Cardiac Fibroblasts Mediate a Sexually Dimorphic Fibrotic Response to ß-Adrenergic Stimulation.

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 ß-adrenergic receptor (ß-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, ß-AR mRNA and PKA (protein kinase A) activity were measured. In response to ISO treatment, male CFs increased expression of ß1- and ß2-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 ß-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 ß-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.

Atypical ALPK2 kinase is not essential for cardiac development and function.

Protein kinases play an integral role in cardiac development, function, and disease. Recent experimental and clinical data have implied that protein kinases belonging to a family of atypical α-protein kinases, including α-protein kinase 2 (ALPK2), are important for regulating cardiac development and maintaining function via regulation of WNT signaling. A recent study in zebrafish reported that loss of ALPK2 leads to severe cardiac defects; however, the relevance of ALPK2 has not been studied in a mammalian animal model. To assess the role of ALPK2 in the mammalian heart, we generated two independent global Alpk2-knockout (Alpk2-gKO) mouse lines, using CRISPR/Cas9 technology. We performed physiological and biochemical analyses of Alpk2-gKO mice to determine the functional, morphological, and molecular consequences of Alpk2 deletion at the organismal level. We found that Alpk2-gKO mice exhibited normal cardiac function and morphology up to one year of age. Moreover, we did not observe altered WNT signaling in neonatal Alpk2-gKO mouse hearts. In conclusion, Alpk2 is dispensable for cardiac development and function in the murine model. Our results suggest that Alpk2 is a rapidly evolving gene that lost its essential cardiac functions in mammals.NEW & NOTEWORTHY Several studies indicated the importance of ALPK2 for cardiac function and development. A recent study in zebrafish report that loss of ALPK2 leads to severe cardiac defects. In contrast, murine Alpk2-gKO models developed in this work display no overt cardiac phenotype. Our results suggest ALPK2, as a rapidly evolving gene, lost its essential cardiac functions in mammals.

Deletion of heat shock protein 60 in adult mouse cardiomyocytes perturbs mitochondrial protein homeostasis and causes heart failure.

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.

The BAG3-dependent and -independent roles of cardiac small heat shock proteins.

Small heat shock proteins (sHSPs) comprise an important protein family that is ubiquitously expressed, is highly conserved among species, and has emerged as a critical regulator of protein folding. While these proteins are functionally important for a variety of tissues, an emerging field of cardiovascular research reveals sHSPs are also extremely important for maintaining normal cardiac function and regulating the cardiac stress response. Notably, numerous mutations in genes encoding sHSPs have been associated with multiple cardiac diseases. sHSPs (HSPB5, HSPB6, and HSPB8) have been described as mediating chaperone functions within the heart by interacting with the cochaperone protein BCL-2-associated anthanogene 3 (BAG3); however, recent reports indicate that sHSPs (HSPB7) can perform other BAG3-independent functions. Here, we summarize the cardiac functions of sHSPs and present the notion that cardiac sHSPs function via BAG3-dependent or -independent pathways.

Inositol 1,4,5-Trisphosphate Receptors in Endothelial Cells Play an Essential Role in Vasodilation and Blood Pressure Regulation.

Background Endothelial NO synthase plays a central role in regulating vasodilation and blood pressure. Intracellular Ca2+ mobilization is a critical modulator of endothelial NO synthase function, and increased cytosolic Ca2+ concentration in endothelial cells is able to induce endothelial NO synthase phosphorylation. Ca2+ release mediated by 3 subtypes of inositol 1,4,5-trisphosphate receptors ( IP 3Rs) from the endoplasmic reticulum and subsequent Ca2+ entry after endoplasmic reticulum Ca2+ store depletion has been proposed to be the major pathway to mobilize Ca2+ in endothelial cells. However, the physiological role of IP 3Rs in regulating blood pressure remains largely unclear. Methods and Results To investigate the role of endothelial IP 3Rs in blood pressure regulation, we first generated an inducible endothelial cell-specific IP 3R1 knockout mouse model and found that deletion of IP 3R1 in adult endothelial cells did not affect vasodilation and blood pressure. Considering all 3 subtypes of IP 3Rs are expressed in mouse endothelial cells, we further generated inducible endothelial cell-specific IP 3R triple knockout mice and found that deletion of all 3 IP 3R subtypes decreased plasma NO concentration and increased basal blood pressure. Furthermore, IP 3R deficiency reduced acetylcholine-induced vasodilation and endothelial NO synthase phosphorylation at Ser1177. Conclusions Our results reveal that IP 3R-mediated Ca2+ release in vascular endothelial cells plays an important role in regulating vasodilation and physiological blood pressure.

Deletion of IP3R1 by Pdgfrb-Cre in mice results in intestinal pseudo-obstruction and lethality.

BACKGROUND: Inositol 1,4,5-trisphosphate receptors (IP3Rs) are a family of intracellular Ca2+ release channels located on the membrane of endoplasmic reticulum, which have been shown to play critical roles in various cellular and physiological functions. However, their function in regulating gastrointestinal (GI) tract motility in vivo remains unknown. Here, we investigated the physiological function of IP3R1 in the GI tract using genetically engineered mouse models. METHODS: Pdgfrb-Cre mice were bred with homozygous Itpr1 floxed (Itpr1f/f) mice to generate conditional IP3R1 knockout (pcR1KO) mice. Cell lineage tracing was used to determine where Pdgfrb-Cre-mediated gene deletion occurred in the GI tract. Isometric tension recording was used to measure the effects of IP3R1 deletion on muscle contraction. RESULTS: In the mouse GI tract, Itpr1 gene deletion by Pdgfrb-Cre occurred in smooth muscle cells, enteric neurons, and interstitial cells of Cajal. pcR1KO mice developed impaired GI motility, with prolonged whole-gut transit time and abdominal distention. pcR1KO mice also exhibited lethality as early as 8 weeks of age and 50% of pcR1KO mice were dead by 40 weeks after birth. The frequency of spontaneous contractions in colonic circular muscles was dramatically decreased and the amplitude of spontaneous contractions was increased in pcR1KO mice. Deletion of IP3R1 in the GI tract also reduced the contractile response to the muscarinic agonist, carbachol, as well as to electrical field stimulation. However, KCl-induced contraction and expression of smooth muscle-specific contractile genes were not significantly altered in pcR1KO mice. CONCLUSIONS: Here, we provided a novel mouse model for impaired GI motility and demonstrated that IP3R1 plays a critical role in regulating physiological function of GI tract in vivo.

Ushering in the cardiac role of Ubiquilin1.

Protein quality control (PQC) mechanisms are essential for maintaining cardiac function, and alterations in this pathway influence multiple forms of heart disease. Since heart disease is the leading cause of death worldwide, understanding how the delicate balance between protein synthesis and degradation is regulated in the heart demands attention. The study by Hu et al. reveals that the extraproteasomal ubiquitin receptor Ubiquilin1 (Ubqln1) plays an important role in cardiac ubiquitination-proteasome coupling, particularly in response to myocardial ischemia/reperfusion injury, thereby suggesting that this may be a new avenue for therapeutics.

Transcriptome and Functional Profile of Cardiac Myocytes Is Influenced by Biological Sex.

BACKGROUND: Although cardiovascular disease is the primary killer of women in the United States, 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 cardiovascular disease. 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 patients with cardiovascular disease. METHODS AND 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 protein kinase A activity, consistent with pathway enrichment identified through RNA sequencing. In addition, activating the protein kinase A 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 protein kinase A pathway, and could potentially be responsible for differences in cardiovascular disease presentation and outcomes.