The heart beats approximately 100,000 times per day in humans, imposing substantial energetic demands on cardiac muscle. Adenosine triphosphate (ATP) is an essential energy source for normal function of cardiac muscle during each beat, as it powers ion transport, intracellular Ca2+ handling, and actin-myosin cross-bridge cycling. Despite this, the impact of excitation-contraction coupling on the intracellular ATP concentration ([ATP]i) in myocytes is poorly understood. Here, we conducted real-time measurements of [ATP]i in ventricular myocytes using a genetically encoded ATP fluorescent reporter. Our data reveal rapid beat-to-beat variations in [ATP]i. Notably, diastolic [ATP]i was <1 mM, which is eightfold to 10-fold lower than previously estimated. Accordingly, ATP-sensitive K+ (KATP) channels were active at physiological [ATP]i. Cells exhibited two distinct types of ATP fluctuations during an action potential: net increases (Mode 1) or decreases (Mode 2) in [ATP]i. Mode 1 [ATP]i increases necessitated Ca2+ entry and release from the sarcoplasmic reticulum (SR) and were associated with increases in mitochondrial Ca2+. By contrast, decreases in mitochondrial Ca2+ accompanied Mode 2 [ATP]i decreases. Down-regulation of the protein mitofusin 2 reduced the magnitude of [ATP]i fluctuations, indicating that SR-mitochondrial coupling plays a crucial role in the dynamic control of ATP levels. Activation of β-adrenergic receptors decreased [ATP]i, underscoring the energetic impact of this signaling pathway. Finally, our work suggests that cross-bridge cycling is the largest consumer of ATP in a ventricular myocyte during an action potential. These findings provide insights into the energetic demands of EC coupling and highlight the dynamic nature of ATP concentrations in cardiac muscle.

BACKGROUND: Microvascular dysfunction (MVD) is a hallmark feature of chronic graft dysfunction in patients that underwent orthotopic heart transplantation (OHT) and is the main contributor to impaired long-term graft survival. The aim of this study was to determine the effect of MVD on functional and structural properties of cardiomyocytes isolated from ventricular biopsies of OHT patients.
METHODS: We included 14 patients post-OHT, who had been transplanted for 8.1 years [5.0; 15.7 years]. Mean age was 49.6 ± 14.3 years; 64% were male. Coronary microvasculature was assessed using guidewire-based coronary flow reserve(CFR)/index of microvascular resistance (IMR) measurements. Ventricular myocardial biopsies were obtained and cardiomyocytes were isolated using enzymatic digestion. Cells were electrically stimulated and subcellular Ca2+ signalling as well as mitochondrial density were measured using confocal imaging.
RESULTS: MVD measured by IMR was present in 6 of 14 patients with a mean IMR of 53±10 vs. 12±2 in MVD vs. controls (CTRL), respectively. CFR did not differ between MVD and CTRL. Ca2+ transients during excitation-contraction coupling in isolated ventricular cardiomyocytes from a subset of patients showed unaltered amplitudes. In addition, Ca2+ release and Ca2+ removal were not significantly different between MVD and CTRL. However, mitochondrial density was significantly increased in MVD vs. CTRL (34±1 vs. 29±2%), indicating subcellular changes associated with MVD.
CONCLUSIONS: In-vivo ventricular microvascular dysfunction post OHT is associated with preserved excitation-contraction coupling in-vitro, potentially owing to compensatory changes on the mitochondrial level or due to the potentially reversible cause of the disease.

Voltage-gated calcium channels (VGCCs) are the major conduits for calcium ions (Ca2+) within excitable cells. Recent studies have highlighted the non-ionotropic functionality of VGCCs, revealing their capacity to activate intracellular pathways independently of ion flow. This non-ionotropic signaling mode plays a pivotal role in excitation-coupling processes, including gene transcription through excitation-transcription (ET), synaptic transmission via excitation-secretion (ES), and cardiac contraction through excitation-contraction (EC). However, it is noteworthy that these excitation-coupling processes require extracellular calcium (Ca2+) and Ca2+ occupancy of the channel ion pore. Analogous to the \"non-canonical\" characterization of the non-ionotropic signaling exhibited by the N-methyl-D-aspartate receptor (NMDA), which requires extracellular Ca2+ without the influx of ions, VGCC activation requires depolarization-triggered conformational change(s) concomitant with Ca2+ binding to the open channel. Here, we discuss the contributions of VGCCs to ES, ET, and EC coupling as Ca2+ binding macromolecules that transduces external stimuli to intracellular input prior to elevating intracellular Ca2+. We emphasize the recognition of calcium ion occupancy within the open ion-pore and its contribution to the excitation coupling processes that precede the influx of calcium. The non-ionotropic activation of VGCCs, triggered by the upstroke of an action potential, provides a conceptual framework to elucidate the mechanistic aspects underlying the microseconds nature of synaptic transmission, cardiac contractility, and the rapid induction of first-wave genes.

Dysfunctional Ca2+ signaling affects the myocardial systole and diastole, may trigger arrhythmia and cause transcriptomic and proteomic modifications in heart failure. Thus, synchronous real-time measurement of Ca2+ and force is essential to investigate the relationship between contractility and Ca2+ signaling and the alteration of excitation-contraction coupling (ECC) in human failing myocardium. Here, we present a method for synchronized acquisition of intracellular Ca2+ and contraction force in long-term cultivated slices of human failing myocardium. Synchronous time series of contraction force and intracellular Ca2+ were used to calculate force-calcium loops and to analyze the dynamic alterations of ECC in response to various pacing frequencies, post-pause potentiation, high mechanical preload and pharmacological interventions in human failing myocardium. We provide an approach to simultaneously and repeatedly investigate alterations of contractility and Ca2+ signals in long-term cultured myocardium, which will allow detecting the effects of electrophysiological or pharmacological interventions on human myocardial ECC.

The ability to fight or flee from a threat relies on an acute adrenergic surge that augments cardiac output, which is dependent on increased cardiac contractility and heart rate. This cardiac response depends on β-adrenergic-initiated reversal of the small RGK G protein Rad-mediated inhibition of voltage-gated calcium channels (CaV) acting through the Cavβ subunit. Here, we investigate how Rad couples phosphorylation to augmented Ca2+ influx and increased cardiac contraction. We show that reversal required phosphorylation of Ser272 and Ser300 within Rad\'s polybasic, hydrophobic C-terminal domain (CTD). Phosphorylation of Ser25 and Ser38 in Rad\'s N-terminal domain (NTD) alone was ineffective. Phosphorylation of Ser272 and Ser300 or the addition of 4 Asp residues to the CTD reduced Rad\'s association with the negatively charged, cytoplasmic plasmalemmal surface and with CaVβ, even in the absence of CaVα, measured here by FRET. Addition of a posttranslationally prenylated CAAX motif to Rad\'s C-terminus, which constitutively tethers Rad to the membrane, prevented the physiological and biochemical effects of both phosphorylation and Asp substitution. Thus, dissociation of Rad from the sarcolemma, and consequently from CaVβ, is sufficient for sympathetic upregulation of Ca2+ currents.

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CACNA1S-related myopathy, due to pathogenic variants in the CACNA1S gene, is a recently described congenital muscle disease. Disease associated variants result in loss of gene expression and/or reduction of Cav1.1 protein stability. There is an incomplete understanding of the underlying disease pathomechanisms and no effective therapies are currently available. A barrier to the study of this myopathy is the lack of a suitable animal model that phenocopies key aspects of the disease. To address this barrier, we generated knockouts of the two zebrafish CACNA1S paralogs, cacna1sa and cacna1sb. Double knockout fish exhibit severe weakness and early death, and are characterized by the absence of Cav1.1 α1 subunit expression, abnormal triad structure, and impaired excitation-contraction coupling, thus mirroring the severe form of human CACNA1S-related myopathy. A double mutant (cacna1sa homozygous, cacna1sb heterozygote) exhibits normal development, but displays reduced body size, abnormal facial structure, and cores on muscle pathologic examination, thus phenocopying the mild form of human CACNA1S-related myopathy. In summary, we generated and characterized the first cacna1s zebrafish loss-of-function mutants, and show them to be faithful models of severe and mild forms of human CACNA1S-related myopathy suitable for future mechanistic studies and therapy development.

Heart failure is a serious global health challenge, affecting more than 6.2 million people in the United States and is projected to reach over 8 million by 2030. Independent of etiology, failing hearts share common features, including defective calcium (Ca2+) handling, mitochondrial Ca2+ overload, and oxidative stress. In cardiomyocytes, Ca2+ not only regulates excitation-contraction coupling, but also mitochondrial metabolism and oxidative stress signaling, thereby controlling the function and actual destiny of the cell. Understanding the mechanisms of mitochondrial Ca2+ uptake and the molecular pathways involved in the regulation of increased mitochondrial Ca2+ influx is an ongoing challenge in order to identify novel therapeutic targets to alleviate the burden of heart failure. In this review, we discuss the mechanisms underlying altered mitochondrial Ca2+ handling in heart failure and the potential therapeutic strategies.

Earlier work has shown that ventricular ryanodine receptors (RyR2) within a cluster rearrange on phosphorylation as well as with a number of other stimuli. Using dSTORM, we investigated the effects of 300 nmol/liter isoproterenol on RyR2 clusters. In rat ventricular cardiomyocytes, there was a symmetrical enlargement of RyR2 cluster areas, a decrease in the edge-to-edge nearest neighbor distance, and distribution changes that suggested movement to increase the cluster areas by coalescence. The surface area covered by the phosphorylated clusters was significantly greater than in the control cells, as was the cluster density. This latter change was accompanied by a decreased cluster fragmentation, implying that new tetramers were brought into the sarcoplasmic reticulum. We propose a possible mechanism to explain these changes. We also visualized individual RyR2 tetramers and confirmed our earlier electron-tomographic finding that the tetramers are in a disorganized but non-random array occupying about half of the cluster area. Multiclusters, cluster groups defined by the maximum distance between their members, were analyzed for various distances. At 100 nm, the areas occupied by the multiclusters just exceeded those of the single clusters, and more than half of the multiclusters had only a single subcluster that could initiate a spark. Phosphorylation increased the size of the multiclusters, markedly so for distances >100 nm. There was no relationship between the number of subclusters in a group and the area covered by it. We conclude that isoproterenol induces rapid, significant, changes in the molecular architecture of excitation-contraction coupling.

Calcium (Ca2+) sparks are the elementary events of excitation-contraction coupling, yet they are not explicitly represented in human ventricular myocyte models. A stochastic ventricular cardiomyocyte human model that adapts to intracellular Ca2+ ([Ca2+]i) dynamics, spark regulation, and frequency-dependent changes in the form of locally controlled Ca2+ release was developed. The 20,000 CRUs in this model are composed of 9 individual LCCs and 49 RyRs that function as couplons. The simulated action potential duration at 1 Hz steady-state pacing is ~0.280 s similar to human ventricular cell recordings. Rate-dependence experiments reveal that APD shortening mechanisms are largely contributed by the L-type calcium channel inactivation, RyR open fraction, and [Ca2+]myo concentrations. The dynamic slow-rapid-slow pacing protocol shows that RyR open probability during high pacing frequency (2.5 Hz) switches to an adapted \"nonconducting\" form of Ca2+-dependent transition state. The predicted force was also observed to be increased in high pacing, but the SR Ca2+ fractional release was lower due to the smaller difference between diastolic and systolic [Ca2+]SR. Restitution analysis through the S1S2 protocol and increased LCC Ca2+-dependent activation rate show that the duration of LCC opening helps modulate its effects on the APD restitution at different diastolic intervals. Ultimately, a longer duration of calcium sparks was observed in relation to the SR Ca2+ loading at high pacing rates. Overall, this study demonstrates the spontaneous Ca2+ release events and ion channel responses throughout various stimuli.