To maintain healthy mitochondrial enzyme content and function, mitochondria possess a complex protein quality control system, which is composed of different endogenous sets of chaperones and proteases. Heat shock protein 60 (HSP60) is one of these mitochondrial molecular chaperones and has been proposed to play a pivotal role in the regulation of protein folding and the prevention of protein aggregation. However, the physiological function of HSP60 in mammalian tissues is not fully understood.Here we generated an inducible cardiac-specific HSP60 knockout mouse model, and demonstrated that HSP60 deletion in adult mouse hearts altered mitochondrial complex activity, mitochondrial membrane potential, and ROS production, and eventually led to dilated cardiomyopathy, heart failure, and lethality. Proteomic analysis was performed in purified control and mutant mitochondria before mutant hearts developed obvious cardiac abnormalities, and revealed a list of mitochondrial-localized proteins that rely on HSP60 (HSP60-dependent) for correctly folding in mitochondria. We also utilized an in vitro system to assess the effects of HSP60 deletion on mitochondrial protein import and protein stability after import, and found that both HSP60-dependent and HSP60-independent mitochondrial proteins could be normally imported in mutant mitochondria. However, the former underwent degradation in mutant mitochondria after import, suggesting that the protein exhibited low stability in mutant mitochondria. Interestingly, the degradation could be almost fully rescued by a non-specific LONP1 and proteasome inhibitor, MG132, in mutant mitochondria. Therefore, our results demonstrated that HSP60 plays an essential role in maintaining normal cardiac morphology and function by regulating mitochondrial protein homeostasis and mitochondrial function.

Background Although cardiovascular disease (CVD) is the primary killer of women in the U.S., women and female animals have traditionally been omitted from research studies. In reports that do include both sexes, significant sexual dimorphisms have been demonstrated in development, presentation and outcome of CVD. However, there is little understanding of the mechanisms underlying these observations. A more thorough understanding of sex-specific cardiovascular differences both at baseline and in disease is required to effectively consider and treat all CVD patients. Methods & Results We analyzed contractility in the whole rat heart, adult rat ventricular myocytes (ARVMs) and myofibrils from both sexes of rats and observed functional sex differences at all levels. Hearts and ARVMs from female rats displayed greater fractional shortening than males, and female ARVMs and myofibrils took longer to relax. To define factors underlying these functional differences, we performed an RNA-sequencing experiment on ARVMs from male and female rats and identified ~600 genes were expressed in a sexually dimorphic manner. Further analysis revealed sex-specific enrichment of signaling pathways and key regulators. At the protein level, female ARVMs exhibited higher PKA activity, consistent with pathway enrichment identified through RNA-seq. Additionally, activating the PKA pathway diminished the contractile sexual dimorphisms previously observed. Conclusions These data support the notion that sex-specific gene expression differences at baseline influence cardiac function, particularly through the PKA pathway, and could potentially be responsible for differences in CVD presentation and outcomes.

Background Endothelial NO synthase plays a central role in regulating vasodilation and blood pressure. Intracellular Ca 2+ mobilization is a critical modulator of endothelial NO synthase function, and increased cytosolic Ca 2+ concentration in endothelial cells is able to induce endothelial NO synthase phosphorylation. Ca 2+ release mediated by 3 subtypes of inositol 1,4,5‐trisphosphate receptors ( IP 3 Rs) from the endoplasmic reticulum and subsequent Ca 2+ entry after endoplasmic reticulum Ca 2+ store depletion has been proposed to be the major pathway to mobilize Ca 2+ in endothelial cells. However, the physiological role of IP 3 Rs in regulating blood pressure remains largely unclear. Methods and Results To investigate the role of endothelial IP 3 Rs in blood pressure regulation, we first generated an inducible endothelial cell–specific IP 3 R1 knockout mouse model and found that deletion of IP 3 R1 in adult endothelial cells did not affect vasodilation and blood pressure. Considering all 3 subtypes of IP 3 Rs are expressed in mouse endothelial cells, we further generated inducible endothelial cell–specific IP 3 R triple knockout mice and found that deletion of all 3 IP 3 R subtypes decreased plasma NO concentration and increased basal blood pressure. Furthermore, IP 3 R deficiency reduced acetylcholine‐induced vasodilation and endothelial NO synthase phosphorylation at Ser1177. Conclusions Our results reveal that IP 3 R‐mediated Ca 2+ release in vascular endothelial cells plays an important role in regulating vasodilation and physiological blood pressure.

Background Biological sex is an important modifier of cardiovascular disease and women generally have better outcomes compared with men. However, the contribution of cardiac fibroblasts (CFs) to this sexual dimorphism is relatively unexplored. Methods and Results Isoproterenol (ISO) was administered to rats as a model for chronic β‐adrenergic receptor (β‐AR)‐mediated cardiovascular disease. ISO‐treated males had higher mortality than females and also developed fibrosis whereas females did not. Gonadectomy did not abrogate this sex difference. To determine the cellular contribution to this phenotype, CFs were studied. CFs from both sexes had increased proliferation in vivo in response to ISO, but CFs from female hearts proliferated more than male cells. In addition, male CFs were significantly more activated to myofibroblasts by ISO. To investigate potential regulatory mechanisms for the sexually dimorphic fibrotic response, β‐AR mRNA and PKA (protein kinase A) activity were measured. In response to ISO treatment, male CFs increased expression of β1‐ and β2‐ARs, whereas expression of both receptors decreased in female CFs. Moreover, ISO‐treated male CFs had higher PKA activity relative to vehicle controls, whereas ISO did not activate PKA in female CFs. Conclusions Chronic in vivo β‐AR stimulation causes fibrosis in male but not female rat hearts. Male CFs are more activated than female CFs, consistent with elevated fibrosis in male rat hearts and may be caused by higher β‐AR expression and PKA activation in male CFs. Taken together, our data suggest that CFs play a substantial role in mediating sex differences observed after cardiac injury.

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