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Background The neonatal heart maintains its entire regeneration capacity within days after birth. Using quantitative phosphoproteomics technology, we identified that SGK3 (serine/threonine‐protein kinase 3) in the neonatal heart is highly expressed and activated after myocardial infarction. This study aimed to uncover the function and related mechanisms of SGK3 on cardiomyocyte proliferation and cardiac repair after apical resection or ischemia/reperfusion injury. Methods and Results The effect of SGK3 on proliferation and oxygen glucose deprivation/reoxygenation– induced apoptosis in isolated cardiomyocytes was evaluated using cardiomyocyte‐specific SGK3 overexpression or knockdown adenovirus5 vector. In vivo, gain‐ and loss‐of‐function experiments using cardiomyocyte‐specific adeno‐associated virus 9 were performed to determine the effect of SGK3 in cardiomyocyte proliferation and cardiac repair after apical resection or ischemia/reperfusion injury. In vitro, overexpression of SGK3 enhanced, whereas knockdown of SGK3 decreased, the cardiomyocyte proliferation ratio. In vivo, inhibiting the expression of SGK3 shortened the time window of cardiac regeneration after apical resection in neonatal mice, and overexpression of SGK3 significantly promoted myocardial repair and cardiac function recovery after ischemia/reperfusion injury in adult mice. Mechanistically, SGK3 promoted cardiomyocyte regeneration and myocardial repair after cardiac injury by inhibiting GSK‐3β (glycogen synthase kinase‐3β) activity and upregulating β‐catenin expression. SGK3 also upregulated the expression of cell cycle promoting genes G1/S‐specific cyclin‐D1, c‐myc (cellular‐myelocytomatosis viral oncogene), and cdc20 (cell division cycle 20), but downregulated the expression of cell cycle negative regulators cyclin kinase inhibitor P 21 and cyclin kinase inhibitor P 27. Conclusions Our study reveals a key role of SGK3 on cardiac repair after apical resection or ischemia/reperfusion injury, which may reopen a novel therapeutic option for myocardial infarction.

Background Apoptosis plays a pivotal role in cardiac rupture after myocardial infarction (MI), and p53 is a key molecule in apoptosis during cardiac rupture. Hif‐1α (hypoxia‐inducible factor‐1α), upregulated under hypoxia, is a known p53 inducer. However, the role of Hif‐1α in the regulatory mechanisms underlying p53 upregulation, apoptosis, and cardiac rupture after MI is unclear. Methods and Results We induced MI in mice by ligating the left anterior descending artery. Hif‐1α and p53 expressions were upregulated in the border zone at day 5 after MI, accompanied by apoptosis. In rat neonatal cardiomyocytes, treatment with cobalt chloride (500 μmol/L), which mimics severe hypoxia by inhibiting PHD (prolyl hydroxylase domain‐containing protein), increased Hif‐1α and p53, accompanied by myocyte death with caspase‐3 cleavage. Silencing Hif‐1α or p53 inhibited caspase‐3 cleavage, and completely prevented myocyte death under PHD inhibition. In cardiac‐specific Hif‐1α hetero‐knockout mice, expression of p53 and cleavage of caspase‐3 and poly (ADP‐ribose) polymerase were reduced, and apoptosis was suppressed on day 5. Furthermore, the cleavage of caspase‐8 and IL‐1β (interleukin‐1β) was also suppressed in hetero knockout mice, accompanied by reduced macrophage infiltration and matrix metalloproteinase/tissue inhibitor of metalloproteinase activation. Although there was no intergroup difference in infarct size, the cardiac rupture and survival rates were significantly improved in the hetero knockout mice until day 10 after MI. Conclusions Hif‐1α plays a pivotal role in apoptosis, inflammation, and cardiac rupture after MI, in which p53 is a critical mediator, and may be a prospective therapeutic target for preventing cardiac rupture.

Background Current right ventricular (RV) volume overload (VO) is established in adult mice. There are no neonatal mouse VO models and how VO affects postnatal RV development is largely unknown. Methods and Results Neonatal VO was induced by the fistula between abdominal aorta and inferior vena cava on postnatal day 7 and confirmed by abdominal ultrasound, echocardiography, and hematoxylin and eosin staining. The RNA‐sequencing results showed that the top 5 most enriched gene ontology terms in normal RV development were energy derivation by oxidation of organic compounds, generation of precursor metabolites and energy, cellular respiration, striated muscle tissue development, and muscle organ development. Under the influence of VO, the top 5 most enriched gene ontology terms were angiogenesis, regulation of cytoskeleton organization, regulation of vasculature development, regulation of mitotic cell cycle, and regulation of the actin filament‐based process. The top 3 enriched signaling pathways for the normal RV development were PPAR signaling pathway, citrate cycle (Tricarboxylic acid cycle), and fatty acid degradation. VO changed the signaling pathways to focal adhesion, the PI3K‐Akt signaling pathway, and pathways in cancer. The RNA sequencing results were confirmed by the examination of the markers of metabolic and cardiac muscle maturation and the markers of cell cycle and angiogenesis. Conclusions A neonatal mouse VO model was successfully established, and the main processes of postnatal RV development were metabolic and cardiac muscle maturation, and VO changed that to angiogenesis and cell cycle regulation.

Background Sarcomere gene mutations lead to cardiomyocyte hypertrophy and pathological myocardial remodeling. However, there is considerable phenotypic heterogeneity at both the cellular and the organ level, suggesting modifiers regulate the effects of these mutations. We hypothesized that sarcomere dysfunction leads to cardiomyocyte genotoxic stress, and this modifies pathological ventricular remodeling. Methods and Results Using a murine model deficient in the sarcomere protein, Mybpc3 −/− (cardiac myosin‐binding protein 3), we discovered that there was a surge in cardiomyocyte nuclear DNA damage during the earliest stages of cardiomyopathy. This was accompanied by a selective increase in ataxia telangiectasia and rad3‐related phosphorylation and increased p53 protein accumulation. The cause of the DNA damage and DNA damage pathway activation was dysregulated cardiomyocyte DNA synthesis, leading to replication stress. We discovered that selective inhibition of ataxia telangiectasia and rad3 related or cardiomyocyte deletion of p53 reduced pathological left ventricular remodeling and cardiomyocyte hypertrophy in Mybpc3 −/− animals. Mice and humans harboring other types of sarcomere gene mutations also had evidence of activation of the replication stress response, and this was associated with cardiomyocyte aneuploidy in all models studied. Conclusions Collectively, our results show that sarcomere mutations lead to activation of the cardiomyocyte replication stress response, which modifies pathological myocardial remodeling in sarcomeric cardiomyopathy.

Background In complex congenital heart disease patients such as those with tetralogy of Fallot, the right ventricle (RV) is subject to pressure overload, leading to RV hypertrophy and eventually RV failure. The mechanisms that promote the transition from stable RV hypertrophy to RV failure are unknown. We evaluated the role of mitochondrial bioenergetics in the development of RV failure. Methods and Results We created a murine model of RV pressure overload by pulmonary artery banding and compared with sham‐operated controls. Gene expression by RNA‐sequencing, oxidative stress, mitochondrial respiration, dynamics, and structure were assessed in pressure overload‐induced RV failure. RV failure was characterized by decreased expression of electron transport chain genes and mitochondrial antioxidant genes (aldehyde dehydrogenase 2 and superoxide dismutase 2) and increased expression of oxidant stress markers (heme oxygenase, 4‐hydroxynonenal). The activities of all electron transport chain complexes decreased with RV hypertrophy and further with RV failure (oxidative phosphorylation: sham 552.3±43.07 versus RV hypertrophy 334.3±30.65 versus RV failure 165.4±36.72 pmol/(s×mL), P Conclusions Pressure overload‐induced RV failure is characterized by decreased transcription and activity of electron transport chain complexes and increased oxidative stress which are associated with decreased energy generation. An improved understanding of the complex processes of energy generation could aid in developing novel therapies to mitigate mitochondrial dysfunction and delay the onset of RV failure.

Background The overall goal of this longitudinal study was to determine if the Black population has decreased myocardial function, which has the potential to lead to the early development of congestive heart failure, compared with the White population. Methods and Results A total of 673 subjects were evaluated over a period of 30 years including similar percentages of Black and White participants. Left ventricular systolic function was probed using the midwall fractional shortening (MFS). A longitudinal analysis of the MFS using a mixed effect growth curve model was performed. Black participants had greater body mass index, higher blood pressure readings, and greater left ventricular mass compared with White participants (all P Conclusions Changes in myocardial function mirror the race‐dependent variations in blood pressure, afterload, and cardiac mass, suggesting that myocardial function depression occurs early in childhood in populations at high cardiovascular risk such as Black participants.

Background The mutated α‐B‐Crystallin (CryAB R120G ) mouse model of desmin‐related myopathy (DRM) shows an age‐dependent onset of pathologic cardiac remodeling and progression of heart failure. CryAB R120G expression in cardiomyocytes affects the mitochondrial spatial organization within the myofibrils, but the molecular perturbation within the mitochondria in the relation of the overall course of the proteotoxic disease remains unclear. Methods and Results CryAB R120G mice show an accumulation of electron‐dense aggregates and myofibrillar degeneration associated with the development of cardiac dysfunction. Though extensive studies demonstrated that these altered ultrastructural changes cause cardiac contractility impairment, the molecular mechanism of cardiomyocyte death remains elusive. Here, we explore early pathological processes within the mitochondria contributing to the contractile dysfunction and determine the pathogenic basis for the heart failure observed in the CryAB R120G mice. In the present study, we report that the CryAB R120G mice transgenic hearts undergo altered mitochondrial dynamics associated with increased level of dynamin‐related protein 1 and decreased level of optic atrophy type 1 as well as mitofusin 1 over the disease process. In association with these changes, an altered level of the components of mitochondrial oxidative phosphorylation and pyruvate dehydrogenase complex regulatory proteins occurs before the manifestation of pathologic adverse remodeling in the CryAB R120G hearts. Mitochondria isolated from CryAB R120G transgenic hearts without visible pathology show decreased electron transport chain complex activities and mitochondrial respiration. Taken together, we demonstrated the involvement of mitochondria in the pathologic remodeling and progression of DRM‐associated cellular dysfunction. Conclusions Mitochondrial dysfunction in the form of altered mitochondrial dynamics, oxidative phosphorylation and pyruvate dehydrogenase complex proteins level, abnormal electron transport chain complex activities, and mitochondrial respiration are evident on the CryAB R120G hearts before the onset of detectable pathologies and development of cardiac contractile dysfunction.

BACKGROUND The novel severe acute respiratory syndrome coronavirus 2 threatens human health, and the mortality rate is higher in patients who develop myocardial damage. However, the possible risk factors for myocardial damage in patients with coronavirus disease 2019 (COVID‐19) are not fully known. METHODS AND RESULTS Critical type patients were selected randomly from 204 confirmed COVID‐19 cases occurring in Renmin Hospital of Wuhan University from February 1, 2020 to February 24, 2020. Univariate analyses were used to compare the 2 groups: the myocardial damage group and the non–myocardial damage group. A total of 82 critical patients with COVID‐19 were recruited: 34 with myocardial damage and 48 without myocardial damage. A total of 30 patients died in the myocardial damage group, and 20 died in the non–myocardial damage group. In univariate analysis, the proportion of elderly patients (>70 years old, 70.59% versus 37.50%; P =0.003) and patients with cardiovascular disease (41.18% versus 12.50%; P =0.003) was higher among myocardial damage patients than among non–myocardial damage patients. Multivariate analysis showed that age >70 years old (hazard ratio [HR], 2.44; 95% CI, 1.01–5.40), CRP (C‐reactive protein) >100 mg/L (HR, 1.92; 95% CI, 0.94–3.92), lactate dehydrogenase >300 U/L (HR, 2.67; 95% CI, 1.03–6.90), and lactic acid >3 mmol/L (HR, 3.25; 95% CI, 1.57–6.75) were independent risk factors for myocardial damage in patients with COVID‐19. CONCLUSIONS Old age (>70 years old), CRP >100 mg/L, lactate dehydrogenase >300 U/L, and lactic acid >3 mmol/L are high‐risk factors related to myocardial damage in critical patients with COVID‐19.

Background Trimethylamine N‐oxide (TMAO) may have prothrombotic properties. We examined the association of TMAO quartiles with major adverse cardiovascular events (MACE) and the effect of TMAO on the efficacy of ticagrelor. Methods and Results PEGASUS‐TIMI 54 (Prevention of Cardiovascular Events in Patients With Prior Heart Attack Using Ticagrelor Compared to Placebo on a Background of Aspirin ‐ Thrombolysis in Myocardial Infarction 54) randomized patients with prior myocardial infarction to ticagrelor or placebo (median follow‐up 33 months). Baseline plasma concentrations of TMAO were measured in a nested case‐control study of 597 cases with cardiovascular death, myocardial infarction, or stroke (MACE) and 1206 controls matched for age, sex, and estimated glomerular filtration rate [eGFR]. Odds ratios (OR) were used for the association between TMAO quartiles and MACE, adjusting for baseline clinical characteristics (age, sex, eGFR, region, body mass index, hypertension, hypercholesterolemia, diabetes mellitus, smoking, peripheral artery disease, index event, aspirin dosage and treatment arm), and cardiovascular biomarkers (hs‐TnT [high‐sensitivity troponin T], hs‐CRP [high‐sensitivity C‐reactive protein], NT‐proBNP [N‐terminal‐pro‐B‐type natriuretic peptide]). Higher TMAO quartiles were associated with risk of MACE (OR for quartile 4 versus quartile 1, 1.43, 95% CI, 1.06–1.93, P trend=0.015). The association was driven by cardiovascular death (OR 2.25, 95% CI, 1.28–3.96, P trend=0.003) and stroke (OR 2.68, 95% CI, 1.39–5.17, P trend

Background Pregnancy complications such as preterm birth and fetal growth restriction are associated with altered prenatal and postnatal cardiac development. We studied whether there were changes related specifically to pregnancy hypertension. Methods and Results Left and right ventricular volumes, mass, and function were assessed at birth and 3 months of age by echocardiography in 134 term‐born infants. Fifty‐four had been born to mothers who had normotensive pregnancy and 80 had a diagnosis of preeclampsia or pregnancy‐induced hypertension. Differences between groups were interpreted, taking into account severity of pregnancy disorder, sex, body size, and blood pressure. Left and right ventricular mass indexed to body surface area (LVMI and RVMI) were similar in both groups at birth (LVMI 20.9±3.7 versus 20.6±4.0 g/m2, P=0.64, RVMI 17.5±3.7 versus 18.1±4.7 g/m2, P=0.57). However, right ventricular end diastolic volume index was significantly smaller in those born to hypertensive pregnancy (16.8±5.3 versus 12.7±4.7 mL/m2, P=0.001), persisting at 3 months of age (16.4±3.2 versus 14.4±4.8 mL/m2, P=0.04). By 3 months of age these infants also had significantly greater LVMI and RVMI (LVMI 24.9±4.6 versus 26.8±4.9 g/m2, P=0.04; RVMI 17.1±4.2 versus 21.1±3.9 g/m2, P

Background Few reports have addressed the mechanism by which microRNA miR‐10b‐5p regulates post–myocardial infarction (post‐ MI ) cardiomyocyte apoptosis under hypoxic conditions. Methods and Results C57 BL /6 mice underwent surgical ligation of the left anterior descending artery to create an MI or ischemia/reperfusion animal model. The expression of miR‐10b‐5p, PTEN (phosphatase and tensin homolog), and HIF‐1α (hypoxia‐inducible factor 1α) was detected in infarct border zone tissues at various time points. After precordial injections of the negative control or miR‐10b‐5p, overexpression lentiviruses were made in the areas surrounding the MI sites at 1 week, and myocardial infarct size, cardiac function, and cardiomyocyte apoptosis were examined. A miR‐10b‐5p mimic was transfected into primary mouse cardiomyocytes to analyze its effects on cardiomyocyte apoptosis and PTEN expression. Meanwhile, PTEN as a target of miR‐10b‐5p was verified via luciferase reporter gene assays. Cotransfection of miR‐10b‐5 and PTEN verified the relationship between miR‐10b‐5 and PTEN . Under hypoxic stress, the expression of HIF ‐1α and miR‐10b‐5p was examined. The results showed that miR‐10b‐5p expression was markedly reduced in the infarct border zone. Overexpression of miR‐10b‐5p in the murine model of MI significantly reduced MI size, improved cardiac function, and inhibited apoptosis. Overexpression of miR‐10b‐5p in vitro antagonized hypoxia‐induced cardiomyocyte apoptosis and specifically inhibited the expression of the apoptosis‐related gene PTEN , but overexpression of PTEN weakened these effects. We also found that hypoxia‐induced accumulation of HIF ‐1α resulted in decreased expression of miR‐10b‐5p. Interfering with the activation of the HIF ‐1α signaling pathway promoted Pri‐miR‐10b and miR‐10b‐5p expression and inhibited PTEN expression. Conclusions MicroRNA miR‐10b‐5p antagonizes hypoxia‐induced cardiomyocyte apoptosis, indicating that miR‐10b‐5p may serve as a potential future clinical target for the treatment of MI .

Background Myocardial infarction results in a large‐scale cardiomyocyte loss and heart failure due to subsequent pathological remodeling. Whereas zebrafish and neonatal mice have evident cardiomyocyte expansion following injury, adult mammalian cardiomyocytes are principally nonproliferative. Despite historical presumptions of stem cell–mediated cardiac regeneration, numerous recent studies using advanced lineage‐tracing methods demonstrated that the only source of cardiomyocyte renewal originates from the extant myocardium; thus, the augmented proliferation of preexisting adult cardiomyocytes remains a leading therapeutic approach toward cardiac regeneration. In the present study we investigate the significance of suppressing cell cycle inhibitors Rb1 and Meis2 to promote adult cardiomyocyte reentry to the cell cycle. Methods and Results In vitro experiments with small interfering RNA –mediated simultaneous knockdown of Rb1 and Meis2 in both adult rat cardiomyocytes, isolated from 12‐week‐old Fischer rats, and human induced pluripotent stem cell–derived cardiomyocytes showed a significant increase in cell number, a decrease in cell size, and an increase in mononucleated cardiomyocytes. In vivo, a hydrogel‐based delivery method for small interfering RNA –mediated silencing of Rb1 and Meis2 is utilized following myocardial infarction. Immunofluorescent imaging analysis revealed a significant increase in proliferation markers 5‐ethynyl‐2′‐deoxyuridine, PH 3, KI 67, and Aurora B in adult cardiomyocytes as well as improved cell survivability with the additional benefit of enhanced peri‐infarct angiogenesis. Together, this intervention resulted in a reduced infarct size and improved cardiac function post–myocardial infarction. Conclusions Silencing of senescence‐inducing pathways in adult cardiomyocytes via inhibition of Rb1 and Meis2 results in marked cardiomyocyte proliferation and increased protection of cardiac function in the setting of ischemic injury.

Background An increase in serum cortisol has been identified as a risk factor for cardiac failure, which highlights the impact of glucocorticoid signaling in cardiomyocytes and its influence in the progression of failure. Dexamethasone, a synthetic glucocorticoid, is sufficient for induction of cardiomyocyte hypertrophy, but little is known of the glucocorticoid receptor (GR) genome‐binding and ‐dependent transcriptional changes that mediate this phenotype. Methods and Results In this study using high‐resolution sequencing, we identified genomic targets of GR and associated change in the transcriptome after 1 and 24 hours of dexamethasone treatment. We showed that GR associates with 6482 genes in the cardiac genome, with differential regulation of 738 genes. Interestingly, alignment of the chromatin immunoprecipitation and RNA sequencing data show that, after 1 hour, 69% of differentially regulated genes are associated with GR and identify as regulators of RNA pol II–dependent transcription. Conversely, after 24 hours only 45% of regulated genes are associated with GR and involved in dilated and hypertrophic cardiomyopathies as well as other growth‐related pathways. In addition, our data also reveal that a majority of genes (76.42%) associated with GR show incremental changes in transcript abundance and are genes involved in basic cellular processes that might be regulated by the dynamics of promoter‐paused RNA pol II, as seen in hearts undergoing hypertrophy. In vivo administration of dexamethasone resulted in similar changes in the cardiac transcriptome, as seen in isolated cardiomyocytes. Conclusions Our data reveal genome‐wide GR binding sites in cardiomyocytes, identify novel targets and GR‐dependent change in the transcriptome that induces and contributes to cardiomyocyte hypertrophy.

Background Mutations in αB‐crystallin result in proteotoxic cardiomyopathy with desmin mislocalization to protein aggregates. Intermittent fasting (IF) is a novel approach to activate transcription factor EB (TFEB), a master regulator of the autophagy‐lysosomal pathway, in the myocardium. We tested whether TFEB activation can be harnessed to treat advanced proteotoxic cardiomyopathy. Methods and Results Mice overexpressing the R120G mutant of αB‐crystallin in cardiomyocytes (Myh6‐CryABR120G) were subjected to IF or ad‐lib feeding, or transduced with adeno‐associated virus–TFEB or adeno‐associated virus—green fluorescent protein after development of advanced proteotoxic cardiomyopathy. Adeno‐associated virus–short hairpin RNA–mediated knockdown of TFEB and HSPB8 was performed simultaneously with IF. Myh6‐CryABR120G mice demonstrated impaired autophagic flux, reduced lysosome abundance, and mammalian target of rapamycin activation in the myocardium. IF resulted in mammalian target of rapamycin inhibition and nuclear translocation of TFEB with restored lysosome abundance and autophagic flux; and reduced aggregates with normalized desmin localization. IF also attenuated left ventricular dilation and myocardial hypertrophy, increased percentage fractional shortening, and increased survival. Adeno‐associated virus–TFEB transduction was sufficient to rescue cardiomyopathic manifestations, and resulted in reduced aggregates and normalized desmin localization in Myh6‐CryABR120G mice. CryABR120G‐expressing hearts demonstrated increased interaction of desmin with αB‐crystallin and reduced interaction with chaperone protein, HSPB8, compared with wild type, which was reversed by both IF and TFEB transduction. TFEB stimulated autophagic flux to remove protein aggregates and transcriptionally upregulated HSPB8, to restore normal desmin localization in CryABR120G‐expressing cardiomyocytes. Short hairpin RNA–mediated knockdown of TFEB and HSPB8 abrogated IF effects, in vivo. Conclusions IF and TFEB activation are clinically relevant therapeutic strategies to rescue advanced R120G αB‐crystallin mutant‐induced cardiomyopathy by normalizing desmin localization via autophagy‐dependent and autophagy‐independent mechanisms., See Editorial by Mukai et al

Background Particulate matter (particles < 2.5 μm [PM 2.5]) exposure during the in utero and postnatal developmental periods causes cardiac dysfunction during adulthood. Here, we investigated the potential priming effects of preconception exposure of PM 2.5 on cardiac function in adult offspring. Methods and Results Male and female friend leukemia virus b (FVB) mice were exposed to either filtered air (FA) or PM 2.5 at an average concentration of 38.58 μg/m3 for 6 hours/day, 5 days/week for 3 months. Mice were then crossbred into 2 groups: (1) FA male×FA female (both parents were exposed to FA preconception) and, (2) PM 2.5male×PM 2.5female (both parents were exposed to PM 2.5 preconception). Male offspring were divided: (1) preconception FA (offspring born to FA exposed parents) and, (2) preconception PM 2.5 (offspring born to PM 2.5 exposed parents) and analyzed at 3 months of age. Echocardiography identified increased left ventricular end systolic volume and reduced posterior wall thickness, reduced %fractional shortening and %ejection fraction in preconception PM 2.5 offspring. Cardiomyocytes isolated from preconception PM 2.5 offspring showed reduced %peak shortening, −dL/dT, TPS90 and slower calcium reuptake (tau). Gene and protein expression revealed modifications in markers of inflammation (IL‐6, IL‐15, TNFα, NFқB, CRP, CD26E, CD26P, intercellular adhesion molecule 1, and monocyte chemoattractant protein‐1) profibrosis (collagen type III alpha 1 chain), oxidative stress (NOS2), antioxidants (Nrf2, SOD, catalase), Ca2+ regulatory proteins (SERCA2a, p‐PLN, NCX), and epigenetic regulators (Dnmt1, Dnmt3a, Dnmt3b, Sirt1, and Sirt2) in preconception PM 2.5 offspring. Conclusions Preconception exposure to PM 2.5 results in global cardiac dysfunction in adult offspring, suggesting that abnormalities during development are not limited to the prenatal or postnatal periods but can also be determined before conception., See Editorial by Stapleton

Background The Sigma 1 receptor (Sigmar1) functions as an interorganelle signaling molecule and elicits cytoprotective functions. The presence of Sigmar1 in the heart was first reported on the basis of a ligand‐binding assay, and all studies to date have been limited to pharmacological approaches using less‐selective ligands for Sigmar1. However, the physiological function of cardiac Sigmar1 remains unknown. We investigated the physiological function of Sigmar1 in regulating cardiac hemodynamics using the Sigmar1 knockout mouse (Sigmar1 −/− ). Methods and Results Sigmar1 −/− hearts at 3 to 4 months of age showed significantly increased contractility as assessed by left ventricular catheterization with stimulation by increasing doses of a β 1 ‐adrenoceptor agonist. Noninvasive echocardiographic measurements were also used to measure cardiac function over time, and the data showed the development of cardiac contractile dysfunction in Sigmar1 −/− hearts as the animals aged. Histochemistry demonstrated significant cardiac fibrosis, collagen deposition, and increased periostin in the Sigmar1 −/− hearts compared with wild‐type hearts. Ultrastructural analysis of Sigmar1 −/− cardiomyocytes revealed an irregularly shaped, highly fused mitochondrial network with abnormal cristae. Mitochondrial size was larger in Sigmar1 −/− hearts, resulting in decreased numbers of mitochondria per microscopic field. In addition, Sigmar1 −/− hearts showed altered expression of mitochondrial dynamics regulatory proteins. Real‐time oxygen consumption rates in isolated mitochondria showed reduced respiratory function in Sigmar1 −/− hearts compared with wild‐type hearts. Conclusions We demonstrate a potential function of Sigmar1 in regulating normal mitochondrial organization and size in the heart. Sigmar1 loss of function led to mitochondrial dysfunction, abnormal mitochondrial architecture, and adverse cardiac remodeling, culminating in cardiac contractile dysfunction.

Background The healthy heart has a dynamic capacity to respond and adapt to changes in nutrient availability. Diabetes mellitus disrupts this metabolic flexibility and promotes cardiomyopathy through mechanisms that are not completely understood. Phosphofructokinase 2 (PFK‐2) is a primary regulator of cardiac glycolysis and substrate selection, yet its regulation under normal and pathological conditions is unknown. This study was undertaken to determine how changes in insulin signaling affect PFK‐2 content, activity, and cardiac metabolism. Methods and Results Streptozotocin‐induced diabetes mellitus, high‐fat diet feeding, and fasted mice were used to identify how decreased insulin signaling affects PFK‐2 and cardiac metabolism. Primary adult cardiomyocytes were used to define the mechanisms that regulate PFK‐2 degradation. Both type 1 diabetes mellitus and a high‐fat diet induced a significant decrease in cardiac PFK‐2 protein content without affecting its transcript levels. Overnight fasting also induced a decrease in PFK‐2, suggesting it is rapidly degraded in the absence of insulin signaling. An unbiased metabolomic study demonstrated that decreased PFK‐2 in fasted animals is accompanied by an increase in glycolytic intermediates upstream of phosphofructokianse‐1, whereas those downstream are diminished. Mechanistic studies using cardiomyocytes showed that, in the absence of insulin signaling, PFK‐2 is rapidly degraded via both proteasomal‐ and chaperone‐mediated autophagy. Conclusions The loss of PFK‐2 content as a result of reduced insulin signaling impairs the capacity to dynamically regulate glycolysis and elevates the levels of early glycolytic intermediates. Although this may be beneficial in the fasted state to conserve systemic glucose, it represents a pathological impairment in diabetes mellitus.

Background Guanylyl cyclase, a heme‐containing α1β1 heterodimer ( GC 1), produces cGMP in response to Nitric oxide ( NO ) stimulation. The NO ‐ GC1 ‐ cGMP pathway negatively regulates cardiomyocyte contractility and protects against cardiac hypertrophy–related remodeling. We recently reported that the β1 subunit of GC 1 is detected at the intercalated disc with connexin 43 (Cx43). Cx43 forms gap junctions ( GJs ) at the intercalated disc that are responsible for electrical propagation. We sought to determine whether there is a functional association between GC 1 and Cx43 and its role in cardiac homeostasis. Methods and Results GC 1 and Cx43 immunostaining at the intercalated disc and coimmunoprecipitation from membrane fraction indicate that GC 1 and Cx43 are associated. Mice lacking the α subunit of GC 1 ( GC α1 knockout mice) displayed a significant decrease in GJ function (dye‐spread assay) and Cx43 membrane lateralization. In a cardiac‐hypertrophic model, angiotensin II treatment disrupted the GC1‐Cx43 association and induced significant Cx43 membrane lateralization, which was exacerbated in GC α1 knockout mice. Cx43 lateralization correlated with decreased Cx43‐containing GJs at the intercalated disc, predictors of electrical dysfunction. Accordingly, an ECG revealed that angiotensin II –treated GC α1 knockout mice had impaired ventricular electrical propagation. The phosphorylation level of Cx43 at serine 365, a protein‐kinase A upregulated site involved in trafficking/assembly of GJs , was decreased in these models. Conclusions GC 1 modulates ventricular Cx43 location, hence GJ function, and partially protects from electrical dysfunction in an angiotensin II hypertrophy model. Disruption of the NO ‐ cGMP pathway is associated with cardiac electrical disturbance and abnormal Cx43 phosphorylation. This previously unknown NO /Cx43 signaling could be a protective mechanism against stress‐induced arrhythmia.

Background Cardiac fibrosis occurs because of disruption of the extracellular matrix network leading to myocardial dysfunction. Angiotensin II has been implicated in the development of cardiac fibrosis. Recently, micro RNA s have been identified as an attractive target for therapeutic intervention in cardiac pathologies; however, the underlying mechanism of micro RNA s in cardiac fibrosis remains unclear. Micro RNA ‐130a (miR‐130a) has been shown to participate in angiogenesis and cardiac arrhythmia; however, its role in cardiac fibrosis is unknown. Methods and Results In this study, we found that miR‐130a was significantly upregulated in angiotensin II ‐infused mice. The in vivo inhibition of miR‐130a by locked nucleic acid– based anti‐miR‐130a in mice significantly reduced angiotensin II ‐induced cardiac fibrosis. Upregulation of miR‐130a was confirmed in failing human hearts. Overexpressing miR‐130a in cardiac fibroblasts promoted profibrotic gene expression and myofibroblasts differentiation, and the inhibition of miR‐130a reversed the processes. Using the constitutive and dominant negative constructs of peroxisome proliferator‐activated receptor γ 3‐′untranslated region (UTR), data revealed that the protective mechanism was associated with restoration of peroxisome proliferator‐activated receptor γ level leading to the inhibition of angiotensin II ‐induced cardiac fibrosis. Conclusions Our findings provide evidence that miR‐130a plays a critical role in cardiac fibrosis by directly targeting peroxisome proliferator‐activated receptor γ. We conclude that inhibition of miR‐130a would be a promising strategy for the treatment of cardiac fibrosis.

Background Cardiomyocytes differentiated from human pluripotent stem cells can serve as an unexhausted source for a cellular cardiac disease model. Although small molecule–mediated cardiomyocyte differentiation methods have been established, the differentiation efficiency is relatively unsatisfactory in multiple lines due to line‐to‐line variation. Additionally, hurdles including line‐specific low expression of endogenous growth factors and the high apoptotic tendency of human pluripotent stem cells also need to be overcome to establish robust and efficient cardiomyocyte differentiation. Methods and Results We used the H9–human cardiac troponin T–e GFP reporter cell line to screen for small molecules that promote cardiac differentiation in a monolayer‐based and growth factor–free differentiation model. We found that collaterally treating human pluripotent stem cells with rapamycin and CHIR 99021 during the initial stage was essential for efficient and reliable cardiomyocyte differentiation. Moreover, this method maintained consistency in efficiency across different human embryonic stem cell and human induced pluripotent stem cell lines without specifically optimizing multiple parameters (the efficiency in H7, H9, and UQ 1 human induced pluripotent stem cells is 98.3%, 93.3%, and 90.6%, respectively). This combination also increased the yield of cardiomyocytes (1:24) and at the same time reduced medium consumption by about 50% when compared with the previous protocols. Further analysis indicated that inhibition of the mammalian target of rapamycin allows efficient cardiomyocyte differentiation through overcoming p53‐dependent apoptosis of human pluripotent stem cells during high‐density monolayer culture via blunting p53 translation and mitochondrial reactive oxygen species production. Conclusions We have demonstrated that mammalian target of rapamycin exerts a stage‐specific and multifaceted regulation over cardiac differentiation and provides an optimized approach for generating large numbers of functional cardiomyocytes for disease modeling and in vitro drug screening.

The immune system plays a pivotal role in myocardial homeostasis and response to injury. Interleukins-4 and -13 are anti-inflammatory type-2 cytokines, signaling via the common interleukin-13 receptor α1 chain and the type-2 interleukin-4 receptor. The role of interleukin-13 receptor α1 in the heart is unknown.We analyzed myocardial samples from human donors (n=136) and patients with end-stage heart failure (n=177). We found that the interleukin-13 receptor α1 is present in the myocardium and, together with the complementary type-2 interleukin-4 receptor chainThe results of our studies in humans and mice indicate, for the first time, a role of interleukin-13 receptor α1 in myocardial homeostasis and heart failure and suggests a new therapeutic target to treat heart disease.