Excitation-contraction Coupling Process Research Articles

Task-Dependent Mechanisms Underlying Prolonged Low-Frequency Force Depression

Abstract Prolonged low-frequency force depression (PLFFD) is an intramuscular phenomenon involving the slow recovery of submaximal muscle strength following strenuous exercise. We hypothesize that the contribution of impaired excitation-contraction coupling processes to PLFFD are task-dependent, and that they will be different between metabolically and mechanically demanding exercises. We also discuss evidence of the effectiveness of interventions to mitigate PLFFD.

Recording and Interpretation of Active Calcium Transients in Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Calcium plays a pivotal role in the excitation-contraction coupling process in cardiomyocytes, a critical multi-parametric event leading to rhythmic contraction. Over the past few decades, calcium signaling in cardiomyocytes has been extensively studied in cardiovascular sciences. However, a standard methodology is needed not only to trace the calcium within cells but also to remove signal processing biases and to accurately interpret the features of calcium transient signals in relation to cardiomyocyte electrophysiology. This article outlines the use of genetically encoded calcium indicator (GCaMP) human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to record calcium transients. These cells express a green fluorescent signal when calcium binds to intracellular calmodulin, a key regulator of calcium signaling. The extraction and processing of calcium transient waveforms are performed using ImageJ and MATLAB software. Key features of these waveforms are then identified and categorized based on their physiological relevance to cardiomyocyte function. Additionally, this work includes a Support Protocol for the successful replating of cardiomyocytes onto non-traditional culture platforms, such as metallic sensors and polymer-based substrates, to facilitate data multiplexing. The three Basic Protocols outlined here provide a comprehensive approach for maintaining, expanding, and differentiating the GCaMP hiPSCs, video recording of calcium transients, and the subsequent signal extraction, preprocessing, analysis, and data visualization. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Maintenance, expansion, and differentiation of genetically encoded calcium indicator hiPSCs Support Protocol: Replating GCaMP hiPSC-CMs for stimulation and multielectrode array studies Basic Protocol 2: Video recording from calcium transients of GCaMP hiPSC-CMs Basic Protocol 3: Signal extraction, preprocessing, analysis, and data visualization.

Preventing Site-Specific Calpain Proteolysis of Junctophilin-2 Protects Against Stress-Induced Excitation-Contraction Uncoupling and Heart Failure Development.

Excitation-contraction (E-C) coupling processes become disrupted in heart failure (HF), resulting in abnormal Ca2+ homeostasis, maladaptive structural and transcriptional remodeling, and cardiac dysfunction. Junctophilin-2 (JP2) is an essential component of the E-C coupling apparatus but becomes site-specifically cleaved by calpain, leading to disruption of E-C coupling, plasmalemmal transverse tubule degeneration, abnormal Ca2+ homeostasis, and HF. However, it is not clear whether preventing site-specific calpain cleavage of JP2 is sufficient to protect the heart against stress-induced pathological cardiac remodeling in vivo. Calpain-resistant JP2 knock-in mice (JP2CR) were generated by deleting the primary JP2 calpain cleavage site. Stress-dependent JP2 cleavage was assessed through in vitro cleavage assays and in isolated cardiomyocytes treated with 1 μmol/L isoproterenol by immunofluorescence. Cardiac outcomes were assessed in wild-type and JP2CR mice 5 weeks after transverse aortic constriction compared with sham surgery using echocardiography, histology, and RNA-sequencing methods. E-C coupling efficiency was measured by in situ confocal microscopy. E-C coupling proteins were evaluated by calpain assays and Western blotting. The effectiveness of adeno-associated virus gene therapy with JP2CR, JP2, or green fluorescent protein to slow HF progression was evaluated in mice with established cardiac dysfunction. JP2 proteolysis by calpain and in response to transverse aortic constriction and isoproterenol was blocked in JP2CR cardiomyocytes. JP2CR hearts are more resistant to pressure-overload stress, having significantly improved Ca2+ homeostasis and transverse tubule organization with significantly attenuated cardiac dysfunction, hypertrophy, lung edema, fibrosis, and gene expression changes relative to wild-type mice. JP2CR preserves the integrity of calpain-sensitive E-C coupling-related proteins, including ryanodine receptor 2, CaV1.2, and sarcoplasmic reticulum calcium ATPase 2a, by attenuating transverse aortic constriction-induced increases in calpain activity. Furthermore, JP2CR gene therapy after the onset of cardiac dysfunction was found to be effective at slowing the progression of HF and superior to wild-type JP2. The data presented here demonstrate that preserving JP2-dependent E-C coupling by prohibiting the site-specific calpain cleavage of JP2 offers multifaceted beneficial effects, conferring cardiac protection against stress-induced proteolysis, hypertrophy, and HF. Our data also indicate that specifically targeting the primary calpain cleavage site of JP2 by gene therapy approaches holds great therapeutic potential as a novel precision medicine for treating HF.

Cardiac arrhythmogenesis: roles of ion channels and their functional modification.

Cardiac arrhythmias cause significant morbidity and mortality and pose a major public health problem. They arise from disruptions in the normally orderly propagation of cardiac electrophysiological activation and recovery through successive cardiomyocytes in the heart. They reflect abnormalities in automaticity, initiation, conduction, or recovery in cardiomyocyte excitation. The latter properties are dependent on surface membrane electrophysiological mechanisms underlying the cardiac action potential. Their disruption results from spatial or temporal instabilities and heterogeneities in the generation and propagation of cellular excitation. These arise from abnormal function in their underlying surface membrane, ion channels, and transporters, as well as the interactions between them. The latter, in turn, form common regulatory targets for the hierarchical network of diverse signaling mechanisms reviewed here. In addition to direct molecular-level pharmacological or physiological actions on these surface membrane biomolecules, accessory, adhesion, signal transduction, and cytoskeletal anchoring proteins modify both their properties and localization. At the cellular level of excitation-contraction coupling processes, Ca2+ homeostatic and phosphorylation processes affect channel activity and membrane excitability directly or through intermediate signaling. Systems-level autonomic cellular signaling exerts both acute channel and longer-term actions on channel expression. Further upstream intermediaries from metabolic changes modulate the channels both themselves and through modifying Ca2+ homeostasis. Finally, longer-term organ-level inflammatory and structural changes, such as fibrotic and hypertrophic remodeling, similarly can influence all these physiological processes with potential pro-arrhythmic consequences. These normal physiological processes may target either individual or groups of ionic channel species and alter with particular pathological conditions. They are also potentially alterable by direct pharmacological action, or effects on longer-term targets modifying protein or cofactor structure, expression, or localization. Their participating specific biomolecules, often clarified in experimental genetically modified models, thus constitute potential therapeutic targets. The insights clarified by the physiological and pharmacological framework outlined here provide a basis for a recent modernized drug classification. Together, they offer a translational framework for current drug understanding. This would facilitate future mechanistically directed therapeutic advances, for which a number of examples are considered here. The latter are potentially useful for treating cardiac, in particular arrhythmic, disease.

UNDERSTANDING MUSCLE CONTRACTION: MECHANISMS AND IMPLICATIONS

Muscle contraction is a fundamental physiological process critical for movement, stability, and various bodily functions. This research paper delves into the intricate mechanisms underlying muscle contraction, exploring the role of actin, myosin, calcium ions, and regulatory proteins in the sliding filament theory. Additionally, it discusses the physiological and biochemical aspects of muscle contraction, including the excitation-contraction coupling process and the energy requirements for muscle activity. Furthermore, the paper explores the implications of muscle contraction dysfunction in conditions such as muscular dystrophy and the potential therapeutic interventions. By comprehensively understanding muscle contraction, researchers and clinicians can advance treatments for muscle-related disorders and optimize performance in various fields.

Cadence Modulation during Eccentric Cycling Affects Perception of Effort But Not Neuromuscular Alterations.

A recent study showed that cadence modulation during short eccentric cycling exercise affects oxygen consumption (V̇O 2 ), muscular activity (EMG), and perception of effort (PE). This study examined the effect of cadence on V̇O 2 , EMG, and PE during prolonged eccentric cycling and exercise-induced neuromuscular alterations. Twenty-two participants completed three sessions 2-3 wk apart: 1) determination of the maximal concentric peak power output, familiarization with eccentric cycling at two cadences (30 and 60 rpm at 60% peak power output), and neuromuscular testing procedure; 2) and 3) 30 min of eccentric cycling exercise at a cadence of 30 or 60 rpm. PE, cardiorespiratory parameters, and vastus lateralis and rectus femoris EMG were collected during exercise. The knee extensors' maximal voluntary contraction torque, the torque evoked by double stimulations at 100 Hz (Dt100) and 10 Hz (Dt10), and the voluntary activation level were evaluated before and after exercise. V̇O 2 , EMG, and PE were greater at 30 than 60 rpm (all P < 0.05). Maximal voluntary contraction torque, evoked torque, and Dt10/Dt100 ratio decreased (all P < 0.01) without cadence effect (all P > 0.28). Voluntary activation level remained constant after both eccentric cycling exercises ( P = 0.87). When performed at the same power output, eccentric cycling exercise at 30 rpm elicited a greater PE, EMG, and cardiorespiratory demands than pedaling at 60 rpm. Exercise-induced fatigability was similar in both eccentric cycling conditions without neural impairments, suggesting that eccentric cycling seemed to alter more specifically muscular function, such as the excitation-contraction coupling process. In a rehabilitation context, eccentric cycling at 60 rpm seems more appropriate because it will induce lower PE for similar strength loss compared with 30 rpm.

Abstract P3130: Preventing Site-specific Calpain Cleavage Of Junctophilin-2 Protects Against Stress-induced E-c Coupling Dysfunction And Heart Failure Development In Mice

In heart failure (HF), excitation-contraction (E-C) coupling processes become disrupted, resulting in abnormal Ca 2+ homeostasis that triggers cardiac dysfunction. Junctophilin-2 (JP2) is an essential E-C coupling constituent for maintaining cardiomyocyte integrity and cardiac function both at baseline and in response to stress. We previously showed that calpain-mediated proteolytic cleavage of JP2 at arginine 565 / threonine 566 (R 565 /T 566 ) is key to its downregulation in the diseased heart. However, it remains unclear whether preventing site-specific proteolysis of JP2 can prevent cardiac remodeling and HF progression following stress. To investigate this, calpain-resistant mutant JP2 knockin mice (JP2 CR ) were generated by deleting residues 563-568 spanning the primary calpain cleavage site. Studies in cultured cardiomyocytes treated with isoproterenol (ISO) showed that JP2 CR is resistant to stress-induced loss of JP2 integrity and T-tubule disorganization compared to wild-type (WT) cardiomyocytes. Further, in vivo studies demonstrate JP2 CR mice are more resistant to pressure overload stress having significantly less abnormal Ca 2+ homeostasis, cardiac dysfunction, hypertrophy, lung edema and fibrosis relative to WT mice. Western blot analysis revealed JP2 CR hearts have preserved expression levels of several key E-C coupling proteins, including RyR2, SERCA2a and Ca V 1.2. RNA-Sequencing analysis further revealed attenuated pressure overload induced hypertrophic and heart failure-related transcriptional reprogramming in JP2 CR hearts. These findings indicate that preserving JP2-dependent E-C coupling by prohibiting the site-specific calpain-cleavage of JP2 offers cardiac protection against stress-induced hypertrophy and heart failure. Our data indicate that preventing JP2 cleavage by gene therapy approaches may hold great therapeutic potential for treating heart failure.

Inositol 1,4,5-trisphosphate receptors in cardiomyocyte physiology and disease.

The contraction of cardiac muscle underlying the pumping action of the heart is mediated by the process of excitation-contraction coupling (ECC). While triggered by Ca2+ entry across the sarcolemma during the action potential, it is the release of Ca2+ from the sarcoplasmic reticulum (SR) intracellular Ca2+ store via ryanodine receptors (RyRs) that plays the major role in induction of contraction. Ca2+ also acts as a key intracellular messenger regulating transcription underlying hypertrophic growth. Although Ca2+ release via RyRs is by far the greatest contributor to the generation of Ca2+ transients in the cardiomyocyte, Ca2+ is also released from the SR via inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs). This InsP3-induced Ca2+ release modifies Ca2+ transients during ECC, participates in directing Ca2+ to the mitochondria, and stimulates the transcription of genes underlying hypertrophic growth. Central to these specific actions of InsP3Rs is their localization to responsible signalling microdomains, the dyad, the SR-mitochondrial interface and the nucleus. In this review, the various roles of InsP3R in cardiac (patho)physiology and the mechanisms by which InsP3 signalling selectively influences the different cardiomyocyte cell processes in which it is involved will be presented.This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’.

Diabetes-induced chronic heart failure is due to defects in calcium transporting and regulatory contractile proteins: cellular and molecular evidence.

Heart failure (HF) is a major deteriorating disease of the myocardium due to weak myocardial muscles. As such, the heart is unable to pump blood efficiently around the body to meet its constant demand. HF is a major global health problem with more than 7 million deaths annually worldwide, with some patients dying suddenly due to sudden cardiac death (SCD). There are several risk factors which are associated with HF and SCD which can negatively affect the heart synergistically. One major risk factor is diabetes mellitus (DM) which can cause an elevation in blood glucose level or hyperglycaemia (HG) which, in turn, has an insulting effect on the myocardium. This review attempted to explain the subcellular, cellular and molecular mechanisms and to a lesser extent, the genetic factors associated with the development of diabetes- induced cardiomyopathy due to the HG which can subsequently lead to chronic heart failure (CHF) and SCD. The study first explained the structure and function of the myocardium and then focussed mainly on the excitation-contraction coupling (ECC) processes highlighting the defects of calcium transporting (SERCA, NCX, RyR and connexin) and contractile regulatory (myosin, actin, titin and troponin) proteins. The study also highlighted new therapies and those under development, as well as preventative strategies to either treat or prevent diabetic cardiomyopathy (DCM). It is postulated that prevention is better than cure.

Molecular interactions of STAC proteins with skeletal muscle dihydropyridine receptor and excitation-contraction coupling.

Excitation‐contraction coupling (ECC) is the physiological process in which an electrical signal originating from the central nervous system is converted into muscle contraction. In skeletal muscle tissue, the key step in the molecular mechanism of ECC initiated by the muscle action potential is the cooperation between two Ca2+ channels, dihydropyridine receptor (DHPR; voltage‐dependent L‐type calcium channel) and ryanodine receptor 1 (RyR1). These two channels were originally postulated to communicate with each other via direct mechanical interactions; however, the molecular details of this cooperation have remained ambiguous. Recently, it has been proposed that one or more supporting proteins are in fact required for communication of DHPR with RyR1 during the ECC process. One such protein that is increasingly believed to play a role in this interaction is the SH3 and cysteine‐rich domain‐containing protein 3 (STAC3), which has been proposed to bind a cytosolic portion of the DHPR α1S subunit known as the II–III loop. In this work, we present direct evidence for an interaction between a small peptide sequence of the II–III loop and several residues within the SH3 domains of STAC3 as well as the neuronal isoform STAC2. Differences in this interaction between STAC3 and STAC2 suggest that STAC3 possesses distinct biophysical features that are potentially important for its physiological interactions with the II–III loop. Therefore, this work demonstrates an isoform‐specific interaction between STAC3 and the II–III loop of DHPR and provides novel insights into a putative molecular mechanism behind this association in the skeletal muscle ECC process.

Effect of the Knee and Hip Angles on Knee Extensor Torque: Neural, Architectural, and Mechanical Considerations.

This study examined the influence of knee extensors’ hip and knee angle on force production capacity and their neuromuscular and architectural consequences. Sixteen healthy men performed sub-maximal and maximal voluntary isometric contractions (MVIC) of knee extensors with four different combinations of the knee and hip angles. Muscle architecture, excitation-contraction coupling process, muscular activity, and corticospinal excitability were evaluated on the vastus lateralis (VL) and rectus femoris (RF) muscles. MVIC and evoked peak twitch (Pt) torques of knee extensors increased significantly (p < 0.05) by 42 ± 12% and 47 ± 16% on average, respectively, under knee flexed positions (110° flexion, 0° = full extension) compared to knee extended positions (20° flexion) but were not different between hip positions (i.e., 0° or 60° flexion). Knee flexion also affected VL and RF muscle and fascicle lengths toward greater length than under knee extended position, while pennation angle decreased for both muscles with knee flexion. Pennation angles of the VL muscle were also lower under extended hip positions. Alternatively, no change in maximal muscle activation or corticospinal activity occurred for the VL and RF muscles across the different positions. Altogether these findings evidenced that MVIC torque of knee extensors depended particularly upon peripheral contractile elements, such as VL and RF muscle and fascicle lengths, but was unaffected by central factors (i.e., muscle activation). Furthermore, the hip position can affect the pennation angle of the VL, while VL muscle length can affect the pennation angle of the RF muscle. These elements suggest that the VL and RF muscles exert a mutual influence on their architecture, probably related to the rectus-vastus aponeurosis.

Development an ovine myocardial infarction- induced heart failure model and characterisation of altered calcium homeostasis mechanisms

Abstract Introduction Ventricular arrhythmias (VA) and heart failure (HF) are the major complications following myocardial infarction (MI). In both conditions, there is a key role for perturbed calcium homeostasis of which the underlying mechanisms remain unclear. A preclinical model that faithfully presents most of the features of MI-induced HF has been lacking. The complexity of this syndrome means that animal modelling is difficult. As the hearts of large animals share many electrophysiological similarities to humans, ovine modelling of cardiac diseases could better reflect human pathologies than in small mammals. Question Is it possible to develop a clinically relevant ovine model with moderate cardiac dysfunction following myocardial infarction? Methods MI was induced in sheep by inflating an angioplasty balloon distal to the second diagonal branch of the left anterior descending artery for 90 min. Cardiac function was monitored for 20 weeks using electrocardiography (ECG), echocardiography, blood biochemical analysis, and subjective signs of cardiac deterioration (lethargy, dyspnoea, and cough). 20 weeks post-MI, the animals were humanely killed and single left ventricular myocytes were isolated from the infarct border zone. Changes in cellular electrophysiology and intracellular calcium concentration were monitored using whole-cell patch technique in voltage-clamp mode and the calcium sensitive fluorescent indicator Fura-2 (K5 salt). Results By using minimally invasive procedures, we obtained a survival rate of 80% (n=15). During surgery, our data show clinical features of ischaemia, including changes in the ECG features (elevation of the ST and T segment, left bundle branch block and/or pathological Q waves) and elevation of the cardiac biomarker such as troponin I. Following MI, we observed a decline in ejection fraction (−25±3%, p&amp;lt;0.0001) and an increase in whole animal arrhythmias (incidence of VA 72 hours post-MI, ∼70%). On cardiac removal apico-septal transmural necrosis / scarring was evident. Importantly, the L-type calcium current (ICaL) was decreased in MI cells compared to healthy cells (−1.87±0.73 pA/pF, p&amp;lt;0.05), but isoprenaline had no effet on ICaL (0.48±1.2 pA/pF, p=0.70) in MI cells. Moreover, the amplitude of the systolic calcium transient (−0.33±0.1 F/F0, p&amp;lt;0.05) and the sarcoplasmic reticulum calcium content (−23±7 μmol/L, p&amp;lt;0.01) were also decreased. The shortening velocity of the sarcomere was also decreased in MI cells (−0.55±0.18 μm/s, p&amp;lt;0.01). Conclusion We successfully established an ovine MI model using minimally invasive procedure which displays a moderately impaired cardiac function, reduced contractility, and pro-arrhythmic electrophysiological remodelling. Future analysis will examine the role of the L-type calcium channel with respect to the excitation-contraction coupling process and myocyte contractility and how we can improve therapeutic strategies towards VA and HF. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

Alcohol Use Disorders and Their Harmful Effects on the Contractility of Skeletal, Cardiac and Smooth Muscles.

Alcohol misuse has deleterious effects on personal health, family, societal units, and global economies. Moreover, alcohol misuse usually leads to several diseases and conditions, including alcoholism, which is a chronic condition and a form of addiction. Alcohol misuse, whether as acute intoxication or alcoholism, adversely affects skeletal, cardiac and/or smooth muscle contraction. Ethanol (ethyl alcohol) is the main effector of alcohol-induced dysregulation of muscle contractility, regardless of alcoholic beverage type or the ethanol metabolite (with acetaldehyde being a notable exception). Ethanol, however, is a simple and “promiscuous” ligand that affects many targets to mediate a single biological effect. In this review, we firstly summarize the processes of excitation-contraction coupling and calcium homeostasis which are critical for the regulation of contractility in all muscle types. Secondly, we present the effects of acute and chronic alcohol exposure on the contractility of skeletal, cardiac, and vascular/ nonvascular smooth muscles. Distinctions are made between in vivo and in vitro experiments, intoxicating vs. sub-intoxicating ethanol levels, and human subjects vs. animal models. The differential effects of alcohol on biological sexes are also examined. Lastly, we show that alcohol-mediated disruption of muscle contractility, involves a wide variety of molecular players, including contractile proteins, their regulatory factors, membrane ion channels and pumps, and several signaling molecules. Clear identification of these molecular players constitutes a first step for a rationale design of pharmacotherapeutics to prevent, ameliorate and/or reverse the negative effects of alcohol on muscle contractility.

Calcium administration In patients undergoing CardiAc suRgery under cardiopulmonary bypasS (ICARUS trial): Rationale and design of a randomized controlled trial

IntroductionWeaning from cardiopulmonary bypass (CPB) is a critical step of any cardiac surgical procedure and often requires pharmacologic intervention. Calcium ions are pivotal elements for the excitation-contraction coupling process of cardiac myocytes. Thus, calcium administration might be helpful during weaning from CPB. MethodsWe describe a multicenter, placebo-controlled, double blind randomized clinical trial to assess the effect of calcium chloride on the need for inotropic support among adult patients during weaning from CPB. The experimental group (409 patients) will receive 15 mg/kg of calcium chloride. The control group (409 patients) will receive an equivalent volume of 0.9% sodium chloride. Both drugs will be administered intravenously as a bolus at the beginning of weaning from CPB. ResultsThe primary outcome will be the need for inotropic support between termination of CPB and completion of surgery. Secondary outcomes will be: duration of inotropic support, vasoactive-inotropic score 30 min after transfer to intensive care unit and on postoperative day 1, plasma alpha-amylase on postoperative day 1, plasma Ca2+ concentration immediately before and 10–15 min after calcium chloride administration, non-fatal myocardial infarction, blood loss on postoperative day 1, need for transfusion of red blood cells, signs of myocardial ischemia on electrocardiogram after arrival to intensive care unit, all-cause mortality at 30 days or during hospital stay if this is longer than 30 days. DiscussionThis trial is designed to assess whether intravenous calcium chloride administration could reduce the need for inotropic support after cardiopulmonary bypass weaning among adults undergoing cardiac surgery.

Excitation-contraction coupling and calcium release in atrial muscle.

In cardiac muscle, the process of excitation-contraction coupling (ECC) describes the chain of events that links action potential induced myocyte membrane depolarization, surface membrane ion channel activation, triggering of Ca2+ induced Ca2+ release from the sarcoplasmic reticulum (SR) Ca2+ store to activation of the contractile machinery that is ultimately responsible for the pump function of the heart. Here we review similarities and differences of structural and functional attributes of ECC between atrial and ventricular tissue. We explore a novel "fire-diffuse-uptake-fire" paradigm of atrial ECC and Ca2+ release that assigns a novel role to the SR SERCA pump and involves a concerted "tandem" activation of the ryanodine receptor Ca2+ release channel by cytosolic and luminal Ca2+. We discuss the contribution of the inositol 1,4,5-trisphosphate (IP3) receptor Ca2+ release channel as an auxiliary pathway to Ca2+ signaling, and we review IP3 receptor-induced Ca2+ release involvement in beat-to-beat ECC, nuclear Ca2+ signaling, and arrhythmogenesis. Finally, we explore the topic of electromechanical and Ca2+ alternans and its ramifications for atrial arrhythmia.

Preserved Ca2+ handling and excitation-contraction coupling in muscle fibres from diet-induced obese mice.

Disrupted intracellular Ca2+ handling is known to play a role in diabetic cardiomyopathy but it has also been postulated to contribute to obesity- and type 2 diabetes-associated skeletal muscle dysfunction. Still, there is so far very limited functional insight into whether, and if so to what extent, muscular Ca2+ homeostasis is affected in this situation, so as to potentially determine or contribute to muscle weakness. In differentiated muscle, force production is under the control of the excitation-contraction coupling process: upon plasma membrane electrical activity, the CaV1.1 voltage sensor/Ca2+ channel in the plasma membrane triggers opening of the ryanodine receptor Ca2+ release channel in the sarcoplasmic reticulum (SR) membrane. Opening of the ryanodine receptor triggers the rise in cytosolic Ca2+, which activates contraction while Ca2+ uptake by the SR ATPase Ca2+-pump promotes relaxation. These are the core mechanisms underlying the tight control of muscle force by neuronal electrical activity. This study aimed at characterising their inherent physiological function in a diet-induced mouse model of obesity and type 2 diabetes. Intact muscle fibres were isolated from mice fed either with a standard chow diet or with a high-fat, high-sucrose diet generating obesity, insulin resistance and glucose intolerance. Properties of muscle fibres were investigated with a combination of whole-cell voltage-clamp electrophysiology and confocal fluorescence imaging. The integrity and density of the plasma membrane network (transverse tubules) that carries the membrane excitation throughout the muscle fibres was assessed with the dye Di-8-ANEPPS. CaV1.1 Ca2+ channel activity was studied by measuring the changes in current across the plasma membrane elicited by voltage-clamp depolarising pulses of increasing amplitude. SR Ca2+ release through ryanodine receptors was simultaneously detected with the Ca2+-sensitive dye Rhod-2 in the cytosol. CaV1.1 voltage-sensing activity was separately characterised from the properties of intra-plasma-membrane charge movement produced by short voltage-clamp depolarising pulses. Spontaneous Ca2+ release at rest was assessed with the Ca2+-sensitive dye Fluo-4. The rate of SR Ca2+ uptake was assessed from the time course of cytosolic Ca2+ recovery after the end of voltage excitation using the Ca2+-sensitive dye Fluo-4FF. The response to a fatigue-stimulation protocol was determined from the time course of decline of the peak Fluo-4FF Ca2+ transients elicited by 30 trains of 5-ms-long depolarising pulses delivered at 100Hz. The transverse tubule network architecture and density were well preserved in the fibres from the obese mice. The CaV1.1 Ca2+ current and voltage-sensing properties were also largely unaffected with mean values for maximum conductance and maximum amount of charge of 234 ± 12 S/F and 30.7 ± 1.6 nC/μF compared with 196 ± 13 S/F and 32.9 ± 2.0 nC/μF in fibres from mice fed with the standard diet, respectively. Voltage-activated SR Ca2+ release through ryanodine receptors also exhibited very similar properties in the two groups with mean values for maximum rate of Ca2+ release of 76.0 ± 6.5 and 78.1 ± 4.4μmoll-1ms-1, in fibres from control and obese mice, respectively. The response to a fatigue protocol was also largely unaffected in fibres from the obese mice, and so were the rate of cytosolic Ca2+ removal and the spontaneous Ca2+ release activity at rest. The functional properties of the main mechanisms involved in the control of muscle Ca2+ homeostasis are well preserved in muscle fibres from obese mice, at the level of both the plasma membrane and of the SR. We conclude that intracellular Ca2+ handling and excitation-contraction coupling in skeletal muscle fibres are not primary targets of obesity and type 2 diabetes. Graphical abstract.

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Update on antiarrhythmic drug pharmacology.

Cardiac arrhythmias constitute a major public health problem. Pharmacological intervention remains mainstay to their clinical management. This, in turn, depends upon systematic drug classification schemes relating their molecular, cellular, and systems effects to clinical indications and therapeutic actions. This approach was first pioneered in the 1960s Vaughan-Williams classification. Subsequent progress in cardiac electrophysiological understanding led to a lag between the fundamental science and its clinical translation, partly addressed by The working group of the European Society of Cardiology (1991), which, however, did not emerge with formal classifications. We here utilize the recent Revised Oxford Classification Scheme to review antiarrhythmic drug pharmacology. We survey drugs and therapeutic targets offered by the more recently characterized ion channels, transporters, receptors, intracellular Ca2+ handling, and cell signaling molecules. These are organized into their strategic roles in cardiac electrophysiological function. Following analysis of the arrhythmic process itself, we consider (a) pharmacological agents directly targeting membrane function, particularly the Na+ and K+ ion channels underlying depolarizing and repolarizing events in the cardiac action potential. (b) We also consider agents that modify autonomic activity that, in turn, affects both the membrane and (c) the Ca2+ homeostatic and excitation-contraction coupling processes linking membrane excitation to contractile activation. Finally, we consider (d) drugs acting on more upstream energetic and structural remodeling processes currently the subject of clinical trials. Such systematic correlations of drug actions and arrhythmic mechanisms at different molecular to systems levels of cardiac function will facilitate current and future antiarrhythmic therapy.

Dynamics of triadin, a muscle-specific triad protein, within sarcoplasmic reticulum subdomains.

In skeletal muscle, proteins of the calcium release complex responsible for the excitation-contraction (EC) coupling are exclusively localized in specific reticulum–plasma membrane (ER-PM) contact points named triads. The CRC protein triadin (T95) is localized in the sarcoplasmic reticulum (SR) subdomain of triads where it forms large multimers. However, the mechanisms leading to the steady-state accumulation of T95 in these specific areas of SR are largely unknown. To visualize T95 dynamics, fluorescent chimeras were expressed in triadin knockout myotubes, and their mobility was compared with the mobility of Sec61β, a membrane protein of the SR unrelated to the EC coupling process. At all stages of skeletal muscle cells differentiation, we show a permanent flux of T95 diffusing in the SR membrane. Moreover, we find evidence that a longer residence time in the ER-PM contact point is due to the transmembrane domain of T95 resulting in an overall triad localization.

Structure and Pharmacology of Voltage-Gated Sodium and Calcium Channels.

Voltage-gated sodium and calcium channels are evolutionarily related transmembrane signaling proteins that initiate action potentials, neurotransmission, excitation-contraction coupling, and other physiological processes. Genetic or acquired dysfunction of these proteins causes numerous diseases, termed channelopathies, and sodium and calcium channels are the molecular targets for several major classes of drugs. Recent advances in the structural biology of these proteins using X-ray crystallography and cryo-electron microscopy have given new insights into the molecular basis for their function and pharmacology. Here we review this recent literature and integrate findings on sodium and calcium channels to reveal the structural basis for their voltage-dependent activation, fast and slow inactivation, ion conductance and selectivity, and complex pharmacology at the atomic level. We conclude with the theme that new understanding of the diseases and therapeutics of these channels will be derived from application of the emerging structural principles from these recent structural analyses.