Reduced cardiac antioxidant defenses mediate increased susceptibility to workload-induced myocardial injury in males with genetic cardiomyopathy.

Ongoing cardiomyocyte injury is a major mechanism in the progression of heart failure, particularly in dystrophic hearts. Due to the poor regenerative capacity of the adult heart, cardiomyocyte death results in the permanent loss of functional myocardium. Understanding the factors contributing to myocyte injury is essential for the development of effective heart failure therapies. As a model of persistent cardiac injury, we examined mice lacking ß-sarcoglycan (ß-SG), a key component of the dystrophin glycoprotein complex (DGC). The loss of the sarcoglycan complex markedly compromises sarcolemmal integrity in this ß-SG-/- model. Our studies aim to characterize the mechanisms underlying dramatic sex differences in susceptibility to cardiac injury in ß-SG-/- mice. Male ß-SG-/- hearts display significantly greater myocardial injury and death following isoproterenol-induced cardiac stress than female ß-SG-/- hearts. This protection of females was independent of ovarian hormones. Male ß-SG-/- hearts displayed increased susceptibility to exogenous oxidative stress and were significantly protected by angiotensin II type 1 receptor (AT1R) antagonism. Increasing general antioxidative defenses or increasing the levels of S-nitrosylation both provided protection to the hearts of ß-SG-/- male mice. Here we demonstrate that increased susceptibility to oxidative damage leads to an AT1R-mediated amplification of workload-induced myocardial injury in male ß-SG-/- mice. Improving oxidative defenses, specifically by increasing S-nitrosylation, provided protection to the male ß-SG-/- heart from workload-induced injury. These studies describe a unique susceptibility of the male heart to injury and may contribute to the sex differences in other forms of cardiac injury.

Prenatal arsenite exposure alters maternal cardiac remodeling during late pregnancy.

Exposure to inorganic arsenic through drinking water is widespread and has been linked to many chronic diseases, including cardiovascular disease. Arsenic exposure has been shown to alter hypertrophic signaling in the adult heart, as well as in utero offspring development. However, the effect of arsenic on maternal cardiac remodeling during pregnancy has not been studied. As such, there is a need to understand how environmental exposure contributes to adverse pregnancy-related cardiovascular events. This study seeks to understand the impact of trivalent inorganic arsenic exposure during gestation on maternal cardiac remodeling in late pregnancy, as well as offspring outcomes. C57BL/6 J mice were exposed to 0 (control), 100 or 1000 µg/L sodium arsenite (NaAsO2) beginning at embryonic day (E) 2.5 and continuing through E17.5. Maternal heart function and size were assessed via transthoracic echocardiography, gravimetric measurement, and histology. Transcript levels of hypertrophic markers were probed via qRT-PCR and confirmed by western blot. Offspring outcomes were assessed through echocardiography and gravimetric measurement. We found that maternal heart size was smaller and transcript levels of Esr1 (estrogen receptor alpha), Pgrmc1 (progesterone receptor membrane component 1) and Pgrmc2 (progesterone receptor membrane component 2) reduced during late pregnancy with exposure to 1000 µg/L iAs vs. non-exposed pregnant controls. Both 100 and 1000 µg/L iAs also reduced transcription of Nppa (atrial natriuretic peptide). Akt protein expression was also significantly reduced after 1000 µg/L iAs exposure in the maternal heart with no change in activating phosphorylation. This significant abrogation of maternal cardiac hypertrophy suggests that arsenic exposure during pregnancy can potentially contribute to cardiovascular disease. Taken together, our findings further underscore the importance of reducing arsenic exposure during pregnancy and indicate that more research is needed to assess the impact of arsenic and other environmental exposures on the maternal heart and adverse pregnancy events.

Nitric Oxide Modulates Ca2+ Leak and Arrhythmias via S-Nitrosylation of CaMKII.

BACKGROUND: Nitric oxide (NO) has been identified as a signaling molecule generated during ß-adrenergic receptor stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of CaMKIIδ (Ca2+/calmodulin kinase II delta) is emerging. NO donors are routinely used clinically for their cardioprotective effects on the heart, but it is unknown how NO donors modulate the proarrhythmic CaMKII to alter cardiac arrhythmia incidence. We test the role of S-nitrosylation of CaMKIIδ at the Cysteine-273 inhibitory site and cysteine-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during ß-adrenergic receptor stimulation. METHODS: We measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at cysteine-273 or cysteine-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 µM), sodium nitroprusside (200 µM), and ß-adrenergic agonist isoproterenol (100 nmol/L). RESULTS: Both WT and CaMKIIδ-KO cardiomyocytes responded to isoproterenol with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from isoproterenol-induced Ca2+ sparks and waves was mimicked by GSNO pretreatment in WT cardiomyocytes but lost in CaMKIIδ-C273S cardiomyocytes. When GSNO was applied after isoproterenol, this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pretreatment limited isoproterenol-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after isoproterenol sustained or exacerbated arrhythmic events. CONCLUSIONS: We conclude that prior S-nitrosylation of CaMKIIδ at cysteine-273 can limit subsequent ß-adrenergic receptor-induced arrhythmias, but that S-nitrosylation at cysteine-290 might worsen or sustain ß-adrenergic receptor-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Cardioprotective O-GlcNAc signaling is elevated in murine female hearts via enhanced O-GlcNAc transferase activity.

The post-translational modification of intracellular proteins by O-linked ß-GlcNAc (O-GlcNAc) has emerged as a critical regulator of cardiac function. Enhanced O-GlcNAcylation activates cytoprotective pathways in cardiac models of ischemia-reperfusion (I/R) injury; however, the mechanisms underpinning O-GlcNAc cycling in response to I/R injury have not been comprehensively assessed. The cycling of O-GlcNAc is regulated by the collective efforts of two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which catalyze the addition and hydrolysis of O-GlcNAc, respectively. It has previously been shown that baseline heart physiology and pathophysiology are impacted by sex. Here, we hypothesized that sex differences in molecular signaling may target protein O-GlcNAcylation both basally and in ischemic hearts. To address this question, we subjected male and female WT murine hearts to ex vivo ischemia or I/R injury. We assessed hearts for protein O-GlcNAcylation, abundance of OGT, OGA, and glutamine:fructose-6-phosphate aminotransferase (GFAT2), activity of OGT and OGA, and UDP-GlcNAc levels. Our data demonstrate elevated O-GlcNAcylation in female hearts both basally and during ischemia. We show that OGT activity was enhanced in female hearts in all treatments, suggesting a mechanism for these observations. Furthermore, we found that ischemia led to reduced O-GlcNAcylation and OGT-specific activity. Our findings provide a foundation for understanding molecular mechanisms that regulate O-GlcNAcylation in the heart and highlight the importance of sex as a significant factor when assessing key regulatory events that control O-GlcNAc cycling. These data suggest the intriguing possibility that elevated O-GlcNAcylation in females contributes to reduced ischemic susceptibility.

Prenatal Arsenite Exposure Alters Maternal Cardiac Remodeling During Late Pregnancy.

Exposure to inorganic arsenic through drinking water is widespread and has been linked to many chronic diseases, including cardiovascular disease. Arsenic exposure has been shown to alter hypertrophic signaling in the adult heart, as well as in-utero offspring development. However, the effect of arsenic on maternal cardiac remodeling during pregnancy has not been studied. As such, there is a need to understand how environmental exposure contributes to adverse pregnancy-related cardiovascular events. This study seeks to understand the impact of trivalent inorganic arsenic exposure during gestation on maternal cardiac remodeling in late pregnancy, as well as offspring outcomes. C57BL/6J mice were exposed to 0 (control), 100 or 1000 µg/L sodium arsenite (NaAsO 2 ) beginning at embryonic day (E) 2.5 and continuing through E17.5. Maternal heart function and size were assessed via transthoracic echocardiography, gravimetric measurement, and histology. Transcript levels of hypertrophic markers were probed via qRT-PCR and confirmed by western blot. Offspring outcomes were assessed through echocardiography and gravimetric measurement. We found that exposure to 1000 µg/L iAs abrogated normal physiologic growth of the maternal heart during late pregnancy and reduced transcript levels of estrogen receptor alpha (ERα), progesterone receptor membrane component 1 (Pgrmc1) and progesterone receptor membrane component 2 (Pgrmc2). Both 100 and 1000 µg/L iAs also reduced transcription of protein kinase B (Akt) and atrial natriuretic peptide (ANP). Akt protein expression was also significantly reduced after 1000 µg/L iAs exposure in the maternal heart with no change in activating phosphorylation. This significant abrogation of maternal cardiac hypertrophy suggests that arsenic exposure during pregnancy can potentially contribute to cardiovascular disease. Taken together, our findings further underscore the importance of reducing arsenic exposure during pregnancy and indicate that more research is needed to assess the impact of arsenic and other environmental exposures on the maternal heart and adverse pregnancy events.

Nitric Oxide modulates spontaneous Ca2+ release and ventricular arrhythmias during ß-adrenergic signalling through S-nitrosylation of Calcium/Calmodulin dependent kinase II.

Rationale: Nitric oxide (NO) has been identified as a signalling molecule generated during ß-adrenergic receptor (AR) stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of Ca2+/calmodulin kinase II delta (CaMKIIδ) is emerging. NO donors are routinely used clinically for their cardioprotective effects in the heart, but it is unknown how NO donors modulate the pro-arrhythmic CaMKII to alter cardiac arrhythmia incidence. Objective: We test the role of S-nitrosylation of CaMKIIδ at the Cys-273 inhibitory site and Cys-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during ß-AR stimulation. Methods and Results: We measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at Cys-273 or Cys-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 µM), sodium nitroprusside (SNP; 200 µM) and/or ß-adrenergic agonist isoproterenol (ISO; 100 nM). WT and CaMKIIδ-KO cardiomyocytes treated with GSNO showed no change in Ca2+ transient or spark properties under baseline conditions (0.5 Hz stimulation frequency). Both WT and CaMKIIδ-KO cardiomyocytes responded to ISO with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from ISO-induced Ca2+ sparks and waves was mimicked by GSNO pre-treatment in WT cardiomyocytes, but lost in CaMKIIδ-C273S cardiomyocytes that displayed a robust increase in Ca2+ waves. This observation is consistent with CaMKIIδ-C273 S-nitrosylation being critical in limiting ISO-induced arrhythmogenic sarcoplasmic reticulum Ca2+ leak. When GSNO was applied after ISO this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pre-treatment limited ISO-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after ISO sustained or exacerbated arrhythmic events. Conclusions: We conclude that prior S-nitrosylation of CaMKIIδ at Cys-273 can limit subsequent ß-AR induced arrhythmias, but that S-nitrosylation at Cys-290 might worsen or sustain ß-AR-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Differential Detection of O-GlcNAcylated proteins in the heart using antibodies.

Thousands of mammalian intracellular proteins are dynamically modified by O-linked ß-N-acetylglucosamine (O-GlcNAc). Global changes in O-GlcNAcylation have been associated with the development of cardiomyopathy, heart failure, hypertension, and neurodegenerative disease. Levels of O-GlcNAc in cells and tissues can be detected using numerous approaches; however, immunoblotting using GlcNAc-specific antibodies and lectins is commonplace. The goal of this study was to optimize the detection of O-GlcNAc in heart lysates by immunoblotting. Using a combination of tissue fractionation, immunoblotting, and galactosyltransferase labeling, as well as hearts from wild-type and O-GlcNAc transferase transgenic mice, we demonstrate that contractile proteins in the heart are differentially detected by two commercially available antibodies (CTD110.6 and RL2). As CTD110.6 displays poor reactivity toward contractile proteins, and as these proteins represent a major fraction of the heart proteome, a better assessment of cardiac O-GlcNAcylation is obtained in total tissue lysates with RL2. The data presented highlight tissue lysis approaches that should aid the assessment of the cardiac O-GlcNAcylation by immunoblotting.

Long-term effects of prenatal arsenic exposure from gestational day 9 to birth on lung, heart, and immune outcomes in the C57BL/6 mouse model.

Prenatal arsenic exposure is a major public health concern, associated with altered birth outcomes and increased respiratory disease risk. However, characterization of the long-term effects of mid-pregnancy (second trimester) arsenic exposure on multiple organ systems is scant. This study aimed to characterize the long-term impact of mid-pregnancy inorganic arsenic exposure on the lung, heart, and immune system, including infectious disease response using the C57BL/6 mouse model. Mice were exposed from gestational day 9 till birth to either 0 or 1000 µg/L sodium (meta)arsenite in drinking water. Male and female offspring assessed at adulthood (10-12 weeks of age) did not show significant effects on recovery outcomes after ischemia reperfusion injury but did exhibit increased airway hyperresponsiveness compared to controls. Flow cytometric analysis revealed significantly greater total numbers of cells in arsenic-exposed lungs, lower MHCII expression in natural killer cells, and increased percentages of dendritic cell populations. Activated interstitial (IMs) and alveolar macrophages (AMs) isolated from arsenic-exposed male mice produced significantly less IFN-γ than controls. Conversely, activated AMs from arsenic-exposed females produced significantly more IFN-γ than controls. Although systemic cytokine levels were higher upon Mycobacterium tuberculosis (Mtb) infection in prenatally arsenic-exposed offspring there was no difference in lung Mtb burden compared to controls. This study highlights significant long-term impacts of prenatal arsenic exposure on lung and immune cell function. These effects may contribute to the elevated risk of respiratory diseases associated with prenatal arsenic exposure in epidemiology studies and point to the need for more research into mechanisms driving these maintained responses.

Oxidization of optic atrophy 1 cysteines occurs during heart ischemia-reperfusion and amplifies cell death by oxidative stress.

During cardiac ischemia-reperfusion, excess reactive oxygen species can damage mitochondrial, cellular and organ function. Here we show that cysteine oxidation of the mitochondrial protein Opa1 contributes to mitochondrial damage and cell death caused by oxidative stress. Oxy-proteomics of ischemic-reperfused hearts reveal oxidation of the C-terminal C786 of Opa1 and treatment of perfused mouse hearts, adult cardiomyocytes, and fibroblasts with H2O2 leads to the formation of a reduction-sensitive ∼180 KDa Opa1 complex, distinct from the ∼270 KDa one antagonizing cristae remodeling. This Opa1 oxidation process is curtailed by mutation of C786 and of the other 3 Cys residues of its C-terminal domain (Opa1TetraCys). When reintroduced in Opa1-/- cells, Opa1TetraCys is not efficiently processed into short Opa1TetraCys and hence fails to fuse mitochondria. Unexpectedly, Opa1TetraCys restores mitochondrial ultrastructure in Opa1-/- cells and protects them from H2O2-induced mitochondrial depolarization, cristae remodeling, cytochrome c release and cell death. Thus, preventing the Opa1 oxidation occurring during cardiac ischemia-reperfusion reduces mitochondrial damage and cell death induced by oxidative stress independent of mitochondrial fusion.

Cadmium exposure induces a sex-dependent decline in left ventricular cardiac function.

AIMS: Cadmium exposure is a worldwide problem that has been linked to the development of cardiovascular disease. This study aimed to elucidate mechanistic details of chronic cadmium exposure on the structure and function of the heart. MAIN METHODS: Male and female mice were exposed to cadmium chloride (CdCl2) via drinking water for eight weeks. Serial echocardiography and blood pressure measurements were performed. Markers of hypertrophy and fibrosis were assessed, along with molecular targets of Ca2+-handling. KEY FINDINGS: Males exhibited a significant reduction in left ventricular ejection fraction and fractional shortening with CdCl2 exposure, along with increased ventricular volume at end-systole, and decreased interventricular septal thickness at end-systole. Interestingly, no changes were detected in females. Experiments in isolated cardiomyocytes revealed that CdCl2-induced contractile dysfunction was also present at the cellular level, showing decreased Ca2+ transient and sarcomere shortening amplitude with CdCl2 exposure. Further mechanistic investigation uncovered a decrease in sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) protein expression and phosphorylated phospholamban levels in male hearts with CdCl2 exposure. SIGNIFICANCE: The findings of our novel study provide important insight into how cadmium exposure may act as a sex-specific driver of cardiovascular disease, and further underscore the importance of reducing human exposure to cadmium.

Inorganic arsenic induces sex-dependent pathological hypertrophy in the heart.

Arsenic exposure though drinking water is widespread and well associated with adverse cardiovascular outcomes, yet the pathophysiological mechanisms by which iAS induces these effects are largely unknown. Recently, an epidemiological study in an American population with a low burden of cardiovascular risk factors found that iAS exposure was associated with altered left ventricular geometry. Considering the possibility that iAS directly induces cardiac remodeling independently of hypertension, we investigated the impact of an environmentally relevant iAS exposure on the structure and function of male and female hearts. Adult male and female C56BL/6J mice were exposed to 615 µg/L iAS for 8 wk. Males exhibited increased systolic blood pressure via tail cuff photoplethysmography, left ventricular wall thickening via transthoracic echocardiography, and increased plasma atrial natriuretic peptide via enzyme immunoassay. RT-qPCR revealed increased myocardial RNA transcripts of Acta1, Myh7, and Nppa and decreased Myh6, providing evidence of pathological hypertrophy in the male heart. Similar changes were not detected in females, and nitric oxide-dependent mechanisms of cardioprotection in the heart appeared to remain intact. Further investigation found that Rcan1 was upregulated in male hearts and that iAS activated NFAT in HEK-293 cells via luciferase assay. Interestingly, iAS induced similar hypertrophic gene expression changes in neonatal rat ventricular myocytes, which were blocked by calcineurin inhibition, suggesting that iAS may induce pathological cardiac hypertrophy in part by targeting the calcineurin-NFAT pathway. As such, these results highlight iAS exposure as an independent cardiovascular risk factor and provide biological impetus for its removal from human consumption.NEW & NOTEWORTHY This investigation provides the first mechanistic link between an environmentally relevant dose of inorganic arsenic (iAS) and pathological hypertrophy in the heart. By demonstrating that iAS exposure may cause pathological cardiac hypertrophy not only by increasing systolic blood pressure but also by potentially activating calcineurin-nuclear factor of activated T cells and inducing fetal gene expression, these results provide novel mechanistic insight into the theat of iAS exposure to the heart, which is necessary to identify targets for medical and public health intervention.

Nuclear-mitochondrial communication involving miR-181c plays an important role in cardiac dysfunction during obesity.

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, miR-181c, 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.

An emerging perspective on sex differences: Intersecting S-nitrosothiol and aldehyde signaling in the heart.

Cardiovascular disease is the leading cause of the death for both men and women. Although baseline heart physiology and the response to disease are known to differ by sex, little is known about sex differences in baseline molecular signaling, especially with regard to redox biology. In this review, we describe current research on sex differences in cardiac redox biology with a focus on the regulation of nitric oxide and aldehyde signaling. Furthermore, we argue for a new perspective on cardiovascular sex differences research, one that focuses on baseline redox biology without the elimination or disruption of sex hormones.

miR-181c Activates Mitochondrial Calcium Uptake by Regulating MICU1 in the Heart.

Background Translocation of miR-181c into cardiac mitochondria downregulates the mitochondrial gene, mt-COX1. miR-181c/d-/- hearts experience less oxidative stress during ischemia/reperfusion (I/R) and are protected against I/R injury. Additionally, miR-181c overexpression can increase mitochondrial matrix Ca2+ ([Ca2+]m), but the mechanism by which miR-181c regulates [Ca2+]m is unknown. Methods and Results By RNA sequencing and analysis, here we show that hearts from miR-181c/d-/- mice overexpress nuclear-encoded Ca2+ regulatory and metabolic pathway genes, suggesting that alterations in miR-181c and mt-COX1 perturb mitochondria-to-nucleus retrograde signaling and [Ca2+]m regulation. Quantitative polymerase chain reaction validation of transcription factors that are known to initiate retrograde signaling revealed significantly higher Sp1 (specificity protein) expression in the miR-181c/d-/- hearts. Furthermore, an association of Sp1 with the promoter region of MICU1 was confirmed by chromatin immunoprecipitation-quantitative polymerase chain reaction and higher expression of MICU1 was found in the miR-181c/d-/- hearts. Conversely, downregulation of Sp1 by small interfering RNA decreased MICU1 expression in neonatal mouse ventricular myocytes. Changes in PDH activity provided evidence for a change in [Ca2+]m via the miR-181c/MICU1 axis. Moreover, this mechanism was implicated in the pathology of I/R injury. When MICU1 was knocked down in the miR-181c/d-/- heart by lentiviral expression of a short-hairpin RNA against MICU1, cardioprotective effects against I/R injury were abrogated. Furthermore, using an in vitro I/R model in miR-181c/d-/- neonatal mouse ventricular myocytes, we confirmed the contribution of both Sp1 and MICU1 in ischemic injury. Conclusions miR-181c regulates mt-COX1, which in turn regulates MICU1 expression through the Sp1-mediated mitochondria-to-nucleus retrograde pathway. Loss of miR-181c can protect the heart from I/R injury by modulating [Ca2+]m through the upregulation of MICU1.

Unlocking the Secrets of Mitochondria in the Cardiovascular System: Path to a Cure in Heart Failure­A Report from the 2018 National Heart, Lung, and Blood Institute Workshop

Mitochondria have emerged as a central factor in the pathogenesis and progression of heart failure, and other cardiovascular diseases, as well, but no therapies are available to treat mitochondrial dysfunction. The National Heart, Lung, and Blood Institute convened a group of leading experts in heart failure, cardiovascular diseases, and mitochondria research in August 2018. These experts reviewed the current state of science and identified key gaps and opportunities in basic, translational, and clinical research focusing on the potential of mitochondria-based therapeutic strategies in heart failure. The workshop provided short- and long-term recommendations for moving the field toward clinical strategies for the prevention and treatment of heart failure and cardiovascular diseases by using mitochondria-based approaches.

Sustained elevation of MG53 in the bloodstream increases tissue regenerative capacity without compromising metabolic function.

MG53 is a muscle-specific TRIM-family protein that presides over the cell membrane repair response. Here, we show that MG53 present in blood circulation acts as a myokine to facilitate tissue injury-repair and regeneration. Transgenic mice with sustained elevation of MG53 in the bloodstream (tPA-MG53) have a healthier and longer life-span when compared with littermate wild type mice. The tPA-MG53 mice show normal glucose handling and insulin signaling in skeletal muscle, and sustained elevation of MG53 in the bloodstream does not have a deleterious impact on db/db mice. More importantly, the tPA-MG53 mice display remarkable dermal wound healing capacity, enhanced muscle performance, and improved injury-repair and regeneration. Recombinant human MG53 protein protects against eccentric contraction-induced acute and chronic muscle injury in mice. Our findings highlight the myokine function of MG53 in tissue protection and present MG53 as an attractive biological reagent for regenerative medicine without interference with glucose handling in the body.

A knock-in mutation at cysteine 144 of TRIM72 is cardioprotective and reduces myocardial TRIM72 release.

TRIM72 is a membrane repair protein that protects against ischemia reperfusion (I/R) injury. We previously identified Cys144 (C144) on TRIM72 as a site of S-nitrosylation. To study the importance of C144, we generated a knock-in mouse with C144 mutated to a serine (TRIM72 C144S). We subjected ex vivo perfused mouse hearts to 20 min of ischemia followed by 90 min of reperfusion and observed less injury in TRIM72 C144S compared to WT hearts. Infarct size was smaller (54 vs 27% infarct size) and cardiac functional recovery (37 vs 62% RPP) was higher for the TRIM72 C144S mouse hearts. We also demonstrated that TRIM72 C144S hearts were protected against I/R injury using an in vivo LAD occlusion model. As TRIM72 has been reported to be released from muscle we tested whether C144 is involved in TRIM72 release. After I/R there was significantly less TRIM72 in the perfusate normalized to total released protein from the TRIM72 C144S compared to WT hearts, suggesting that C144 of TRIM72 regulates myocardial TRIM72 release during I/R injury. In addition to TRIM72's protective role in I/R injury, TRIM72 has also been implicated in cardiac hypertrophy and insulin resistance, and secreted TRIM72 has recently been shown to impair insulin sensitivity. However, insulin sensitivity (measured by glucose and insulin tolerance) of TRIM72 C144S mice was not impaired. Further, whole body metabolism, as measured using metabolic cages, was not different in WT vs TRIM72 C144S mice and we did not observe enhanced cardiac hypertrophy in the TRIM72 C144S mice. In agreement, protein levels of the TRIM72 ubiquitination targets insulin receptor ß, IRS1, and focal adhesion kinase were similar between WT and TRIM72 C144S hearts. Overall, these data indicate that mutation of TRIM72 C144 is protective during I/R and reduces myocardial TRIM72 release without impairing insulin sensitivity or enhancing the development of hypertrophy.

Inorganic arsenic exposure induces sex-disparate effects and exacerbates ischemia-reperfusion injury in the female heart.

Arsenic is a common contaminant in drinking water throughout the world, and recent studies support a link between inorganic arsenic (iAS) exposure and ischemic heart disease in men and women. Female hearts exhibit an estrogen-dependent reduction in susceptibility to myocardial ischemic injury compared with males, and as such, female hearts may be more susceptible to the endocrine-disrupting effects of iAS exposure. However, iAS exposure and susceptibility to ischemic heart injury have not been examined in mechanistic studies. Male and female mice (8 wk) were exposed to environmentally relevant concentrations of sodium arsenite (0, 10, 100, and 1,000 parts/billion) via drinking water for 4 wk. Pre- and postexposure echocardiography was performed, and postexposure plasma was collected for 17ß-estradiol measurement. Hearts were excised and subjected to ischemia-reperfusion (I/R) injury via Langendorff perfusion. Exposure to 1,000 parts/billion iAS led to sex-disparate effects, such that I/R injury was exacerbated in female hearts but unexpectedly attenuated in males. Assessment of echocardiographic parameters revealed statistically significant structural remodeling in iAS-treated female hearts with no change in function; males showed no change. Plasma 17ß-estradiol levels were not significantly altered by iAS in male or female mice versus nontreated controls. Although total eNOS protein levels did not change in whole heart homogenates from iAS-treated male or female mice, eNOS phosphorylation (Ser1177) was significantly elevated in iAS-treated male hearts. These results suggest that iAS exposure can induce sex-disparate effects and modulate susceptibility to ischemic heart injury by targeting distinct sex-dependent pathways. NEW & NOTEWORTHY This is the first mechanistic study examining iAS exposure on myocardial ischemia-reperfusion injury in male and female mice. Following iAS exposure, ischemia-reperfusion injury was exacerbated in female hearts but attenuated in males. iAS treatment induced statistically significant cardiac remodeling in females, with no change in males. iAS treatment also enhanced phosphorylated eNOS levels at Ser1177, but only in male hearts. These results suggest that iAS alters susceptibility to myocardial I/R injury through distinct sex-dependent pathways.

S-Nitrosoglutathione Reductase Is Essential for Protecting the Female Heart From Ischemia-Reperfusion Injury.

RATIONALE: Protein S-nitros(yl)ation (SNO) has been implicated as an essential mediator of nitric oxide-dependent cardioprotection. Compared with males, female hearts exhibit higher baseline levels of protein SNO and associated with this, reduced susceptibility to myocardial ischemia-reperfusion injury. Female hearts also exhibit enhanced S-nitrosoglutathione reductase (GSNO-R) activity, which would typically favor decreased SNO levels as GSNO-R mediates SNO catabolism. OBJECTIVE: Because female hearts exhibit higher SNO levels, we hypothesized that GSNO-R is an essential component of sex-dependent cardioprotection in females. METHODS AND RESULTS: Male and female wild-type mouse hearts were subjected to ex vivo ischemia-reperfusion injury with or without GSNO-R inhibition (N6022). Control female hearts exhibited enhanced functional recovery and decreased infarct size versus control males. Interestingly, GSNO-R inhibition reversed this sex disparity, significantly reducing injury in male hearts, and exacerbating injury in females. Similar results were obtained with male and female GSNO-R-/- hearts using ex vivo and in vivo models of ischemia-reperfusion injury. Assessment of SNO levels using SNO-resin assisted capture revealed an increase in total SNO levels with GSNO-R inhibition in males, whereas total SNO levels remained unchanged in females. However, we found that although GSNO-R inhibition significantly increased SNO at the cardioprotective Cys39 residue of nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 3 in males, SNO-NADH dehydrogenase subunit 3 levels were surprisingly reduced in N6022-treated female hearts. Because GSNO-R also acts as a formaldehyde dehydrogenase, we examined postischemic formaldehyde levels and found that they were nearly 2-fold higher in N6022-treated female hearts compared with nontreated hearts. Importantly, the mitochondrial aldehyde dehydrogenase 2 activator, Alda-1, rescued the phenotype in GSNO-R-/- female hearts, significantly reducing infarct size. CONCLUSIONS: These striking findings point to GSNO-R as a critical sex-dependent mediator of myocardial protein SNO and formaldehyde levels and further suggest that different therapeutic strategies may be required to combat ischemic heart disease in males and females.

Adenosine A1 receptor activation increases myocardial protein S-nitrosothiols and elicits protection from ischemia-reperfusion injury in male and female hearts.

Nitric oxide (NO) plays an important role in cardioprotection, and recent work from our group and others has implicated protein S-nitrosylation (SNO) as a critical component of NO-mediated protection in different models, including ischemic pre- and post-conditioning and sex-dependent cardioprotection. However, studies have yet to examine whether protein SNO levels are similarly increased with pharmacologic preconditioning in male and female hearts, and whether an increase in protein SNO levels, which is protective in male hearts, is sufficient to increase baseline protection in female hearts. Therefore, we pharmacologically preconditioned male and female hearts with the adenosine A1 receptor agonist N6-cyclohexyl adenosine (CHA). CHA administration prior to ischemia significantly improved functional recovery in both male and female hearts compared to baseline in a Langendorff-perfused heart model of ischemia-reperfusion injury (% of preischemic function ± SE: male baseline: 37.5±3.4% vs. male CHA: 55.3±3.2%; female baseline: 61.4±5.7% vs. female CHA: 76.0±6.2%). In a separate set of hearts, we found that CHA increased p-Akt and p-eNOS levels. We also used SNO-resin-assisted capture with LC-MS/MS to identify SNO proteins in male and female hearts, and determined that CHA perfusion induced a modest increase in protein SNO levels in both male (11.4%) and female (12.3%) hearts compared to baseline. These findings support a potential role for protein SNO in a model of pharmacologic preconditioning, and provide evidence to suggest that a modest increase in protein SNO levels is sufficient to protect both male and female hearts from ischemic injury. In addition, a number of the SNO proteins identified with CHA treatment were also observed with other forms of cardioprotective stimuli in prior studies, further supporting a role for protein SNO in cardioprotection.