The Role of Calpains in Skeletal Muscle Remodeling with Exercise and Inactivity-induced Atrophy

Calpains are cysteine proteases expressed in skeletal muscle fibers and other cells. Although calpain was first reported to act as a kinase activating factor in skeletal muscle, the consensus is now that calpains play a canonical role in protein turnover. However, recent evidence reveals new and exciting roles for calpains in skeletal muscle. This review will discuss the functions of calpains in skeletal muscle remodeling in response to both exercise and inactivity-induced muscle atrophy. Calpains participate in protein turnover and muscle remodeling by selectively cleaving target proteins and creating fragmented proteins that can be further degraded by other proteolytic systems. Nonetheless, an often overlooked function of calpains is that calpain-mediated cleavage of proteins can result in fragmented proteins that are biologically active and have the potential to actively influence cell signaling. In this manner, calpains function beyond their roles in protein turnover and influence downstream signaling effects. This review will highlight both the canonical and noncanonical roles that calpains play in skeletal muscle remodeling including sarcomere transformation, membrane repair, triad junction formation, regulation of excitation-contraction coupling, protein turnover, cell signaling, and mitochondrial function. We conclude with a discussion of key unanswered questions regarding the roles that calpains play in skeletal muscle.

Skeletal muscle triad junction ultrastructure by Focused-Ion-Beam milling of muscle and Cryo-Electron Tomography

Cryo-electron tomography (cryo-ET) has emerged as perhaps the only practical technique for revealing nanometer-level three-dimensional structural details of subcellular macromolecular complexes in their native context, inside the cell. As currently practiced, the specimen should be 0.1- 0.2 microns in thickness to achieve optimal resolution. Thus, application of cryo-ET to intact frozen (vitreous) tissues, such as skeletal muscle, requires that they be sectioned. Cryo-ultramicrotomy is notoriously difficult and artifact-prone when applied to frozen cells and tissue, but a new technique, focused ion beam milling (cryo-FIB), shows great promise for “thinning” frozen biological specimens. Here we describe our initial results in applying cryo-FIB and cryo-ET to triad junctions of skeletal muscle.

Mechanisms of excitation-contraction uncoupling relevant to activity-induced muscle fatigueThis paper is one of a selection of papers published in this Special Issue, entitled 14th International Biochemistry of Exercise Conference – Muscles as Molecular and Metabolic Machines, and has undergone the Journal’s usual peer review process.

If the free [Ca2+] in the cytoplasm of a skeletal muscle fiber is raised substantially for a period of seconds to minutes or to high levels just briefly, it leads to disruption of the normal excitation-contraction (E-C) coupling process and a consequent long-lasting decrease in force production. It appears that the disruption to the coupling occurs at the triad junction, where the voltage-sensor molecules (dihydropyridine receptors) normally interact with and open the Ca2+ release channels (ryanodine receptors) in the adjacent sarcoplasmic reticulum (SR). This disruption results in inadequate release of SR Ca2+ upon stimulation. Such E-C uncoupling may underlie the long-duration low-frequency fatigue that can occur after various types of exercise, as well as possibly being a contributing factor to the muscle weakness in certain muscle diseases. The process or processes causing the disruption of the coupling between the voltage sensors and the release channels is not known with certainty, but might be associated with structural changes at the triad junction, possibly caused by activation of the Ca2+-dependent protease, µ-calpain.

Na+-K+ pumps in the transverse tubular system of skeletal muscle fibers preferentially use ATP from glycolysis

The Na+-K+ pumps in the transverse tubular (T) system of a muscle fiber play a vital role keeping K+ concentration in the T-system sufficiently low during activity to prevent chronic depolarization and consequent loss of excitability. These Na+-K+ pumps are located in the triad junction, the key transduction zone controlling excitation-contraction (EC) coupling, a region rich in glycolytic enzymes and likely having high localized ATP usage and limited substrate diffusion. This study examined whether Na+-K+ pump function is dependent on ATP derived via the glycolytic pathway locally within the triad region. Single fibers from rat fast-twitch muscle were mechanically skinned, sealing off the T-system but retaining normal EC coupling. Intracellular composition was set by the bathing solution and action potentials (APs) triggered in the T-system, eliciting intracellular Ca2+ release and twitch and tetanic force responses. Conditions were selected such that increased Na+-K+ pump function could be detected from the consequent increase in T-system polarization and resultant faster rate of AP repriming. Na+-K+ pump function was not adequately supported by maintaining cytoplasmic ATP concentration at its normal resting level (∼8 mM), even with 10 or 40 mM creatine phosphate present. Addition of as little as 1 mM phospho(enol)pyruvate resulted in a marked increase in Na+-K+ pump function, supported by endogenous pyruvate kinase bound within the triad. These results demonstrate that the triad junction is a highly restricted microenvironment, where glycolytic resynthesis of ATP is critical to meet the high demand of the Na+-K+ pump and maintain muscle excitability.

Long-lasting muscle fatigue: partial disruption of excitation-contraction coupling by elevated cytosolic Ca2+ concentration during contractions

The repeated elevation of cytosolic Ca2+ concentration ([Ca2+]i) above resting levels during contractile activity has been associated with long-lasting muscle fatigue. The mechanism underlying this fatigue appears to involve elevated [Ca2+]i levels that induce disruption of the excitation-contraction (E-C) coupling process at the triad junction. Unclear, however, are which aspects of the activity-related [Ca2+]i changes are responsible for the deleterious effects, in particular whether they depend primarily on the peak [Ca2+]i reached locally at particular sites or on the temporal summation of the increased [Ca2+] in the cytoplasm as a whole. In this study, we used mechanically skinned fibers from rat extensor digitorum longus muscle, in which the normal E-C coupling process remains intact. The [Ca2+]i was raised either by applying a set elevated [Ca2+] throughout the fiber or by using action potential stimulation to induce the release of sarcoplasmic reticulum Ca2+ by the normal E-C coupling system with or without augmentation by caffeine or buffering with BAPTA. Herein we show that elevating [Ca2+]i in the physiological range of 2–20 μM irreversibly disrupts E-C coupling in a concentration-dependent manner but requires exposure for a relatively long time (1–3 min) to cause substantial uncoupling. The effectiveness of Ca2+ released via the endogenous system in disrupting E-C coupling indicates that the relatively high [Ca2+]i attained close to the release site at the triad junction is a more important factor than the increase in bulk [Ca2+]i. Our results suggest that during prolonged vigorous activity, the many repeated episodes of relatively high triadic [Ca2+] can disrupt E-C coupling and lead to long-lasting fatigue.

Enhanced resistance to fatigue and altered calcium handling properties of sarcalumenin knockout mice

Sarcalumenin is a Ca2+-binding protein located in the sarcoplasmic reticulum of striated muscle cells, the physiological function of which has not been fully determined yet. Using sarcalumenin knockout ( sar −/−) mice, we showed that sar ablation altered store-operated Ca2+ entry (SOCE) and enhanced muscle fatigue resistance. Sar−/− mice fatigued less with treadmill exercise, and intact isolated soleus and extensor digitorum longus muscles from sar−/− mice were more resistant to intermittent fatiguing stimulation than those from wild-type mice. Enhanced SOCE was observed in the sar −/− muscles. Biochemical analysis revealed that sar −/− muscles contained significantly elevated expression of mitsugumin 29 (MG29), a synaptophysin-related membrane protein located in the triad junction of skeletal muscle. Because the ablation of mg29 has been shown to cause increased fatigability and dysfunction of SOCE, the enhanced SOCE activity seen in sar −/− muscle may be correlated with the increased expression of MG29. Our data suggest that systemic ablation of sarcalumenin caused enhanced resistance to muscle fatigue by compensatory changes in Ca2+ regulatory proteins that effect SOCE.