Revertant Fibers in the mdx Murine Model of Duchenne Muscular Dystrophy: An Age- and Muscle-Related Reappraisal

Muscles in Duchenne dystrophy patients are characterized by the absence of dystrophin, yet transverse sections show a small percentage of fibers (termed “revertant fibers”) positive for dystrophin expression. This phenomenon, whose biological bases have not been fully elucidated, is present also in the murine and canine models of DMD and can confound the evaluation of therapeutic approaches. We analyzed 11 different muscles in a cohort of 40 mdx mice, the most commonly model used in pre-clinical studies, belonging to four age groups; such number of animals allowed us to perform solid ANOVA statistical analysis. We assessed the average number of dystrophin-positive fibers, both absolute and normalized for muscle size, and the correlation between their formation and the ageing process. Our results indicate that various muscles develop different numbers of revertant fibers, with different time trends; besides, they suggest that the biological mechanism(s) behind dystrophin re-expression might not be limited to the early development phases but could actually continue during adulthood. Importantly, such finding was seen also in cardiac muscle, a fact that does not fit into the current hypothesis of the clonal origin of “revertant” myonuclei from satellite cells. This work represents the largest, statistically significant analysis of revertant fibers in mdx mice so far, which can now be used as a reference point for improving the evaluation of therapeutic approaches for DMD. At the same time, it provides new clues about the formation of revertant fibers/cardiomyocytes in dystrophic skeletal and cardiac muscle.     

Clinical Heterogeneity of Duchenne Muscular Dystrophy (DMD): Definition of Sub-Phenotypes and Predictive Criteria by Long-Term Follow-Up

Background

To explore clinical heterogeneity of Duchenne muscular dystrophy (DMD), viewed as a major obstacle to the interpretation of therapeutic trials

Methodology/Principal Findings

A retrospective single institution long-term follow-up study was carried out in DMD patients with both complete lack of muscle dystrophin and genotyping. An exploratory series (series 1) was used to assess phenotypic heterogeneity and to identify early criteria predicting future outcome; it included 75 consecutive steroid-free patients, longitudinally evaluated for motor, respiratory, cardiac and cognitive functions (median follow-up: 10.5 yrs). A validation series (series 2) was used to test robustness of the selected predictive criteria; it included 34 more routinely evaluated patients (age>12 yrs). Multivariate analysis of series 1 classified 70/75 patients into 4 clusters with distinctive intellectual and motor outcomes: A (early infantile DMD, 20%): severe intellectual and motor outcomes; B (classical DMD, 28%): intermediate intellectual and poor motor outcome; C (moderate pure motor DMD, 22%): normal intelligence and delayed motor impairment; and D (severe pure motor DMD, 30%): normal intelligence and poor motor outcome. Group A patients had the most severe respiratory and cardiac involvement. Frequency of mutations upstream to exon 30 increased from group A to D, but genotype/phenotype correlations were restricted to cognition (IQ>71: OR 7.7, 95%CI 1.6–20.4, p<0.003). Diagnostic accuracy tests showed that combination of “clinical onset <2 yrs” with “mental retardation” reliably assigned patients to group A (sensitivity 0.93, specificity 0.98). Combination of “lower limb MMT score>6 at 8 yrs” with “normal or borderline mental status” reliably assigned patients to group C (sensitivity: 1, specificity: 0.94). These criteria were also predictive of “early infantile DMD” and “moderate pure motor DMD” in series 2.

Conclusions/Significance

DMD can be divided into 4 sub-phenotypes differing by severity of muscle and brain dysfunction. Simple early criteria can be used to include patients with similar outcomes in future therapeutic trials.     

Expression of dystrophin on the cell surface membrane of intrafusal fibers of human skeletal muscle

Summary We examined the morphological expression of dystrophin in the intrafusal muscle fibers in skeletal muscle from normal human and Duchenne muscular dystrophy (DMD) patients, using antisera against the N-terminal and C-terminal regions of dystrophin. The intrafusal fibers of normal muscle express dystrophin on their cell surface membrane, but those of DMD muscle do not.Abbreviation DMD Duchenne muscular dystrophy     

Glycogen-autophagy: Molecular machinery and cellular mechanisms of glycophagy

Autophagy is an essential cellular process involving degradation of superfluous or defective macromolecules and organelles as a form of homeostatic recycling. Initially proposed to be a “bulk” degradation pathway, a more nuanced appreciation of selective autophagy pathways has developed in the literature in recent years. As a glycogen-selective autophagy process, “glycophagy” is emerging as a key metabolic route of transport and delivery of glycolytic fuel substrate. Study of glycophagy is at an early stage. Enhanced understanding of this major noncanonical pathway of glycogen flux will provide important opportunities for new insights into cellular energy metabolism. In addition, glycogen metabolic mishandling is centrally involved in the pathophysiology of several metabolic diseases in a wide range of tissues, including the liver, skeletal muscle, cardiac muscle, and brain. Thus, advances in this exciting new field are of broad multidisciplinary interest relevant to many cell types and metabolic states. Here, we review the current evidence of glycophagy involvement in homeostatic cellular metabolic processes and of molecular mediators participating in glycophagy flux. We integrate information from a variety of settings including cell lines, primary cell culture systems, ex vivo tissue preparations, genetic disease models, and clinical glycogen disease states.     

Quantitation of “autophagic flux” in mature skeletal muscle

Reliable and quantitative assays to measure in vivo autophagy are essential. Currently, there are varied methods for monitoring autophagy; however, it is a challenge to measure “autophagic flux” in an in vivo model system. Conversion and subsequent degradation of the microtubule-associated protein 1 light chain 3 (MAP1-LC3/LC3) to the autophagosome associated LC3-II isoform can be evaluated by immunoblot. However, static levels of endogenous LC3-II protein may render possible misinterpretations since LC3-II levels can increase, decrease or remain unchanged in the setting of autophagic induction. Therefore, it is necessary to measure LC3-II protein levels in the presence and absence of lysomotropic agents that block the degradation of LC3-II, a technique aptly named the “autophagometer.” In order to measure autophagic flux in mouse skeletal muscle, we treated animals with the microtubule depolarizing agent colchicine. Two days of 0.4 mg/kg/day intraperitoneal colchicine blocked autophagosome maturation to autolysosomes and increased LC3-II protein levels in mouse skeletal muscle by >100%. the addition of an autophagic stimulus such as dietary restriction or rapamycin led to an additional increase in LC3-II above that seen with colchicine alone. Moreover, this increase was not apparent in the absence of a “colchicine block.” Using this assay, we evaluated the autophagic response in skeletal muscle upon denervation induced atrophy. Our studies highlight the feasibility of performing an “in vivo autophagometer” study using colchicine in skeletal muscle.Key words: autophagy, rapamycin, skeletal muscle     

Biological Ion Exchanger Resins: I. Quantitative Electrostatic Correspondence of Fixed Charge and Mobile Counter Ion

Utilizing Escherichia coli as the prototype of an ion-accumulating cell, the ion exchange isotherm is introduced as a concise method of characterizing biological ion exchange events. The ion exchange isotherm for the alkali cation exchange, K ↔ Na, is described. The total charge profile of this bacterium is compiled and compared for bacteria in the Na form and in the K form. Macromolecule fixed charge was found to provide 80% of the counter ions that pair with potassium. Therefore, in its physiological state, 80% of the cell potassium in E. coli is associated with an ion exchange site on a macromolecule. The primary cation exchange sites are found to be about equally divided between carboxylate and phosphate sites indicating that E. coli is a bifunctional resin with respect to cation exchange. During substrate-dependent cation accumulation (“active transport”), phosphate esters and organic acids were shown to accumulate. One may conclude that the role of intermediate metabolism in “active transport” is to increase the ion exchange capacity of the biological resin by the production of charged metabolites that sorb to the framework of the resin.     

A Correlative Analysis of Actin Filament Assembly, Structure, and Dynamics

The effect of the type of metal ion (i.e., Ca²⁺, Mg²⁺, or none) bound to the high-affinity divalent cation binding site (HAS) of actin on filament assembly, structure, and dynamics was investigated in the absence and presence of the mushroom toxin phalloidin. In agreement with earlier reports, we found the polymerization reaction of G-actin into F-actin filaments to be tightly controlled by the type of divalent cation residing in its HAS. Moreover, novel polymerization data are presented indicating that LD, a dimer unproductive by itself, does incorporate into growing F-actin filaments. This observation suggests that during actin filament formation, in addition to the obligatory nucleation– condensation pathway involving UD, a productive filament dimer, a facultative, LD-based pathway is implicated whose abundance strongly depends on the exact polymerization conditions chosen. The “ragged” and “branched” filaments observed during the early stages of assembly represent a hallmark of LD incorporation and might be key to producing an actin meshwork capable of rapidly assembling and disassembling in highly motile cells. Hence, LD incorporation into growing actin filaments might provide an additional level of regulation of actin cytoskeleton dynamics. Regarding the structure and mechanical properties of the F-actin filament at steady state, no significant correlation with the divalent cation residing in its HAS was found. However, compared to native filaments, phalloidin-stabilized filaments were stiffer and yielded subtle but significant structural changes. Together, our data indicate that whereas the G-actin conformation is tightly controlled by the divalent cation in its HAS, the F-actin conformation appears more robust than this variation. Hence, we conclude that the structure and dynamics of the Mg–F-actin moiety within the thin filament are not significantly modulated by the cyclic Ca²⁺ release as it occurs in muscle contraction to regulate the actomyosin interaction via troponin.     

Preconditioning improves function and recovery of single muscle fibers during severe hypoxia and reoxygenation

Reperfusion following prolonged ischemia induces cellular damage in whole skeletal muscle models. Ischemic preconditioning attenuates the deleterious effects. We tested whether individual skeletal muscle fibers would be similarly affected by severe hypoxia and reoxygenation (H/R) in the absence of extracellular factors and whether cellular damage could be alleviated by hypoxic preconditioning. Force and free cytosolic Ca2+ ([Ca2+]c) were monitored in Xenopus single muscle fibers (n = 24) contracting tetanically at 0.2 Hz during 5 min of severe hypoxia and 5 min of reoxygenation. Twelve cells were preconditioned by a shorter bout of H/R 1 h before the experimental trial. In preconditioned cells, force relative to initial maximal values (P/P(o)) and relative peak [Ca2+]c fell (P < 0.05) during 5 min of hypoxia and recovered during reoxygenation. In contrast, P/P(o) and relative peak [Ca2+]c fell more during hypoxia (P < 0.05) and recovered less during reoxygenation (P < 0.05) in control cells. The ratio of force to [Ca2+]c was significantly higher in the preconditioned cells during severe hypoxia, suggesting that changes in [Ca2+]c were not solely responsible for the loss in force. We conclude that 1) isolated skeletal muscle fibers contracting in the absence of extracellular factors are susceptible to H/R injury associated with changes in Ca2+ handling; and 2) hypoxic preconditioning improves contractility, Ca2+ handling, and cell recovery during subsequent hypoxic insult.     

Quantitative techniques for steady-state calculation and dynamic integrated modelling of membrane potential and intracellular ion concentrations

The membrane potential (E_m) is a fundamental cellular parameter that is primarily determined by the transmembrane permeabilities and concentration gradients of various ions. However, ion gradients are themselves profoundly influenced by E_m due to its influence upon transmembrane ion fluxes and cell volume (V_c). These interrelationships between E_m, V_c and intracellular ion concentrations make computational modelling useful or necessary in order to guide experimentation and to achieve an integrated understanding of experimental data, particularly in complex, dynamic, multi-compartment systems such as skeletal and cardiac myocytes. A variety of quantitative techniques exist that may assist such understanding, from classical approaches such as the Goldman–Hodgkin–Katz equation and the Gibbs–Donnan equilibrium, to more recent “current-summing” models as exemplified by cardiac myocyte models including those of DiFrancesco & Noble, Luo & Rudy and Puglisi & Bers, or the “charge-difference” modelling technique of Fraser & Huang so far applied to skeletal muscle. In general, the classical approaches provide useful and important insights into the relationships between E_m, V_c and intracellular ion concentrations at steady state, providing their core assumptions are fully understood, while the more recent techniques permit the modelling of changing values of E_m, V_c and intracellular ion concentrations. The present work therefore reviews the various approaches that may be used to calculate E_m, V_c and intracellular ion concentrations with the aim of establishing the requirements for an integrated model that can both simulate dynamic systems and recapitulate the key findings of classical techniques regarding the cellular steady state. At a time when the number of cellular models is increasing at an unprecedented rate, it is hoped that this article will provide a useful and critical analysis of the mathematical techniques fundamental to each of them.     

Transport of electrolytes in muscle

Summary The muscle fiber stands alongside the red blood cell and the giant axon as one of the three classical cell types that have had major application in investigating ion transport processes in cell membranes. Of these three cell types, the muscle fiber was the first to provide definite evidence for a sodium pump. The ability of the sodium pump to produce an electrical potential difference across the cell membrane was also first demonstrated in muscle fibers. This important property of the sodium pump is now known to have physiological significance in many other types of cells.In this review, electrolyte transport investigations in skeletal muscle are traced from their inception to the current state of the field. Applications of major research techniques are discussed and key results are summarized. An overview of electrolyte transport in muscle, this article emphasizes relationships between the muscle fiber membrane potential and ionic transport processes.     

Transport of electrolytes in muscle

The muscle fiber stands alongside the red blood cell and the giant axon as one of the three classical cell types that have had major application in investigating ion transport processes in cell membranes. Of these three cell types, the muscle fiber was the first to provide definite evidence for a sodium pump. The ability of the sodium pump to produce an electrical potential difference across the cell membrane was also first demonstrated in muscle fibers. This important property of the sodium pump is now known to have physiological significance in many other types of cells. In this review, electrolyte transport investigations in skeletal muscle are traced from their inception to the current state of the field. Applications of major research techniques are discussed and key results are summarized. An overview of electrolyte transport in muscle, this article emphasizes relationships between the muscle fiber membrane potential and ionic transport processes.     

The role of super-relaxed myosin in skeletal and cardiac muscle

The super-relaxed (SRX) state of myosin was only recently reported in striated muscle. It is characterised by a sub-population of myosin heads with a highly inhibited rate of ATP turnover. Myosin heads in the SRX state are bound to each other along the thick filament core producing a highly ordered arrangement. Upon activation, these heads project into the interfilament space where they can bind to the actin filaments. Thus far, the population and lifetimes of myosin heads in the SRX state have been characterised in rabbit cardiac, and fast and slow skeletal muscle, as well as in the skeletal muscle of the tarantula. These studies suggest that the role of SRX in cardiac and skeletal muscle regulation is tailored to their specific functions. In skeletal muscle, the SRX modulates the resting metabolic rate. Cardiac SRX represents a “reserve” of inactive myosin heads that may protect the heart during times of stress, e.g. hypoxia and ischaemia. These heads may also be called up when there is a sustained demand for increased power. The SRX in cardiac muscle provides a potential target for novel therapies.     

Mass Transfer in the Cornea: II. Ion Transport and Electrical Properties of a Series Membrane Tissue

The electrical and active transport properties of isolated rabbit cornea are investigated by computer experimentation. The tissue is modeled as a series membrane system and the passive ion fluxes through it are described by the frictional formulation of irreversible thermodynamics. From short-circuit current (SCC) data, it is found that the epithelial sodium pump rate (P) is not appreciably changed when much of the sodium in the solution bathing the anterior corneal surface (concentration = c₁₁) is replaced by choline, with choline-free medium posteriorly. Simulations of open-circuited corneas, using the mean P computed from the SCC data, yield corneal and stromal potentials in agreement with experiment. The stromal fluid is calculated to become more hypotonic as c₁₁ is diminished, a result consistent with posttest measurements of the sodium content of experimental stromata. The apparent decrease in “bound sodium” which accompanies the reduction of c₁₁ is a result of the associated changes in steady stromal hydration; the epithelial sodium pump does not contribute to corneal deturgescence. The inclusion of a simple epithelial structure in the computations changes the value of P but affects neither its constancy nor the calculated behavior of the cornea under open-circuit conditions. A general algebraic relation among pump rates and ion fluxes in short-circuited series membrane systems bathed in complex media is derived and used to construct a relation between P and SCC for the cornea. This equation yields pump rates in good agreement with the computer results and is used to show that (a) P is independent of c₁₁ if d(SCC)/dc₁₁ is a constant related to the over-all corneal permeability to sodium, and (b) a Lineweaver-Burke plot of 1/SCC vs. 1/c₁₁ can appear to be linear at constant P.     

Accumulation of CK-MM is impaired in innervated and contracting cultured muscle fibers of Duchenne muscular dystrophy patients

No specific abnormalities have been reproducibly manifested in aneurally cultured muscle of Duchenne muscular dystrophy (DMD) patients. We now report that the accumulation of the muscle-"specific" isozyme of creatine kinase (CK-MM) was significantly and preferentially impaired in long-term innervated contracting muscle fibers cultured from 4 DMD patients (DMD-InnCMFs) compared to: i) their noninnervated sister-cultured muscle fibers, and ii) innervated contracting control cultured human muscle fibers (Control-InnCHMFs). Accumulation of other muscle-"specific" isozymes (MSIs), viz. glycogen phosphorylase, phosphoglycerate mutase, and lactic dehydrogenase, was not significantly impaired. We have not observed preferentially-impaired CK-MM accumulation in any Control-InnCHMFs from 22 patients (children and adults) with a variety of neuromuscular diseases. There was no apparent difference between DMD-InnCMFs and Control InnCHMFs regarding: acceptance of innervation; neuronally-driven, virtually continuous muscle-fiber contractions; characteristic myofiber organization by phase-contrast microscopy, and increased longevity of the innervated fibers.     

Nemaline Myopathy-Related Skeletal Muscle α-Actin (ACTA1) Mutation,Asp286Gly,Prevents Proper Strong Myosin Binding and Triggers Muscle Weakness

Many mutations in the skeletal muscle α-actin gene (ACTA1) lead to muscle weakness and nemaline myopathy. Despite increasing clinical and scientific interest, the molecular and cellular pathogenesis of weakness remains unclear. Therefore, in the present study, we aimed at unraveling these mechanisms using muscles from a transgenic mouse model of nemaline myopathy expressing the ACTA1 Asp286Gly mutation. We recorded and analyzed the mechanics of membrane-permeabilized single muscle fibers. We also performed molecular energy state computations in the presence or absence of Asp286Gly. Results demonstrated that during contraction, the Asp286Gly acts as a “poison-protein” and according to the computational analysis it modifies the actin-actin interface. This phenomenon is likely to prevent proper myosin cross-bridge binding, limiting the fraction of actomyosin interactions in the strong binding state. At the cell level, this decreases the force-generating capacity, and, overall, induces muscle weakness. To counterbalance such negative events, future potential therapeutic strategies may focus on the inappropriate actin-actin interface or myosin binding.     

IP3 receptor blockade restores autophagy and mitochondrial function in skeletal muscle fibers of dystrophic mice

Duchenne muscular dystrophy (DMD) is characterized by a severe and progressive destruction of muscle fibers associated with altered Ca²⁺ homeostasis. We have previously shown that the IP₃ receptor (IP₃R) plays a role in elevating basal cytoplasmic Ca²⁺ and that pharmacological blockade of IP₃R restores muscle function. Moreover, we have shown that the IP₃R pathway negatively regulates autophagy by controlling mitochondrial Ca²⁺ levels. Nevertheless, it remains unclear whether IP₃R is involved in abnormal mitochondrial Ca²⁺ levels, mitochondrial dynamics, or autophagy and mitophagy observed in adult DMD skeletal muscle. Here, we show that the elevated basal autophagy and autophagic flux levels were normalized when IP₃R was downregulated in mdx fibers. Pharmacological blockade of IP₃R in mdx fibers restored both increased mitochondrial Ca²⁺ levels and mitochondrial membrane potential under resting conditions. Interestingly, mdx mitochondria changed from a fission to an elongated state after IP₃R knockdown, and the elevated mitophagy levels in mdx fibers were normalized. To our knowledge, this is the first study associating IP₃R1 activity with changes in autophagy, mitochondrial Ca²⁺ levels, mitochondrial membrane potential, mitochondrial dynamics, and mitophagy in adult mouse skeletal muscle. Moreover, these results suggest that increased IP₃R activity in mdx fibers plays an important role in the pathophysiology of DMD. Overall, these results lead us to propose the use of specific IP₃R blockers as a new pharmacological treatment for DMD, given their ability to restore both autophagy/mitophagy and mitochondrial function.     

Fast muscle fibers are preferentially affected in Duchenne muscular dystrophy

We show that Duchenne muscular dystrophy (DMD) selectively affects a subset of skeletal muscle fibers specialized for fast contraction. Muscle fiber types were characterized immunohistochemically with monoclonal antibodies that distinguish isoforms of fetal and adult-fast or adult-slow myosin heavy chain present in the same fiber. Fetal myosin expression increased with patient age and was not due to arrested development but rather to de novo synthesis, which served as a sensitive indicator of muscle regeneration. A subset of fast fibers were the first to degenerate (type IIb). Extensive fast fiber regeneration occurred before slow fibers were affected. These results suggest that the DMD gene product has a specific function in a subpopulation of muscle fibers specialized to respond to the highest frequency of neuronal stimulation with maximal rates of contraction.     

Nutrient cycling is an important mechanism for homeostasis in plant cells

Homeostasis in living cells refers to the steady state of internal, physical, and chemical conditions. It is sustained by self-regulation of the dynamic cellular system. To gain insight into the homeostatic mechanisms that maintain cytosolic nutrient concentrations in plant cells within a homeostatic range, we performed computational cell biology experiments. We mathematically modeled membrane transporter systems and simulated their dynamics. Detailed analyses of ‘what-if’ scenarios demonstrated that a single transporter type for a nutrient, irrespective of whether it is a channel or a cotransporter, is not sufficient to calibrate a desired cytosolic concentration. A cell cannot flexibly react to different external conditions. Rather, at least two different transporter types for the same nutrient, which are energized differently, are required. The gain of flexibility in adjusting a cytosolic concentration was accompanied by the establishment of energy-consuming cycles at the membrane, suggesting that these putatively “futile” cycles are not as futile as they appear. Accounting for the complex interplay of transporter networks at the cellular level may help design strategies for increasing nutrient use efficiency of crop plants.

First principles of membrane transport explain why maintaining a constant cytosolic nutrient concentration is often accompanied by the “futile” cycling of the nutrient across the membrane.     

Spontaneous Excitation Patterns Computed for Axons with Injury-like Impairments of Sodium Channels and Na/K Pumps

In injured neurons, “leaky” voltage-gated sodium channels (Nav) underlie dysfunctional excitability that ranges from spontaneous subthreshold oscillations (STO), to ectopic (sometimes paroxysmal) excitation, to depolarizing block. In recombinant systems, mechanical injury to Nav1.6-rich membranes causes cytoplasmic Na⁺-loading and “Nav-CLS”, i.e., coupled left-(hyperpolarizing)-shift of Nav activation and availability. Metabolic injury of hippocampal neurons (epileptic discharge) results in comparable impairment: left-shifted activation and availability and hence left-shifted I_Na-window. A recent computation study revealed that CLS-based I_Na-window left-shift dissipates ion gradients and impairs excitability. Here, via dynamical analyses, we focus on sustained excitability patterns in mildly damaged nodes, in particular with more realistic Gaussian-distributed Nav-CLS to mimic “smeared” injury intensity. Since our interest is axons that might survive injury, pumps (sine qua non for live axons) are included. In some simulations, pump efficacy and system volumes are varied. Impacts of current noise inputs are also characterized. The diverse modes of spontaneous rhythmic activity evident in these scenarios are studied using bifurcation analysis. For “mild CLS injury”, a prominent feature is slow pump/leak-mediated E_Ion oscillations. These slow oscillations yield dynamic firing thresholds that underlie complex voltage STO and bursting behaviors. Thus, Nav-CLS, a biophysically justified mode of injury, in parallel with functioning pumps, robustly engenders an emergent slow process that triggers a plethora of pathological excitability patterns. This minimalist “device” could have physiological analogs. At first nodes of Ranvier and at nociceptors, e.g., localized lipid-tuning that modulated Nav midpoints could produce Nav-CLS, as could co-expression of appropriately differing Nav isoforms.     

Intrinsic Feedback Factors Producing Inertial Compensation in Muscle

An attempt was made to determine the factors causing the load-inertia compensation that has been observed in skeletal muscle. Cat skeletal muscle force output was determined as a function of the two variables, length and stimulus pulse rate. The results were represented in a system diagram from which it becomes apparent that: (a) the length-tension relationship in muscle forms a functional, non-neural servo feedback; (b) the force-velocity curve appears as an oscillation-damping, velocity feedback in the muscle servo; (c) the nonlinear action of pulse rate on response is, in effect, in the input element to the muscle servo system. For purpose of analysis of the motor system it appears that these signal handling characteristics of muscle make it more nearly a “position servo” than a “force motor.”