The uptake of calcium into and extrusion of calcium from the mitochondrial matrix is a fundamental biological process that has critical effects on cellular metabolism, signaling, and survival. Disruption of mitochondrial calcium (mCa2+) cycling is implicated in numerous acquired diseases such as heart failure, stroke, neurodegeneration, diabetes, and cancer, and is genetically linked to several inherited neuromuscular disorders. Understanding the mechanisms responsible for mCa2+ exchange therefore holds great promise for the treatment of these diseases. The past decade has seen the genetic identification of many of the key proteins that mediate mitochondrial calcium uptake and efflux. Here, we present an overview of the phenomenon of mCa2+ transport, and a comprehensive examination of the molecular machinery that mediates calcium flux across the inner mitochondrial membrane: the mitochondrial uniporter complex (consisting of MCU, EMRE, MICU1, MICU2, MICU3, MCUB, and MCUR1), NCLX, LETM1, the mitochondrial ryanodine receptor, and the mitochondrial permeability transition pore. We then consider the physiological implications of mCa2+ flux and evaluate how alterations in mCa2+ homeostasis contribute to human disease. This review concludes by highlighting opportunities and challenges for therapeutic intervention in pathologies characterized by aberrant mCa2+ handling and by summarizing critical unanswered questions regarding the biology of mCa2+ flux.

Patients deficient in dystrophin, a protein that links the cytoskeleton to the extracellular matrix via the dystrophin-glycoprotein complex (DGC), exhibit muscular dystrophy, cardiomyopathy, and impaired muscle nitric oxide (NO) production. We used live-cell NO imaging and in vitro cyclic stretch of isolated adult mouse cardiomyocytes as a model system to investigate if and how the DGC directly regulates the mechanical activation of muscle NO signaling. Acute activation of NO synthesis by mechanical stretch was impaired in dystrophin-deficient mdx cardiomyocytes, accompanied by loss of stretch-induced neuronal NO synthase (nNOS) S1412 phosphorylation. Intriguingly, stretch induced the acute activation of AMP-activated protein kinase (AMPK) in normal cardiomyocytes but not in mdx cardiomyocytes, and specific inhibition of AMPK was sufficient to attenuate mechanoactivation of NO production. Therefore, we tested whether direct pharmacologic activation of AMPK could bypass defective mechanical signaling to restore nNOS activity in dystrophin-deficient cardiomyocytes. Indeed, activation of AMPK with 5-aminoimidazole-4-carboxamide riboside or salicylate increased nNOS S1412 phosphorylation and was sufficient to enhance NO production in mdx cardiomyocytes. We conclude that the DGC promotes the mechanical activation of cardiac nNOS by acting as a mechanosensor to regulate AMPK activity, and that pharmacologic AMPK activation may be a suitable therapeutic strategy for restoring nNOS activity in dystrophin-deficient hearts and muscle.T he muscular dystrophies are a group of muscle wasting disorders characterized by progressive weakening and degeneration of striated muscle. The most common form is Duchenne muscular dystrophy (DMD), an X-linked disorder caused by genetic disruption of dystrophin (1) that affects 1 in 3,500-5,000 males (2, 3). DMD and several other types of muscular dystrophy result from disruption of the dystrophin-glycoprotein complex (DGC), a structure that spans the sarcolemma and forms a mechanical linkage between the cytoskeleton and the extracellular matrix via the association of dystrophin with subsarcolemmal γ-actin and the binding of α-dystroglycan to laminin (4). The generally accepted role for this complex is to act as a molecular shock absorber and stabilize the plasma membrane during muscle contraction. Disruption of the DGC's linkage between the cytoskeleton and extracellular matrix, such as occurs in DMD (1, 5-7) or with the disruption of α-dystroglycan-laminin binding in glycosylation-deficient muscular dystrophies (8, 9), leads to destabilization of the plasma membrane, rendering skeletal muscle fibers and cardiomyocytes susceptible to stretch-or contraction-induced injury and cell death (10)(11)(12)(13)(14).In addition to this structural role, a signaling function for the DGC has been proposed based on its association with several signaling proteins including Grb2-Sos1 (15), MEK and ERK (16), heterotrimeric G protein subunits (17, 18), archvillin (19), and neuronal nitric oxide synthase (n...

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Rationale: Ca 2+ induced Ca 2+ release (CICR) in normal hearts requires close approximation of L-type calcium channels (LTCCs) within the transverse tubules (T-tubules), and Ryanodine receptors (RyR) within the junctional sarcoplasmic reticulum (jSR). CICR is disrupted in cardiac hypertrophy and heart failure, which is associated with loss of T-tubules and disruption of cardiac dyads. In these conditions, LTCCs are redistributed from the T-tubules to disrupt CICR. The molecular mechanism responsible for LTCCs recruitment to and from the T-tubules is not well known. Junctophilin-2 (JPH2) enables close association between T-tubules and the jSR to ensure efficient CICR. JPH2 has a so-called Joining region that is located near domains that interact with T-tubular plasma membrane, where LTCCs are housed. The idea that this Joining region directly interacts with LTCCs and contributes to LTCC recruitment to T-tubules is unknown. Objective: To determine if the Joining region in JPH2 recruits LTCCs to T-tubules through direct molecular interaction in cardiomyocytes to enable efficient CICR. Methods and Results: Modified abundance of JPH2 and redistribution of LTCC were studied in left ventricular hypertrophy in vivo and in cultured adult Feline and rat ventricular myocytes. Protein-protein interaction studies showed that the Joining region in JPH2 interacts with LTCC-α1C subunit and causes LTCCs distribution to the dyads, where they colocalize with RyRs. A JPH2 with induced mutations in the Joining region (mutPG1JPH2) caused T-tubule remodeling and dyad loss, showing that an interaction between LTCC and JPH2 is crucial for T-tubule stabilization. mut PG1JPH2 caused asynchronous Ca 2+ -release with impaired excitation-contraction (EC) coupling after β-adrenergic stimulation. The disturbed Ca 2+ regulation in mut PG1JPH2 overexpressing myocytes caused Calcium/calmodulin-dependent kinase-II activation and altered myocyte bioenergetics. Conclusions: The interaction between LTCC and the Joining region in JPH2 facilitates dyad assembly and maintains normal CIRC in cardiomyocytes.

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