Hydrogen sulfide protects cardiomyocytes from doxorubicin-induced ferroptosis through the SLC7A11/GSH/GPx4 pathway by Keap1 S-sulfhydration and Nrf2 activation.

Recent studies have demonstrated that ferroptosis, a novel form of nonapoptotic regulated cell death plays an important role in doxorubicin (DOX)-induced cardiotoxicity (DoIC). Hydrogen sulfide (H2S) is emerging as the third important gaseous mediator in cardiovascular system. However, whether H2S has an effect on DOX-induced ferroptosis remains unknown. Here, we found that DOX not only triggered cardiomyocyte ferroptosis but also significantly inhibited the synthesis of endogenous H2S in the murine model of chronic DoIC. Application of NaHS, an H2S donor obviously activated the SLC7A11/GSH/GPx4 antioxidant pathway and thus alleviated DOX-induced ferroptosis and cardiac injury in mice. In contrast, cardiac-specific knockout of cystathionine γ-lyase gene (Cse) in mice (Csef/f/Cre+) to abolish the cardiac synthesis of endogenous H2S evidently exacerbated DOX-induced ferroptosis and cardiac dysfunction. A further suppression of SLC7A11/GSH/GPx4 pathway was obtained in Csef/f/Cre+ mice with DoIC, as compared to Csef/f/Cre- mice with DoIC. The aggravation caused by cardiac-specific Cse deficiency was remarkably rescued by exogenous supplementation of NaHS. Moreover, in DOX-stimulated H9c2 cardiomyocytes, pretreatment with NaHS dose-dependently enhanced the activity of SLC7A11/GSH/GPx4 pathway and subsequently mitigated ferroptosis and mitochondrial impairment. On the contrary, transfection with Cse siRNA in DOX-stimulated H9c2 cardiomyocytes markedly inhibited SLC7A11/GSH/GPx4 pathway, thus leading to aggravated ferroptosis and more damage to mitochondrial structure and function. In addition, the protective effect of NaHS on DOX-induced ferroptosis was closely related to the S-sulfhydrated Keap1, which in turn promoted nuclear translocation of Nrf2 and the transcription of SLC7A11 and GPx4. In conclusion, our findings suggest that H2S may exert protective effect on DoIC by inhibiting DOX-induced ferroptosis via Keap1/Nrf2-dependent SLC7A11/GSH/GPx4 antioxidant pathway.

Intercellular mitochondrial component transfer triggers ischemic cardiac fibrosis.

Myocardial fibrosis is the villain of sudden cardiac death. Myocardial ischemia/reperfusion (MI/R) injury induces cardiomyocyte damage or even death, which in turn stimulates fibroblast activation and fibrosis, but the intercellular communication mechanism remains unknown. Recent studies have shown that small extracellular vesicles (sEVs) significantly contribute to intercellular communication. Whether and how sEV might mediate post-MI/R cardiomyocyte/fibroblasts communication remain unknown. Here, in vivo and in vitro MI/R models were established. We demonstrate that sEVs derived from cardiomyocyte (Myo-sEVs) carry mitochondrial components, which enter fibroblasts to initiate myocardial fibrosis. Based on bioinformatics screening and experimental verification, the activating molecule in Beclin1-regulated autophagy protein 1 (autophagy/beclin-1 regulator 1, Ambra1) was found to be a critical component of these sEV and might be a new marker for Myo-sEVs. Interestingly, release of Ambra1+-Myo-sEVs was caused by secretory rather than canonical autophagy after MI/R injury and thereby escaped degradation. In ischemic and peripheral areas, Ambra1+-Myo-sEVs were internalized by fibroblasts, and the delivered mtDNA components to activate the fibroblast cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway to promote fibroblast activation and proliferation. In addition, our data show that Ambra1 is expressed on the EV surface and cardiac-specific Ambra1 down regulation inhibits the Ambra1+-Myo-sEVs release and fibroblast uptake, effectively inhibiting ischemic myocardial fibrosis. This finding newly provides the evidence that myocardial secretory autophagy plays a role in intercellular communication during cardiac fibrosis. Ambra1 is a newly characterized molecule with bioactivity and might be a marker for Myo-sEVs, providing new therapeutic targets for cardiac remodeling.

The Imbalance of p53-Park7 Signaling Axis Induces Iron Homeostasis Dysfunction in Doxorubicin-Challenged Cardiomyocytes.

Doxorubicin (DOX)-induced cardiotoxicity (DoIC) is a major side effect for cancer patients. Recently, ferroptosis, triggered by iron overload, is demonstrated to play a role in DoIC. How iron homeostasis is dysregulated in DoIC remains to be elucidated. Here, the authors demonstrate that DOX challenge exhibits reduced contractile function and induction of ferroptosis-related phenotype in cardiomyocytes, evidenced by iron overload, lipid peroxide accumulation, and mitochondrial dysfunction. Compared to Ferric ammonium citrate (FAC) induced secondary iron overload, DOX-challenged cardiomyocytes show a dysfunction of iron homeostasis, with decreased cytoplasmic and mitochondrial iron-sulfur (FeS) cluster-mediated aconitase activity and abnormal expression of iron homeostasis-related proteins. Mechanistically, mass spectrometry analysis identified DOX-treatment induces p53-dependent degradation of Parkinsonism associated deglycase (Park7) which results in iron homeostasis dysregulation. Park7 counteracts iron overload by regulating iron regulatory protein family transcription while blocking mitochondrial iron uptake. Knockout of p53 or overexpression of Park7 in cardiomyocytes remarkably restores the activity of FeS cluster and iron homeostasis, inhibits ferroptosis, and rescues cardiac function in DOX treated animals. These results demonstrate that the iron homeostasis plays a key role in DoIC ferroptosis. Targeting of the newly identified p53-Park7 signaling axis may provide a new approach to prevent DoIC.

A highly specific CRISPR-Cas12j nuclease enables allele-specific genome editing.

The CRISPR-Cas system can treat autosomal dominant diseases by nonhomologous end joining (NHEJ) gene disruption of mutant alleles. However, many single-nucleotide mutations cannot be discriminated from wild-type alleles by current CRISPR-Cas systems. Here, we functionally screened six Cas12j nucleases and determined Cas12j-8 as an ideal genome editor with a hypercompact size. Cas12j-8 displayed comparable activity to AsCas12a and Un1Cas12f1. Cas12j-8 is a highly specific nuclease sensitive to single-nucleotide mismatches in the protospacer adjacent motif (PAM)-proximal region. We experimentally proved that Cas12j-8 enabled allele-specific disruption of genes with a single-nucleotide polymorphism (SNP). Cas12j-8 recognizes a simple TTN PAM that provides for high target site density. In silico analysis reveals that Cas12j-8 enables allele-specific disruption of 25,931 clinically relevant variants in the ClinVar database, and 485,130,147 SNPs in the dbSNP database. Therefore, Cas12j-8 would be particularly suitable for therapeutic applications.

p53-Dependent Mitochondrial Compensation in Heart Failure With Preserved Ejection Fraction.

Background Heart failure with preserved ejection fraction (HFpEF) accounts for 50% of patients with heart failure. Clinically, HFpEF prevalence shows age and gender biases. Although the majority of patients with HFpEF are elderly, there is an emergence of young patients with HFpEF. A better understanding of the underlying pathogenic mechanism is urgently needed. Here, we aimed to determine the role of aging in the pathogenesis of HFpEF. Methods and Results HFpEF dietary regimen (high-fat diet + Nω-Nitro-L-arginine methyl ester hydrochloride) was used to induce HFpEF in wild type and telomerase RNA knockout mice (second-generation and third-generation telomerase RNA component knockout), an aging murine model. First, both male and female animals develop HFpEF equally. Second, cardiac wall thickening preceded diastolic dysfunction in all HFpEF animals. Third, accelerated HFpEF onset was observed in second-generation telomerase RNA component knockout (at 6 weeks) and third-generation telomerase RNA component knockout (at 4 weeks) compared with wild type (8 weeks). Fourth, we demonstrate that mitochondrial respiration transitioned from compensatory state (normal basal yet loss of maximal respiratory capacity) to dysfunction (loss of both basal and maximal respiratory capacity) in a p53 dosage dependent manner. Last, using myocardial-specific p53 knockout animals, we demonstrate that loss of p53 activation delays the development of HFpEF. Conclusions Here we demonstrate that p53 activation plays a role in the pathogenesis of HFpEF. We show that short telomere animals exhibit a basal level of p53 activation, mitochondria upregulate mtDNA encoded genes as a mean to compensate for blocked mitochondrial biogenesis, and loss of myocardial p53 delays HFpEF onset in high fat diet + Nω-Nitro-L-arginine methyl ester hydrochloride challenged murine model.

Secretion of miRNA-326-3p by senescent adipose exacerbates myocardial metabolism in diabetic mice.

BACKGROUND: Adipose tissue homeostasis is at the heart of many metabolic syndromes such as diabetes. Previously it has been demonstrated that adipose tissues from diabetic patients are senescent but whether this contributes to diabetic cardiomyopathy (DCM) remains to be elucidated. METHODS: The streptozotocin (STZ) type 1 diabetic mice were established as animal model, and adult mouse ventricular myocytes (AMVMs) isolated by langendorff perfusion as well as neonatal mouse ventricular myocytes (NMVMs) were used as cell models. Senescent associated ß galactosidase (SA-ß-gal) staining and RT-qPCR were used to identify the presence of adipose senescence in diabetic adipose tissue. Senescent adipose were removed either by surgery or by senolytic treatment. Large extracellular vesicles (LEVs) derived from adipose tissue and circulation were separated by ultracentrifugation. Cardiac systolic and diastolic function was evaluated through cardiac ultrasound. Cardiomyocytes contraction function was evaluated by the Ionoptix HTS system and live cell imaging, mitochondrial morphology and functions were evaluated by transmission electron microscope, live cell fluorescent probe and seahorse analysis. RNA-seq for AMVMs and miRNA-seq for LEVs were performed, and bioinformatic analysis combined with RT-qPCR and Western blot were used to elucidate underlying mechanism that senescent adipose derives LEVs exacerbates myocardial metabolism. RESULTS: SA-ß-gal staining and RT-qPCR identified the presence of adipose tissue senescence in STZ mice. Through surgical as well as pharmacological means we show that senescent adipose tissue participates in the pathogenesis of DCM in STZ mice by exacerbates myocardial metabolism through secretion of LEVs. Specifically, expression of miRNA-326-3p was up-regulated in LEVs isolated from senescent adipose tissue, circulation, and cardiomyocytes of STZ mice. Up-regulation of miRNA-326-3p coincided with myocardial transcriptomic changes in metabolism. Functionally, we demonstrate that miRNA-326-3p inhibited the expression of Rictor and resulted in impaired mitochondrial and contractile function in cardiomyocytes. CONCLUSION: We demonstrate for the first time that senescent adipose derived LEVs exacerbates myocardial metabolism through up-regulated miRNA-326-3p which inhibits Rictor in cardiomyocytes. Furthermore, reducing senescence burden in adipose tissue is capable of relieving myocardial metabolism disorder in diabetes mellitus.

TECRL deficiency results in aberrant mitochondrial function in cardiomyocytes.

Sudden cardiac death (SCD) caused by ventricular arrhythmias is the leading cause of mortality of cardiovascular disease. Mutation in TECRL, an endoplasmic reticulum protein, was first reported in catecholaminergic polymorphic ventricular tachycardia during which a patient succumbed to SCD. Using loss- and gain-of-function approaches, we investigated the role of TECRL in murine and human cardiomyocytes. Tecrl (knockout, KO) mouse shows significantly aggravated cardiac dysfunction, evidenced by the decrease of ejection fraction and fractional shortening. Mechanistically, TECRL deficiency impairs mitochondrial respiration, which is characterized by reduced adenosine triphosphate production, increased fatty acid synthase (FAS) and reactive oxygen species production, along with decreased MFN2, p-AKT (Ser473), and NRF2 expressions. Overexpression of TECRL induces mitochondrial respiration, in PI3K/AKT dependent manner. TECRL regulates mitochondrial function mainly through PI3K/AKT signaling and the mitochondrial fusion protein MFN2. Apoptosis inducing factor (AIF) and cytochrome C (Cyc) is released from the mitochondria into the cytoplasm after siTECRL infection, as demonstrated by immunofluorescent staining and western blotting. Herein, we propose a previously unrecognized TECRL mechanism in regulating CPVT and may provide possible support for therapeutic target in CPVT.

Hypoglycemia-Exacerbated Mitochondrial Connexin 43 Accumulation Aggravates Cardiac Dysfunction in Diabetic Cardiomyopathy.

Background: Diabetic cardiomyopathy (DCM) is a complex multifaceted disease responsible for elevated heart failure (HF) morbidity and mortality in patients with diabetes mellitus (DM). Patients with DCM exhibit subclinical diastolic dysfunction, progression toward systolic impairment, and abnormal electrophysiology. Hypoglycemia events that occur spontaneously or due to excess insulin administration threaten the lives of patients with DM-with the increased risk of sudden death. However, the molecular underpinnings of this fatal disease remain to be elucidated. Methods and Results: Here, we used the established streptozotocin-induced DCM murine model to investigate how hypoglycemia aggravates DCM progression. We confirmed connexin 43 (Cx43) dissociation from cell-cell interaction and accumulation at mitochondrial inner membrane both in the cardiomyocytes of patients with DM and DCM murine. Here, we observed that cardiac diastolic function, induced by chronic hyperglycemia, was further aggravated upon hypoglycemia challenge. 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 mitogen-activated protein kinase kinase (MAPK kinase) (MEK)/extracellular regulated protein kinase (ERK) and inhibits phosphoinositide 3-kinase (PI3K)/Akt pathways, which results in Cx43 phosphorylation by Src protein and translocation to mitochondria in cardiomyocytes. To determine causality, we overexpressed a mitochondrial targeting Cx43 (mtCx43) using adeno-associated virus serotype 2 (AAV2)/9. At normal blood glucose levels, mtCx43 overexpression recapitulated cardiac diastolic dysfunction as well as 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. Conclusion: DCM presents compensatory adaptation of mild mtCx43 accumulation, yet acute hypoglycemia challenges result in further accumulation of mtCx43 through the MEK/ERK/Src and PI3K/Akt/Src pathways. We provide evidence that Cx43 mislocalization is present in hearts of patients with DM 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.

Association of Metformin with the Mortality and Incidence of Cardiovascular Events in Patients with Pre-existing Cardiovascular Diseases.

INTRODUCTION: Whether metformin reduces all-cause cardiovascular mortality and the incidence of cardiovascular events in patients with pre-existing cardiovascular diseases (CVD) remains inconclusive. Some randomised controlled trials (RCTs) and cohort studies have shown that metformin is associated with an increased risk of mortality and cardiovascular events. METHODS: We conducted a pooling synthesis to assess the effects of metformin in all-cause cardiovascular mortality and incidence of cardiovascular events in patients with CVD. Studies published up to October 2021 in PubMed or Embase with a registration in PROSPERO (CRD42020189905) were collected. Both RCT and cohort studies were included. Hazard ratios (HR) with 95% CI were pooled across various trials using the random-effects model. RESULTS: This study enrolled 35 published studies (in 14 publications) for qualitative synthesis and identified 33 studies (published in 26 publications) for quantitative analysis. We analysed a total of 61,704 patients, among them 58,271 patients were used to calculate all-cause mortality while 12,814 patients were used to calculate cardiovascular mortality. Compared with non-metformin control, metformin usage is associated with a reduction in all-cause mortality (HR: 0.90; 95% CI 0.83, 0.98; p = 0.01), cardiovascular mortality (HR: 0.89; 95% CI 0.85, 0.94; p < 0.0001), incidence of coronary revascularisation (HR: 0.79; 95% CI 0.64, 0.98; p = 0.03), and heart failure (HR: 0.90; 95% CI 0.87, 0.94; p < 0.0001) in patients with pre-existing cardiovascular diseases. CONCLUSION: Metformin use is associated with a reduction in all-cause mortality, cardiovascular mortality, incidence of coronary revascularisation, and heart failure in patients with CVD; however, metformin usage was not associated with reduction in the incidence of myocardial infarction, angina, or stroke.

Generation of two induced pluripotent stem cell lines, SHIPMi001-A from a patient with hypertrophic cardiomyopathy caused by MYBPC3 gene mutation and SHIPMi002-A from a healthy male individual.

Hypertrophic cardiomyopathy is a hereditary disease with high incidence of sudden death and heart failure. Myosin-binding protein C3 (MYBPC3) is the most commonly mutation gene. Here, we report the establishment of two human induced pluripotent stem cell (iPSC) lines: one from a patient carrying a heterozygous c.1377delC mutation in MYBPC3 (c.1377delC: p.L460Wfs) and one from a healthy donor. The generated iPSC lines showed comparable pluripotent genes, demonstrated the capacity to differentiate into derivatives of all three germ layers and normal karyotypes. These lines are valuable for the mechanism research and drug development of hypertrophic cardiomyopathy.

Silencing of MYH7 ameliorates disease phenotypes in human iPSC-cardiomyocytes.

Allele-specific RNA silencing has been shown to be an effective therapeutic treatment in a number of diseases, including neurodegenerative disorders. Studies of allele-specific silencing in hypertrophic cardiomyopathy (HCM) to date have focused on mouse models of disease. We here examine allele-specific silencing in a human-cell model of HCM. We investigate two methods of silencing, short hairpin RNA (shRNA) and antisense oligonucleotide (ASO) silencing, using a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model. We used cellular micropatterning devices with traction force microscopy and automated video analysis to examine each strategy's effects on contractile defects underlying disease. We find that shRNA silencing ameliorates contractile phenotypes of disease, reducing disease-associated increases in cardiomyocyte velocity, force, and power. We find that ASO silencing, while better able to target and knockdown a specific disease-associated allele, showed more modest improvements in contractile phenotypes. These findings are the first exploration of allele-specific silencing in a human HCM model and provide a foundation for further exploration of silencing as a therapeutic treatment for MYH7-mutation-associated cardiomyopathy.

Pharmacological Silencing of MicroRNA-152 Prevents Pressure Overload-Induced Heart Failure.

BACKGROUND: MicroRNAs are small, noncoding RNAs that play a key role in gene expression. Accumulating evidence suggests that aberrant microRNA expression contributes to the heart failure (HF) phenotype; however, the underlying molecular mechanisms are not well understood. A better understanding of the mechanisms of action of microRNAs could potentially lead to targeted therapies that could halt the progression or even reverse HF. METHODS AND RESULTS: We found that microRNA-152 (miR-152) expression was upregulated in the failing human heart and experimental animal models of HF. Transgenic mice with cardiomyocyte-specific miR-152 overexpression developed systolic dysfunction (mean difference, -38.74% [95% CI, -45.73% to -31.74%]; P<0.001) and dilated cardiomyopathy. At the cellular level, miR-152 overexpression perturbed mitochondrial ultrastructure and dysregulated key genes involved in cardiomyocyte metabolism and inflammation. Mechanistically, we identified Glrx5 (glutaredoxin 5), a critical regulator of mitochondrial iron homeostasis and iron-sulfur cluster synthesis, as a direct miR-152 target. Finally, a proof-of-concept of the therapeutic efficacy of targeting miR-152 in vivo was obtained by utilizing a locked nucleic acid-based inhibitor of miR-152 (LNA 152) in a murine model of HF subjected to transverse aortic constriction. We demonstrated that animals treated with LNA-152 (n=10) showed preservation of systolic function when compared with locked nucleic acid-control treated animals (n=9; mean difference, 18.25% [95% CI, 25.10% to 11.39%]; P<0.001). CONCLUSIONS: The upregulation of miR-152 expression in the failing myocardium contributes to HF pathophysiology. Preclinical evidence suggests that miR-152 inhibition preserves cardiac function in a model of pressure overload-induced HF. These findings offer new insights into the pathophysiology of HF and point to miR-152-Glrx5 axis as a potential novel therapeutic target.

Telomere shortening and metabolic compromise underlie dystrophic cardiomyopathy.

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/ß 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.

RUNX3 maintains the mesenchymal phenotype after termination of the Notch signal.

Notch is a critical mediator of endothelial-to-mesenchymal transition (EndMT) during cardiac cushion development. Slug, a transcriptional repressor that is a Notch target, is an important Notch effector of EndMT in the cardiac cushion. Here, we report that the runt-related transcription factor RUNX3 is a novel direct Notch target in the endothelium. Ectopic expression of RUNX3 in endothelium induces Slug expression and EndMT independent of Notch activation. Interestingly, RUNX3 physically interacts with CSL, the Notch-interacting partner in the nucleus, and induces Slug in a CSL-dependent, but Notch-independent manner. Although RUNX3 may not be required for the initial induction of Slug and EndMT by Notch, because RUNX3 has a much longer half-life than Slug, it sustains the expression of Slug thereby maintaining the mesenchymal phenotype. CSL binds to the Runx3 promoter in the atrioventricular canal in vivo, and inhibition of Notch reduces RUNX3 expression in the cardiac cushion of embryonic hearts. Taken together, our results suggest that induction of RUNX3 may be a mechanism to maintain Notch-transformed mesenchymal cells during heart development.