An updated view of the structural basis for dihydropyridine receptors-ryanodine receptors direct molecular interaction in skeletal muscle.

This presentation reviews images of electron micrographs from various skeletal muscles identifying a consistent association of diydropyridine receptors (DHPR) tetrads with  alternate ryanodine receptors. Imaging of the junctional gap in triads from various sources  provide direct evidence for the  association of four diydropyridine receptors (DHPRs), clustered into tetrads, with alternate ryanodine receptors (RyRs). It is not clear whether firing of all four components of a tetrad is necessary to fully activate the opening of the RyR channel.

Ca2+ entry units in a superfast fish muscle.

Over the past two decades, mounting evidence has demonstrated that a mechanism known as store-operated Ca2+ entry (SOCE) plays a crucial role in sustaining skeletal muscle contractility by facilitating Ca2+ influx from the extracellular space during sarcoplasmic reticulum (SR) Ca2+ depletion. We recently demonstrated that, in exercised fast-twitch muscle from mice, the incidence of Ca2+ entry units (CEUs), newly described intracellular junctions between dead-end longitudinal transverse tubular (T-tubule) extensions and stacks of sarcoplasmic reticulum (SR) flat cisternae, strictly correlate with both the capability of fibers to maintain contractions during fatigue and enhanced Ca2+ influx via SOCE. Here, we tested the broader relevance of this result across vertebrates by searching for the presence of CEUs in the vocal muscles of a teleost fish adapted for extended, high-frequency activity. Specifically, we examined active vs. inactive superfast sonic muscles of plainfin midshipman (Porichthys notatus). Interestingly, muscles from actively humming territorial males had a much higher incidence of CEU SR stacks relative to territorial males that were not actively vocalizing, strengthening the concept that assembly of these structures is dynamic and use-dependent, as recently described in exercised muscles from mice. Our results support the hypothesis that CEUs represent a conserved mechanism, across vertebrates, for enabling high levels of repetitive muscle activity, and also provide new insights into the adaptive mechanisms underlying the unique properties of superfast midshipman sonic muscles.

The distal C terminus of the dihydropyridine receptor ß1a subunit is essential for tetrad formation in skeletal muscle.

The skeletal muscle dihydropyridine receptor (DHPR) ß1a subunit is indispensable for full trafficking of DHPRs into triadic junctions (i.e., the close apposition of transverse tubules and sarcoplasmic reticulum [SR]), facilitation of DHPRα1S voltage sensing, and arrangement of DHPRs into tetrads as a consequence of their interaction with ryanodine receptor (RyR1) homotetramers. These three features are obligatory for skeletal muscle excitation­contraction (EC) coupling. Previously, we showed that all four vertebrate ß isoforms (ß1­ß4) facilitate α1S triad targeting and, except for ß3, fully enable DHPRα1S voltage sensing [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488­7493 (2013)]. Consequently, ß3 failed to restore EC coupling despite the fact that both ß3 and ß1a restore tetrads. Thus, all ß-subunits are able to restore triad targeting, but only ß1a restores both tetrads and proper DHPR­RyR1 coupling [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488­7493 (2013)]. To investigate the molecular region(s) of ß1a responsible for the tetradic arrangement of DHPRs and thus DHPR­RyR1 coupling, we expressed loss- and gain-of-function chimeras between ß1a and ß4, with systematically swapped domains in zebrafish strain relaxed (ß1-null) for patch clamp, cytoplasmic Ca2+ transients, motility, and freeze-fracture electron microscopy. ß1a/ß4 chimeras with either N terminus, SH3, HOOK, or GK domain derived from ß4 showed complete restoration of SR Ca2+ release. However, chimera ß1a/ß4(C) with ß4 C terminus produced significantly reduced cytoplasmic Ca2+ transients. Conversely, gain-of-function chimera ß4/ß1a(C) with ß1a C terminus completely restored cytoplasmic Ca2+ transients, DHPR tetrads, and motility. Furthermore, we found that the nonconserved, distal C terminus of ß1a plays a pivotal role in reconstitution of DHPR tetrads and thus allosteric DHPR­RyR1 interaction, essential for skeletal muscle EC coupling.

The location of InsP3 receptors in Purkinje cells of murine cerebellum does not supports a direct interaction in the transfer of calcium ions between ER and mitochondria.

The inositol-3-phosphate receptors (IP3Rs) of cerebellar Purkinje cells are located in abundant, large stacks of endoplasmic reticulum (ER) cisternae. Using thin section electron microscopy, we identify very frequent associations of the ER stacks with mitochondria. The associations have two components: a single, close ER-mitochondria contact on one side to the stack, and multiple layers of ER cisternae decorated by IP3Rs receptors on the side away from the mitochondria. Due to their location in the stacks, IP3Rs are never in contact with the mitochondria, although they are in their vicinity. We conclude that transfer of Ca2+ between ER and mitochondria is not directly mediated by IP3Rs, but is based on mitochondrial Ca2+ uptake from the local cytoplasmic spikes during IP3Rs' activity.

In vivo RyR1 reduction in muscle triggers a core-like myopathy.

Mutations in the RYR1 gene, encoding the skeletal muscle calcium channel RyR1, lead to congenital myopathies, through expression of a channel with abnormal permeability and/or in reduced amount, but the direct functional whole organism consequences of exclusive reduction in RyR1 amount have never been studied. We have developed and characterized a mouse model with inducible muscle specific RYR1 deletion. Tamoxifen-induced recombination in the RYR1 gene at adult age resulted in a progressive reduction in the protein amount reaching a stable level of 50% of the initial amount, and was associated with a progressive muscle weakness and atrophy. Measurement of calcium fluxes in isolated muscle fibers demonstrated a reduction in the amplitude of RyR1-related calcium release mirroring the reduction in the protein amount. Alterations in the muscle structure were observed, with fibers atrophy, abnormal mitochondria distribution and membrane remodeling. An increase in the expression level of many proteins was observed, as well as an inhibition of the autophagy process. This model demonstrates that RyR1 reduction is sufficient to recapitulate most features of Central Core Disease, and accordingly similar alterations were observed in muscle biopsies from Dusty Core Disease patients (a subtype of Central Core Disease), pointing to common pathophysiological mechanisms related to RyR1 reduction.

The structural basis for intermitochondrial communications is fundamentally different in cardiac and skeletal muscle.

NEW FINDINGS: What is the topic for this review? This review summarizes recent discoveries in mitochondrial development and morphology studied with electron microscopy. What advances does it highlight? Although mitochondria are generally considered to be isolated from each other, this review highlights recently discovered evidence for the presence of intermitochondrial communication structures in cardiac and skeletal muscle, in animal models and humans. Within striated muscles, the means of intermitochondrial exchange and the reaction of mitochondria to external stimuli are uniquely dependent on the tissue, and we clearly differentiate between nanotunnels, the active protrusion of cardiac mitochondria, and the connecting ducts of skeletal muscle derived from fusion-fission and elongation events. ABSTRACT: This review focuses on recent discoveries in skeletal and cardiac muscles indicating that mitochondria behave as an interactive cohort with inter-organelle communication and specific reactions to stress signals. Our new finding is that intermitochondrial communications in cardiac and skeletal muscles rely on two distinct methods. In cardiac muscle, mitochondria are discrete entities and are fairly well immobilized in a structural context. The organelles have developed a unique method of communication, via nanotunnels, which allow temporary connection from one mitochondrion to another over distances of up to several micrometres, without overall movement of the individual organelles and loss of their identity. Skeletal muscle mitochondria, in contrast, are dynamic. Through fusion, fission and elongation, they form connections that include constrictions and connecting ducts (distinct from nanotunnels) and lose individual identity in the formation of extensive networks. Connecting elements in skeletal muscle are distinct from nanotunnels in cardiac muscle.

Ultrastructure of primary pacemaking cells in rabbit sino-atrial node cells indicates limited sarcoplasmic reticulum content.

The main mammalian heart pacemakers are spindle-shaped cells compressed into tangles within protective layers of collagen in the sino-atrial node (SAN). Two cell types, "dark" and "light," differ on their high or low content of intermediate filaments, but share scarcity of myofibrils and a high content of glycogen. Sarcoplasmic reticulum (SR) is scarce. The free SR (fSR) occupies 0.04% of the cell volume within ~0.4 µm wide peripheral band. The junctional SR (jSR), constituting peripheral couplings (PCs), occupies 0.03% of the cell volume. Total fSR + jSR volume is 0.07% of cell volume, lower than the SR content of ventricular myocytes. The average distance between PCs is 7.6 µm along the periphery. On the average, 30% of the SAN cells surfaces is in close proximity to others. Identifiable gap junctions are extremely rare, but small sites of close membrane-to-membrane contacts are observed. Possibly communication occurs via these very small sites of contact if conducting channels (connexons) are located within them. There is no obvious anatomical detail that might support ephaptic coupling. These observations have implications for understanding of SAN cell physiology, and require incorporation into biophysically detailed models of SAN cell behavior that currently do not include such features.

Effects of Germline VHL Deficiency on Growth, Metabolism, and Mitochondria.

Mutations in VHL, which encodes von Hippel-Lindau tumor suppressor (VHL), are associated with divergent diseases. We describe a patient with marked erythrocytosis and prominent mitochondrial alterations associated with a severe germline VHL deficiency due to homozygosity for a novel synonymous mutation (c.222C→A, p.V74V). The condition is characterized by early systemic onset and differs from Chuvash polycythemia (c.598C→T) in that it is associated with a strongly reduced growth rate, persistent hypoglycemia, and limited exercise capacity. We report changes in gene expression that reprogram carbohydrate and lipid metabolism, impair muscle mitochondrial respiratory function, and uncouple oxygen consumption from ATP production. Moreover, we identified unusual intermitochondrial connecting ducts. Our findings add unexpected information on the importance of the VHL-hypoxia-inducible factor (HIF) axis to human phenotypes. (Funded by Associazione Italiana Ricerca sul Cancro and others.).

A proposed role for non-junctional transverse tubules in skeletal muscle as flexible segments allowing expansion of the transverse network.

Using a variety of technical approaches, we have detected the presence of continuous triads that cover the entire length of T tubules in the main white body muscles of several small fish. This is in contrast to the discontinuous association of sarcoplasmic reticulum with T tubules in the red muscles from the same fish as well as in all other previously described muscles in a large variety of skeletal muscles. We suggest that continuous triads are permissible only in muscle fibers that are not normally subject to significant changes in sarcomere length during normal in vivo activity, as is the case for white muscles in the trunk of fish.

The binding interactions that maintain excitation-contraction coupling junctions in skeletal muscle.

Calcium for contraction of skeletal muscles is released via tetrameric ryanodine receptor (RYR1) channels of the sarcoplasmic reticulum (SR), which are assembled in ordered arrays called couplons at junctions where the SR abuts T tubules or plasmalemma. Voltage-gated Ca2+ (CaV1.1) channels, found in tubules or plasmalemma, form symmetric complexes called CaV tetrads that associate with and activate underlying RYR tetramers during membrane depolarization by conveying a conformational change. Intriguingly, CaV tetrads regularly skip every other RYR tetramer within the array; therefore, the RYRs underlying tetrads (named V), but not the voltage sensor-lacking (C) RYRs, should be activated by depolarization. Here we hypothesize that the checkerboard association is maintained solely by reversible binary interactions between CaVs and RYRs and test this hypothesis using a quantitative model of the energies that govern CaV1.1-RYR1 binding, which are assumed to depend on number and location of bound CaVs. A Monte Carlo simulation generates large statistical samples and distributions of state variables that can be compared with quantitative features in freeze-fracture images of couplons from various sources. This analysis reveals two necessary model features: (1) the energy of a tetramer must have wells at low and high occupation by CaVs, so that CaVs positively cooperate in binding RYR (an allosteric effect), and (2) a large energy penalty results when two CaVs bind simultaneously to adjacent RYR protomers in adjacent tetramers (a steric clash). Under the hypothesis, V and C channels will eventually reverse roles. Role reversal justifies the presence of sensor-lacking C channels, as a structural and functional reserve for control of muscle contraction.

Elongated mitochondrial constrictions and fission in muscle fatigue.

Mitochondria respond to stress and undergo fusion and fission at variable rates, depending on cell status. To understand mitochondrial behavior during muscle fatigue, we investigated mitochondrial ultrastructure and expression levels of a fission- and stress-related protein in fast-twitch muscle fibers of mice subjected to fatigue testing. Mice were subjected to running at increasing speed until exhaustion at 45 min-1 h. In further experiments, high-intensity muscle stimulation through the sciatic nerve simulated the forced treadmill exercise. We detected a rare phenotype characterized by elongated mitochondrial constrictions (EMCs) connecting two separate segments of the original organelles. EMCs are rare in resting muscles and their frequency increases, albeit still at low levels, in stimulated muscles. The constrictions are accompanied by elevated phosphorylation of Drp1 (Dnm1l) at Ser 616, indicating an increased translocation of Drp1 to the mitochondrial membrane. This is indicative of a mitochondrial stress response, perhaps leading to or facilitating a long-lasting fission event. A close apposition of sarcoplasmic reticulum (SR) to the constricted areas, detected using both transmission and scanning electron microscopy, is highly suggestive of SR involvement in inducing mitochondrial constrictions.

Functionally Driven Modulation of Sarcomeric Structure and Membrane Systems in the Fast Muscles of a Copepod (Gaussia princeps).

Muscles of the mesopelagic copepod Gaussia princeps (Arthropoda, Crustacea, Calanoida) are responsible for repetitive movements of feeding and swimming appendages that are too fast to be followed by eye. This article provides a comparative functional and ultrastructural description of five muscles that have different contraction speeds and are located within different anatomical sites. All are very fast, as indicated by a thick:thin filament ratio of 3:1 and sarcomere lengths that vary between 1 and 3 µm. Measured lengths of thin and thick filaments indicate classification of the muscles into three distinct groups (short, medium, and long) and predict a difference in speed of up to threefold between fibers with the shortest and longest sarcomeres. Indeed, the kicking movement of the posterior legs (with the shortest sarcomere length) is approximately threefold faster than the simultaneous back-folding of the antennae (with the longest length). Thus, a specific relationship between speed of movement and sarcomere length is established, and we can use the latter to predict the former. Regulatory systems of contraction (sarcoplasmic reticulum [SR] and transverse [T] tubules) match the different contractile properties, varying in frequency of distribution and overall content in parallel to sarcomere variations. All muscles from appendages and body musculature show a unique disposition of contractile material, SR, and T tubules found only in copepod muscles; muscle filaments are grouped in large supermyofibrils that are riddled with frequent cylindrical shafts containing SR and T tubules. This arrangement insures a high spatial frequency of regulatory components. Anat Rec, 301:2164-2176, 2018. © 2018 Wiley Periodicals, Inc.

A conserved interaction of the dynein light intermediate chain with dynein-dynactin effectors necessary for processivity.

Cytoplasmic dynein is the major minus-end-directed microtubule-based motor in cells. Dynein processivity and cargo selectivity depend on cargo-specific effectors that, while generally unrelated, share the ability to interact with dynein and dynactin to form processive dynein-dynactin-effector complexes. How this is achieved is poorly understood. Here, we identify a conserved region of the dynein Light Intermediate Chain 1 (LIC1) that mediates interactions with unrelated dynein-dynactin effectors. Quantitative binding studies map these interactions to a conserved helix within LIC1 and to N-terminal fragments of Hook1, Hook3, BICD2, and Spindly. A structure of the LIC1 helix bound to the N-terminal Hook domain reveals a conformational change that creates a hydrophobic cleft for binding of the LIC1 helix. The LIC1 helix competitively inhibits processive dynein-dynactin-effector motility in vitro, whereas structure-inspired mutations in this helix impair lysosomal positioning in cells. The results reveal a conserved mechanism of effector interaction with dynein-dynactin necessary for processive motility.

The relationship between form and function throughout the history of excitation-contraction coupling.

The concept of excitation-contraction coupling is almost as old as Journal of General Physiology It was understood as early as the 1940s that a series of stereotyped events is responsible for the rapid contraction response of muscle fibers to an initial electrical event at the surface. These early developments, now lost in what seems to be the far past for most young investigators, have provided an endless source of experimental approaches. In this Milestone in Physiology, I describe in detail the experiments and concepts that introduced and established the field of excitation-contraction coupling in skeletal muscle. More recent advances are presented in an abbreviated form, as readers are likely to be familiar with recent work in the field.

Increased mitochondrial nanotunneling activity, induced by calcium imbalance, affects intermitochondrial matrix exchanges.

Exchanges of matrix contents are essential to the maintenance of mitochondria. Cardiac mitochondrial exchange matrix content in two ways: by direct contact with neighboring mitochondria and over longer distances. The latter mode is supported by thin tubular protrusions, called nanotunnels, that contact other mitochondria at relatively long distances. Here, we report that cardiac myocytes of heterozygous mice carrying a catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutation (A4860G) show a unique and unusual mitochondrial response: a significantly increased frequency of nanotunnel extensions. The mutation induces Ca2+ imbalance by depressing RyR2 channel activity during excitation-contraction coupling, resulting in random bursts of Ca2+ release probably due to Ca2+ overload in the sarcoplasmic reticulum. We took advantage of the increased nanotunnel frequency in RyR2A4860G+/- cardiomyocytes to investigate and accurately define the ultrastructure of these mitochondrial extensions and to reconstruct the overall 3D distribution of nanotunnels using electron tomography. Additionally, to define the effects of communication via nanotunnels, we evaluated the intermitochondrial exchanges of matrix-targeted soluble fluorescent proteins, mtDsRed and photoactivable mtPA-GFP, in isolated cardiomyocytes by confocal microscopy. A direct comparison between exchanges occurring at short and long distances directly demonstrates that communication via nanotunnels is slower.

Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle.

Calsequestrin, the only known protein with cyclical storage and supply of calcium as main role, is proposed to have other functions, which remain unproven. Voluntary movement and the heart beat require this calcium flow to be massive and fast. How does calsequestrin do it? To bind large amounts of calcium in vitro, calsequestrin must polymerize and then depolymerize to release it. Does this rule apply inside the sarcoplasmic reticulum (SR) of a working cell? We answered using fluorescently tagged calsequestrin expressed in muscles of mice. By FRAP and imaging we monitored mobility of calsequestrin as [Ca2+] in the SR--measured with a calsequestrin-fused biosensor--was lowered. We found that calsequestrin is polymerized within the SR at rest and that it depolymerized as [Ca2+] went down: fully when calcium depletion was maximal (a condition achieved with an SR calcium channel opening drug) and partially when depletion was limited (a condition imposed by fatiguing stimulation, long-lasting depolarization, or low drug concentrations). With fluorescence and electron microscopic imaging we demonstrated massive movements of calsequestrin accompanied by drastic morphological SR changes in fully depleted cells. When cells were partially depleted no remodeling was found. The present results support the proposed role of calsequestrin in termination of calcium release by conformationally inducing closure of SR channels. A channel closing switch operated by calsequestrin depolymerization will limit depletion, thereby preventing full disassembly of the polymeric calsequestrin network and catastrophic structural changes in the SR.

Congenital myopathy results from misregulation of a muscle Ca2+ channel by mutant Stac3.

Skeletal muscle contractions are initiated by an increase in Ca2+ released during excitation-contraction (EC) coupling, and defects in EC coupling are associated with human myopathies. EC coupling requires communication between voltage-sensing dihydropyridine receptors (DHPRs) in transverse tubule membrane and Ca2+ release channel ryanodine receptor 1 (RyR1) in the sarcoplasmic reticulum (SR). Stac3 protein (SH3 and cysteine-rich domain 3) is an essential component of the EC coupling apparatus and a mutation in human STAC3 causes the debilitating Native American myopathy (NAM), but the nature of how Stac3 acts on the DHPR and/or RyR1 is unknown. Using electron microscopy, electrophysiology, and dynamic imaging of zebrafish muscle fibers, we find significantly reduced DHPR levels, functionality, and stability in stac3 mutants. Furthermore, stac3NAM myofibers exhibited increased caffeine-induced Ca2+ release across a wide range of concentrations in the absence of altered caffeine sensitivity as well as increased Ca2+ in internal stores, which is consistent with increased SR luminal Ca2+ These findings define critical roles for Stac3 in EC coupling and human disease.

Skeletal Muscle Microalterations in Patients Carrying Malignant Hyperthermia-Related Mutations of the e-c Coupling Machinery.

We have compared the ultrastructure of skeletal muscle biopsies from patients that have survived a [Malignant Hyperthermia, MH] episode and siblings that test positive for MH susceptibility with those from siblings that tested negatives. The aim is to establish whether life long exposure to the MH-related mutation effects may result in subtle abnormalities even in the absence of active episodes and/or clinically detectable deficiencies. Although a specific ultrastructural signature for MH mutants cannot be demonstrated, an MH related pattern of minor alterations does exist. These include the tendency for micro damage to the contractile apparatus and a higher than normal level of mitochondrial abnormalities.