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Background: Diabetic cardiomyopathy (DCM) is a complex multifaceted disease responsible for elevated hospitalization and mortality in patients with diabetes mellitus (DM). DCM patients exhibit subclinical diastolic dysfunction, progression towards systolic impairment, and abnormal electrophysiology. Hypoglycemia events that occur spontaneously or due to excess insulin administration threaten the lives of DM patients – with the increased risk of sudden death. However, the molecular underpinnings of hypoglycemia-aggravated DCM remain to be elucidated. Methods and Results: Here we used the established streptozotocin-induced type 1 diabetic cardiomyopathy (T1 DCM) murine model to investigate how hypoglycemia aggravates DCM progression. We showed that chronic hyper- or hypoglycemic challenges dampened cardiac diastolic function in vivo as well as myocardial contractility and calcium handling in isolated cardiomyocytes. Similar contractile defects were recapitulated using neonatal mouse ventricular myocytes (NMVMs) under glucose fluctuation challenges. Using immunoprecipitation mass spectrometry, we identified and validated that hypoglycemia challenge activates the MEK/ERK and PI3K/Akt pathways which results in Cx43 phosphorylation by Src protein in cardiomyocytes. Cx43 dissociation and accumulation at mitochondrial inner membrane was confirmed both in human and murine cardiomyocytes. To determine causality, we overexpressed a mitochondrial targeting Cx43 (mtCx43) using AAV2. At normal blood glucose levels, mtCx43 overexpression recapitulated cardiomyocytes contractile deficiencies, cardiac diastolic dysfunction as well as aberrant electrophysiology both in vitro as well as in vivo. Conclusions: Hypoglycemia challenges results in the accumulation of mtCx43 through the MEK/ERK/Src and PI3K/Akt/Src pathways. We provide evidence that Cx43 mislocalization is present in diabetes mellitus patient hearts, STZ-induced DCM murine model, and glucose fluctuation challenged NMVMs. Mechanistically, we demonstrated that mtCx43 is responsible for inducing aberrant contraction and disrupts electrophysiology in cardiomyocytes and our results support targeting of mtCx43 in treating DCM. Translational perspective: Severe hypoglycemia drives cardiac dysfunction and aggressive ventricular arrhythmias in patients with DCM that leads to sudden cardiac death. Here we demonstrate that Cx43 mislocalization to mitochondria occurs upon hypoglycemic challenge and mtCx43 accumulation is responsible for cardiac diastolic dysfunction, cardiomyocyte contractile dysfunction, and aberrant electrophysiology in vivo. Our findings give support for therapeutic targeting of MEK/ERK/Src and PI3K/Akt/Src pathways to prevent mtCx43-driven DCM.

A correction to this paper has been published: https://doi.org/10.1038/s41536-020-00112-0.

The G protein-coupled receptor APJ is a promising therapeutic target for heart failure. Constitutive deletion of APJ in the mouse is protective against the hypertrophy-heart failure transition via elimination of ligand-independent, β-arrestin-dependent stretch transduction. However, the cellular origin of this stretch transduction and the details of its interaction with apelin signaling remain unknown. We generated mice with conditional elimination of APJ in the endothelium (APJendo−/−) and myocardium (APJmyo−/−). No baseline difference was observed in left ventricular function in APJendo−/−, APJmyo−/−, or control (APJendo+/+, APJmyo+/+) mice. After exposure to transaortic constriction, APJendo−/− mice displayed decreased left ventricular systolic function and increased wall thickness, whereas APJmyo−/− mice were protected. At the cellular level, carbon fiber stretch of freshly isolated single cardiomyocytes demonstrated decreased contractile responses to stretch in APJ−/− cardiomyocytes compared with APJ+/+ cardiomyocytes. Ca2+ transients did not change with stretch in either APJ−/− or APJ+/+ cardiomyocytes. Application of apelin to APJ+/+ cardiomyocytes resulted in decreased Ca2+ transients. Furthermore, hearts of mice treated with apelin exhibited decreased phosphorylation in cardiac troponin I NH2-terminal residues (Ser22 and Ser23) consistent with increased Ca2+ sensitivity. These data establish that APJ stretch transduction is mediated specifically by myocardial APJ, that APJ is necessary for stretch-induced increases in contractility, and that apelin opposes APJ’s stretch-mediated hypertrophy signaling by lowering Ca2+ transients while maintaining contractility through myofilament Ca2+ sensitization. These findings underscore apelin’s unique potential as a therapeutic agent that can simultaneously support cardiac function and protect against the hypertrophy-heart failure transition. NEW & NOTEWORTHY These data address fundamental gaps in our understanding of apelin-APJ signaling in heart failure by localizing APJ’s ligand-independent stretch sensing to the myocardium, identifying a novel mechanism of apelin-APJ inotropy via myofilament Ca2+ sensitization, and identifying potential mitigating effects of apelin in APJ stretch-induced hypertrophic signaling.

Duchenne Muscular Dystrophy (DMD) is a X-link disease affecting ~1:3500 boys per year and culminating with heart failure in early adulthood. DMD results from >200 possible genetic mutations on dystrophin. The lack of dystrophin disrupts the anchoring of the cell sarcomere to the extracellular matrix (ECM), affecting the cardiac contraction. With disease progression, in an attempt to mitigate the subcellular defects, the tissue stiffness and ECM composition remodel in association with a dilated cardiomyopathy phenotype and fibrosis. Our hypothesis is that disease progression is accelerating because of this remodelling, through a positive feedback loop involving multiple mechanosensing pathways. Here, we use a single-cell assay platform to model the effect of fibrotic remodelling in DMD. This platform allows measuring the force production of single human-derived pluripotent stem cell (hiPSC) cardiomyocytes (CMs). The substrate stiffness can be controlled to match that of healthy (~10kPa) or fibrotic (~35kPa) tissue. In addition, single iPSC-CMs are patterned in an elongated 1:7 aspect ratio using microcontact printing of ECM protein. This enhances their intracellular structural maturity towards a more mature adult phenotype. This renders our in-vitro model more representative of the human pathology and greatly improves our measurements standardization. Our results show that single iPSC-CMs with DMD mutations have a dramatically reduced ability to produce force on stiffer substrates compared to their isogenic control. This loss of contractile function correlates with an increase in reactive oxygen species (ROS) and mitochondria dysfunction, as well as with other markers of stress response and cellular senescence.We are asking how the remodeling of the ECM stiffness and composition is signaled from the outside-in and affects the progression of the disease phenotype. The further development of our platform and approach will allow for more accurate in-vitro modeling of cardiac diseases and greatly increase our understanding of the underlying biophysics of mechanosensing.

Background Empirical data on conditions that increase risk of coronavirus disease 2019 (COVID-19) progression are needed to identify high risk individuals. We performed a comprehensive quantitative assessment of pre-existing clinical phenotypes associated with COVID-19-related hospitalization. Methods Phenome-wide association study (PheWAS) of SARS-CoV-2-positive patients from an integrated health system (Geisinger) with system-level outpatient/inpatient COVID-19 testing capacity and retrospective electronic health record (EHR) data to assess pre-COVID-19 pandemic clinical phenotypes associated with hospital admission (hospitalization). Results Of 12,971 individuals tested for SARS-CoV-2 with sufficient pre-COVID-19 pandemic EHR data at Geisinger, 1604 were SARS-CoV-2 positive and 354 required hospitalization. We identified 21 clinical phenotypes in 5 disease categories meeting phenome-wide significance (P-4), including: six kidney phenotypes, e.g. end stage renal disease or stage 5 CKD (OR = 11.07, p = 1.96x10-8), six cardiovascular phenotypes, e.g. congestive heart failure (OR = 3.8, p = 3.24x10-5), five respiratory phenotypes, e.g. chronic airway obstruction (OR = 2.54, p = 3.71x10-5), and three metabolic phenotypes, e.g. type 2 diabetes (OR = 1.80, p = 7.51x10-5). Additional analyses defining CKD based on estimated glomerular filtration rate, confirmed high risk of hospitalization associated with pre-existing stage 4 CKD (OR 2.90, 95% CI: 1.47, 5.74), stage 5 CKD/dialysis (OR 8.83, 95% CI: 2.76, 28.27), and kidney transplant (OR 14.98, 95% CI: 2.77, 80.8) but not stage 3 CKD (OR 1.03, 95% CI: 0.71, 1.48). Conclusions This study provides quantitative estimates of the contribution of pre-existing clinical phenotypes to COVID-19 hospitalization and highlights kidney disorders as the strongest factors associated with hospitalization in an integrated US healthcare system.

Aims: Diastolic dysfunction (DD) is common among hypertrophic cardiomyopathy (HCM) patients, causing major morbidity and mortality. Yet its cellular mechanisms are not fully understood, and presently, there is no effective treatment. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold great potential for investigating the mechanisms underlying DD and as a platform for drug discovery. Methods and results: In the present study, beating iPSC-CMs were generated from healthy recruits and HCM patients with evident DD. Micropatterned iPSC-CMs from HCM patients showed impaired diastolic function, as evidenced by prolonged relaxation time, retarded relaxation rate, and shortened diastolic sarcomere length. Ratiometric Ca 2+ imaging indicated elevated diastolic [Ca 2+ ] i and abnormal Ca 2+ handling in HCM iPSC-CMs, which were exacerbated by β-adrenergic challenge. Combining Ca 2+ imaging and traction force microscopy (TFM), we observed enhanced myofilament Ca 2+ sensitivity (measured as dF/Δ[Ca 2+ ] i ) in HCM iPSC-CMs. These results indicated that cytosolic diastolic Ca 2+ overload, slowed [Ca 2+ ] i decline and increased myofilament Ca 2+ sensitivity, collectively impair the relaxation of HCM cells. Treating HCM iPSC-CMs with partial blockade of Ca 2+ or late Na + current reset diastolic Ca 2+ homeostasis, restored diastolic function and improved long-term survival, suggesting disturbed Ca 2+ signaling is an important cellular pathological mechanism of DD. Further investigation showed increased expression of L-type calcium channel (LTCC) and transient receptor potential channels (TRPCs) in HCM iPSC-CMs, which likely contribute to diastolic [Ca 2+ ] i overload. Conclusion: In summary, this study recapitulated DD at the single cell level, and revealed novel mechanisms and potential therapeutic targets of DD using HCM iPSC-CMs.

Duchenne muscular dystrophy (DMD) is an incurable X-linked genetic disease that is caused by a mutation in the dystrophin gene and affects one in every 3,600 boys. We previously showed that long telomeres protect mice from the lethal cardiac disease seen in humans with the same genetic defect, dystrophin deficiency. By generating the mdx4cv/mTRG2 mouse model with “humanized” telomere lengths, the devastating dilated cardiomyopathy phenotype seen in patients with DMD was recapitulated. Here, we analyze the degenerative sequelae that culminate in heart failure and death in this mouse model. We report progressive telomere shortening in developing mouse cardiomyocytes after postnatal week 1, a time when the cells are no longer dividing. This proliferation-independent telomere shortening is accompanied by an induction of a DNA damage response, evident by p53 activation and increased expression of its target gene p21 in isolated cardiomyocytes. The consequent repression of Pgc1α/β leads to impaired mitochondrial biogenesis, which, in conjunction with the high demands of contraction, leads to increased oxidative stress and decreased mitochondrial membrane potential. As a result, cardiomyocyte respiration and ATP output are severely compromised. Importantly, treatment with a mitochondrial-specific antioxidant before the onset of cardiac dysfunction rescues the metabolic defects. These findings provide evidence for a link between short telomere length and metabolic compromise in the etiology of dilated cardiomyopathy in DMD and identify a window of opportunity for preventive interventions.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disease that is result of mutations in the dystrophin gene and is the most common myopathic disease in humans with a prevalence of one in every 3500 males. Dystrophin is crucial for the formation of a dystrophin-glycoprotein complex (DGC), which connects the cytoskeleton of a muscle fiber to the surrounding extracellular matrix in both skeletal and cardiac muscles. In the heart, loss of dystrophin leads to increased fibrosis and death in the third decade of life due to dilated cardiomyopathy. A conundrum in studying and developing therapies for DMD has been the lack of a mouse model that fully recapitulates the clinical phenotype, as mice that lack dystrophin (mdx model), unlike patients, exhibit only mild skeletal muscle defects, essentially no cardiac defects and have a relatively normal lifespan. Our lab reasoned that the difference in the manifestation of the disease in mice and humans could be telomere length, as mice have substantially longer telomeres than humans. We created a novel mouse model with shortened telomere lengths (similar to humans) that fully recapitulates the skeletal muscle (Cell. 2010;143:1059-1071; the mdx/mTRKO model) and cardiac muscle phenotype of DMD (Nat Cell Biol. 2013; 15:895-904; dilated cardiomyopathy). Interestingly, we observed a relative 45% reduction in cardiomyocyte telomere length in our mdx/mTRKO animals (3 animals per group, N = 300-400) as well as patient samples (4 DMD patient samples, N = 40-95). Here we present new evidence of mitochondrial dysfunction and telomere dysfunction.

SummaryThe heart is the most common site of congenital defects, and valvuloseptal defects are the most common of the cardiac anomalies seen in the newborn. The process of endothelial-to-mesenchymal transition (EndMT) in the cardiac cushions is a required step during early valve development, and Notch signaling is required for this process. Here we show that Notch activation induces the transcription of both subunits of the soluble guanylyl cyclase (sGC) heterodimer, GUCY1A3 and GUCY1B3, which form the nitric oxide receptor. In parallel, Notch also promotes nitric oxide (NO) production by inducing Activin A, thereby activating a PI3-kinase/Akt pathway to phosphorylate eNOS. We thus show that the activation of sGC by NO through a Notch-dependent autocrine loop is necessary to drive early EndMT in the developing atrioventricular canal (AVC).