Experimental models of cardiac ischemia/reperfusion injury have served as useful tools in isolating the sequence of events and mechanisms involved following an infarct. The in vitro coverslip ischemia model in neonatal myocytes is key in observing acute cellular and organelle changes during ischemia and in reperfusion. Here we use neonatal mouse ventricular myocytes, and describe two experimental readouts of lactate dehydrogenase assay, for cell damage/injury and measurement of mitochondrial membrane potential.

Stress granules (SGs) are cytoplasmic ribonucleoprotein condensates that form during acute stress through liquid-liquid phase separation. Protein-protein interactions intrinsically disordered regions are important in this process. SGs protect against stress by rewiring translation and sequestering signaling proteins. Previous research found that the addition of O-GlcNAc to cytoplasmic proteins (O-GlcNAcylation), is required for SG formation. SGs have been implicated in neuronal dysfunction, but their role in cardiac pathophysiology is unexplored.

Background: Cardiac risk rises during acute SARS-CoV-2 infection and in long COVID syndrome in humans, but the mechanisms behind COVID-19-linked arrhythmias are unknown. This study explores the acute and long term effects of SARS-CoV-2 on the cardiac conduction system (CCS) in a hamster model of COVID-19. Methods: Radiotelemetry in conscious animals was used to non-invasively record electrocardiograms and subpleural pressures after intranasal SARS-CoV-2 infection. Cardiac cytokines, interferon-stimulated gene expression, and macrophage infiltration of the CCS, were assessed at 4 days and 4 weeks post-infection. A double-stranded RNA mimetic, polyinosinic:polycytidylic acid (PIC), was used in vivo and in vitro to activate viral pattern recognition receptors in the absence of SARS-CoV-2 infection. Results: COVID-19 induced pronounced tachypnea and severe cardiac conduction system (CCS) dysfunction, spanning from bradycardia to persistent atrioventricular block, although no viral protein expression was detected in the heart. Arrhythmias developed rapidly, partially reversed, and then redeveloped after the pulmonary infection was resolved, indicating persistent CCS injury. Increased cardiac cytokines, interferon-stimulated gene expression, and macrophage remodeling in the CCS accompanied the electrophysiological abnormalities. Interestingly, the arrhythmia phenotype was reproduced by cardiac injection of PIC in the absence of virus, indicating that innate immune activation was sufficient to drive the response. PIC also strongly induced cytokine secretion and robust interferon signaling in hearts, human iPSC-derived cardiomyocytes (hiPSC-CMs), and engineered heart tissues, accompanied by alterations in electrical and Ca2+ handling properties. Importantly, the pulmonary and cardiac effects of COVID-19 were blunted by in vivo inhibition of JAK/STAT signaling or by a mitochondrially-targeted antioxidant. Conclusions: The findings indicate that long term dysfunction and immune cell remodeling of the CCS is induced by COVID-19, arising indirectly from oxidative stress and excessive activation of cardiac innate immune responses during infection, with implications for long COVID Syndrome.

The dynamic cycling of O-linked GlcNAc (O-GlcNAc) on and off Ser/Thr residues of intracellular proteins, termed O-GlcNAcylation, is mediated by the conserved enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase. O-GlcNAc cycling is important in homeostatic and stress responses, and its perturbation sensitizes the heart to ischemic and other injuries. Despite considerable progress, many molecular pathways impacted by O-GlcNAcylation in the heart remain unclear. The mitogen-activated protein kinase (MAPK) pathway is a central signaling cascade that coordinates developmental, physiological, and pathological responses in the heart. The developmental or adaptive arm of MAPK signaling is primarily mediated by Erk kinases, while the pathophysiologic arm is mediated by p38 and Jnk kinases. Here, we examine whether O-GlcNAcylation affects MAPK signaling in cardiac myocytes, focusing on Erk1/2 and p38 in basal and hypertrophic conditions induced by phenylephrine. Using metabolic labeling of glycans coupled with alkyne-azide "click" chemistry, we found that Erk1/2 and p38 are O-GlcNAcylated. Supporting the regulation of p38 by O-GlcNAcylation, the OGT inhibitor, OSMI-1, triggers the phosphorylation of p38, an event that involves the NOX2-Ask1-MKK3/6 signaling axis and also the noncanonical activator Tab1. Additionally, OGT inhibition blocks the phenylephrine-induced phosphorylation of Erk1/2. Consistent with perturbed MAPK signaling, OSMI-1-treated cardiomyocytes have a blunted hypertrophic response to phenylephrine, decreased expression of cTnT (key component of the contractile apparatus), and increased expression of maladaptive natriuretic factors Anp and Bnp. Collectively, these studies highlight new roles for O-GlcNAcylation in maintaining a balanced activity of Erk1/2 and p38 MAPKs during hypertrophic growth responses in cardiomyocytes.

The regulated and reversible modification of Ser/Thr residues by O-linked N-Acetylglucosamine (O-GlcNAc) is termed O-GlcNAcylation. O-GlcNAc cycling on and off proteins is regulated by two conserved enzymes, the ‘writer’ O-GlcNAc Transferase (OGT) and the ‘eraser’ O-GlcNAcase (OGA). Crucially, O-GlcNAcylation is implicated in nutrient sensing, cell growth and stress adaptation. Despite these significant roles, the precise pathways impacted by O-GlcNAcylation and what key substrate proteins are involved remain incompletely understood. A hallmark of cardiomyocyte hypertrophy is the rapid production of new proteins that assemble into the contractile machinery of the growing myocyte. eIF2α is a rate-limiting factor in translation initiation. Interestingly, phosphorylation of eIF2α causes the arrest of general mRNA translation and favors the translation of stress-adaptive mRNAs. In the present work we investigated whether altering O-GlcNAcylation affects nascent protein synthesis (NPS) in hypertrophic cardiomyocytes. NPS was significantly elevated by the agonist phenylephrine, and this response was suppressed by OSMI-1 (OGT inhibitor). Moreover, we found that OGT-inhibited cells had increased phosphorylation of eIF2α. Additionally, we found increased protein levels of the stress adaptive factors Atf4 and Chop, whose mRNA translation is favored when eIF2α is phosphorylated. ISRIB, a small molecule designed to suppress the effects of phospho-eIF2α in altering translation, significantly prevented OSMI-1-induced accumulation of Atf4. Furthermore, we used metabolic labeling of glycans in cells, coupled with alkyne-azide ‘click’ chemistry and streptavidin pull-down, to show that eIF2α can be O-GlcNAcylated in cardiomyocytes. Furthermore, protein enrichment followed by mass spectrometry identified potential sites of O-GlcNAcylation on eIF2α. Taken together, these results highlight a novel role of O-GlcNAcylation on cardiomyocyte protein synthesis through the regulation of eIF2α. Future investigations will characterize the upstream mechanisms that promote eIF2α phosphorylation in OGT-inhibited cells and investigate the implications of direct eIF2α O-GlcNAcylation on hypertrophic protein synthesis in cardiomyocytes.

Introduction: Acute and long-term cardiovascular complications of COVID-19 have been reported, ranging from arrhythmias, ischemic heart disease, and heart failure to cerebrovascular and cardiometabolic disorders, yet the underlying molecular mechanisms are not well understood. Here, we examine the effects of stimulating the double-stranded RNA (dsRNA) pattern recognition receptor pathway, which is strongly induced to activate interferon-stimulated gene (ISG) expression in the heart during COVID-19, on excitation-contraction coupling and mitochondrial function. While providing antiviral protection, we hypothesize that, even in the absence of cardiac viral replication, this pathway might contribute to COVID-19-related cardiac dysfunction. Results: We used the viral dsRNA mimetic polyinosinic: polycytidylic (PIC) to induce a type I interferon response in iPSC-CM or A549 (lung epithelial) cells. In iPSC-CM, PIC induced robust protein expression of STAT1/pSTAT1, OAS2, OAS3, IRF7, pIRF7, IRF9 and MX1. A marked increase in cytokine protein secretion was also observed, with significant increases in CXCL-10 (~160 fold), IL6, CCL5 (~20-fold) IL8 (15-fold), and CXCL1 (~10-fold), and a 2-5-fold increase in ~30 other cytokines. Negative effects on excitation-contraction coupling were observed after 72 hours of PIC treatment, manifested as a 25% reduction in iPSC-CM Ca2+ transient amplitude. Microelectrode array analysis of iPSC-CM monolayers revealed electrophysiological dysfunction: PIC treatment increased cardiac field potential duration and spike amplitude, but reduced beat periods. A549 cells also showed increases in interferon pathway genes (STAT1, pSTAT1, IRF3, pIRF3, OAS1,2,3 and IFNb1), along with increased rates of RNA degradation after PIC treatment, consistent with activation of the OAS/RNAseL dsRNA sensor pathway. PIC-treated A549 cells also showed a large decrease in oxidative phosphorylation (maximal respiration and spare respiratory capacity), which was prevented by the mitochondrial antioxidant, mitoTEMPO. Conclusions: Activation of the viral dsRNA innate immune sensor pathway, even in the absence of active cardiac viral replication, might contribute to mitochondrial and cardiac dysfunction in COVID-19.

Physiologic Ca2+ entry via the Mitochondrial Calcium Uniporter (MCU) participates in energetic adaption to workload but may also contribute to cell death during ischemia/reperfusion (I/R) injury. The MCU has been identified as the primary mode of Ca2+ import into mitochondria. Several groups have tested the hypothesis that Ca2+ import via MCU is detrimental during I/R injury using genetically-engineered mouse models, yet the results from these studies are inconclusive. Furthermore, mitochondria exhibit unstable or oscillatory membrane potentials (ΔΨm) when subjected to stress, such as during I/R, but it is unclear if the primary trigger is an excess influx of mitochondrial Ca2+ (mCa2+), reactive oxygen species (ROS) accumulation, or other factors. Here, we critically examine whether MCU-mediated mitochondrial Ca2+ uptake during I/R is involved in ΔΨm instability, or sustained mitochondrial depolarization, during reperfusion by acutely knocking out MCU in neonatal mouse ventricular myocyte (NMVM) monolayers subjected to simulated I/R. Unexpectedly, we find that MCU knockout does not significantly alter mCa2+ import during I/R, nor does it affect ΔΨm recovery during reperfusion. In contrast, blocking the mitochondrial sodium-calcium exchanger (mNCE) suppressed the mCa2+ increase during Ischemia but did not affect ΔΨm recovery or the frequency of ΔΨm oscillations during reperfusion, indicating that mitochondrial ΔΨm instability on reperfusion is not triggered by mCa2+. Interestingly, inhibition of mitochondrial electron transport or supplementation with antioxidants stabilized I/R-induced ΔΨm oscillations. The findings are consistent with mCa2+ overload being mediated by reverse-mode mNCE activity and supporting ROS-induced ROS release as the primary trigger of ΔΨm instability during reperfusion injury.

Intro: Cardiac risk rises during acute SARS-CoV-2 infection and in long COVID syndrome, but the mechanisms behind COVID-19-linked arrhythmias are unknown. Here, we test the hypothesis that innate immune activation and mitochondrial ROS contribute to cardiac conduction abnormalities and pulmonary dysfunction in a COVID-19 hamster model. Results: ECGs and subpleural pressures were recorded by radiotelemetry over a 4-week timespan after SARS-CoV-2 infection. Multiple cardiac arrhythmias were observed, including bradycardia, sinus pauses, and atrioventricular block (AVB). RR intervals and sinus arrhythmia (RR> mean+100ms, RR100) increased transiently 3-5 days after SARS-CoV-2 infection (baseline/peak: RR, 167±3ms/241±7ms; RR100, 5.5±0.4min -1 /22.7±3.0min -1 ) and returned to levels ≤ baseline by 7 dpi. After the acute phase, these measures increased above baseline over 3 weeks (at 28dpi: RR, 190±2ms, p<0.05; RR100, 13.4±1.0 min -1 , p<0.01). AVB count peaked at 3.1±0.1 min -1 at 1 dpi and gradually decreased to 0.4±0.2 min -1 by 28 dpi (vs. 0.004±0.002 at 0 dpi, p<0.05). These parameters did not change in Mock-infected controls. RT-PCR showed that RIG-I, CXCL-10, OAS1, IL-1b and CCL-2 expression increased 18, 7, 2, 3, and 2-fold, respectively, over Mock controls at 4 dpi. IF analysis of the His-bundle region showed increased abundance of inflammatory IBA-1 + macrophages and decreased anti-inflammatory CD163 + macrophages. Ruxolitinib (Jak/Stat inhibitor) or mitochondrial antioxidant (mitoTEMPO) treatments were administered via implanted osmotic pumps. Subpleural pressure recordings indicated that infection induced tachypnea peaking at 7 dpi (272±11 versus 81±17 breaths/min at 0 dpi, p<0.0001), which was attenuated by either treatment (peak: mitoTEMPO, 170±10; Ruxo,184±14). MitoTEMPO, but not Ruxolitinib, suppressed the increases in RR, RR100, and AVB during the acute phase and prevented the secondary changes in these measures in the post-acute phase (up to 28 dpi). Conclusions: The hamster model recapitulates a subset of arrhythmias observed in COVID-19 patients. Jak/Stat inhibition or mitoTEMPO treatment both mitigated acute respiratory distress, but only the latter suppressed arrhythmias during the acute and late phases of COVID-19.

Formate (FM) is a substrate in one-carbon metabolism, and recent studies implicate its function in both health and disease, but a role in the heart remains unexplored. Research from our group demonstrates that the loss of formaldehyde dehydrogenase, which oxidizes formaldehyde into FM, ameliorates sex-dependent cardioprotection against ischemia-reperfusion (I/R) injury in the female mouse heart. Considering that FM depletion may be detrimental, we tested the hypothesis that FM yields cardioprotective benefit in a model of I/R injury. Hearts from male and female mice (n=5-10/group) were subjected to ex vivo I/R injury with or without FM via Langendorff perfusion. FM significantly enhanced post-ischemic functional recovery in male hearts (27.1%, 95% CI 10.8 to 45.9, p=0.0023; females: 9.5%, 95% CI -6.9 to 27.8, p=0.2020) and decreased infarct size (males: -24.6%, 95% CI -35.1 to -12.2, p<0.0001; females: -3.7%, 95% CI -9.6 to -0.3, p=0.1128), indicating that FM protects the myocardium against I/R injury. Although FM had a greater protective effect in male hearts than in female hearts, plasma FM levels were greater in female mice, suggesting that FM-mediated cardioprotection may already exist near steady state in females. Mechanistically, cardiomyoblasts stimulated with FM exhibited a significant increase in total protein S-nitrosylation (SNO) without changing nitric oxide synthase (NOS) phosphorylation or protein expression; this was suppressed by NOS inhibition, suggesting that FM contributes to NOS-dependent NO cycling in the heart. FM-treated cardiomyocytes and cardiomyoblasts also exhibited significant increases in mitochondrial marker protein expression, metabolic activity, and maximal oxygen consumption, demonstrating an enhanced respiratory capacity to respond to increased energetic demand. Furthermore, FM treatment significantly increased phospho-activation of the energy regulator AMPKα and the protein expression of PGC-1α targets, indicating a potential upregulation in mitochondrial biogenesis. Taken together, these data suggest that FM may stimulate protein SNO and mitochondrial activity to protect the heart against I/R injury.

Background: The COVID-19 pandemic has highlighted how little is known about how double stranded RNA (dsRNA) viruses like the SARS-CoV-2 impact cardiac physiology acutely and long-term. While it is unclear if the cardiac effects are direct or indirect, adverse effects in patients are common, including cardiac inflammation, electrophysiological changes in QT interval, atrial fibrillation, bradyarrhythmia, and ventricular tachycardia. Mechanisms underlying these COVID-19-associated arrhythmias are unknown. Methods: We assess the effects of SARS-CoV-2 infection on cardiac electrophysiology and inflammatory pathway activation in a hamster model over a 4-week timespan. We further test if activation of antiviral innate immune responses with a dsRNA mimetic, Poly I:C (PI:C) induces similar effects in uninfected hearts and in human iPSC-derived cardiomyocytes (hiPSC-CMs), examining the determinants of RNAseL activation, interferon (IFN) signaling, and cytokine release in vivo and in vitro. Results: Intranasal SARS-CoV-2 USA-WA1 infection of hamsters caused ECG abnormalities including bradycardia and cardiac conduction defects including sinus pauses, 2 nd and 3 rd degree Atrio-Ventricular block. Bradycardia peaked at 4-5 days after infection. Mean RR interval increased from 182±4ms pre- to 233±6ms post-infection, without significant increases in mock-infected hamsters. In the absence of viral infection, ventricular injection of PI:C induced similar arrhythmias and induction of IFN-stimulated genes. PI:C treatment of hiPSC-CMs induced robust increases in type-I IFN pathway proteins STAT1/pSTAT1, OAS2 (48% increase) and OAS3 (32% increase) and a robust cytokine response. Negative effects on excitation-contraction coupling after 72hr of PI:C treatment, manifested as a 25% reduction in hiPSC-CM Ca 2+ transient peaks. In vitro studies showed increased RNA degradation after PI:C, consistent with activation of the OAS/RNAseL dsRNA sensor pathway. Conclusions: COVID-19 in the hamster model recapitulates a subset of arrhythmias observed in COVID-19 patients. Activation of the viral dsRNA innate immune sensor pathway, even in the absence of active cardiac viral replication, may contribute to SARS-CoV-2-associated cardiac dysfunction.

Mitochondria exhibit unstable inner membrane potentials (ΔΨm) when subjected to stress, such as during ischemia/reperfusion (I/R). Understanding the mechanism of ΔΨm instability involves characterizing and quantifying this phenomenon in an unbiased and reproducible manner. Here, we describe a simple analytical workflow called "MitoWave" that combines wavelet transform methods and image segmentation to unravel dynamic ΔΨm changes in the cardiac mitochondrial network during I/R. In vitro ischemia was affected by placing a glass coverslip on a monolayer of neonatal mouse ventricular myocytes for 1 h and removing the coverslip to allow for reperfusion, revealing complex oscillatory ΔΨm. MitoWave analysis was then used to identify individual mitochondrial clusters within the cells and track their intrinsic oscillation frequencies over the course of reperfusion. Responses segregated into five typical behaviors were quantified by MitoWave that were corroborated by visual inspection of the time series. Statistical analysis of the distribution of oscillating mitochondrial clusters during reperfusion showed significant differences between the five different outcomes. Features such as the time point of ΔΨm depolarization during I/R, area of mitochondrial clusters, and time-resolved frequency components during reperfusion were determined per cell and per mitochondrial cluster. Mitochondria from neonatal mouse ventricular myocytes subjected to I/R oscillate in the frequency range of 8.6-45 mHz, with a mean of 8.73 ± 4.35 mHz. Oscillating clusters had smaller areas ranging from 49.8 ± 1.2 μm2, whereas nonoscillating clusters had larger areas 66 ± 1.5 μm2. A negative correlation between frequency and mitochondrial cluster area was observed. We also observed that late ΔΨm loss during ischemia correlated with early ΔΨm stabilization after oscillation on reperfusion. Thus, MitoWave analysis provides a semiautomated method to quantify complex time-resolved mitochondrial behavior in an easy-to-follow workflow, enabling unbiased, reproducible quantitation of complex nonstationary cellular phenomena.

Central obesity with cardiometabolic syndrome (CMS) is a major global contributor to human disease, and effective therapies are needed. Here, we show that cyclic GMP-selective phosphodiesterase 9A inhibition (PDE9-I) in both male and ovariectomized female mice suppresses preestablished severe diet-induced obesity/CMS with or without superimposed mild cardiac pressure load. PDE9-I reduces total body, inguinal, hepatic, and myocardial fat; stimulates mitochondrial activity in brown and white fat; and improves CMS, without significantly altering activity or food intake. PDE9 localized at mitochondria, and its inhibition in vitro stimulated lipolysis in a PPARα-dependent manner and increased mitochondrial respiration in both adipocytes and myocytes. PPARα upregulation was required to achieve the lipolytic, antiobesity, and metabolic effects of PDE9-I. All these PDE9-I-induced changes were not observed in obese/CMS nonovariectomized females, indicating a strong sexual dimorphism. We found that PPARα chromatin binding was reoriented away from fat metabolism-regulating genes when stimulated in the presence of coactivated estrogen receptor-α, and this may underlie the dimorphism. These findings have translational relevance given that PDE9-I is already being studied in humans for indications including heart failure, and efficacy against obesity/CMS would enhance its therapeutic utility.

The mitogen activated protein kinase (MAPK) p38 is important in cardiac hypertrophic responses and p38 inhibition has been tested as a potential therapeutic approach to heart failure. p38 is tightly regulated by upstream kinases and phosphatases. While p38 inhibitors suppress cardiac hypertrophy in vitro and in animal models, the partial efficacy of p38 inhibitors in clinical trials for heart failure illustrates the need for a deeper understanding of p38-regulatory mechanisms. O -linked N-Acetylglucosamine ( O -GlcNAc) on Ser/Thr residues is a ubiquitous intracellular modification ( O -GlcNAcylation) that participates in intracellular signaling, often occurring in counterpoint to phosphorylation. O -GlcNAcylation is catalyzed by O -GlcNAc Transferase (OGT) and removed by O -GlcNAc-Ase (OGA). Given the crucial regulation of p38 activity by phosphorylation, we hypothesized that O -GlcNAcylation regulates p38 phosphorylation during basal and hypertrophic cardiomyocyte signaling. Treating neonatal rat ventricular myocytes (NRVM) with OSMI-1 (inhibitor of OGT) significantly decreased O -GlcNAcylation (0.48 ± 0.02, P <0.001 vs. vehicle), whereas treatment with Thiamet-G (inhibitor of OGA) significantly increased O -GlcNAcylation (3.0-fold increase ± 0.35, P <0.05 vs. vehicle). OSMI1 treatment induced the phosphorylation of p38 at its activation site (3.9-fold increase ± 0.46, P <0.001 vs. vehicle) and promoted the phosphorylation of the downstream target, heat shock protein Hsp27 (8-fold increase ± 1.3, P <0.0001 vs. vehicle) and transcription factor Creb (3.3-fold increase ± 0.12, P <0.001 vs. vehicle). OSMI-1 had an additive effect in inducing p38 and Creb phosphorylation following hypertrophic stimulation by phenylephrine (3.1-fold and 1.4-fold increase vs. phenylephrine respectively, P <0.05). Treatment with the p38 inhibitor SB202190 abolished the phosphorylation of Hsp27 and Creb that was induced by OSMI-1. Canonical upstream activators of p38 include the MAP3Ks, TAK1 and ASK1. However, we found that treatment with ASK1 or TAK1 inhibitors (GS-444217 and Takinib, respectively) either alone, or in combination, did not negate the phosphorylation of p38 by OSMI-1. We conclude that regulation of p38 by OGT activity could occur at a level downstream of canonical MAP3Ks or through non-canonical pathways.

Ca2+ serves as a ubiquitous second messenger mediating a variety of cellular processes including electrical excitation, contraction, gene expression, secretion, cell death and others. The identification of the molecular components of the mitochondrial Ca2+ influx and efflux pathways has created a resurgent interest in the regulation of mitochondrial Ca2+ balance and its physiological and pathophysiological roles. While the pace of discovery has quickened with the availability of new cellular and animal models, many fundamental questions remain to be answered regarding the regulation and functional impact of mitochondrial Ca2+ in health and disease. This review highlights several experimental observations pertaining to key aspects of mitochondrial Ca2+ homeostasis that remain enigmatic, particularly whether mitochondrial Ca2+ signaling is depressed or excessive in heart failure, which will determine the optimal approach to therapeutic intervention.

ABSTRACTCentral obesity with cardiometabolic syndrome (CMS) is a major global contributor to human disease, and effective therapies are needed. Here, we show inhibiting cyclic-GMP selective phosphodiesterase-9A (PDE9-I) suppresses established diet-induced obesity and CMS in ovariectomized female and male mice. PDE9-I reduces abdominal, hepatic, and myocardial fat accumulation, stimulates mitochondrial activity in brown and white fat, and improves CMS, without altering activity or food intake. PDE9 localizes to mitochondria, and its inhibition stimulates lipolysis and mitochondrial respiration coupled to PPARα-dependent gene regulation. PPARα upregulation is required for PDE9-I metabolic efficacy and is absent in non-ovariectomized females that also display no metabolic benefits from PDE9-I. The latter is compatible with estrogen receptor-α altering PPARα chromatin binding identified by ChIPSeq. In humans with heart failure and preserved ejection fraction, myocardial expression ofPPARAand its regulated genes is reduced versus control. These findings support testing PDE9-I to treat obesity/CMS in men and postmenopausal women.SummaryOral inhibition of phosphodiesterase type 9 stimulates mitochondrial fat metabolism and lipolysis, reducing central obesity without changing appetite

Ca 2+ entry via the Mitochondrial Calcium Uniporter (MCU) participates in energetic adaption to workload under physiological conditions but is thought to contribute to cell death during ischemia-reperfusion (I/R) injury. We have previously shown that mitochondrial membrane potential (ΔΨm) instability contributes to early-reperfusion arrhythmias and contractile dysfunction; however, the role of mitochondrial Ca 2+ (mCa 2+ ) uptake in triggering ΔΨm oscillation is unclear. Here, by acutely knocking out MCU, we examine whether MCU-mediated mCa 2+ uptake is required to trigger ΔΨm loss or oscillation during early reperfusion in neonatal mouse ventricular myocyte (NMVM) monolayers. We monitored mCa 2+ (with MitoCam) and ΔΨm (with TMRM) in WT(MCU fl/fl ) and MCU-KO (MCU fl/fl +AdCre) NMVMs during in vitro I/R (previously described by Solhjoo et al JMCC, 2015) by confocal microscopy. Image sequences were analyzed for changes in mCa 2+ and ΔΨm by segmenting individual cells in ImageJ. To quantify ΔΨm oscillations in mitochondrial clusters during reperfusion, a new wavelet-transform-based analysis was developed using MATLAB’s wavelet toolbox. Surprisingly, our findings demonstrate that MCU knockout does not significantly alter mCa 2+ import during I/R, nor does it affect ΔΨm recovery during Reperfusion. In fact, MCU-KO moderately shortened the latency to Ischemic ΔΨm depolarization. In contrast, blocking the mitochondrial sodium-calcium exchanger (mNCLX) with CGP-37157 suppressed the mCa 2+ increase during Ischemia. Moreover, blocking mNCLX also did not affect ΔΨm recovery during Reperfusion or the frequency of ΔΨ m oscillations, confirming that mitochondrial ΔΨm instability on reperfusion is not triggered by mCa 2+ . Interestingly, inhibition of mitochondrial electron transport stabilized ΔΨm oscillations during reperfusion. The findings are consistent with mCa 2+ overload being mediated by reverse-mode mNCLX activity and support ROS-induced ROS release as the primary trigger of ΔΨm instability during reperfusion injury.

Aims: In cardiomyocytes, there is microRNA (miR) in the mitochondria that originates from the nuclear genome and matures in the cytoplasm before translocating into the mitochondria. Overexpression of one such miR, miR181c, can lead to heart failure by stimulating reactive oxygen species (ROS) production and increasing mitochondrial calcium level ([Ca2+](m)). Mitochondrial calcium uptake 1 protein (MICU1), a regulatory protein in the mitochondrial calcium uniporter complex, plays an important role in regulating [Ca2+](m). Obesity results in miR-181c overexpression and a decrease in MICU1. We hypothesize that lowering miR-181c would protect against obesity-induced cardiac dysfunction. Methods and results: We used an in vivo mouse model of high-fat diet (HFD) for 18 weeks and induced high lipid load in H9c2 cells with oleate-conjugated bovine serum albumin in vitro. We tested the cardioprotective role of lowering miR-181c by using miR-181c/d(-/-) mice (in vivo) and AntagomiR against miR-181c (in vitro). HFD significantly upregulated heart levels of miR-181c and led to cardiac hypertrophy in wild-type mice, but not in miR-181c/d(-/-) mice. HFD also increased ROS production and pyruvate dehydrogenase activity (a surrogate for [Ca2+](m), but the increases were alleviated in miR-181c/d(-/-) mice. Moreover, miR-181c/d-mice fed a HFD had higher levels of MICU1 than did wild-type mice fed a HFD, attenuating the rise in [Ca2+](m). Overexpression of miR-181c in neonatal ventricular cardiomyocytes (NMVM) caused increased ROS production, which oxidized transcription factor Sp1 and led to a loss of Sp1, thereby slowing MICU1 transcription. Hence, miR-181c increases [Ca2+](m) through Sp1 oxidation and downregulation of MICU1, suggesting that the cardioprotective effect of miR-181c/d(-/-) results from inhibition of Sp1 oxidation. Conclusion: This study has identified a unique nuclear-mitochondrial communication mechanism in the heart orchestrated by miR-181c. Obesity-induced overexpression of miR-181c increases [Ca2+](m) via downregulation of MICU1 and leads to cardiac injury. A strategy to inhibit miR-181c in cardiomyocytes can preserve cardiac function during obesity by improving mitochondrial function. Altering miR-181c expression may provide a pharmacologic approach to improve cardiomyopathy in individuals with obesity/type 2 diabetes.