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Prediabetic myocardium, induced by fructose-rich diet (FRD), is prone to increased sarcoplasmic reticulum (SR)-Ca 2+ leak and arrhythmias due to increased activity of the Ca 2+ /calmodulin protein kinase II (CaMKII). However, little is known about the role of SR-mitochondria microdomains, mitochondrial structure, and mitochondrial metabolisms. To address this knowledge gap we measured SR-mitochondrial proximity, intracellular Ca 2+ , and mitochondrial metabolism in wild type (WT) and AC3-I transgenic mice, with myocardial-targeted CaMKII inhibition, fed with control diet (CD) or with FRD. Confocal images showed significantly increased spontaneous Ca 2+ release events in FRD vs. CD WT cardiomyocytes. [ 3 H]-Ryanodine binding assay revealed higher [ 3 H]Ry binding in FRD than CD WT hearts. O 2 consumption at State 4 and hydrogen peroxide (H 2 O 2 ) production rate were increased, while respiratory control rate (RCR) and Ca 2+ retention capacity (CRC) were decreased in FRD vs. CD WT isolated mitochondria. Transmission Electron Microscopy (TEM) images showed increased proximity at the SR-mitochondria microdomains, associated with increased tethering proteins, Mfn2, Grp75, and VDAC in FRD vs. CD WT. Mitochondria diameter was decrease and roundness and density were increased in FRD vs. CD WT specimens. The fission protein, Drp1 was significantly increased while the fusion protein, Opa1 was unchanged in FRD vs. CD WT hearts. These differences were prevented in AC3-I mice. We conclude that SR-mitochondria microdomains are subject to CaMKII-dependent remodeling, involving SR-Ca 2+ leak and mitochondria fission, in prediabetic mice induced by FRD. We speculate that CaMKII hyperactivity induces SR-Ca 2+ leak by RyR2 activation which in turn increases mitochondria Ca 2+ content due to the enhanced SR-mitochondria tethering, decreasing CRC.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) stimulates calcium release from acidic stores such as lysosomes and is a highly potent calcium-mobilising second messenger. NAADP plays an important role in calcium signalling in the heart under basal conditions and following β-adrenergic stress. Nevertheless, the spatial interaction of acidic stores with other parts of the calcium signalling apparatus in cardiac myocytes is unknown. We present evidence that lysosomes are intimately associated with the sarcoplasmic reticulum (SR) in ventricular myocytes; a median separation of 20 nm in 2D electron microscopy and 3.3 nm in 3D electron tomography indicates a genuine signalling microdomain between these organelles. Fourier analysis of immunolabelled lysosomes suggests a sarcomeric pattern (dominant wavelength 1.80 μm). Furthermore, we show that lysosomes form close associations with mitochondria (median separation 6.2 nm in 3D studies) which may provide a basis for the recently-discovered role of NAADP in reperfusion-induced cell death. The trigger hypothesis for NAADP action proposes that calcium release from acidic stores subsequently acts to enhance calcium release from the SR. This work provides structural evidence in cardiac myocytes to indicate the formation of microdomains between acidic and SR calcium stores, supporting emerging interpretations of NAADP physiology and pharmacology in heart.

Imatinib mesylate (Gleevec) is a small-molecule inhibitor of the fusion protein Bcr-Abl, the causal agent in chronic myelogenous leukemia. Here we report ten individuals who developed severe congestive heart failure while on imatinib and we show that imatinib-treated mice develop left ventricular contractile dysfunction. Transmission electron micrographs from humans and mice treated with imatinib show mitochondrial abnormalities and accumulation of membrane whorls in both vacuoles and the sarco- (endo-) plasmic reticulum, findings suggestive of a toxic myopathy. With imatinib treatment, cardiomyocytes in culture show activation of the endoplasmic reticulum (ER) stress response, collapse of the mitochondrial membrane potential, release of cytochrome c into the cytosol, reduction in cellular ATP content and cell death. Retroviral gene transfer of an imatinib-resistant mutant of c-Abl, alleviation of ER stress or inhibition of Jun amino-terminal kinases, which are activated as a consequence of ER stress, largely rescues cardiomyocytes from imatinib-induced death. Thus, cardiotoxicity is an unanticipated side effect of inhibition of c-Abl by imatinib.

Store-operated Ca 2+ entry (SOCE), a ubiquitous mechanism that allows recovery of Ca 2+ ions from the extracellular space, has been proposed to limit fatigue during repetitive skeletal muscle activity. However, the subcellular location for SOCE in muscle fibers has not been unequivocally identified. Here we show that exercise drives a significant remodeling of the sarcotubular system to form previously unidentified junctions between the sarcoplasmic reticulum (SR) and transverse-tubules (TTs). We also demonstrate that these new SR-TT junctions contain the molecular machinery that mediate SOCE: stromal interaction molecule-1 (STIM1), which functions as the SR Ca 2+ sensor, and Orai1, the Ca 2+ -permeable channel in the TT. In addition, EDL muscles isolated from exercised mice exhibit an increased capability of maintaining contractile force during repetitive stimulation in the presence of 2.5 mM extracellular Ca 2+ , compared to muscles from control mice. This functional difference is significantly reduced by either replacement of extracellular Ca 2+ with Mg 2+ or the addition of SOCE inhibitors (BTP-2 and 2-APB). We propose that the new SR-TT junctions formed during exercise, and that contain STIM1 and Orai1, function as Ca 2+ Entry Units (CEUs) , structures that provide a pathway to rapidly recover Ca 2+ ions from the extracellular space during repetitive muscle activity.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia characterized by syncope and sudden death occurring during exercise or acute emotion. CPVT is caused by abnormal intracellular Ca2+ handling resulting from mutations in the RyR2 or CASQ2 genes. Because CASQ2 and RyR2 are involved in different aspects of the excitation‐contraction coupling process, we hypothesized that these mutations are associated with different functional and intracellular Ca²+ abnormalities. To test the hypothesis we generated induced Pluripotent Stem Cell‐derived cardiomyocytes (iPSC‐CM) from CPVT1 and CPVT2 patients carrying the RyR2R420Q and CASQ2D307H mutations, respectively, and investigated in CPVT1 and CPVT2 iPSC‐CM (compared to control): (i) The ultrastructural features; (ii) the effects of isoproterenol, caffeine and ryanodine on the [Ca2+]i transient characteristics. Our major findings were: (i) Ultrastructurally, CASQ2 and RyR2 mutated cardiomyocytes were less developed than control cardiomyocytes. (ii) While in control iPSC‐CM isoproterenol caused positive inotropic and lusitropic effects, in the mutated cardiomyocytes isoproterenol was either ineffective, caused arrhythmias, or markedly increased diastolic [Ca2+]i. Importantly, positive inotropic and lusitropic effects were not induced in mutated cardiomyocytes. (iii) The effects of caffeine and ryanodine in mutated cardiomyocytes differed from control cardiomyocytes. Our results show that iPSC‐CM are useful for investigating the similarities/differences in the pathophysiological consequences of RyR2 versus CASQ2 mutations underlying CPVT1 and CPVT2 syndromes.

Skeletal muscle contraction is triggered by the excitation-contraction (E-C) coupling machinery residing at the triad, a membrane structure formed by the juxtaposition of T-tubules and sarcoplasmic reticulum (SR) cisternae. The formation and maintenance of this structure is key for muscle function but is not well characterized. We have investigated the mechanisms leading to X-linked myotubular myopathy (XLMTM), a severe congenital disorder due to loss of function mutations in the MTM1 gene, encoding myotubularin, a phosphoinositide phosphatase thought to have a role in plasma membrane homeostasis and endocytosis. Using a mouse model of the disease, we report that Mtm1-deficient muscle fibers have a decreased number of triads and abnormal longitudinally oriented T-tubules. In addition, SR Ca²⁺ release elicited by voltage-clamp depolarizations is strongly depressed in myotubularin-deficient muscle fibers, with myoplasmic Ca²⁺ removal and SR Ca²⁺ content essentially unaffected. At the molecular level, Mtm1-deficient myofibers exhibit a 3-fold reduction in type 1 ryanodine receptor (RyR1) protein level. These data reveal a critical role of myotubularin in the proper organization and function of the E-C coupling machinery and strongly suggest that defective RyR1-mediated SR Ca²⁺ release is responsible for the failure of muscle function in myotubular myopathy.

During aging reduction in muscle mass (sarcopenia) and decrease in physical activity lead to partial loss of muscle force and increased fatigability. Deficiency in the essential trace element selenium might augment these symptoms as it can cause muscle pain, fatigue, and proximal weakness. Average voluntary daily running, maximal twitch and tetanic force, and calcium release from the sarcoplasmic reticulum (SR) decreased while reactive oxygen species (ROS) production associated with tetanic contractions increased in aged – 22-month-old – as compared to young – 4-month-old – mice. These changes were accompanied by a decline in the ryanodine receptor type 1 (RyR1) and Selenoprotein N content and the increased amount of a degraded RyR1. Both lifelong training and selenium supplementation, but not the presence of an increased muscle mass at young age, were able to compensate for the reduction in muscle force and SR calcium release with age. Selenium supplementation was also able to significantly enhance the Selenoprotein N levels in aged mice. Our results describe, for the first time, the beneficial effects of selenium supplementation on calcium release from the SR and muscle force in old age while point out that increased muscle mass does not improve physical performance with aging.

Induced pluripotent stem cells (iPSC) are generated from fully differentiated somatic cells that were reprogrammed into a pluripotent state. Human iPSC which can be obtained from various types of somatic cells such as fibroblasts or keratinocytes can differentiate into cardiomyocytes (iPSC‐CM), which exhibit cardiac‐like transmembrane action potentials, intracellular Ca2+ transients and contractions. While major features of the excitation‐contraction coupling of iPSC‐CM have been well‐described, very little is known on the ultrastructure of these cardiomyocytes. The ultrastructural features of 31‐day‐old (post‐plating) iPSC‐CM generated from human hair follicle keratinocytes (HFKT‐iPSC‐CM) were analysed by electron microscopy, and compared with those of human embryonic stem‐cell‐derived cardiomyocytes (hESC‐CM). The comparison showed that cardiomyocytes from the two sources share similar proprieties. Specifically, HFKT‐iPSC‐CM and hESC‐CM, displayed ultrastructural features of early and immature phenotype: myofibrils with sarcomeric pattern, large glycogen deposits, lipid droplets, long and slender mitochondria, free ribosomes, rough endoplasmic reticulum, sarcoplasmic reticulum and caveolae. Noteworthy, the SR is less developed in HFKT‐iPSC‐CM. We also found in both cell types: (1) ‘Ca2+‐release units’, which connect the peripheral sarcoplasmic reticulum with plasmalemma; and (2) intercellular junctions, which mimic intercalated disks (desmosomes and fascia adherens). In conclusion, iPSC and hESC differentiate into cardiomyocytes of comparable ultrastructure, thus supporting the notion that iPSC offer a viable option for an autologous cell source for cardiac regenerative therapy.

The BAR domain protein superfamily is involved in membrane invagination and endocytosis, but its role in organizing membrane proteins has not been explored. In particular, the membrane scaffolding protein BIN1 functions to initiate T-tubule genesis in skeletal muscle cells. Constitutive knockdown of BIN1 in mice is perinatal lethal, which is associated with an induced dilated hypertrophic cardiomyopathy. However, the functional role of BIN1 in cardiomyocytes is not known. An important function of cardiac T-tubules is to allow L-type calcium channels (Cav1.2) to be in close proximity to sarcoplasmic reticulum-based ryanodine receptors to initiate the intracellular calcium transient. Efficient excitation-contraction (EC) coupling and normal cardiac contractility depend upon Cav1.2 localization to T-tubules. We hypothesized that BIN1 not only exists at cardiac T-tubules, but it also localizes Cav1.2 to these membrane structures. We report that BIN1 localizes to cardiac T-tubules and clusters there with Cav1.2. Studies involve freshly acquired human and mouse adult cardiomyocytes using complementary immunocytochemistry, electron microscopy with dual immunogold labeling, and co-immunoprecipitation. Furthermore, we use surface biotinylation and live cell confocal and total internal fluorescence microscopy imaging in cardiomyocytes and cell lines to explore delivery of Cav1.2 to BIN1 structures. We find visually and quantitatively that dynamic microtubules are tethered to membrane scaffolded by BIN1, allowing targeted delivery of Cav1.2 from the microtubules to the associated membrane. Since Cav1.2 delivery to BIN1 occurs in reductionist non-myocyte cell lines, we find that other myocyte-specific structures are not essential and there is an intrinsic relationship between microtubule-based Cav1.2 delivery and its BIN1 scaffold. In differentiated mouse cardiomyocytes, knockdown of BIN1 reduces surface Cav1.2 and delays development of the calcium transient, indicating that Cav1.2 targeting to BIN1 is functionally important to cardiac calcium signaling. We have identified that membrane-associated BIN1 not only induces membrane curvature but can direct specific antegrade delivery of microtubule-transported membrane proteins. Furthermore, this paradigm provides a microtubule and BIN1-dependent mechanism of Cav1.2 delivery to T-tubules. This novel Cav1.2 trafficking pathway should serve as an important regulatory aspect of EC coupling, affecting cardiac contractility in mammalian hearts.

Heart muscle excitation-contraction (E-C) coupling is governed by Ca²⁺ release units (CRUs) whereby Ca²⁺ influx via L-type Ca²⁺ channels (Cav1.2) triggers Ca²⁺ release from juxtaposed Ca²⁺ release channels (RyR2) located in junctional sarcoplasmic reticulum (jSR). Although studies suggest that the jSR protein triadin anchors cardiac calsequestrin (Casq2) to RyR2, its contribution to E-C coupling remains unclear. Here, we identify the role of triadin using mice with ablation of the Trdn gene (Trdn⁻/⁻). The structure and protein composition of the cardiac CRU is significantly altered in Trdn⁻/⁻ hearts. jSR proteins (RyR2, Casq2, junctin, and junctophilin 1 and 2) are significantly reduced in Trdn⁻/⁻ hearts, whereas Cav1.2 and SERCA2a remain unchanged. Electron microscopy shows fragmentation and an overall 50% reduction in the contacts between jSR and T-tubules. Immunolabeling experiments show reduced colocalization of Cav1.2 with RyR2 and substantial Casq2 labeling outside of the jSR in Trdn⁻/⁻ myocytes. CRU function is impaired in Trdn⁻/⁻ myocytes, with reduced SR Ca²⁺ release and impaired negative feedback of SR Ca²⁺ release on Cav1.2 Ca²⁺ currents (ICa). Uninhibited Ca²⁺ influx via ICa likely contributes to Ca²⁺ overload and results in spontaneous SR Ca²⁺ releases upon β-adrenergic receptor stimulation with isoproterenol in Trdn⁻/⁻ myocytes, and ventricular arrhythmias in Trdn⁻/⁻ mice. We conclude that triadin is critically important for maintaining the structural and functional integrity of the cardiac CRU; triadin loss and the resulting alterations in CRU structure and protein composition impairs E-C coupling and renders hearts susceptible to ventricular arrhythmias.