Search

BackgroundAltered kinase localization is gaining appreciation as a mechanism of cardiovascular disease. Previous work suggests GSK-3β (glycogen synthase kinase 3β) localizes to and regulates contractile function of the myofilament. We aimed to discover GSK-3β's in vivo role in regulating myofilament function, the mechanisms involved, and the translational relevance.MethodsInducible cardiomyocyte-specific GSK-3β knockout mice and left ventricular myocardium from nonfailing and failing human hearts were studied.ResultsSkinned cardiomyocytes from knockout mice failed to exhibit calcium sensitization with stretch indicating a loss of length-dependent activation (LDA), the mechanism underlying the Frank-Starling Law. Titin acts as a length sensor for LDA, and knockout mice had decreased titin stiffness compared with control mice, explaining the lack of LDA. Knockout mice exhibited no changes in titin isoforms, titin phosphorylation, or other thin filament phosphorylation sites known to affect passive tension or LDA. Mass spectrometry identified several z-disc proteins as myofilament phospho-substrates of GSK-3β. Agreeing with the localization of its targets, GSK-3β that is phosphorylated at Y216 binds to the z-disc. We showed pY216 was necessary and sufficient for z-disc binding using adenoviruses for wild-type, Y216F, and Y216E GSK-3β in neonatal rat ventricular cardiomyocytes. One of GSK-3β's z-disc targets, abLIM-1 (actin-binding LIM protein 1), binds to the z-disc domains of titin that are important for maintaining passive tension. Genetic knockdown of abLIM-1 via siRNA in human engineered heart tissues resulted in enhancement of LDA, indicating abLIM-1 may act as a negative regulator that is modulated by GSK-3β. Last, GSK-3β myofilament localization was reduced in left ventricular myocardium from failing human hearts, which correlated with depressed LDA.ConclusionsWe identified a novel mechanism by which GSK-3β localizes to the myofilament to modulate LDA. Importantly, z-disc GSK-3β levels were reduced in patients with heart failure, indicating z-disc localized GSK-3β is a possible therapeutic target to restore the Frank-Starling mechanism in patients with heart failure.

Left ventricular (LV) heart failure (HF) is a significant and increasing cause of death worldwide. HF is characterized by myocardial remodeling and excessive fibrosis. Transcriptional co-activator Yes-associated protein (Yap), the downstream effector of HIPPO signaling pathway, is an essential factor in cardiomyocyte survival; however, its status in human LV HF is not entirely elucidated. Here, we report that Yap is elevated in LV tissue of patients with HF, and is associated with down-regulation of its upstream inhibitor HIPPO component large tumor suppressor 1 (LATS1) activation as well as upregulation of the fibrosis marker connective tissue growth factor (CTGF). Applying the established profibrotic combined stress of TGFβ and hypoxia to human ventricular cardiac fibroblasts in vitro increased Yap protein levels, down-regulated LATS1 activation, increased cell proliferation and collagen I production, and decreased ribosomal protein S6 and S6 kinase phosphorylation, a hallmark of mTOR activation, without any significant effect on mTOR and raptor protein expression or phosphorylation of mTOR or 4E-binding protein 1 (4EBP1), a downstream effector of mTOR pathway. As previously reported in various cell types, TGFβ/hypoxia also enhanced cardiac fibroblast Akt and ERK1/2 phosphorylation, which was similar to our observation in LV tissues from HF patients. Further, depletion of Yap reduced TGFβ/hypoxia-induced cardiac fibroblast proliferation and Akt phosphorylation at Ser 473 and Thr308, without any significant effect on TGFβ/hypoxia-induced ERK1/2 activation or reduction in S6 and S6 kinase activities. Taken together, these data demonstrate that Yap is a mediator that promotes human cardiac fibroblast proliferation and suggest its possible contribution to remodeling of the LV, opening the door to further studies to decipher the cell-specific roles of Yap signaling in human HF.

Heart failure is a leading cause of mortality, yet our understanding of the genetic interactions underlying this disease remains incomplete. Here, we harvest 1352 healthy and failing human hearts directly from transplant center operating rooms, and obtain genome-wide genotyping and gene expression measurements for a subset of 313. We build failing and non-failing cardiac regulatory gene networks, revealing important regulators and cardiac expression quantitative trait loci (eQTLs). PPP1R3A emerges as a regulator whose network connectivity changes significantly between health and disease. RNA sequencing after PPP1R3A knockdown validates network-based predictions, and highlights metabolic pathway regulation associated with increased cardiomyocyte size and perturbed respiratory metabolism. Mice lacking PPP1R3A are protected against pressure-overload heart failure. We present a global gene interaction map of the human heart failure transition, identify previously unreported cardiac eQTLs, and demonstrate the discovery potential of disease-specific networks through the description of PPP1R3A as a central regulator in heart failure.

BackgroundMitochondrial oxidation is a major source of reactive oxygen species (ROS) and mitochondrial dysfunction plays a central role in development of heart failure (HF). Paraoxonase 2 deficient (PON2-def) mitochondria are impaired in function. In this study, we tested whether PON2-def aggravates HF progression.Methods and resultsUsing qPCR, immunoblotting and lactonase activity assay, we demonstrate that PON2 activity was significantly decreased in failing hearts despite increased PON2 expression. To determine the cardiac-specific function of PON2, we performed heart transplantations in which PON2-def and wild type (WT) donor hearts were implanted into WT recipient mice. Beating scores of the donor hearts, assessed at 4 weeks post-transplantation, were significantly decreased in PON2-def hearts when compared to WT donor hearts. By using a transverse aortic constriction (TAC) model, we found PON2 deficiency significantly exacerbated left ventricular remodeling and cardiac fibrosis post-TAC. We further demonstrated PON2 deficiency significantly enhanced ROS generation in heart tissues post-TAC. ROS generation was measured through dihydroethidium (DHE) using high-pressure liquid chromatography (HPLC) with a fluorescent detector. By using neonatal cardiomyocytes treated with CoCl2 to mimic hypoxia, we found PON2 deficiency dramatically increased ROS generation in the cardiomyocytes upon CoCl2 treatment. In response to a short CoCl2 exposure, cell viability and succinate dehydrogenase (SDH) activity assessed by MTT assay were significantly diminished in PON2-def cardiomyocytes compared to those in WT cardiomyocytes. PON2-def cardiomyocytes also had lower baseline SDH activity. By using adult mouse cardiomyocytes and mitochondrial ToxGlo assay, we found impaired cellular ATP generation in PON2-def cells compared to that in WT cells, suggesting that PON2 is necessary for proper mitochondrial function.ConclusionOur study suggests a cardioprotective role for PON2 in both experimental and human heart failure, which may be associated with the ability of PON2 to improve mitochondrial function and diminish ROS generation.

Corticotropin-releasing factor (CRF) and the CRF-related peptides, urocortin (Ucn)-1, Ucn2, and Ucn3 signal through receptors CRFR1 and CRFR2 to restore homeostasis in response to stress. The Ucns exert potent cardioprotective effects and may have clinical utility in heart failure. To explore the activity of this system in the heart, we measured the levels of myocardial gene expression of the CRF/Ucn family of ligands/receptors and investigated genetic variation and alternative splicing of CRFR1 in 110 heart failure patients and 108 heart donors. Using quantitative real-time PCR, we detected CRFR1, CRFR2, CRF, Ucn1, Ucn2, and Ucn3 in all samples. CRFR2α was the most abundant receptor and Ucn3 the most abundant ligand, both in patients and donors. Compared with donors, cardiac expression of CRFR1, CRF, and Ucn3 was higher (P < .001) and CRFR2α lower (P = .012) in patients. In patients and donors, genetic variation within CRFR1, represented by the chromosome 17q21.31 inversion polymorphism, was associated with markedly higher CRFR1 expression (P < .001), making CRFR1 and CRFR2α expression almost equivalent in some patients. A novel, truncated splice variant of CRFR1, designated CRFR1j, was identified and shown to exert a dominant-negative effect on CRFR1 signaling in vitro. The novel variant was expressed in a greater proportion of patients (60%) than donors (3%, P < .001). In summary, cardiac expression of CRFR1, CRF, and Ucn3 genes is elevated in heart failure and may contribute to the activation of the CRF/Ucn system in these patients. A common variant within the CRFR1 gene and a novel CRFR1 splice variant may modulate CRFR1 expression and signaling.

SCN5A encodes the voltage-gated [Na.sup.+] channel [Na.sub.v]1.5 that is responsible for depolarization of the cardiac action potential and rapid intercellular conduction. Mutations disrupting the SCN5A coding sequence cause inherited arrhythmias and cardiomyopathy, and single-nucleotide polymorphisms (SNPs) linked to SCN5A splicing, localization, and function associate with heart failure-related sudden cardiac death. However, the clinical relevance of SNPs that modulate SCN5A expression levels remains understudied. We recently generated a transcriptome-wide map of microRNA (miR) binding sites in human heart, evaluated their overlap with common SNPs, and identified a synonymous SNP (rs1805126) adjacent to a miR-24 site within the SCN5A coding sequence. This SNP was previously shown to reproducibly associate with cardiac electrophysiological parameters, but was not considered to be causal. Here, we show that miR-24 potently suppresses SCN5A expression and that rs1805126 modulates this regulation. We found that the rs1805126 minor allele associates with decreased cardiac SCN5A expression and that heart failure subjects homozygous for the minor allele have decreased ejection fraction and increased mortality, but not increased ventricular tachyarrhythmias. In mice, we identified a potential basis for this in discovering that decreased 5cn5a expression leads to accumulation of myocardial reactive oxygen species. Together, these data reiterate the importance of considering the mechanistic significance of synonymous SNPs as they relate to miRs and disease, and highlight a surprising link between SCN5A expression and nonarrhythmic death in heart failure., Introduction The onset and clinical course of heart failure is shaped by a complex interplay of environmental and genetic factors that influence a host of biological processes, including gene regulatory [...]

It is not clear whether endothelial cell (EC) activation by the hormone angiotensin II (Ang II) modulates contraction of vascular smooth muscle ceils (VSMCs) in the vasculature and whether impairment of this regulation in vivo contributes to hypertension. Delineation of the actions of Ang II through the type 1 receptor (AT1R) on ECs in the blood vessels has been a challenging problem because of the predominance of the AT1R functions in VSMCs that lie underneath the endothelium. We have obviated this limitation by generating transgenic (TG) mice engineered to target expression of the constitutively active N111G mutant AT1R only in ECs. In these TG mice, the enhanced angiotensinergic signal in ECs without infusion of Ang II resulted in hypotension and bradycardia. The pressor response to acute infusion of Ang II was significantly reduced. Increased expression of endothelial nitric oxide synthase and production of hypotensive mediators, nitric oxide and cyclic guanosine monophosphate, cause these phenotypes. Hypotension and bradycardia observed in the TG mice could be rescued by treatment with an AT1R-selective antagonist. Our results imply that the Ang II action by means of EC-AT1R is antagonistic to vasoconstriction in general, and it may moderate the magnitude of functional response to Ang II in VSMCs. This control mechanism in vivo most likely is a determinant of altered hemodynamic regulation involved in endothelial dysfunction in hypertensive cardiovascular disease. angiotensin II receptor | endothelial nitric oxide synthase | hypertension | nitric oxide | angiotensin receptor blockers

Spontaneously hypertensive rats (SHR) are used to evaluate the myofilament calcium sensitivity-associated alteration in troponin-1 (Tn-1) phosphorylation and the expression of troponin isoforms during the evolution to decompensated cardiac hypertrophy. Results indicate that Tn-1 phosphorylation upon beta adrenergic stimulation of myocytes decreased among SHRs. The implications of these findings are discussed.