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Cardiovascular disease is a leading cause of morbidity and mortality, and exercise-training (TRN) is known to reduce risk factors and protect the heart from ischemia and reperfusion injury. Though the cardioprotective effects of exercise are well-documented, underlying mechanisms are not well understood. This review highlights recent findings and focuses on cardiac factors with emphasis on K + channel control of the action potential duration (APD), β-adrenergic and adenosine regulation of cardiomyocyte function, and mitochondrial Ca 2+ regulation. TRN-induced prolongation and shortening of the APD at low and high activation rates, respectively, is discussed in the context of a reduced response of the sarcolemma delayed rectifier potassium channel (IK) and increased content and activation of the sarcolemma K ATP channel. A proposed mechanism underlying the latter is presented, including the phosphatidylinositol-3kinase/protein kinase B pathway. TRN induced increases in cardiomyocyte contractility and the response to adrenergic agonists are discussed. The TRN-induced protection from reperfusion injury is highlighted by the increased content and activation of the sarcolemma K ATP channel and the increased phosphorylated glycogen synthase kinase-3β, which aid in preventing mitochondrial Ca 2+ overload and mitochondria-triggered apoptosis. Finally, a brief section is presented on the increased incidences of atrial fibrillation associated with age and in life-long exercisers., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Mitochondria within skeletal muscle cells are located either between the muscle contractile apparatus (interfibrillar mitochondria, IFM) or beneath the cell membrane (subsarcolemmal mitochondria, SSM), with several structural and functional differences reported between IFM and SSM. However, recent 3D imaging studies demonstrate that mitochondria are particularly concentrated in the proximity of capillaries embedded in sarcolemmal grooves rather than in proximity to the sarcolemma itself (paravascular mitochondria, PVM). To evaluate the impact of capillary vs. sarcolemmal proximity, we compared the structure and function of skeletal muscle mitochondria located either lateral to embedded capillaries (PVM), adjacent to the sarcolemma but not in PVM pools (SSM) or interspersed between sarcomeres (IFM). Mitochondrial morphology and interactions were assessed by 3D electron microscopy coupled with machine learning segmentation, whereas mitochondrial energy conversion was assessed by two-photon microscopy of mitochondrial membrane potential, content, calcium, NADH redox and flux in live, intact cells. Structurally, although PVM and SSM were similarly larger than IFM, PVM were larger, rounder and had more physical connections to neighbouring mitochondria compared to both IFM and SSM. Functionally, PVM had similar or greater basal NADH flux compared to SSM and IFM, respectively, despite a more oxidized NADH pool and a greater membrane potential, signifying a greater activation of the electron transport chain in PVM. Together, these data indicate that proximity to capillaries has a greater impact on resting mitochondrial energy conversion and distribution in skeletal muscle than the sarcolemma alone. KEY POINTS: Capillaries have a greater impact on mitochondrial energy conversion in skeletal muscle than the sarcolemma. Paravascular mitochondria are larger, and the outer mitochondrial membrane is more connected with neighbouring mitochondria. Interfibrillar mitochondria are longer and have greater contact sites with other organelles (i.e. sarcoplasmic reticulum and lipid droplets). Paravascular mitochondria have greater activation of oxidative phosphorylation than interfibrillar mitochondria at rest, although this is not regulated by calcium., (© 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Galectin-1 is a small (14.5 kDa) multifunctional protein with cell-cell and cell-ECM adhesion due to interactions with the carbohydrate recognition domain (CRD). In two types of muscular dystrophies, this lectin protein has shown therapeutic properties, including positive regulation of skeletal muscle differentiation and regeneration. Both Duchenne and limb-girdle muscular dystrophy 2B (LGMD2B) are subtypes of muscular dystrophies characterized by deficient membrane repair, muscle weakness, and eventual loss of ambulation. This chapter explains confocal techniques such as laser injury, calcium imaging, and galectin-1 localization to examine the effects of galectin-1 on membrane repair in injured LGMD2B models., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Isolated myofibers are commonly used to understand the function of skeletal muscle in vivo . This can involve single isolated myofibers obtained from dissection or from enzymatic dissociation. Isolation via dissection allows control of sarcomere length and preserves tendon attachment but is labor-intensive, time-consuming and yields few viable myofibers. In contrast, enzymatic dissociation is fast and facile, produces hundreds of myofibers, and more importantly reduces the number of muscles/animals needed for studies. Biomechanical properties of the sarcolemma have been studied using myofibers from the extensor digitorum longus, but this has been limited to dissected myofibers, making data collection slow and difficult. We have modified this tool to perform biomechanical measurements of the sarcolemma in dissociated myofibers from the flexor digitorum brevis.

Cardiac myocytes have multiple cell autonomous mechanisms that facilitate stabilization and repair of damaged sarcolemmal membranes following myocardial injury. Dysferlin is a protein which facilitates membrane repair by promoting membrane resealing. Although prior studies have shown that dysferlin-deficient (Dysf -/- ) mouse hearts have an impaired recovery from acute ischemia/reperfusion (I/R) injury ex vivo, the role of dysferlin in mediating the recovery from myocardial injury in vivo is unknown. Here we show that Dysf -/- mice develop adverse LV remodeling following I/R injury secondary to the collateral damage from sustained myocardial inflammation within the infarct zone. Backcrossing Dysf -/- mice with mice lacking signaling through the Toll-Interleukin 1 Receptor Domain-Containing Adaptor Protein (Tirap -/- ), attenuated inflammation and abrogated adverse LV remodeling following I/R injury. Subsequent studies using Poloxamer 188 (P188), a membrane resealing reagent, demonstrated that P188 did not attenuate inflammation nor prevent adverse LV remodeling in Dysf -/- mice following I/R injury. Viewed together these studies reveal a previously unappreciated role for the importance of membrane sealing and the resolution of inflammation following myocardial injury.

The skeletal muscle T-tubule is a specialized membrane domain essential for coordinated muscle contraction. However, in the absence of genetically tractable systems the mechanisms involved in T-tubule formation are unknown. Here, we use the optically transparent and genetically tractable zebrafish system to probe T-tubule development in vivo. By combining live imaging of transgenic markers with three-dimensional electron microscopy, we derive a four-dimensional quantitative model for T-tubule formation. To elucidate the mechanisms involved in T-tubule formation in vivo, we develop a quantitative screen for proteins that associate with and modulate early T-tubule formation, including an overexpression screen of the entire zebrafish Rab protein family. We propose an endocytic capture model involving firstly, formation of dynamic endocytic tubules at transient nucleation sites on the sarcolemma, secondly, stabilization by myofibrils/sarcoplasmic reticulum and finally, delivery of membrane from the recycling endosome and Golgi complex.

Transverse and axial tubules (TATS) are an essential ingredient of the excitation-contraction machinery that allow the effective coupling of L-type Calcium Channels (LCC) and ryanodine receptors (RyR2). They form a regular network in ventricular cells, while their presence in atrial myocytes is variable regionally and among animal species We have studied the effect of variations in the TAT network using a bidomain computational model of an atrial myocyte with variable density of tubules. At each z-line the t-tubule length is obtained from an exponential distribution, with a given mean penetration length. This gives rise to a distribution of t-tubules in the cell that is characterized by the fractional area (F.A.) occupied by the t-tubules. To obtain consistent results, we average over different realizations of the same mean penetration length. To this, in some simulations we add the effect of a network of axial tubules. Then we study global properties of calcium signaling, as well as regional heterogeneities and local properties of sparks and RyR2 openings. In agreement with recent experiments in detubulated ventricular and atrial cells, we find that detubulation reduces the calcium transient and synchronization in release. However, it does not affect sarcoplasmic reticulum (SR) load, so the decrease in SR calcium release is due to regional differences in Ca2+ release, that is restricted to the cell periphery in detubulated cells. Despite the decrease in release, the release gain is larger in detubulated cells, due to recruitment of orphaned RyR2s, i.e, those that are not confronting a cluster of LCCs. This probably provides a safeguard mechanism, allowing physiological values to be maintained upon small changes in the t-tubule density. Finally, we do not find any relevant change in spark properties between tubulated and detubulated cells, suggesting that the differences found in experiments could be due to differential properties of the RyR2s in the membrane and in the t-tubules, not incorporated in the present model. This work will help understand the effect of detubulation, that has been shown to occur in disease conditions such as heart failure (HF) in ventricular cells, or atrial fibrillation (AF) in atrial cells., Competing Interests: The authors declare that no competing interests exist.

Objective: Hypokalaemic periodic paralysis (HypoPP) is caused by mutations of Ca v 1.1, and Na v 1.4 which result in an aberrant gating pore current. Hyperkalaemic periodic paralysis (HyperPP) is due to a gain-of-function mutation of the main alpha pore of Na v 1.4. This study used muscle velocity recovery cycles (MVRCs) to investigate changes in interictal muscle membrane properties in vivo., Methods: MVRCs and responses to trains of stimuli were recorded in tibialis anterior and compared in patients with HyperPP(n = 7), HypoPP (n = 10), and normal controls (n = 26)., Results: Muscle relative refractory period was increased, and early supernormality reduced in HypoPP, consistent with depolarisation of the interictal resting membrane potential. In HyperPP the mean supernormality and residual supernormality to multiple conditioning stimuli were increased, consistent with increased inward sodium current and delayed repolarisation, predisposing to spontaneous myotonic discharges., Conclusions: The in vivo findings suggest the interictal resting membrane potential is depolarized in HypoPP, and mostly normal in HyperPP. The MVRC findings in HyperPP are consistent with presence of a window current, previously proposed on the basis of in vitro expression studies. Although clinically similar, HyperPP was electrophysiologically distinct from paramyotonia congenita., Significance: MVRCs provide important in vivo data that complements expression studies of ion channel mutations., Competing Interests: Declaration of Competing Interest Professor Hugh Bostock receives royalties from the sales of Qtrac Software used for the muscle excitability studies. The remaining authors have no conflicts of interest., (Copyright © 2020 International Federation of Clinical Neurophysiology. Published by Elsevier B.V. All rights reserved.)

The skeletal muscle and myocardial cells present highly specialized structures; for example, the close interaction between the sarcoplasmic reticulum (SR) and mitochondria-responsible for excitation-metabolism coupling-and the junction that connects the SR with T-tubules, critical for excitation-contraction (EC) coupling. The mechanisms that underlie EC coupling in these two cell types, however, are fundamentally distinct. They involve the differential expression of Ca 2+ channel subtypes: Ca V 1.1 and RyR1 (skeletal), vs. Ca V 1.2 and RyR2 (cardiac). The Ca V channels transform action potentials into elevations of cytosolic Ca 2+ , by activating RyRs and thus promoting SR Ca 2+ release. The high levels of Ca 2+ , in turn, stimulate not only the contractile machinery but also the generation of mitochondrial reactive oxygen species (ROS). This forward signaling is reciprocally regulated by the following feedback mechanisms: Ca 2+ -dependent inactivation (of Ca 2+ channels), the recruitment of Na + /Ca 2+ exchanger activity, and oxidative changes in ion channels and transporters. Here, we summarize both well-established concepts and recent advances that have contributed to a better understanding of the molecular mechanisms involved in this bidirectional signaling.

The cardiomyocyte plasma membrane, termed the sarcolemma, is fundamental for regulating a myriad of cellular processes. For example, the structural integrity of the cardiomyocyte sarcolemma is essential for mediating cardiac contraction by forming microdomains such as the t-tubular network, caveolae and the intercalated disc. Significantly, remodelling of these sarcolemma microdomains is a key feature in the development and progression of heart failure (HF). However, despite extensive characterisation of the associated molecular and ultrastructural events there is a lack of clarity surrounding the mechanisms driving adverse morphological rearrangements. The sarcolemma also provides protection, and is the cell's first line of defence, against external stresses such as oxygen and nutrient deprivation, inflammation and oxidative stress with a loss of sarcolemma viability shown to be a key step in cell death via necrosis. Significantly, cumulative cell death is also a feature of HF, and is linked to disease progression and loss of cardiac function. Herein, we will review the link between structural and molecular remodelling of the sarcolemma associated with the progression of HF, specifically considering the evidence for: (i) Whether intrinsic, evolutionary conserved, plasma membrane injury-repair mechanisms are in operation in the heart, and (ii) if deficits in key 'wound-healing' proteins (annexins, dysferlin, EHD2 and MG53) may play a yet to be fully appreciated role in triggering sarcolemma microdomain remodelling and/or necrosis. Cardiomyocytes are terminally differentiated with very limited regenerative capability and therefore preserving cell viability and cardiac function is crucially important. This review presents a novel perspective on sarcolemma remodelling by considering whether targeting proteins that regulate sarcolemma injury-repair may hold promise for developing new strategies to attenuate HF progression.

Objective: To study patients with sporadic inclusion body myositis (sIBM) with muscle velocity recovery cycles (MVRC) to assess muscle membrane excitability, pathophysiological mechanisms and potential biomarkers of this disorder., Methods: MVRC were recorded from 20 individuals with sIBM from tibialis anterior (TA) and rectus femoris (RF) muscles. Excitability parameters were compared with MVRC data obtained from 22 normal controls >50 years., Results: Muscle relative refractory period was prolonged in both TA (6.4 ms vs 4.4 ms, P < 0.001) and RF (7.1 ms vs 3.9 ms, P < 0.001) of sIBM affected muscle when compared to controls. Early supernormality was reduced in both TA (3.6% vs 8.8% P = 0.001) and in RF (mean 5.4% vs 13% P < 0.001). Late supernormality was only decreased significantly in sIBM affected TA (1.8% vs 3.6% P = 0.001) but not in RF. No consistent correlations between MVRC parameters and clinical markers of sIBM disease severity were found., Conclusion: The resting sarcolemmal muscle membrane potential of sIBM muscle is depolarized relative to that of normal controls, which may be related to intramuscular amyloid deposition in sIBM., Significance: Sarcolemmal depolarization may play a role in muscle dysfunction and weakness observed in sIBM patients., (Copyright © 2019 International Federation of Clinical Neurophysiology. Published by Elsevier B.V. All rights reserved.)

Introduction: The sarcolemmal resting membrane potential (RMP) affects muscle excitability, contractility, and force generation. However, there are limited In vivo data on the normal RMP of the human sarcolemma between muscles. We hypothesize that the in vivo RMP may differ between human muscles with different physiological roles., Methods: Muscle velocity recovery cycles were recorded from a proximal antigravity muscle, the rectus femoris, and compared with paired recordings from a distal non-antigravity muscle, the tibialis anterior, in 34 normal individuals., Results: Significant differences in muscle relative refractory period (3.55 millseconds vs 3.87 milliseconds, P = .002), early supernormality (14.22% vs 10.50%, P < .0001), and late supernormality (5.43% vs 3.50%, P < .0001) were observed., Discussion: The results strongly suggest a less negative RMP in tibialis anterior vs rectus femoris and attest to intermuscle differences in normal excitability and physiology. This novel finding employing an in vivo methodology highlights the need for muscle-specific normative data in future studies., (© 2019 Wiley Periodicals, Inc.)

α2-adrenoceptor (α2-AR) isoforms, abundant in sympathetic synapses and noradrenergic neurons of the central nervous system, are integral in the presynaptic feed-back loop mechanism that moderates norepinephrine surges. We recently identified that postsynaptic α2-ARs, found in the myocellular sarcolemma, also contribute to a muscle-delimited feedback control capable of attenuating mobilization of intracellular Ca 2+ and myocardial contractility. This previously unrecognized α2-AR-dependent rheostat is able to counteract competing adrenergic receptor actions in cardiac muscle. Specifically, in ventricular myocytes, nitric oxide (NO) and cGMP are the intracellular messengers of α2-AR signal transduction pathways that gauge the kinase-phosphatase balance and manage cellular Ca 2+ handling preventing catecholamine-induced Ca 2+ overload. Moreover, α2-AR signaling counterbalances phospholipase C - PKC-dependent mechanisms underscoring a broader cardioprotective potential under sympathoadrenergic and angiotensinergic challenge. Recruitment of such tissue-specific features of α2-AR under sustained sympathoadrenergic drive may, in principle, be harnessed to mitigate or prevent cardiac malfunction. However, cardiovascular disease may compromise peripheral α2-AR signaling limiting pharmacological targeting of these receptors. Prospective cardiac-specific gene or cell-based therapeutic approaches aimed at repairing or improving stress-protective α2-AR signaling may offer an alternative towards enhanced preservation of cardiac muscle structure and function., (Copyright © 2019. Published by Elsevier Inc.)

Key Points: Prevailing dogma holds that activation of the β-adrenergic receptor/cAMP/protein kinase A signalling pathway leads to enhanced L-type Ca V 1.2 channel activity, resulting in increased Ca 2+ influx into ventricular myocytes and a positive inotropic response. However, the full mechanistic and molecular details underlying this phenomenon are incompletely understood. Ca V 1.2 channel clusters decorate T-tubule sarcolemmas of ventricular myocytes. Within clusters, nanometer proximity between channels permits Ca 2+ -dependent co-operative gating behaviour mediated by physical interactions between adjacent channel C-terminal tails. We report that stimulation of cardiomyocytes with isoproterenol, evokes dynamic, protein kinase A-dependent augmentation of Ca V 1.2 channel abundance along cardiomyocyte T-tubules, resulting in the appearance of channel 'super-clusters', and enhanced channel co-operativity that amplifies Ca 2+ influx. On the basis of these data, we suggest a new model in which a sub-sarcolemmal pool of pre-synthesized Ca V 1.2 channels resides in cardiomyocytes and can be mobilized to the membrane in times of high haemodynamic or metabolic demand, to tune excitation-contraction coupling., Abstract: Voltage-dependent L-type Ca V 1.2 channels play an indispensable role in cardiac excitation-contraction coupling. Activation of the β-adrenergic receptor (βAR)/cAMP/protein kinase A (PKA) signalling pathway leads to enhanced Ca V 1.2 activity, resulting in increased Ca 2+ influx into ventricular myocytes and a positive inotropic response. Ca V 1.2 channels exhibit a clustered distribution along the T-tubule sarcolemma of ventricular myocytes where nanometer proximity between channels permits Ca 2+ -dependent co-operative gating behaviour mediated by dynamic, physical, allosteric interactions between adjacent channel C-terminal tails. This amplifies Ca 2+ influx and augments myocyte Ca 2+ transient and contraction amplitudes. We investigated whether βAR signalling could alter Ca V 1.2 channel clustering to facilitate co-operative channel interactions and elevate Ca 2+ influx in ventricular myocytes. Bimolecular fluorescence complementation experiments reveal that the βAR agonist, isoproterenol (ISO), promotes enhanced Ca V 1.2-Ca V 1.2 physical interactions. Super-resolution nanoscopy and dynamic channel tracking indicate that these interactions are expedited by enhanced spatial proximity between channels, resulting in the appearance of Ca V 1.2 'super-clusters' along the z-lines of ISO-stimulated cardiomyocytes. The mechanism that leads to super-cluster formation involves rapid, dynamic augmentation of sarcolemmal Ca V 1.2 channel abundance after ISO application. Optical and electrophysiological single channel recordings confirm that these newly inserted channels are functional and contribute to overt co-operative gating behaviour of Ca V 1.2 channels in ISO stimulated myocytes. The results of the present study reveal a new facet of βAR-mediated regulation of Ca V 1.2 channels in the heart and support the novel concept that a pre-synthesized pool of sub-sarcolemmal Ca V 1.2 channel-containing vesicles/endosomes resides in cardiomyocytes and can be mobilized to the sarcolemma to tune excitation-contraction coupling to meet metabolic and/or haemodynamic demands., (© 2019 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Background: Niemann-Pick disease type A (NPDA), a disease caused by mutations in acid sphingomyelinase (ASM), involves severe neurodegeneration and early death. Intracellular lipid accumulation and plasma membrane alterations are implicated in the pathology. ASM is also linked to the mechanism of plasma membrane repair, so we investigated the impact of ASM deficiency in skeletal muscle, a tissue that undergoes frequent cycles of injury and repair in vivo., Methods: Utilizing the NPDA/B mouse model ASM -/- and wild type (WT) littermates, we performed excitation-contraction coupling/Ca 2+ mobilization and sarcolemma injury/repair assays with isolated flexor digitorum brevis fibers, proteomic analyses with quadriceps femoris, flexor digitorum brevis, and tibialis posterior muscle and in vivo tests of the contractile force (maximal isometric torque) of the quadriceps femoris muscle before and after eccentric contraction-induced muscle injury., Results: ASM -/- flexor digitorum brevis fibers showed impaired excitation-contraction coupling compared to WT, a defect expressed as reduced tetanic [Ca 2+ ] i in response to electrical stimulation and early failure in sustaining [Ca 2+ ] i during repeated tetanic contractions. When injured mechanically by needle passage, ASM -/- flexor digitorum brevis fibers showed susceptibility to injury similar to WT, but a reduced ability to reseal the sarcolemma. Proteomic analyses revealed changes in a small group of skeletal muscle proteins as a consequence of ASM deficiency, with downregulation of calsequestrin occurring in the three different muscles analyzed. In vivo, the loss in maximal isometric torque of WT quadriceps femoris was similar immediately after and 2 min after injury. The loss in ASM -/- mice immediately after injury was similar to WT, but was markedly larger at 2 min after injury., Conclusions: Skeletal muscle fibers from ASM -/- mice have an impairment in intracellular Ca 2+ handling that results in reduced Ca 2+ mobilization and a more rapid decline in peak Ca 2+ transients during repeated contraction-relaxation cycles. Isolated fibers show reduced ability to repair damage to the sarcolemma, and this is associated with an exaggerated deficit in force during recovery from an in vivo eccentric contraction-induced muscle injury. Our findings uncover the possibility that skeletal muscle functional defects may play a role in the pathology of NPDA/B disease.

A prominent theory of cell death in myocardial ischemia/reperfusion (I/R) posits that the primary and pivotal step of irreversible cell injury is the opening of the mitochondrial permeability transition (MPT) pore. However, the predominantly positive evidence of protection against infarct afforded by the MPT inhibitor, Cyclosporine A (CsA), in experimental studies is in stark contrast with the overall lack of benefit found in clinical trials of CsA. One reason for the discrepancy might be the fact that relatively short experimental ischemic episodes (<1 hour) do not represent clinically-realistic durations, usually exceeding one hour. Here we tested the hypothesis that MPT is not the primary event of cell death after prolonged (60-80 min) episodes of global ischemia. We used confocal microcopy in Langendorff-perfused rabbit hearts treated with the electromechanical uncoupler, 2,3-Butanedione monoxime (BDM, 20 mM) to allow tracking of MPT and sarcolemmal permeabilization (SP) in individual ventricular myocytes. The time of the steepest drop in fluorescence of mitochondrial membrane potential (ΔΨm)-sensitive dye, TMRM, was used as the time of MPT (TMPT). The time of 20% uptake of the normally cell-impermeable dye, YO-PRO1, was used as the time of SP (TSP). We found that during reperfusion MPT and SP were tightly coupled, with MPT trending slightly ahead of SP (TSP-TMPT = 0.76±1.31 min; p = 0.07). These coupled MPT/SP events occurred in discrete myocytes without crossing cell boundaries. CsA (0.2 μM) did not reduce the infarct size, but separated SP and MPT events, such that detectable SP was significantly ahead of MPT (TSP -TMPT = -1.75±1.28 min, p = 0.006). Mild permeabilization of cells with digitonin (2.5-20 μM) caused coupled MPT/SP events which occurred in discrete myocytes similar to those observed in Control and CsA groups. In contrast, deliberate induction of MPT by titration with H2O2 (200-800 μM), caused propagating waves of MPT which crossed cell boundaries and were uncoupled from SP. Taken together, these findings suggest that after prolonged episodes of ischemia, SP is the primary step in myocyte death, of which MPT is an immediate and unavoidable consequence., Competing Interests: The authors have declared that no competing interests exist.

Introduction: The exact mechanisms underlying the loss of skeletal muscle bulk and power with normal human aging are not well established. Recording of muscle velocity recovery cycles (MVRCs) is an in-vivo neurophysiologic technique we employed to assess the impact of age on sarcolemmal excitability., Methods: MVRC recordings were obtained from tibialis anterior (n = 74) and rectus femoris (n = 32) muscles in 74 healthy subjects (18-84 years, median age 35 years, interquartile range 29-55 years)., Results: Increasing age was linearly associated with longer muscle relative refractory period (MRRP) and reduced early supernormality (ESN) in both tibialis anterior (MRRP: r 2  = 0.38, P < 0.001; ESN: r 2  = 0.33, P < 0.001) and rectus femoris (MRRP: r 2  = 0.30, P = 0.002; ESN: r 2  = 0.19, P = 0.01) muscles., Discussion: The results are consistent with progressive depolarization of the resting sarcolemmal potential with normal aging. This may be an important mechanism in explaining age-related muscle decline. Muscle Nerve 57: 981-988, 2018., (© 2018 Wiley Periodicals, Inc.)

The complex membrane structure of the tubular system (t-system) in skeletal muscle fibers is open to the extracellular environment, which prevents measurements of H + movement across its interface with the cytoplasm by conventional methods. Consequently, little is known about the t-system's role in the regulation of cytoplasmic pH, which is different from extracellular pH. Here we describe a novel approach to measure H + -flux measurements across the t-system of fast-twitch fibers under different conditions. The approach involves loading the t-system of intact rat fast-twitch fibers with a strong pH buffer (20 mM HEPES) and pH-sensitive fluorescent probe (10 mM HPTS) before the t-system is sealed off. The pH changes in the t-system are then tracked by confocal microscopy after rapid changes in cytoplasmic ionic conditions. T-system sealing is achieved by removing the sarcolemma by microdissection (mechanical skinning), which causes the tubules to pinch off and seal tight. After this procedure, the t-system repolarizes to physiological levels and can be electrically stimulated when placed in K + -based solutions of cytosolic-like ionic composition. Using this approach, we show that the t-system of fast-twitch skeletal fibers displays amiloride-sensitive Na + /H + exchange (NHE), which decreases markedly at alkaline cytosolic pH and has properties similar to that in mammalian cardiac myocytes. We observed mean values for NHE density and proton permeability coefficient of 339 pmol/m 2 of t-system membrane and 158 µm/s, respectively. We conclude that the cytosolic pH in intact resting muscle can be quantitatively explained with respect to extracellular pH by assuming that these values apply to the t-system membrane and the sarcolemma., (© 2018 Launikonis et al.)

Genetic disruption of the dystrophin complex produces muscular dystrophy characterized by a fragile muscle plasma membrane leading to excessive muscle degeneration. Two genetic modifiers of Duchenne Muscular Dystrophy implicate the transforming growth factor β (TGFβ) pathway, osteopontin encoded by the SPP1 gene and latent TGFβ binding protein 4 (LTBP4). We now evaluated the functional effect of these modifiers in the context of muscle injury and repair to elucidate their mechanisms of action. We found that excess osteopontin exacerbated sarcolemmal injury, and correspondingly, that loss of osteopontin reduced injury extent both in isolated myofibers and in muscle in vivo. We found that ablation of osteopontin was associated with reduced expression of TGFβ and TGFβ-associated pathways. We identified that increased TGFβ resulted in reduced expression of Anxa1 and Anxa6, genes encoding key components of the muscle sarcolemma resealing process. Genetic manipulation of Ltbp4 in dystrophic muscle also directly modulated sarcolemmal resealing, and Ltbp4 alleles acted in concert with Anxa6, a distinct modifier of muscular dystrophy. These data provide a model in which a feed forward loop of TGFβ and osteopontin directly impacts the capacity of muscle to recover from injury, and identifies an intersection of genetic modifiers on muscular dystrophy.

Dyslipidemia is associated with greater risk of ventricular tachyarrhythmias in patients with cardiovascular diseases. We aimed to examine whether the most electronegative subfraction of low-density lipoprotein (LDL), L5, is correlated with QTc prolongation in patients with coronary artery disease (CAD) and investigate the effects of human L5 on the electrophysiological properties of cardiomyocytes in relation to the lectin-like oxidized LDL receptor (LOX-1). L5 was isolated from the plasma of 40 patients with angiography documented CAD and 13 patients with no CAD to correlate the QTc interval respectively. The mean concentration of L5 was higher and correlated with QTc in patients with CAD compared to controls. To examine the direct effect of L5 on QTc, mice were intravenously injected with L5 or L1. L5-injected wild-type but not LOX-1 -/- mice showed longer QTc compared to L1-injected animals in vivo with corresponding longer action potential duration (APD) in cardiomyocytes incubated with L5 in vitro. The APD prolongation was mediated by an increase of L-type calcium current and a decrease of transient outward potassium current. We show that L5 was positively correlated with QTc prolongation in patients with ischemic heart disease. L5 can modulate cardiac repolarization via LOX-1-mediated alteration sarcolemmal ionic currents.

Caveolae are signal transduction centers, yet their subcellular distribution and preservation in cardiac myocytes after cell isolation are not well documented. Here, we quantify caveolae located within 100 nm of the outer cell surface membrane in rabbit single-ventricular cardiomyocytes over 8 h post-isolation and relate this to the presence of caveolae in intact tissue. Hearts from New Zealand white rabbits were either chemically fixed by coronary perfusion or enzymatically digested to isolate ventricular myocytes, which were subsequently fixed at 0, 3, and 8 h post-isolation. In live cells, the patch-clamp technique was used to measure whole-cell plasma membrane capacitance, and in fixed cells, caveolae were quantified by transmission electron microscopy. Changes in cell-surface topology were assessed using scanning electron microscopy. In fixed ventricular myocardium, dual-axis electron tomography was used for three-dimensional reconstruction and analysis of caveolae in situ. The presence and distribution of surface-sarcolemmal caveolae in freshly isolated cells matches that of intact myocardium. With time, the number of surface-sarcolemmal caveolae decreases in isolated cardiomyocytes. This is associated with a gradual increase in whole-cell membrane capacitance. Concurrently, there is a significant increase in area, diameter, and circularity of sub-sarcolemmal mitochondria, indicative of swelling. In addition, electron tomography data from intact heart illustrate the regular presence of caveolae not only at the surface sarcolemma, but also on transverse-tubular membranes in ventricular myocardium. Thus, caveolae are dynamic structures, present both at surface-sarcolemmal and transverse-tubular membranes. After cell isolation, the number of surface-sarcolemmal caveolae decreases significantly within a time frame relevant for single-cell research. The concurrent increase in cell capacitance suggests that membrane incorporation of surface-sarcolemmal caveolae underlies this, but internalization and/or micro-vesicle loss to the extracellular space may also contribute. Given that much of the research into cardiac caveolae-dependent signaling utilizes isolated cells, and since caveolae-dependent pathways matter for a wide range of other study targets, analysis of isolated cell data should take the time post-isolation into account., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Glucocorticoid steroids such as prednisone are prescribed for chronic muscle conditions such as Duchenne muscular dystrophy, where their use is associated with prolonged ambulation. The positive effects of chronic steroid treatment in muscular dystrophy are paradoxical because these steroids are also known to trigger muscle atrophy. Chronic steroid use usually involves once-daily dosing, although weekly dosing in children has been suggested for its reduced side effects on behavior. In this work, we tested steroid dosing in mice and found that a single pulse of glucocorticoid steroids improved sarcolemmal repair through increased expression of annexins A1 and A6, which mediate myofiber repair. This increased expression was dependent on glucocorticoid response elements upstream of annexins and was reinforced by the expression of forkhead box O1 (FOXO1). We compared weekly versus daily steroid treatment in mouse models of acute muscle injury and in muscular dystrophy and determined that both regimens provided comparable benefits in terms of annexin gene expression and muscle repair. However, daily dosing activated atrophic pathways, including F-box protein 32 (Fbxo32), which encodes atrogin-1. Conversely, weekly steroid treatment in mdx mice improved muscle function and histopathology and concomitantly induced the ergogenic transcription factor Krüppel-like factor 15 (Klf15) while decreasing Fbxo32. These findings suggest that intermittent, rather than daily, glucocorticoid steroid regimen promotes sarcolemmal repair and muscle recovery from injury while limiting atrophic remodeling.

Well-coordinated activation of all cardiomyocytes must occur on every heartbeat. At the cell level, a complex network of sarcolemmal invaginations, called the transverse-axial tubular system (TATS), propagates membrane potential changes to the cell core, ensuring synchronous and uniform excitation-contraction coupling. Although myocardial conduction of excitation has been widely described, the electrical properties of the TATS remain mostly unknown. Here, we exploit the formal analogy between diffusion and electrical conductivity to link the latter with the diffusional properties of TATS. Fluorescence recovery after photobleaching (FRAP) microscopy is used to probe the diffusion properties of TATS in isolated rat cardiomyocytes: A fluorescent dextran inside TATS lumen is photobleached, and signal recovery by diffusion of unbleached dextran from the extracellular space is monitored. We designed a mathematical model to correlate the time constant of fluorescence recovery with the apparent diffusion coefficient of the fluorescent molecules. Then, apparent diffusion is linked to electrical conductivity and used to evaluate the efficiency of the passive spread of membrane depolarization along TATS. The method is first validated in cells where most TATS elements are acutely detached by osmotic shock and then applied to probe TATS electrical conductivity in failing heart cells. We find that acute and pathological tubular remodeling significantly affect TATS electrical conductivity. This may explain the occurrence of defects in action potential propagation at the level of single T-tubules, recently observed in diseased cardiomyocytes., Competing Interests: The authors declare no conflict of interest.

The fibroblast growth factor (FGF) homologous factor FGF13, a noncanonical FGF, has been best characterized as a voltage-gated Na + channel auxiliary subunit. Other cellular functions have been suggested, but not explored. In inducible, cardiac-specific Fgf13 knockout mice, we found-even in the context of the expected reduction in Na + channel current-an unanticipated protection from the maladaptive hypertrophic response to pressure overload. To uncover the underlying mechanisms, we searched for components of the FGF13 interactome in cardiomyocytes and discovered the complete set of the cavin family of caveolar coat proteins. Detailed biochemical investigations showed that FGF13 acts as a negative regulator of caveolae abundance in cardiomyocytes by controlling the relative distribution of cavin 1 between the sarcolemma and cytosol. In cardiac-specific Fgf13 knockout mice, cavin 1 redistribution to the sarcolemma stabilized the caveolar structural protein caveolin 3. The consequent increase in caveolae density afforded protection against pressure overload-induced cardiac dysfunction by two mechanisms: ( i ) enhancing cardioprotective signaling pathways enriched in caveolae, and ( ii ) increasing the caveolar membrane reserve available to buffer membrane tension. Thus, our results uncover unexpected roles for a FGF homologous factor and establish FGF13 as a regulator of caveolae-mediated mechanoprotection and adaptive hypertrophic signaling., Competing Interests: The authors declare no conflict of interest.

Cholesterol regulates numerous cellular processes. Depleting its synthesis in skeletal myofibers induces vacuolization and contraction impairment. However, little is known about how cholesterol reduction affects cardiomyocyte behavior. Here, we deplete cholesterol by incubating neonatal cardiomyocytes with methyl-beta-cyclodextrin. Traction force microscopy shows that lowering cholesterol increases the rate of cell contraction and generates defects in cell relaxation. Cholesterol depletion also increases membrane tension, Ca 2+ spikes frequency and intracellular Ca 2+ concentration. These changes can be correlated with modifications in caveolin-3 and L-Type Ca 2+ channel distributions across the sarcolemma. Channel regulation is also compromised since cAMP-dependent PKA activity is enhanced, increasing the probability of L-Type Ca 2+ channel opening events. Immunofluorescence reveals that cholesterol depletion abrogates sarcomeric organization, changing spacing and alignment of α-actinin bands due to increase in proteolytic activity of calpain. We propose a mechanism in which cholesterol depletion triggers a signaling cascade, culminating with contraction impairment and myofibril disruption in cardiomyocytes.

The characterization of the membrane repair machinery in human skeletal muscle has become crucial, since it has been shown that some muscular dystrophies result from a defect of this fundamental physiological process. Deciphering membrane repair mechanism requires the development of methodologies allowing studying the response of skeletal muscle cells to sarcolemma damage and identifying candidate proteins playing a role in the membrane repair machinery. Here, we describe a protocol that is based on the creation of cell membrane disruption by infrared laser irradiation in human myotubes. Membrane disruption and repair are assayed by monitoring the incorporation into myotubes of the membrane probe FM1-43. This methodology has recently enabled us to show that Annexin-A5 is required for membrane repair in human skeletal muscle cells (Carmeille et al., Biochim Biophys Acta 1863:2267-2279, 2016).

Age-related loss of skeletal muscle mass and function, referred to as sarcopenia, is mitigated by lifelong calorie restriction as well as exercise. In aged skeletal muscle fibers there is compromised integrity of the cell membrane that may contribute to sarcopenia. The purpose of this study was to determine if lifelong mild (8%) caloric restriction (CR) and lifelong CR+voluntary wheel running (WR) could ameliorate disruption of membrane scaffolding and signaling proteins during the aging process, thus maintaining a favorable, healthy membrane environment in plantaris muscle fibers. Fischer-344 rats were divided into four groups: 24-month old adults fed ad libitum (OAL); 24-month old on 8% caloric restriction (OCR); 24month old 8% caloric restriction+wheel running (OCRWR); and 6-month old sedentary adults fed ad libitum (YAL) were used to determine age-related changes. Aging resulted in discontinuous membrane expression of dystrophin glycoprotein complex (DGC) proteins: dystrophin and α-syntrophin. Older muscle also displayed decreased content of neuronal nitric oxide synthase (nNOS), a key DGC signaling protein. In contrast, OCR and OCRWR provided significant protection against age-related DGC disruption. In conjunction with the age-related decline in membrane DGC patency, key membrane repair proteins (MG53, dysferlin, annexin A6, and annexin A2) were significantly increased in the OAL plantaris. However, lifelong CR and CRWR interventions were effective at maintaining membrane repair proteins near YAL levels of. OAL fibers also displayed reduced protein content of NADPH oxidase isoform 2 (Nox2) subunits (p67phox and p47phox), consistent with a perturbed sarcolemmal environment. Loss of Nox2 subunits was prevented by lifelong CR and CRWR. Our results are therefore consistent with the hypothesis that lifelong CR and WR are effective countermeasures against age-related alterations in the myofiber membrane environment., Competing Interests: Conflicts of Interest: None declared, (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Duchenne muscular dystrophy is a fatal cardiac and skeletal muscle disease resulting from mutations in the dystrophin gene. We have previously demonstrated that a dystrophin-associated protein, sarcospan (SSPN), ameliorated Duchenne muscular dystrophy skeletal muscle degeneration by activating compensatory pathways that regulate muscle cell adhesion (laminin-binding) to the extracellular matrix. Conversely, loss of SSPN destabilized skeletal muscle adhesion, hampered muscle regeneration, and reduced force properties. Given the importance of SSPN to skeletal muscle, we investigated the consequences of SSPN ablation in cardiac muscle and determined whether overexpression of SSPN into mdx mice ameliorates cardiac disease symptoms associated with Duchenne muscular dystrophy cardiomyopathy., Methods and Results: SSPN-null mice exhibited cardiac enlargement, exacerbated cardiomyocyte hypertrophy, and increased fibrosis in response to β-adrenergic challenge (isoproterenol; 0.8 mg/day per 2 weeks). Biochemical analysis of SSPN-null cardiac muscle revealed reduced sarcolemma localization of many proteins with a known role in cardiomyopathy pathogenesis: dystrophin, the sarcoglycans (α-, δ-, and γ-subunits), and β1D integrin. Transgenic overexpression of SSPN in Duchenne muscular dystrophy mice (mdx(TG)) improved cardiomyofiber cell adhesion, sarcolemma integrity, cardiac functional parameters, as well as increased expression of compensatory transmembrane proteins that mediate attachment to the extracellular matrix., Conclusions: SSPN regulates sarcolemmal expression of laminin-binding complexes that are critical to cardiac muscle function and protects against transient and chronic injury, including inherited cardiomyopathy., (© 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Arterial smooth muscle (SM) cells respond autonomously to changes in intravascular pressure, adjusting tension to maintain vessel diameter. The values of membrane potential (Vm) and sarcoplasmic Ca(2+) concentration (Ca(in)) within minutes of a change in pressure are the results of two opposing pathways, both of which use Ca(2+) as a signal. This works because the two Ca(2+)-signaling pathways are confined to distinct microdomains in which the Ca(2+) concentrations needed to activate key channels are transiently higher than Ca(in). A mathematical model of an isolated arterial SM cell is presented that incorporates the two types of microdomains. The first type consists of junctions between cisternae of the peripheral sarcoplasmic reticulum (SR), containing ryanodine receptors (RyRs), and the sarcolemma, containing voltage- and Ca(2+)-activated K(+) (BK) channels. These junctional microdomains promote hyperpolarization, reduced Ca(in), and relaxation. The second type is postulated to form around stretch-activated nonspecific cation channels and neighboring Ca(2+)-activated Cl(-) channels, and promotes the opposite (depolarization, increased Ca(in), and contraction). The model includes three additional compartments: the sarcoplasm, the central SR lumen, and the peripheral SR lumen. It incorporates 37 protein components. In addition to pressure, the model accommodates inputs of α- and β-adrenergic agonists, ATP, 11,12-epoxyeicosatrienoic acid, and nitric oxide (NO). The parameters of the equations were adjusted to obtain a close fit to reported Vm and Ca(in) as functions of pressure, which have been determined in cerebral arteries. The simulations were insensitive to ± 10% changes in most of the parameters. The model also simulated the effects of inhibiting RyR, BK, or voltage-activated Ca(2+) channels on Vm and Ca(in). Deletion of BK β1 subunits is known to increase arterial-SM tension. In the model, deletion of β1 raised Ca(in) at all pressures, and these increases were reversed by NO., (© 2015 Karlin.)

We examined whether a Ca(2+)-K(+) interaction was a potential mechanism operating during fatigue with repeated tetani in isolated mouse muscles. Raising the extracellular Ca(2+) concentration ([Ca(2+)]o) from 1.3 to 10 mM in K(+)-depressed slow-twitch soleus and/or fast-twitch extensor digitorum longus muscles caused the following: 1) increase of intracellular K(+) activity by 20-60 mM (raised intracellular K(+) content, unchanged intracellular fluid volume), so that the K(+)-equilibrium potential increased by ∼10 mV and resting membrane potential repolarized by 5-10 mV; 2) large restoration of action potential amplitude (16-54 mV); 3) considerable recovery of excitable fibers (∼50% total); and 4) restoration of peak force with the peak tetanic force-extracellular K(+) concentration ([K(+)]o) relationship shifting rightward toward higher [K(+)]o. Double-sigmoid curve-fitting to fatigue profiles (125 Hz for 500 ms, every second for 100 s) showed that prior exposure to raised [K(+)]o (7 mM) increased, whereas lowered [K(+)]o (2 mM) decreased, the rate and extent of force loss during the late phase of fatigue (second sigmoid) in soleus, hence implying a K(+) dependence for late fatigue. Prior exposure to 10 mM [Ca(2+)]o slowed late fatigue in both muscle types, but was without effect on the extent of fatigue. These combined findings support our notion that a Ca(2+)-K(+) interaction is plausible during severe fatigue in both muscle types. We speculate that a diminished transsarcolemmal K(+) gradient and lowered [Ca(2+)]o contribute to late fatigue through reduced action potential amplitude and excitability. The raised [Ca(2+)]o-induced slowing of fatigue is likely to be mediated by a higher intracellular K(+) activity, which prolongs the time before stimulation-induced K(+) efflux depolarizes the sarcolemma sufficiently to interfere with action potentials., (Copyright © 2015 the American Physiological Society.)

Ischemia and heart failure-related cardiac arrhythmias, both atrial (e.g., atrial fibrillation) and ventricular (e.g., malignant tachyarrhythmias) represent a leading cause of morbidity and mortality worldwide. Despite the progress made in the last decade in understanding their pathophysiological mechanisms there is still an unmet need for safer and more efficacious pharmacological treatment, especially when considering the drawbacks and complications of implantable devices. Cardiac ATP-sensitive potassium channels located in the sarcolemmal membrane (sarcKATP) and the inner mitochondrial membrane (mitoKATP) have emerged as crucial controllers of several key cellular functions. In the past three decades a tremendous amount of research led to their structural and functional characterization unveiling both a protective role in cardiac adaptive responses to metabolic stress and a seemingly paradoxical role in promoting as well as protecting against atrial and ventricular arrhythmias. On the other hand, several KATP inhibitors have emerged as potential ischemia selective antiarrhythmic drugs. In this respect, cardioselective, chamber specific and combined sarcKATP and mitoKATP modulators currently represent a promising field for drug development.

In sports medicine, muscle enzymes in the blood are frequently used as an indicator of muscle damage. It is commonly assumed that mechanical stress disrupts plasma membrane to an extent that allows large molecules, such as enzymes, to leak into the extracellular space. However, this does not appear to fully explain changes in muscle enzyme activity in the blood after exercise. Apart from this mechanically induced membrane damage, we hypothesize that, under critical metabolic conditions, ATP consuming enzymes like creatine kinase (CK) are "volitionally" expulsed by muscle cells in order to prevent cell death. This would put themselves into a situation comparable to that of CK deficient muscle fibers, which have been shown in animal experiments to be virtually infatigable at the expense of muscle strength. Additionally we expand on this hypothesis with the idea that membrane blebbing is a way for the muscle fibers to store CK in fringe areas of the muscle fiber or to expulse CK from the cytosol by detaching the blebs from the plasma membrane. The blebbing has been shown to occur in heart muscle cells under ischaemic conditions and has been speculated to be an alternative pathway for the expulsion of troponin. The blebbing has also been seen skeletal muscle cells when intracellular calcium concentration increases. Cytoskeletal damage, induced by reactive oxygen species (ROS) or by calcium activated proteases in concert with increasing intracellular pressure, seems to provoke this type of membrane reaction. If these hypotheses are confirmed by future investigations, our current understanding of CK as a blood muscle damage marker will be fundamentally affected., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

ATP-sensitive potassium (KATP) channels have the unique ability to adjust membrane excitability and functions in accordance with the metabolic status of the cell. Skeletal muscles are primary sites of activity-related energy consumption and have KATP channels expressed in very high density. Previously, we demonstrated that transgenic mice with skeletal muscle-specific disruption of KATP channel function consume more energy than wild-type littermates. However, how KATP channel activation modulates skeletal muscle resting and action potentials under physiological conditions, particularly low-intensity workloads, and how this can be translated to muscle energy expenditure are yet to be determined. Here, we developed a technique that allows evaluation of skeletal muscle excitability in situ, with minimal disruption of the physiological environment. Isometric twitching of the tibialis anterior muscle at 1 Hz was used as a model of low-intensity physical activity in mice with normal and genetically disrupted KATP channel function. This workload was sufficient to induce KATP channel opening, resulting in membrane hyperpolarization as well as reduction in action potential overshoot and duration. Loss of KATP channel function resulted in increased calcium release and aggravated activity-induced heat production. Thus, this study identifies low-intensity workload as a trigger for opening skeletal muscle KATP channels and establishes that this coupling is important for regulation of myocyte function and thermogenesis. These mechanisms may provide a foundation for novel strategies to combat metabolic derangements when energy conservation or dissipation is required.

A characteristic histological feature of striated muscle cells is the presence of deep invaginations of the plasma membrane (sarcolemma), most commonly referred to as T-tubules or the transverse-axial tubular system (TATS). TATS mediates the rapid spread of the electrical signal (action potential) to the cell core triggering Ca(2+) release from the sarcoplasmic reticulum, ultimately inducing myofilament contraction (excitation-contraction coupling). T-tubules, first described in vertebrate skeletal muscle cells, have also been recognized for a long time in mammalian cardiac ventricular myocytes, with a structure and a function that in recent years have been shown to be far more complex and pivotal for cardiac function than initially thought. Renewed interest in T-tubule function stems from the loss and disorganization of T-tubules found in a number of pathological conditions including human heart failure (HF) and dilated and hypertrophic cardiomyopathies, as well as in animal models of HF, chronic ischemia and atrial fibrillation. Disease-related remodeling of the TATS leads to asynchronous and inhomogeneous Ca(2+)-release, due to the presence of orphan ryanodine receptors that have lost their coupling with the dihydropyridine receptors and are either not activated or activated with a delay. Here, we review the physiology of the TATS, focusing first on the relationship between function and structure, and then describing T-tubular remodeling and its reversal in disease settings and following effective therapeutic approaches.

Rationale: In ventricular myocytes of large mammals with low T-tubule density, a significant number of ryanodine receptors (RyRs) are not coupled to the sarcolemma; cardiac remodeling increases noncoupled RyRs., Objective: Our aim was to test the hypothesis that coupled and noncoupled RyRs have distinct microdomain-dependent modulation., Methods and Results: We studied single myocytes from pig left ventricle. The T-tubule network was analyzed in 3-dimension (3D) to measure distance to membrane of release sites. The rising phase of the Ca(2+) transient was correlated with proximity to the membrane (confocal imaging, whole-cell voltage-clamp, K5fluo-4 as Ca(2+) indicator). Ca(2+) sparks after stimulation were thus identified as resulting from coupled or noncoupled RyRs. We used high-frequency stimulation as a known activator of Ca(2+)/calmodulin-dependent kinase II. Spark frequency increased significantly more in coupled than in noncoupled RyRs. This specific modulation of coupled RyRs was abolished by the Ca(2+)/calmodulin-dependent kinase II blockers autocamtide-2-related inhibitory peptide and KN-93, but not by KN-92. Colocalization of Ca(2+)/calmodulin-dependent kinase II and RyR was not detectably different for coupled and noncoupled sites, but the F-actin disruptor cytochalasin D prevented the specific modulation of coupled RyRs. NADPH oxidase 2 inhibition by diphenyleneiodonium or apocynin, or global reactive oxygen species scavenging, also prevented coupled RyR modulation. During stimulated Ca(2+) transients, frequency-dependent increase of the rate of Ca(2+) rise was seen in coupled RyR regions only and abolished by autocamtide-2-related inhibitory peptide. After myocardial infarction, selective modulation of coupled RyR was lost., Conclusions: Coupled RyRs have a distinct modulation by Ca(2+)/calmodulin-dependent kinase II and reactive oxygen species, dependent on an intact cytoskeleton and consistent with a local Ca(2+)/reactive oxygen species microdomain, and subject to modification with disease.

Diazoxide has been identified over the past 50years to have a number of physiological effects, including lowering the blood pressure and rectifying hypoglycemia. Today it is used clinically to treat these conditions. More recently, another important mode of action emerged: diazoxide has powerful protective properties against cardiac ischemia. The heart has intrinsic protective mechanisms against ischemia injury; one of which is ischemic preconditioning. Diazoxide mimics ischemic preconditioning. The purpose of this treatise is to review the literature in an attempt to identify the many effectors of diazoxide and discuss how they may contribute to diazoxide's cardioprotective properties. Particular emphasis is placed on the concentration ranges in which diazoxide affects its different targets and how this compares with the concentrations commonly used to study cardioprotection. It is concluded that diazoxide may have several potential effectors that may potentially contribute to cardioprotection, including KATP channels in the pancreas, smooth muscle, endothelium, neurons and the mitochondrial inner membrane. Diazoxide may also affect other ion channels and ATPases and may directly regulate mitochondrial energetics. It is possible that the success of diazoxide lies in this promiscuity and that the compound acts to rebalance multiple physiological processes during cardiac ischemia., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aims: Transverse tubules (TTs) provide the basic subcellular structures that facilitate excitation-contraction (EC) coupling, the essential process that underlies normal cardiac contractility. Previous studies have shown that TTs develop within the first few weeks of life in mammals but the molecular determinants of this development have remained elusive. This study aims to elucidate the role of junctophilin-2 (JPH2), a junctional membrane complex protein, in the maturation of TTs in cardiomyocytes., Methods and Results: Using a novel cardiac-specific short-hairpin-RNA-mediated JPH2 knockdown mouse model (Mus musculus; αMHC-shJPH2), we assessed the effects of the loss of JPH2 on the maturation of the ventricular TT structure. Between embryonic day (E) 10.5 and postnatal day (P) 10, JPH2 mRNA and protein levels were reduced by >70% in αMHC-shJPH2 mice. At P8 and P10, knockdown of JPH2 significantly inhibited the maturation of TTs, while expression levels of other genes implicated in TT development remained mostly unchanged. At the same time, intracellular Ca(2+) handling was disrupted in ventricular myocytes from αMHC- shJPH2 mice, which developed heart failure by P10 marked by reduced ejection fraction, ventricular dilation, and premature death. In contrast, JPH2 transgenic mice exhibited accelerated TT maturation by P8., Conclusion: Our findings suggest that JPH2 is necessary for TT maturation during postnatal cardiac development in mice. In particular, JPH2 may be critical in anchoring the invaginating sarcolemma to the sarcoplasmic reticulum, thereby enabling the maturation of the TT network.

Aims: Emerging evidence indicates a critical role for junctophilin-2 (JP2) in T-tubule integrity and assembly of cardiac dyads in adult ventricular myocytes. In the postnatal stage, one of the critical features of myocyte maturation is development of the T-tubule system, though the mechanisms remain poorly understood. In this study, we aim to determine whether JP2 is required for normal cardiac T-tubule maturation., Methods and Results: Using in situ confocal imaging of intact murine hearts, we found T-tubules were absent in both left- and right-ventricular myocytes at postnatal Day 8 and did not appear until Day 10. Quantification of T-tubule structural integrity using the T-tubule power (TT(power)) index revealed a progressive increase in TT(power) between postnatal Days 10 and 19. By postnatal Day 19, TT(power) was similar to that in adult murine cardiomyocytes, indicative of a nearly matured T-tubule network. JP2 levels increased dramatically during development, reaching levels observed in adult hearts by postnatal Day 14. Deficiency of JP2, using a mouse model in which a JP2-specific shRNA is expressed during embryonic development, severely impaired T-tubule maturation, with equivalent decreases in the left- and right-ventricular TT(power). We also detected a gradual increase in the density of transverse but not longitudinal tubules during development, and JP2 deficiency abolished the increase in the density of transverse elements. Alterations in T-tubules caused significant reduction in Ca(2+) transient amplitude and marked increase in Ca(2+) release dyssynchrony, Ca(2+) alternans, and spontaneous Ca(2+) waves, leading to contractile failure., Conclusion: Our data identify a critical role for JP2 in T-tubule and excitation-contraction coupling maturation during development.

Introduction: Duchenne muscular dystrophy (DMD) is caused by a genetic defect resulting in absent dystrophin, yet children are able to walk when small and young but lose this ability as they grow. The mdx mouse has absent dystrophin yet does not exhibit significant disability., Methods: Allometric modeling of linearly increasing load per muscle fiber and stress on the sarcolemma with growth and exponential decline associated with loss of muscle fibers correlated with case studies and animal models of DMD., Results: Smaller species or breeds are predictably less affected than large as follows: mdx mice < small golden retriever muscular dystrophy (GRMD) dogs < large GRMD dogs < humans. Case reports of combined growth hormone and dystrophin deficiency show a relatively benign course of disease., Conclusions: Future therapeutic trials in DMD might include specific growth inhibitors in combination with standard of care treatments to delay the clinical onset and reduce the severity of disease and disability., (Copyright © 2013 Wiley Periodicals, Inc.)

Myotonia congenita and periodic paralyses are hereditary skeletal muscle channelopathies. In these disorders, various channel defects in the sarcolemma lead to a severely disturbed membrane excitability of the affected skeletal muscles. The clinical picture can range from severe myotonic reactions (e.g., masseter spasm, opisthotonus) to attacks of weakness and paralysis. Provided here is a short overview of the pathomechanisms behind such wide-ranging phenotypic presentations in these patients, followed by recommendations concerning the management of anesthesia in such populations., (© 2013 John Wiley & Sons Ltd.)

Glucose is an important fuel for contracting muscle, and normal glucose metabolism is vital for health. Glucose enters the muscle cell via facilitated diffusion through the GLUT4 glucose transporter which translocates from intracellular storage depots to the plasma membrane and T-tubules upon muscle contraction. Here we discuss the current understanding of how exercise-induced muscle glucose uptake is regulated. We briefly discuss the role of glucose supply and metabolism and concentrate on GLUT4 translocation and the molecular signaling that sets this in motion during muscle contractions. Contraction-induced molecular signaling is complex and involves a variety of signaling molecules including AMPK, Ca(2+), and NOS in the proximal part of the signaling cascade as well as GTPases, Rab, and SNARE proteins and cytoskeletal components in the distal part. While acute regulation of muscle glucose uptake relies on GLUT4 translocation, glucose uptake also depends on muscle GLUT4 expression which is increased following exercise. AMPK and CaMKII are key signaling kinases that appear to regulate GLUT4 expression via the HDAC4/5-MEF2 axis and MEF2-GEF interactions resulting in nuclear export of HDAC4/5 in turn leading to histone hyperacetylation on the GLUT4 promoter and increased GLUT4 transcription. Exercise training is the most potent stimulus to increase skeletal muscle GLUT4 expression, an effect that may partly contribute to improved insulin action and glucose disposal and enhanced muscle glycogen storage following exercise training in health and disease.

Aims: Arrhythmogenic Ca(2+) waves result from uncontrolled Ca(2+) release from the sarcoplasmic reticulum (SR) that occurs with increased Ca(2+) sensitivity of the ryanodine receptor (RyR) or excessive Ca(2+) accumulation during β-adrenergic stimulation. We hypothesized that inhibition of the L-type Ca(2+) current (I(CaL)) could prevent such Ca(2+) waves in both situations., Methods and Results: Ca(2+) waves were induced in mouse left ventricular cardiomyocytes by isoproterenol combined with caffeine to increase RyR Ca(2+) sensitivity. I(CaL) inhibition by verapamil (0.5 µM) reduced Ca(2+) wave probability in cardiomyocytes during electrostimulation, and during a 10 s rest period after ceasing stimulation. A separate type of Ca(2+) release events occurred during the decay phase of the Ca(2+) transient and was not prevented by verapamil. Verapamil decreased Ca(2+) spark frequency, but not in permeabilized cells, indicating that this was not due to direct effects on RyR. The antiarrhythmic effect of verapamil was due to reduced SR Ca(2+) content following I(CaL) inhibition. Computational modelling supported that the level of I(CaL) inhibition obtained experimentally was sufficient to reduce the SR Ca(2+) content. Ca(2+) wave prevention through reduced SR Ca(2+) content was also effective in heterozygous ankyrin B knockout mice with excessive SR Ca(2+) accumulation during β-adrenergic stimulation., Conclusion: I(CaL) inhibition prevents diastolic Ca(2+) waves caused by increased Ca(2+) sensitivity of RyR or excessive SR Ca(2+) accumulation during β-adrenergic stimulation. In contrast, unstimulated early Ca(2+) release during the decay of the Ca(2+) transient is not prevented, and merits further study to understand the full antiarrhythmic potential of I(CaL) inhibition.

Membrane acid extrusion by Na(+)/H(+) exchange (NHE1) and Na(+)-HCO3(-) co-transport (NBC) is essential for maintaining a low cytoplasmic [H(+)] (∼60 nm, equivalent to an intracellular pH (pHi) of 7.2). This protects myocardial function from the high chemical reactivity of H(+) ions, universal end-products of metabolism. We show here that, in rat ventricular myocytes, fluorescent antibodies map the NBC isoforms NBCe1 and NBCn1 to lateral sarcolemma, intercalated discs and transverse tubules (t-tubules), while NHE1 is absent from t-tubules. This unexpected difference matches functional measurements of pHi regulation (using AM-loaded SNARF-1, a pH fluorophore). Thus, myocyte detubulation (by transient exposure to 1.5 m formamide) reduces global acid extrusion on NBC by 40%, without affecting NHE1. Similarly, confocal pHi imaging reveals that NBC stimulation induces spatially uniform pHi recovery from acidosis, whereas NHE1 stimulation induces pHi non-uniformity during recovery (of ∼0.1 units, for 2-3 min), particularly at the ends of the cell where intercalated discs are commonly located, and where NHE1 immunostaining is prominent. Mathematical modelling shows that this induction of local pHi microdomains is favoured by low cytoplasmic H(+) mobility and long H(+) diffusion distances, particularly to surface NHE1 transporters mediating high membrane flux. Our results provide the first evidence for a spatial localisation of [H(+)]i regulation in ventricular myocytes, suggesting that, by guarding pHi, NHE1 preferentially protects gap junctional communication at intercalated discs, while NBC locally protects t-tubular excitation-contraction coupling.

Goldfish (Carassius auratus L.) are highly tolerant of environmental hypoxia, and with appropriate acclimation may survive and remain active for several days in the complete absence of oxygen. Previous work suggests that the hypoxia-induced activation of cardiac ATP-sensitive potassium (KATP) channels serves to increase tolerance of low oxygen in many species. For goldfish, we have previously characterized a nitric oxide (NO)- and cGMP-dependent pathway by which this channel activation occurs in acute hypoxia. The purpose of the present study was to resolve alterations in KATP channel activity and relevant gene expression in response to acclimation under moderately hypoxic conditions (2.6mg O2/L for seven days at 22°C). Intracellular action potential duration in excised ventricles from hypoxia-acclimated animals was significantly (p<0.05) reduced at both 50% and 90% of full repolarization relative to those from normoxia-acclimated fish. In cell-attached ventricular membrane patches from hypoxia-acclimated goldfish, sarcolemmal KATP channel open probability (NPo) was significantly enhanced vs. control. Of the two genes coding for the pore-forming subunits of cardiac KATP channels (Kir6.1 and Kir6.2), mRNA transcription of kcnj8 (revealed by quantitative real-time PCR) was unchanged while kcnj11 was downregulated in response to chronic low oxygen. The mRNA levels for hif1a (hypoxia inducible factor 1α) in the hearts of hypoxia-acclimated fish were significantly enhanced, as was nitric oxide synthase (nos2) and the sulfonylurea receptor regulatory subunit (sur2, abcc9). These data suggest that prior whole-animal acclimation to chronic hypoxia enhances cardioprotective sarcolemmal KATP currents by altering transcription of regulatory proteins., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Years ago a debate arose as to whether two functionally different mitochondrial subpopulations, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), exist in heart muscle. Nowadays potential differences are often ignored. Presumably, SSM are providing ATP for basic cell function, whereas IFM provide energy for the contractile apparatus. We speculated that two distinguishable subpopulations exist that are differentially affected by pressure overload. Male Sprague-Dawley rats were subjected to transverse aortic constriction for 20 wk or sham operation. Contractile function was assessed by echocardiography. Heart tissue was analyzed by electron microscopy. Mitochondria were isolated by differential centrifugation, and respiratory capacity was analyzed using a Clark electrode. Pressure overload induced left ventricular hypertrophy with increased posterior wall diameter and impaired contractile function. Mitochondrial state 3 respiration in control was 50% higher in IFM than in SSM. Pressure overload significantly impaired respiratory rates in both IFM and SSM, but in SSM to a lower extent. As a result, there were no differences between SSM and IFM after 20 wk of pressure overload. Pressure overload reduced total citrate synthase activity, suggesting reduced total mitochondrial content. Electron microscopy revealed normal morphology of mitochondria but reduced total mitochondrial volume density. In conclusion, IFM show greater respiratory capacity in the healthy rat heart and a greater depression of respiratory capacity by pressure overload than SSM. The differences in respiratory capacity of cardiac IFM and SSM in healthy hearts are eliminated with pressure overload-induced heart failure. The strong effect of pressure overload on IFM together with the simultaneous appearance of mitochondrial and contractile dysfunction may support the notion of IFM primarily producing ATP for contractile function.

Rationale: The ability of a cell to independently regulate nuclear and cytosolic Ca(2+) signaling is currently attributed to the differential distribution of inositol 1,4,5-trisphosphate receptor channel isoforms in the nucleoplasmic versus the endoplasmic reticulum. In cardiac myocytes, T-tubules confer the necessary compartmentation of Ca(2+) signals, which allows sarcomere contraction in response to plasma membrane depolarization, but whether there is a similar structure tunneling extracellular stimulation to control nuclear Ca(2+) signals locally has not been explored., Objective: To study the role of perinuclear sarcolemma in selective nuclear Ca(2+) signaling., Methods and Results: We report here that insulin-like growth factor 1 triggers a fast and independent nuclear Ca(2+) signal in neonatal rat cardiac myocytes, human embryonic cardiac myocytes, and adult rat cardiac myocytes. This fast and localized response is achieved by activation of insulin-like growth factor 1 receptor signaling complexes present in perinuclear invaginations of the plasma membrane. The perinuclear insulin-like growth factor 1 receptor pool connects extracellular stimulation to local activation of nuclear Ca(2+) signaling and transcriptional upregulation through the perinuclear hydrolysis of phosphatidylinositol 4,5-biphosphate inositol 1,4,5-trisphosphate production, nuclear Ca(2+) release, and activation of the transcription factor myocyte-enhancing factor 2C. Genetically engineered Ca(2+) buffers--parvalbumin--with cytosolic or nuclear localization demonstrated that the nuclear Ca(2+) handling system is physically and functionally segregated from the cytosolic Ca(2+) signaling machinery., Conclusions: These data reveal the existence of an inositol 1,4,5-trisphosphate-dependent nuclear Ca(2+) toolkit located in direct apposition to the cell surface, which allows the local control of rapid and independent activation of nuclear Ca(2+) signaling in response to an extracellular ligand.

Dynamic remodeling of mitochondrial morphology through membrane dynamics are linked to changes in mitochondrial and cellular function. Although mitochondrial membrane fusion/fission events are frequent in cell culture models, whether mitochondrial membranes dynamically interact in postmitotic muscle fibers in vivo remains unclear. Furthermore, a quantitative assessment of mitochondrial morphology in intact muscle is lacking. Here, using electron microscopy (EM), we provide evidence of interacting membranes from adjacent mitochondria in intact mouse skeletal muscle. Electron-dense mitochondrial contact sites consistent with events of outer mitochondrial membrane tethering are also described. These data suggest that mitochondrial membranes interact in vivo among mitochondria, possibly to induce morphology transitions, for kiss-and-run behavior, or other processes involving contact between mitochondrial membranes. Furthermore, a combination of freeze-fracture scanning EM and transmission EM in orthogonal planes was used to characterize and quantify mitochondrial morphology. Two subpopulations of mitochondria were studied: subsarcolemmal (SS) and intermyofibrillar (IMF), which exhibited significant differences in morphological descriptors, including form factor (means ± SD for SS: 1.41 ± 0.45 vs. IMF: 2.89 ± 1.76, P < 0.01) and aspect ratio (1.97 ± 0.83 vs. 3.63 ± 2.13, P < 0.01) and circularity (0.75 ± 0.16 vs. 0.45 ± 0.22, P < 0.01) but not size (0.28 ± 0.31 vs. 0.27 ± 0.20 μm(2)). Frequency distributions for mitochondrial size and morphological parameters were highly skewed, suggesting the presence of mechanisms to influence mitochondrial size and shape. In addition, physical continuities between SS and IMF mitochondria indicated mixing of both subpopulations. These data provide evidence that mitochondrial membranes interact in vivo in mouse skeletal muscle and that factors may be involved in regulating skeletal muscle mitochondrial morphology.

The present study further identified factors involved in the cardioprotective phenomenon of remote preconditioning of trauma (RPCT) with special emphasis on the role of the epoxyeicosatrienoic acids (EETs) in mediating this phenomenon. Remote preconditioning of trauma was produced by an abdominal incision only through the skin. Subsequently, all rats were subjected to 30 minutes of left coronary artery occlusion followed by 2 hours of reperfusion and the infarct size was determined. Remote preconditioning of trauma produced a reduction in infarct size expressed as a percentage of the area at risk from 63.0% ± 1.1% to 44.7% ± 1.4%; P < .01 versus control. To test the 3 major triggers of classical preconditioning in mediating RPCT, blockers of the bradykinin B2 receptor (B2BK), (S)-4-[2-[Bis(cyclohexylamino)methyleneamino]-3-(2-naphthalenyl)-1-oxopropylamino]benzyl tributyl phosphonium (WIN 64338, 1 mg/kg, iv), or HOE 140 (50 μg/kg, iv), the nonselective opioid receptor blocker, naloxone (3 mg/kg, iv), or the adenosine A1 receptor blocker, 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 mg/kg, iv) were administered 10 minutes prior to RPCT. Only the 2 B2BK selective antagonists blocked RPCT (60.2% ± 1.1%, WIN 64338; 62.3% ± 2.0%, HOE 140). To test EETs in RPCT, we administered the EET receptor antagonist 14,15-Epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 2.5 mg/kg, iv) or the EET synthesis inhibitor, N-(Methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MSPPOH, 3.0 mg/kg, iv) 10 minutes prior to RPCT. In both groups, the EET antagonists completely blocked RPCT (62.0% ± 0.8%, 14,15-EEZE; 61.8% ± 1.0%, MSPPOH). The EET antagonists also blocked the effect of B2BK activation. We also determined whether the sarcolemmal K(ATP) or the mitochondrial K(ATP) channel mediate RPCT by pretreating rats with 1-[5-[2-(5-Chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl-3 methylthiourea, sodium salt (HMR 1098) or 5-hydroxydecanoic acid (5-HD), respectively. Interestingly, 5-HD blocked RPCT (64.7% ± 1.3%), whereas, HMR 1098 did not (50.3% ± 1.3%). The 2 EET antagonists completely blocked capsaicin-induced cardioprotection. These results clearly suggest that EETs mediate RPCT-, bradykinin- and capsaicin-induced cardioprotection in rat hearts.