronemia (Bishop et al. 2010). |
Table 3.
Mouse mutants altered in HS biosynthesisGene | Enzyme | Phenotype |
---|---|---|
Xt1 | Xylosyltransferase-1 | No mutants reported. |
Xt2 | Xylosyltransferase-2 | Null allele: viable; polycystic kidney and livers (Condac et al. 2007). |
GalTI (β4GalT7) | Galactosyltransferase I | Human mutants: defective chondroitin substitution of decorin and biglycan in an Ehlers–Danlos patient (Gotte and Kresse 2005; Seidler et al. 2006). |
GalTII (β3GalT6) | Galactosyltransferase II | No mutants reported. |
Glcat1 | Glucuronyltransferase I | Null allele: embryonic lethal (4–8-cell stage) (Izumikawa et al. 2010). |
Extl3 | N-acetylglucosaminyl transferase I | Floxed allele: Inactivation in islets decreases growth and insulin secretion (Takahashi et al. 2009). |
Ext1/Ext2 | HS Copolymerase (N-acetylglucosaminyl-glucuronyltransferase) | Null allele: embryonic lethal (E6-7.5); lack of mesoderm differentiation (Lin et al. 2000; Stickens et al. 2005); heterozygotes develop rib growth plate exostoses (Stickens et al. 2005; Zak et al. 2011); unaltered vascular permeability in heterozygous mice (Xu et al. 2010a). |
Floxed allele of Ext1: defective brain morphogenesis and midline axon guidance after nestin-Cre inactivation (Inatani et al. 2003); no effect on adaptive immune response in CD15Cre mice (Garner et al. 2008); altered T-cell and dendritic cell homing to lymph nodes in Tie2Cre mice (Bao et al. 2010); rib growth plate exostosis formation in Col2Cre mice (Jones et al. 2010; Matsumoto et al. 2010; Zak et al. 2011). | ||
Ndst1 | N-acetylglucosaminyl N-deacetylase/N-sulfotransferase-1 | Null allele: Perinatal lethal; lung hypoplasia, defective forebrain, lens, and skull development (Fan et al. 2000; Ringvall et al. 2000; Grobe et al. 2005; Pan et al. 2006). |
Floxed allele: decreased chemokine transcytosis and presentation and neutrophil infiltration in Tie2Cre mice (Wang et al. 2005); decreased allergen-induced airway hyperresponsiveness and inflammation because of reduction in recruitment of eosinophils, macrophages, neutrophils, and lymphocytes in Tie2Cre mice (Zuberi et al. 2009); decreased pathological angiogenesis in Tie2Cre mice (Fuster et al. 2007); decreased vascular VEGF-induced hyperpermeability (Xu et al. 2010a); decreased vascular smooth muscle cell proliferation, vessel size, and vascular remodeling after arterial injury in SM22αCre mice (Adhikari et al. 2010a); mild effect on T-cell response in Tie2Cre;Ndst2−/−mice (Garner et al. 2008); defective lacrimal gland development and Fgf10-Fgfr2b complex formation and signaling in LeCre mice (Pan et al. 2008); defective lobuloalveolar development in mammary gland (Crawford et al. 2010). | ||
Ndst2 | N-acetylglucosaminyl N-deacetylase/N-sulfotransferase-2 | Null allele: viable; mast cell deficiency and defective storage of proteases (Forsberg et al. 1999; Humphries et al. 1999); compounding mutation with Ndst1 reduces l-selectin interactions (Wang et al. 2005). |
Ndst3 | N-acetylglucosaminyl N-deacetylase/N-sulfotransferase-3 | Null allele: viable; floxed allele available (Pallerla et al. 2008). |
Ndst4 | N-acetylglucosaminyl N-deacetylase/N-sulfotransferase-4 | No mutants reported. |
Glce | Uronyl C5 epimerase | Null allele: perinatal lethal; renal agenesis (Li et al. 2003). |
H2st | Uronyl 2-O-sulfotransferase | Null allele: perinatal lethal; renal agenesis; skeletal and ocular defects (Bullock et al. 1998; Merry et al. 2001); defective cerebral cortical development (McLaughlin et al. 2003); altered lacrimal gland development (Qu et al. 2011). |
Floxed allele: altered lipoprotein clearance in AlbCre mice (Stanford et al. 2010); altered branching morphogenesis in the mammary gland (Garner et al. 2011). | ||
H3st1 | Glucosaminyl 3-O-sulfotransferase 1 | Null allele: partially penetrant lethality; no alteration in coagulation (HajMohammadi et al. 2003); fertility defects because of impaired ovarian function and placenta development (Shworak et al. 2002; HajMohammadi et al. 2003). |
H3st2 | Glucosaminyl 3-O-sulfotransferase 2 | Null allele; viable; no neuronal phenotype (Hasegawa and Wang 2008). |
H3st3 | Glucosaminyl 3-O-sulfotransferase 3 | No mutants reported. |
H3st4 | Glucosaminyl 3-O-sulfotransferase 4 | No mutants reported. |
H3st5 | Glucosaminyl 3-O-sulfotransferase 5 | No mutants reported. |
H3st6 | Glucosaminyl 3-O-sulfotransferase 6 | No mutants reported. |
H6st1 | Glucosaminyl 6-O-sulfotransferase 1 | Null allele: embryonic lethal (Habuchi et al. 2007; Sugaya et al. 2008). |
Gene trap allele: embryonic lethal; retinal axon guidance defects (Pratt et al. 2006). | ||
Floxed allele: systemic inactivation embryonic lethal (Izvolsky et al. 2008); no change in plasma triglycerides in AlbCre mice (Stanford et al. 2010). | ||
H6st2 | Glucosaminyl 6-O-sulfotransferase 2 | Null allele: viable (Sugaya et al. 2008); HS6ST-2, but not HS6ST-1, morphants in zebrafish show abnormalities in the branching morphogenesis of the caudal vein (Chen et al. 2005). |
H6st3 | Glucosaminyl 6-O-sulfotransferase 3 | No mutants reported. |
Hpa | Heparanase, transgene | Accelerated wound angiogenesis, enhanced delayed type hypersensitivity response (Zcharia et al. 2005; Edovitsky et al. 2006; Ilan et al. 2006); accumulation of intracellular crystals of protein Ym1 in macrophages (Waern et al. 2010); resistance to amyloid protein A amyloidosis (Li et al. 2005); age-related enlargement of lymphoid tissue and altered leukocyte composition (Wernersson et al. 2009). |
Hpa | Heparanase | Null allele: viable; altered MMP-2 and MMP-14 expression (Zcharia et al. 2009). |
Sulf1 | Endo-6-sulfatase 1 | Null allele: viable; esophageal defect (Ai et al. 2007; Ratzka et al. 2008); enhanced osteoarthritis, MMP-13, ADAMTS-5, and noggin elevated, col2a1 and aggrecan reduced in cartilage and chondrocytes (Otsuki et al. 2010). |
Sulf2 | Endo-6-sulfatase 2 | Null allele: viable; behavioral defects (Lamanna et al. 2006); enhanced osteoarthritis, MMP-13, ADAMTS-5, and noggin elevated, col2a1 and aggrecan reduced in cartilage and chondrocytes (Otsuki et al. 2010). |
Gene trap allele: Sulf2GT(pGT1TMpfs)155Ska, no phenotype (Lum et al. 2007). |
- HSPGs are present in basement membranes (perlecan, agrin, and collagen XVIII), where they collaborate with other matrix components to define basement membrane structure and to provide a matrix for cell migration.
- HSPGs are found in secretory vesicles, most notably serglycin, which plays a role in packaging granular contents, maintaining proteases in an active state, and regulating various biological activities after secretion such as coagulation, host defense, and wound repair.
- HSPGs can bind cytokines, chemokines, growth factors, and morphogens, protecting them against proteolysis. These interactions provide a depot of regulatory factors that can be liberated by selective degradation of the HS chains. They also facilitate the formation of morphogen gradients essential for cell specification during development and chemokine gradients involved in leukocyte recruitment and homing.
- HSPGs can act as receptors for proteases and protease inhibitors regulating their spatial distribution and activity.
- Membrane proteoglycans cooperate with integrins and other cell adhesion receptors to facilitate cell-ECM attachment, cell–cell interactions, and cell motility.
- Membrane HSPGs act as coreceptors for various tyrosine kinase-type growth factor receptors, lowering their activation threshold or changing the duration of signaling reactions.
- Membrane HSPGs act as endocytic receptors for clearance of bound ligands, which is especially relevant in lipoprotein metabolism in the liver and perhaps in the formation of morphogen gradients during development.
2.
Alex Lewalle Kenneth S. Campbell Stuart G. Campbell Gregory N. Milburn Steven A. Niederer 《The Journal of general physiology》2022,154(2)
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 Reference System Intact Skinning method [Mg2+] (mM) Ionic strength (mM) pH Reuben et al. (1971) Crayfish EGTA - 300 7.0 Winegard (1971) Frog cardiac EDTA 1 - 6.5–7.0 Matsubara and Elliott (1972) Frog skeletal X Dissection 1 - 7.0 Godt (1974) Frog skeletal Dissection 5 150 7.3 Wood et al. (1975) Human skeletal EGTA 2–4 - 7.0 Moisescu (1976) Frog skeletal Dissection 1 150 7.1 Godt and Maughan (1977) Frog skeletal X Dissection 3 150 7.0 Best et al. (1977) Rat cardiac Homogenization 0.05, 1 150 7.0 Trube (1978) Mouse cardiac Dissection (partial) 4 132 7.0 Gordon (1978) Rabbit smooth Triton X-100 1.0–6.9 130 7.0 Stienen et al. (1983) Frog skeletal Freeze drying 1.1 160 7.0 Fabiato and Fabiato (1975, 1978a, 1978b) Rat cardiac Dissection 0.32 160 7.0 Fabiato and Fabiato (1978a) Frog skeletal Dissection 0.32 160 7.0 Fabiato (1981) Rat cardiac X EGTA 1 160 7.1 Fabiato (1981) Rabbit cardiac X EGTA 1 160 7.1 Fabiato (1985b) Canine cardiac Dissection 3 170 7.1 Hibberd and Jewell (1982) Rat cardiac Brij-58 0.3 200 7.0 Solaro et al. (1971, 1976); Solaro and Rüegg (1982) Canine cardiac Triton X-100 Var 100 7.0 Donaldson (1985) Rabbit skeletal Dissection 1 150 7.0 Kentish et al. (1986) Rat cardiac X Triton X-100 3 200 7.0 Fill and Best (1988) Frog skeletal Dissection 1 150 7.0 Lues et al. (1988) Various cardiac Triton X-100 - 140 6.7 Roos and Brady (1989) Rat cardiac X Triton X-100 - 160 7.1 Scheld et al. (1989) Human cardiac Lubrol PX - 140 6.7 Harrison and Bers (1989) Rabbit cardiac Triton X-100 2.2 - 7.0 Lamb and Stephenson (1990) Toad skeletal Dissection 1 - 7.10 Gwathmey and Hajjar (1990) Human cardiac X Saponin 3 160 7.1 Sweitzer and Moss (1990) Rat cardia, rabbit skeletal Triton X-100 1 180 7.0 Millar and Homsher (1990) Rabbit skeletal EGTA 1 200 7.1 De Beer et al. (1992) Rabbit skeletal Freeze drying - - - Gross et al. (1993) Guinea pig cardiac Triton X-100 - - 7.4 Gao et al. (1994) Rat cardiac X Triton X-100 1.2 - 7.0 Wolff et al. (1995a) Canine cardiac Triton X-100 1 180 7.0 Edes et al. (1995) Guinea pig cardiac Saponin - 160 7.4 Araujo and Walker (1996) Rat cardiac Triton X-100 1 180 - Allen et al. (2000) Rat cardiac Triton X-100 1–8 150 7.0 Posterino et al. (2000) Rat skeletal Dissection 1 - 7.1 Irving et al. (2000) Rat trabeculae X Triton X-100 - 200 7.35 Patel et al. (2001) Mouse cardiac Saponin + Triton X-100 - 180 7.0 Konhilas et al. (2002) Rat trabeculae Triton X-100 1 180 - Luo et al. (2002) Rabbit skeletal Triton X-100 1 180 7.0 Fukuda et al. (2003) Bovine cardiac Triton X-100 1 180 7.0 Prado et al. (2005) Rabbit skeletal X Triton X-100 - 180 7.0 Fukuda et al. (2005) Bovine and rat cardiac Triton X-100 1 180 7.0 Stelzer et al. (2006) Mouse cardiac Saponin + Triton X-100 1 180 7.0 Terui et al. (2010) Pig cardiac Triton X-100 1 180 7.0 Gillis and Klaiman (2011) Fish cardiac Triton X-100 1 170 7.0 Curtin et al. (2015) Rabbit skeletal X Triton X-100 2 200 7.1 Li et al. (2016) Rabbit skeletal Triton X-100 - 180 7.0 Land et al. (2017) Human cardiac Triton X-100 1 200 7.1 Stehle (2017) Guinea pig cardiac Triton X-100 - 170 7.0 Breithaupt et al. (2019) Rat cardiac Glycerol + Triton X-100 1 200 7.0 Giles et al. (2019) Mouse cardiac Saponin + Triton X-100 1 180 7.0 Azimi et al. (2020) Rat skeletal Dissection 1 - 7.1 Rebbeck et al. (2020) Human and rat skeletal Dissection 1 - 7.4 Palmer et al. (2020) Mouse cardiac Triton X-100 1 200 7.0