Excitation-contraction Coupling In Skeletal Muscle Research Articles

Significance of Zn2+ in RyR1 for Structural Integrity and Ligand Binding: Insight from Molecular Dynamics.

Ryanodine receptor type 1 (RyR1) is a Ca2+-release channel central to skeletal muscle excitation-contraction (EC) coupling. RyR1's cryo-EM structures reveal a zinc-finger motif positioned within the cytoplasmic C-terminal domain (CTD). Yet, owing to limitations in cryo-EM resolution, RyR1 structures lack precision in detailing the metal coordination structure, prompting the need for an accurate model. In this study, we employed molecular dynamics (MD) simulations and the density functional theory (DFT) method to refine the binding characteristics of Zn2+ in the zinc-finger site of the RyR1 channel. Our findings also highlight substantial conformational changes in simulations conducted in the absence of Zn2+. Notably, we observed a loss of contact at the interface between protein domains proximal to the zinc-finger site, indicating a crucial role of Zn2+ in maintaining structural integrity and interdomain interactions within RyR1. Furthermore, this study provides valuable insights into the modulation of ATP, Ca2+, and caffeine binding, shedding light on the intricate relationship between Zn2+ coordination and the dynamic behavior of RyR1. Our integrative approach combining MD simulations and DFT calculations enhances our understanding of the molecular mechanisms governing ligand binding in RyR1.

Dissecting the functions of multiple interactions of STAC3 in skeletal muscle excitation-contraction coupling

Dissecting the functions of multiple interactions of STAC3 in skeletal muscle excitation-contraction coupling

Biophysical reviews top five: voltage-dependent charge movement in nerve and muscle.

The discovery of gating currents and asymmetric charge movement in the early 1970s represented a remarkable leap forward in our understanding of the biophysical basis of voltage-dependent events that underlie electrical signalling that is vital for nerve and muscle function. Gating currents and charge movement reflect a fundamental process in which charged amino acid residues in an ion channel protein move in response to a change in the membrane electrical field and therefore activate the specific voltage-dependent response of that protein. The detection of gating currents and asymmetric charge movement over the past 50 years has been pivotal in unraveling the multiple molecular and intra-molecular processes which lead to action potentials in excitable tissues and excitation-contraction (EC) coupling in skeletal muscle. The recording of gating currents and asymmetric charge movement remains an essential component of investigations into the basic molecular mechanisms of neuronal conduction and muscle contraction.

Dissecting the functions of multiple interactions of STAC3 in skeletal muscle excitation–contraction coupling

Dissecting the functions of multiple interactions of STAC3 in skeletal muscle excitation–contraction coupling

Feedback contributions to excitation-contraction coupling in native functioning striated muscle.

Skeletal and cardiac muscle excitation-contraction coupling commences with Nav1.4/Nav1.5-mediated, surface and transverse (T-) tubular, action potential generation. This initiates feedforward, allosteric or Ca2+-mediated, T-sarcoplasmic reticular (SR) junctional, voltage sensor-Cav1.1/Cav1.2 and ryanodine receptor-RyR1/RyR2 interaction. We review recent structural, physiological and translational studies on possible feedback actions of the resulting SR Ca2+ release on Nav1.4/Nav1.5 function in native muscle. Finite-element modelling predicted potentially regulatory T-SR junctional [Ca2+]TSR domains. Nav1.4/Nav1.5, III-IV linker and C-terminal domain structures included Ca2+ and/or calmodulin-binding sites whose mutations corresponded to specific clinical conditions. Loose-patch-clamped native murine skeletal muscle fibres and cardiomyocytes showed reduced Na+ currents (INa) following SR Ca2+ release induced by the Epac and direct RyR1/RyR2 activators, 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate and caffeine, abrogated by the RyR inhibitor dantrolene. Conversely, dantrolene and the Ca2+-ATPase inhibitor cyclopiazonic acid increased INa. Experimental, catecholaminergic polymorphic ventricular tachycardic RyR2-P2328S and metabolically deficient Pgc1β-/- cardiomyocytes also showed reduced INa accompanying [Ca2+]i abnormalities rescued by dantrolene- and flecainide-mediated RyR block. Finally, hydroxychloroquine challenge implicated action potential (AP) prolongation in slowing AP conduction through modifying Ca2+ transients. The corresponding tissue/organ preparations each showed pro-arrhythmic, slowed AP upstrokes and conduction velocities. We finally extend discussion of possible Ca2+-mediated effects to further, Ca2+, K+ and Cl-, channel types. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Voltage-dependent transitions between resting and activated states of the four Cav1.1 voltage sensors observed in MD simulation.

The sliding helix hypothesis describes how, upon depolarization and repolarization of the membrane, the positive gating charges of the S4 helices of voltage-sensing domains (VSDs) one by one move across the membrane electrical field and thereby effect the opening and closing of ion channels. Voltage-gated calcium channels contain four VSDs, which together regulate gating of a common pore. Although these four VSDs are highly homologous, they differ from each other, e.g. in the number of gating charges and in the number and position of their countercharges. Recent atomic-resolution structures of voltage-gated calcium and sodium channels delineate the endpoints of the VSD movement upon depolarization, showing the S4 helix in the activated up-state. While the first structures of VSDs in resting states have been solved for homotetrameric (prokaryotic) channels, resting state structures of the (pseudo-)heterotetrameric eukaryotic sodium and calcium channels are still lacking. Accordingly, structural models showing the transitions from the resting to the activated state and back are still elusive. Here we addressed this problem using enhanced sampling molecular dynamics simulations of the CaV1.1 calcium channel exposed to a strong membrane potential. Indeed, starting from the known up-state, the S4 helices of the four VSDs moved down to positions closely matching the experimentally determined resting state of the bacterial NaVAb channel. Importantly, the trajectories, the transiently formed ionic interactions between gating- and countercharges, and the speed of the S4 transitions differed substantially between the four VSDs. This is consistent with data from recent mutagenesis and voltage-clamp fluorometry studies on CaV1.1 and allows relating the individual VSD movements with their putative roles in the regulation of distinct channel gating properties, as well as their role in the activation of excitation-contraction coupling in skeletal muscle.

CaV1.1 Calcium Channel Signaling Complexes in Excitation-Contraction Coupling: Insights from Channelopathies.

In skeletal muscle, excitation-contraction (EC) coupling relies on the mechanical coupling between two ion channels: the L-type voltage-gated calcium channel (CaV1.1), located in the sarcolemma and functioning as the voltage sensor of EC coupling, and the ryanodine receptor 1 (RyR1), located on the sarcoplasmic reticulum serving as the calcium release channel. To this day, the molecular mechanism by which these two ion channels are linked remains elusive. However, recently, skeletal muscle EC coupling could be reconstituted in heterologous cells, revealing that only four proteins are essential for this process: CaV1.1, RyR1, and the cytosolic proteins CaVβ1a and STAC3. Due to the crucial role of these proteins in skeletal muscle EC coupling, any mutation that affects any one of these proteins can have devastating consequences, resulting in congenital myopathies and other pathologies.Here, we summarize the current knowledge concerning these four essential proteins and discuss the pathophysiology of the CaV1.1, RyR1, and STAC3-related skeletal muscle diseases with an emphasis on the molecular mechanisms. Being part of the same signalosome, mutations in different proteins often result in congenital myopathies with similar symptoms or even in the same disease.

A reconstituted depolarization-induced Ca2+ release platform for validation of skeletal muscle disease mutations and drug discovery.

In skeletal muscle excitation-contraction (E-C) coupling, depolarization of the plasma membrane triggers Ca2+ release from the sarcoplasmic reticulum (SR), referred to as depolarization-induced Ca2+ release (DICR). DICR occurs through the type 1 ryanodine receptor (RyR1), which physically interacts with the dihydropyridine receptor Cav1.1 subunit in specific machinery formed with additional essential components including β1a, Stac3 adaptor protein, and junctophilins. Exome sequencing has accelerated the discovery of many novel mutations in genes encoding DICR machinery in various skeletal muscle diseases. However, functional validation is time-consuming because it must be performed in a skeletal muscle environment. In this study, we established a platform of the reconstituted DICR in HEK293 cells. The essential components were effectively transduced into HEK293 cells expressing RyR1 using baculovirus vectors, and Ca2+ release was quantitatively measured with R-CEPIA1er, a fluorescent ER Ca2+ indicator, without contaminant of extracellular Ca2+ influx. In these cells, [K+]-dependent Ca2+ release was triggered by chemical depolarization with the aid of inward rectifying potassium channel, indicating a successful reconstitution of DICR. Using the platform, we evaluated several Cav1.1 mutations that are implicated in malignant hyperthermia and myopathy. We also tested several RyR1 inhibitors; whereas dantrolene and Cpd1 inhibited DICR, procaine had no effect. Furthermore, twitch potentiators such as perchlorate and thiocyanate shifted the voltage dependence of DICR to more negative potentials without affecting Ca2+-induced Ca2+ release. These results well reproduced the findings with the muscle fibers and the cultured myotubes. Since the procedure is simple and reproducible, the reconstituted DICR platform will be highly useful for the validation of mutations and drug discovery for skeletal muscle diseases.

Targeted transcript analysis in muscles from patients with genetically diverse congenital myopathies.

Congenital myopathies are a group of early onset muscle diseases of variable severity often with characteristic muscle biopsy findings and involvement of specific muscle types. The clinical diagnosis of patients typically relies on histopathological findings and is confirmed by genetic analysis. The most commonly mutated genes encode proteins involved in skeletal muscle excitation–contraction coupling, calcium regulation, sarcomeric proteins and thin–thick filament interaction. However, mutations in genes encoding proteins involved in other physiological functions (for example mutations in SELENON and MTM1, which encode for ubiquitously expressed proteins of low tissue specificity) have also been identified. This intriguing observation indicates that the presence of a genetic mutation impacts the expression of other genes whose product is important for skeletal muscle function. The aim of the present investigation was to verify if there are common changes in transcript and microRNA expression in muscles from patients with genetically heterogeneous congenital myopathies, focusing on genes encoding proteins involved in excitation–contraction coupling and calcium homeostasis, sarcomeric proteins, transcription factors and epigenetic enzymes. Our results identify RYR1, ATPB2B and miRNA-22 as common transcripts whose expression is decreased in muscles from congenital myopathy patients. The resulting protein deficiency may contribute to the muscle weakness observed in these patients. This study also provides information regarding potential biomarkers for monitoring disease progression and response to pharmacological treatments in patients with congenital myopathies.

STAC3 related congenital myopathy: A case series of seven Comorian patients

The Bailey-Bloch congenital myopathy, also known as Native American myopathy (NAM), is an autosomal recessive congenital myopathy first reported in the Lumbee tribe people settled in North Carolina (USA), and characterized by congenital weakness and arthrogryposis, cleft palate, ptosis, short stature, kyphoscoliosis, talipes deformities, and susceptibility to malignant hyperthermia (MH) triggered by anesthesia. NAM is linked to STAC3 gene coding for a component of excitation-contraction coupling in skeletal muscles. A homozygous missense variant (c.851G > C; p.Trp284Ser) in STAC3 segregated with NAM in the Lumbee families. Non-Native American patients with STAC3 related congenital myopathy, and with other various variants of STAC3 have been reported. Here, we present seven patients from the Comoros Islands (located in the Mozambique Channel) diagnosed with STAC3 related congenital myopathy and having the recurrent variant identified in the Lumbee people. The series is the second largest series of patients having STAC3 related congenital myopathy with a shared ethnicity after le Lumbee series. Local history and geography may explain the overrepresentation of NAM in the Comorian Archipelago with a founder effect. Further researches would be necessary for the understanding of the onset of the NAM in Comorian population as search of the “classical” STAC3 variant in East African population, and haplotypes comparison between Comorian and Lumbee patients.

The distal C terminus of the dihydropyridine receptor β1a subunit is essential for tetrad formation in skeletal muscle

The skeletal muscle dihydropyridine receptor (DHPR) β1a subunit is indispensable for full trafficking of DHPRs into triadic junctions (i.e., the close apposition of transverse tubules and sarcoplasmic reticulum [SR]), facilitation of DHPRα1S voltage sensing, and arrangement of DHPRs into tetrads as a consequence of their interaction with ryanodine receptor (RyR1) homotetramers. These three features are obligatory for skeletal muscle excitation–contraction (EC) coupling. Previously, we showed that all four vertebrate β isoforms (β1–β4) facilitate α1S triad targeting and, except for β3, fully enable DHPRα1S voltage sensing [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488–7493 (2013)]. Consequently, β3 failed to restore EC coupling despite the fact that both β3 and β1a restore tetrads. Thus, all β-subunits are able to restore triad targeting, but only β1a restores both tetrads and proper DHPR–RyR1 coupling [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488–7493 (2013)]. To investigate the molecular region(s) of β1a responsible for the tetradic arrangement of DHPRs and thus DHPR–RyR1 coupling, we expressed loss- and gain-of-function chimeras between β1a and β4, with systematically swapped domains in zebrafish strain relaxed (β1-null) for patch clamp, cytoplasmic Ca2+ transients, motility, and freeze-fracture electron microscopy. β1a/β4 chimeras with either N terminus, SH3, HOOK, or GK domain derived from β4 showed complete restoration of SR Ca2+ release. However, chimera β1a/β4(C) with β4 C terminus produced significantly reduced cytoplasmic Ca2+ transients. Conversely, gain-of-function chimera β4/β1a(C) with β1a C terminus completely restored cytoplasmic Ca2+ transients, DHPR tetrads, and motility. Furthermore, we found that the nonconserved, distal C terminus of β1a plays a pivotal role in reconstitution of DHPR tetrads and thus allosteric DHPR–RyR1 interaction, essential for skeletal muscle EC coupling.

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.

High-throughput platform of reconstituted skeletal muscle depolarization-induced Ca2+release in HEK293 cells

In skeletal muscle excitation-contraction coupling, depolarization of transverse tubule membrane causes conformational change in dihydropyridine receptor (DHPR), which in turn opens type 1 ryanodine receptor (RyR1) to release Ca2+ from sarcoplasmic reticulum. This 'depolarization-induced Ca2+ release' (DICR) occurs through physical interaction between DHPR and RyR1 and requires additional components (β1a, Stac3, junctophilin-2). However, it remains so far unclear about molecular mechanism of DICR, especially conformational change in RyR1.

Therapeutic effects of novel type1 ryanodine receptor inhibitor on skeletal muscle diseases

Type 1 ryanodine receptor (RyR1) plays a key role in Ca2+ release from the sarcoplasmic reticulum (SR) during excitation-contraction coupling of skeletal muscle. Mutations in RyR1 hyperactivate the channel to cause malignant hyperthermia (MH). MH is a serious complication characterized by skeletal muscle rigidity and elevated body temperature in response to commonly used inhalational anesthetics. Thus far, more than 300 mutations in RyR1 gene have been reported in patients with MH. Some heat stroke triggered by exercise or environmental heat stress is also related to MH mutations in the RyR1 gene. The only drug approved for ameliorating the symptoms of MH is dantrolene, which has been first developed in 1960s as a muscle relaxant. However, dantrolene has several disadvantages for clinical use: poor water solubility which makes rapid preparation difficult in emergency situations and long plasma half-life, which causes long-lasting side effects such as muscle weakness. Here we show that a novel RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively rescues MH and heat stroke in new mouse model relevant to MH. Cpd1 has great advantages of higher water solubility and shorter plasma half-life compared to dantrolene. Our data suggest that Cpd1 has the potential to be a promising new candidate for effective treatment of patients carrying RyR1 mutations.

Acute exposure to extracellular BTP2 does not inhibit Ca2+ release during EC coupling in intact skeletal muscle fibers.

The inhibitor of store-operated Ca2+ entry (SOCE) BTP2 was reported to inhibit ryanodine receptor Ca2+ leak and electrically evoked Ca2+ release from the sarcoplasmic reticulum when introduced into mechanically skinned muscle fibers. However, it is unclear how effects of intracellular application of a highly lipophilic drug like BTP2 on Ca2+ release during excitation-contraction (EC) coupling compare with extracellular exposure in intact muscle fibers. Here, we address this question by quantifying the effect of short- and long-term exposure to 10 and 20 µM BTP2 on the magnitude and kinetics of electrically evoked Ca2+ release in intact mouse flexor digitorum brevis muscle fibers. Our results demonstrate that neither the magnitude nor the kinetics of electrically evoked Ca2+ release evoked during repetitive electrical stimulation were altered by brief exposure (2 min) to either BTP2 concentration. However, BTP2 did reduce the magnitude of electrically evoked Ca2+ release in intact fibers when applied extracellularly for a prolonged period of time (30 min at 10 µM or 10 min at 20 µM), consistent with slow diffusion of the lipophilic drug across the plasma membrane. Together, these results indicate that the time course and impact of BTP2 on Ca2+ release during EC coupling in skeletal muscle depends strongly on whether the drug is applied intracellularly or extracellularly. Further, these results demonstrate that electrically evoked Ca2+ release in intact muscle fibers is unaltered by extracellular application of 10 µM BTP2 for <25 min, validating this use to assess the role of SOCE in the absence of an effect on EC coupling.

NEW GENES AND DISEASES: EP.304 Early onset lower limp assymmetry: a new clinical phenotype of calsequestrin mutation

Calsequestrin is a protein that handles calcium in sarcoplasmic reticulum (SR),Ca2+ transients are crucial in skeletal muscle excitation-contraction coupling. Synchronous Ca2+ release from the SR terminal cisternae into the cytoplasm and its reuptake into the SR store are granted by a structural and functional unit and their defect causes myopathy. In an 11-year-old girl, affected by lower limb asymmetry (R limb shorter of 3 cm), upper limb hypotrophy and winging scapulae were found; she had high CK:2900 U/l, her sister presented myalgias and CK:1500 U/l, the mother CK 500 U/l and grandmother had both congenital club foot. We investigated the causes of the high CK by EMG that was myopathic, muscle biopsy that showed few atrophic fibres, in ATPase rare 2C fibres, some increased NADH-TR stain at the periphery, increased central nuclei and rare basophilic fibres. We performed immunohistochemistry for dystrophin, caveolin-3, alpha-sarcoglycan, western blot for dysferlin: all normal. The girl was operated at 15 years for elongation of right femur and by epiphysiodesis left femur and the limb asymmetry was reduced to only half a centimeter: however she complained of fatiguability, especially after physical education at school, and high CK (2186 U). She presented with scoliosis, difficulty walking with calf hypertrophy. A search for the causes for hyperCKemia was done with WGS at TIGEM and resulted in detection of a pathogenetic variant of CASQ1 gene (p.ASP244Gly), coding for calsequestrin, which allowed the solution of this puzzling familial case. In 7 members of 4 Italian families with vacuolar myopathy with CASQ1 mutation calsequestrin aggregates and a similar variant was found. In CASQ1 myopathy in several cases hyperCKemia, myalgia or other vague symptoms are present: in 22 CASQ1-mutated patients from 12 families 21 sharing the same variant of our case presented symptoms in the sixth decade with exercise intolerance and myalgias and later developed mild to moderate, slowly progressive proximal weakness with quadriceps atrophy and scapular winging. In the present family, clubfoot, early onset, limb asymmetry were the predominant clinical symptoms, they could be the effect of somatic mosaicism. In this respect our patient presents an early onset and a new clinical phenotype of lower limb asymmetry that is unique. The present familial case expands the clinical phenotype of CASQ1-myopathy.

High-resolution structure of the membrane-embedded skeletal muscle ryanodine receptor

The type 1 ryanodine receptor (RyR)/calcium release channel on the sarcoplasmic reticulum (SR) is required for skeletal muscle excitation-contraction coupling and is the largest known ion channel, composed of four 565-kDa protomers. Cryogenic electron microscopy (cryo-EM) studies of the RyR have primarily used detergent to solubilize the channel; in the present study, we have used cryo-EM to solve high-resolution structures of the channel in liposomes using a gel-filtration approach with on-column detergent removal to form liposomes and incorporate the channel simultaneously. This allowed us to resolve the structure of the channel in the primed and open states at 3.4 and 4.0Å, respectively, with a single dataset. This method offers validation for detergent-based structures of the RyR and offers a starting point for utilizing a chemical gradient mimicking the SR, where Ca2+ concentrations are millimolar in the lumen and nanomolar in the cytosol.

Pore mutation N617D in the skeletal muscle DHPR blocks Ca2+ influx due to atypical high-affinity Ca2+ binding.

Skeletal muscle excitation-contraction (EC) coupling roots in Ca2+-influx-independent inter-channel signaling between the sarcolemmal dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1) in the sarcoplasmic reticulum. Although DHPR Ca2+ influx is irrelevant for EC coupling, its putative role in other muscle-physiological and developmental pathways was recently examined using two distinct genetically engineered mouse models carrying Ca2+ non-conducting DHPRs: DHPR(N617D) (Dayal et al., 2017) and DHPR(E1014K) (Lee et al., 2015). Surprisingly, despite complete block of DHPR Ca2+-conductance, histological, biochemical, and physiological results obtained from these two models were contradictory. Here, we characterize the permeability and selectivity properties and henceforth the mechanism of Ca2+ non-conductance of DHPR(N617). Our results reveal that only mutant DHPR(N617D) with atypical high-affinity Ca2+ pore-binding is tight for physiologically relevant monovalent cations like Na+ and K+. Consequently, we propose a molecular model of cooperativity between two ion selectivity rings formed by negatively charged residues in the DHPR pore region.

High Resolution Structure of the Membrane Embedded Skeletal Muscle Ryanodine Receptor

The type 1 ryanodine receptor (RyR1)/calcium release channel on the sarcoplasmic reticulum (SR) is required for skeletal muscle excitation-contraction coupling and is the largest known ion channel, comprised of four 565 kDa protomers. Cryogenic electron microscopy (cryoEM) studies of the RyR have primarily used detergent to solubilize the channel; in the present study, we have used cryoEM to solve high-resolution structures of the channel in liposomes using a gel-filtration approach with on-column detergent removal to form liposomes and incorporate the channel simultaneously, improving the incorporation rate by >20-fold compared to a dialysis-based approach. This allowed us to resolve the structure of the channel in the closed and open states at 3.36 and 3.98 A, respectively. This method offers validation for detergent-based structures of the RyR and offers a method for utilizing an electrochemical gradient mimicking the SR, where Ca2+ concentrations are millimolar in the lumen and nanomolar in the cytosol.

Effects of extracellular potassium on calcium handling and force generation in a model of excitation-contraction coupling in skeletal muscle

It is well-established that extracellular potassium (Ko+) accumulation reduces muscle fiber excitability, however the effects of Ko+ on the excitation–contraction coupling (ECC) pathway are less understood. In vivo and in vitro studies following fatiguing stimulation protocols are limited in their ability to capture the effects of Ko+ on force production in combination with other simultaneously changing factors. To address this, a computational model of ECC for slow and fast twitch muscle is presented to explore the relative contributions of excitability-induced and metabolic-induced changes in force generation in response to increasing K+o. The model incorporates mechanisms previously unexplored in modelling studies, including the effects of extracellular calcium on excitability, calcium-dependent inhibition of calcium release, ATP-dependent ionic pumping, and the contribution of ATP hydrolysis to intracellular phosphate accumulation rate. The model was able to capture the frequency-dependent biphasic Force-K+o response observed experimentally. Force potentiation for moderately elevated K+o was driven by increased action potential duration, myoplasmic calcium potentiation, and phosphate accumulation rate, while attenuation of force at higher K+o was due to action potential failure resulting in reduced calcium release. These results suggest that altered calcium release and phosphate accumulation work together with elevated Ko+ to affect force during sustained contractions.