Sexual dimorphism in bidirectional SR-mitochondria crosstalk in ventricular cardiomyocytes.

Calcium transfer into the mitochondrial matrix during sarcoplasmic reticulum (SR) Ca2+ release is essential to boost energy production in ventricular cardiomyocytes (VCMs) and match increased metabolic demand. Mitochondria from female hearts exhibit lower mito-[Ca2+] and produce less reactive oxygen species (ROS) compared to males, without change in respiration capacity. We hypothesized that in female VCMs, more efficient electron transport chain (ETC) organization into supercomplexes offsets the deficit in mito-Ca2+ accumulation, thereby reducing ROS production and stress-induced intracellular Ca2+ mishandling. Experiments using mitochondria-targeted biosensors confirmed lower mito-ROS and mito-[Ca2+] in female rat VCMs challenged with ß-adrenergic agonist isoproterenol compared to males. Biochemical studies revealed decreased mitochondria Ca2+ uniporter expression and increased supercomplex assembly in rat and human female ventricular tissues vs male. Importantly, western blot analysis showed higher expression levels of COX7RP, an estrogen-dependent supercomplex assembly factor in female heart tissues vs males. Furthermore, COX7RP was decreased in hearts from aged and ovariectomized female rats. COX7RP overexpression in male VCMs increased mitochondrial supercomplexes, reduced mito-ROS and spontaneous SR Ca2+ release in response to ISO. Conversely, shRNA-mediated knockdown of COX7RP in female VCMs reduced supercomplexes and increased mito-ROS, promoting intracellular Ca2+ mishandling. Compared to males, mitochondria in female VCMs exhibit higher ETC subunit incorporation into supercomplexes, supporting more efficient electron transport. Such organization coupled to lower levels of mito-[Ca2+] limits mito-ROS under stress conditions and lowers propensity to pro-arrhythmic spontaneous SR Ca2+ release. We conclude that sexual dimorphism in mito-Ca2+ handling and ETC organization may contribute to cardioprotection in healthy premenopausal females.

Microfibrillar-Associated Protein 4 Regulates Stress-Induced Cardiac Remodeling.

[Figure: see text].

Protein acetylation in cardiac aging.

Biological aging is attributed to progressive dysfunction in systems governing genetic and metabolic integrity. At the cellular level, aging is evident by accumulated DNA damage and mutation, reactive oxygen species, alternate lipid and protein modifications, alternate gene expression programs, and mitochondrial dysfunction. These effects sum to drive altered tissue morphology and organ dysfunction. Protein-acylation has emerged as a critical mediator of age-dependent changes in these processes. Despite decades of research focus from academia and industry, heart failure remains a leading cause of death in the United States while the 5 year mortality rate for heart failure remains over 40%. Over 90% of heart failure deaths occur in patients over the age of 65 and heart failure is the leading cause of hospitalization in Medicare beneficiaries. In 1931, Cole and Koch discovered age-dependent accumulation of phosphates in skeletal muscle. These and similar findings provided supporting evidence for, now well accepted, theories linking metabolism and aging. Nearly two decades later, age-associated alterations in biochemical molecules were described in the heart. From these small beginnings, the field has grown substantially in recent years. This growing research focus on cardiac aging has, in part, been driven by advances on multiple public health fronts that allow population level clinical presentation of aging related disorders. It is estimated that by 2030, 25% of the worldwide population will be over the age of 65. This review provides an overview of acetylation-dependent regulation of biological processes related to cardiac aging and introduces emerging non-acetyl, acyl-lysine modifications in cardiac function and aging.

DUSP5-mediated inhibition of smooth muscle cell proliferation suppresses pulmonary hypertension and right ventricular hypertrophy.

Pulmonary hypertension (PH) is associated with structural remodeling of pulmonary arteries (PAs) because of excessive proliferation of fibroblasts, endothelial cells, and smooth muscle cells (SMCs). The peptide hormone angiotensin II (ANG II) contributes to pulmonary vascular remodeling, in part, through its ability to trigger extracellular signal-regulated kinase (ERK1/2) activation. Here, we demonstrate that the ERK1/2 phosphatase, dual-specificity phosphatase 5 (DUSP5), functions as a negative regulator of ANG II-mediated SMC proliferation and PH. In contrast to wild-type controls, Dusp5 null mice infused with ANG II developed PH and right ventricular (RV) hypertrophy. PH in Dusp5 null mice was associated with thickening of the medial layer of small PAs, suggesting an in vivo role for DUSP5 as a negative regulator of ANG II-dependent SMC proliferation. Consistent with this, overexpression of DUSP5 blocked ANG II-mediated proliferation of cultured human pulmonary artery SMCs (hPASMCs) derived from patients with idiopathic PH or from failed donor controls. Collectively, the data support a role for DUSP5 as a feedback inhibitor of ANG II-mediated ERK signaling and PASMC proliferation and suggest that disruption of this circuit leads to adverse cardiopulmonary remodeling.NEW & NOTEWORTHY Dual-specificity phosphatases (DUSPs) serve critical roles in the regulation of mitogen-activated protein kinases, but their functions in the cardiovascular system remain poorly defined. Here, we provide evidence that DUSP5, which resides in the nucleus and specifically dephosphorylates extracellular signal-regulated kinase (ERK1/2), blocks pulmonary vascular smooth muscle cell proliferation. In response to angiotensin II infusion, mice lacking DUSP5 develop pulmonary hypertension and right ventricular cardiac hypertrophy. These findings illustrate DUSP5-mediated suppression of ERK signaling in the lungs as a protective mechanism.

Dynamic Chromatin Targeting of BRD4 Stimulates Cardiac Fibroblast Activation.

RATIONALE: Small molecule inhibitors of the acetyl-histone binding protein BRD4 have been shown to block cardiac fibrosis in preclinical models of heart failure (HF). However, since the inhibitors target BRD4 ubiquitously, it is unclear whether this chromatin reader protein functions in cell type-specific manner to control pathological myocardial fibrosis. Furthermore, the molecular mechanisms by which BRD4 stimulates the transcriptional program for cardiac fibrosis remain unknown. OBJECTIVE: We sought to test the hypothesis that BRD4 functions in a cell-autonomous and signal-responsive manner to control activation of cardiac fibroblasts, which are the major extracellular matrix-producing cells of the heart. METHODS AND RESULTS: RNA-sequencing, mass spectrometry, and cell-based assays employing primary adult rat ventricular fibroblasts demonstrated that BRD4 functions as an effector of TGF-ß (transforming growth factor-ß) signaling to stimulate conversion of quiescent cardiac fibroblasts into Periostin (Postn)-positive cells that express high levels of extracellular matrix. These findings were confirmed in vivo through whole-transcriptome analysis of cardiac fibroblasts from mice subjected to transverse aortic constriction and treated with the small molecule BRD4 inhibitor, JQ1. Chromatin immunoprecipitation-sequencing revealed that BRD4 undergoes stimulus-dependent, genome-wide redistribution in cardiac fibroblasts, becoming enriched on a subset of enhancers and super-enhancers, and leading to RNA polymerase II activation and expression of downstream target genes. Employing the Sertad4 (SERTA domain-containing protein 4) locus as a prototype, we demonstrate that dynamic chromatin targeting of BRD4 is controlled, in part, by p38 MAPK (mitogen-activated protein kinase) and provide evidence of a critical function for Sertad4 in TGF-ß-mediated cardiac fibroblast activation. CONCLUSIONS: These findings define BRD4 as a central regulator of the pro-fibrotic cardiac fibroblast phenotype, establish a p38-dependent signaling circuit for epigenetic reprogramming in heart failure, and uncover a novel role for Sertad4. The work provides a mechanistic foundation for the development of BRD4 inhibitors as targeted anti-fibrotic therapies for the heart.

Dietary natural products as epigenetic modifiers in aging-associated inflammation and disease.

Covering: up to 2020Chronic, low-grade inflammation is linked to aging and has been termed "inflammaging". Inflammaging is considered a key contributor to the development of metabolic dysfunction and a broad spectrum of diseases or disorders including declines in brain and heart function. Genome-wide association studies (GWAS) coupled with epigenome-wide association studies (EWAS) have shown the importance of diet in the development of chronic and age-related diseases. Moreover, dietary interventions e.g. caloric restriction can attenuate inflammation to delay and/or prevent these diseases. Common themes in these studies entail the use of phytochemicals (plant-derived compounds) or the production of short chain fatty acids (SCFAs) as epigenetic modifiers of DNA and histone proteins. Epigenetic modifications are dynamically regulated and as such, serve as potential therapeutic targets for the treatment or prevention of age-related disease. In this review, we will focus on the role for natural products that include phytochemicals and short chain fatty acids (SCFAs) as regulators of these epigenetic adaptations. Specifically, we discuss regulators of methylation, acetylation and acylation, in the protection from chronic inflammation driven metabolic dysfunction and deterioration of neurocognitive and cardiac function.

Epigenetics and vascular diseases.

Cardiovascular disease remains the number one cause of death and disability worldwide despite significant improvements in diagnosis, prevention, and early intervention efforts. There is an urgent need for improved understanding of cardiovascular processes responsible for disease development in order to develop more effective therapeutic strategies. Recent knowledge gleaned from the study of epigenetic mechanisms in the vasculature has uncovered new potential targets for intervention. Herein, we provide an overview of epigenetic mechanism, and review recent findings related to epigenetics in vascular diseases, highlighting classical epigenetic mechanism such as DNA methylation and histone modification as well as the newly discovered non-coding RNA mechanisms.

Class I HDACs control a JIP1-dependent pathway for kinesin-microtubule binding in cardiomyocytes.

Class I histone deacetylase (HDAC) inhibitors block hypertrophy and fibrosis of the heart by suppressing pathological signaling and gene expression programs in cardiac myocytes and fibroblasts. The impact of HDAC inhibition in unstressed cardiac cells remains poorly understood. Here, we demonstrate that treatment of cultured cardiomyocytes with small molecule HDAC inhibitors leads to dramatic induction of c-Jun amino-terminal kinase (JNK)-interacting protein-1 (JIP1) mRNA and protein expression. In contrast to prior findings, elevated levels of endogenous JIP1 in cardiomyocytes failed to significantly alter JNK signaling or cardiomyocyte hypertrophy. Instead, HDAC inhibitor-mediated induction of JIP1 was required to stimulate expression of the kinesin heavy chain family member, KIF5A. We provide evidence for an HDAC-dependent regulatory circuit that promotes formation of JIP1:KIF5A:microtubule complexes that regulate intracellular transport of cargo such as autophagosomes. These findings define a novel role for class I HDACs in the control of the JIP1/kinesin axis in cardiomyocytes, and suggest that HDAC inhibitors could be used to alter microtubule transport in the heart.

Overlapping and Divergent Actions of Structurally Distinct Histone Deacetylase Inhibitors in Cardiac Fibroblasts.

Inhibitors of zinc-dependent histone deacetylases (HDACs) profoundly affect cellular function by altering gene expression via changes in nucleosomal histone tail acetylation. Historically, investigators have employed pan-HDAC inhibitors, such as the hydroxamate trichostatin A (TSA), which simultaneously targets members of each of the three zinc-dependent HDAC classes (classes I, II, and IV). More recently, class- and isoform-selective HDAC inhibitors have been developed, providing invaluable chemical biology probes for dissecting the roles of distinct HDACs in the control of various physiologic and pathophysiological processes. For example, the benzamide class I HDAC-selective inhibitor, MGCD0103 [N-(2-aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl] benzamide], was shown to block cardiac fibrosis, a process involving excess extracellular matrix deposition, which often results in heart dysfunction. Here, we compare the mechanisms of action of structurally distinct HDAC inhibitors in isolated primary cardiac fibroblasts, which are the major extracellular matrix-producing cells of the heart. TSA, MGCD0103, and the cyclic peptide class I HDAC inhibitor, apicidin, exhibited a common ability to enhance histone acetylation, and all potently blocked cardiac fibroblast cell cycle progression. In contrast, MGCD0103, but not TSA or apicidin, paradoxically increased expression of a subset of fibrosis-associated genes. Using the cellular thermal shift assay, we provide evidence that the divergent effects of HDAC inhibitors on cardiac fibroblast gene expression relate to differential engagement of HDAC1- and HDAC2-containing complexes. These findings illustrate the importance of employing multiple compounds when pharmacologically assessing HDAC function in a cellular context and during HDAC inhibitor drug development.

Epigenetic regulation of cardiac fibrosis.

Fibrosis is defined as excess deposition of extracellular matrix (ECM), resulting in tissue scarring and organ dysfunction. In the heart, fibrosis may be reparative, replacing areas of myocyte loss with a structural scar following infarction, or reactive, which is triggered in the absence of cell death and involves interstitial ECM deposition in response to long-lasting stress. Interstitial fibrosis can increase the passive stiffness of the myocardium, resulting in impaired relaxation and diastolic dysfunction. Additionally, fibrosis can lead to disruption of electrical conduction in the heart, causing arrhythmias, and can limit myocyte oxygen availability and thus exacerbate myocardial ischemia. Here, we review recent studies that have illustrated key roles for epigenetic events in the control of pro-fibrotic gene expression, and highlight the potential of small molecules that target epigenetic regulators as a means of treating fibrotic cardiac diseases.

Discovery of novel small molecule inhibitors of cardiac hypertrophy using high throughput, high content imaging.

Chronic cardiac hypertrophy is maladaptive and contributes to the pathogenesis of heart failure. The objective of this study was to identify small molecule inhibitors of pathological cardiomyocyte hypertrophy. High content screening was performed with primary neonatal rat ventricular myocytes (NRVMs) cultured on 96-well plates and treated with a library of 3241 distinct small molecules. Non-toxic hit compounds that blocked hypertrophy in response to phenylephrine (PE) and phorbol myristate acetate (PMA) were identified based on their ability to reduce cell size and inhibit expression of atrial natriuretic factor (ANF), which is a biomarker of pathological cardiac hypertrophy. Many of the hit compounds are existing drugs that have not previously been evaluated for benefit in the setting of cardiovascular disease. One such compound, the anti-malarial drug artesunate, blocked left ventricular hypertrophy (LVH) and improved cardiac function in adult mice subjected to transverse aortic constriction (TAC). These findings demonstrate that phenotypic screening with primary cardiomyocytes can be used to discover anti-hypertrophic lead compounds for heart failure drug discovery. Using annotated libraries of compounds with known selectivity profiles, this screening methodology also facilitates chemical biological dissection of signaling networks that control pathological growth of the heart.

Acetyl-lysine erasers and readers in the control of pulmonary hypertension and right ventricular hypertrophy.

Acetylation of lysine residues within nucleosomal histone tails provides a crucial mechanism for epigenetic control of gene expression. Acetyl groups are coupled to lysine residues by histone acetyltransferases (HATs) and removed by histone deacetylases (HDACs), which are also commonly referred to as "writers" and "erasers", respectively. In addition to altering the electrostatic properties of histones, lysine acetylation often creates docking sites for bromodomain-containing "reader" proteins. This review focuses on epigenetic control of pulmonary hypertension (PH) and associated right ventricular (RV) cardiac hypertrophy and failure. Effects of small molecule HDAC inhibitors in pre-clinical models of PH are highlighted. Furthermore, we describe the recently discovered role of bromodomain and extraterminal (BET) reader proteins in the control of cardiac hypertrophy, and provide evidence suggesting that one member of this family, BRD4, contributes to the pathogenesis of RV failure. Together, the data suggest intriguing potential for pharmacological epigenetic therapies for the treatment of PH and right-sided heart failure.

Reversal of severe angioproliferative pulmonary arterial hypertension and right ventricular hypertrophy by combined phosphodiesterase-5 and endothelin receptor inhibition.

BACKGROUND: Patients with pulmonary arterial hypertension (PAH) are treated with vasodilators, including endothelin receptor antagonists (ERAs), phosphodiesterase-5 (PDE-5) inhibitors, soluble guanylyl cyclase activators, and prostacyclin. Despite recent advances in pharmacotherapy for individuals with PAH, morbidity and mortality rates in this patient population remain unacceptably high. Here, we tested the hypothesis that combination therapy with two PAH drugs that target distinct biochemical pathways will provide superior efficacy relative to monotherapy in the rat SU5416 plus hypoxia (SU-Hx) model of severe angioproliferative PAH, which closely mimics the human condition. METHODS: Male Sprague Dawley rats were injected with a single dose of SU5416, which is a VEGF receptor antagonist, and exposed to hypobaric hypoxia for three weeks. Rats were subsequently housed at Denver altitude and treated daily with the PDE-5 inhibitor, tadalafil (TAD), the type A endothelin receptor (ETA) antagonist, ambrisentan (AMB), or a combination of TAD and AMB for four additional weeks. RESULTS: Monotherapy with TAD or AMB led to modest reductions in pulmonary arterial pressure (PAP) and right ventricular (RV) hypertrophy. In contrast, echocardiography and invasive hemodynamic measurements revealed that combined TAD/AMB nearly completely reversed pulmonary hemodynamic impairment, RV hypertrophy, and RV functional deficit in SU-Hx rats. Efficacy of TAD/AMB was associated with dramatic reductions in pulmonary vascular remodeling, including suppression of endothelial cell plexiform lesions, which are common in human PAH. CONCLUSIONS: Combined therapy with two vasodilators that are approved for the treatment of human PAH provides unprecedented efficacy in the rat SU-Hx preclinical model of severe, angioproliferative PAH.

BET acetyl-lysine binding proteins control pathological cardiac hypertrophy.

Cardiac hypertrophy is an independent predictor of adverse outcomes in patients with heart failure, and thus represents an attractive target for novel therapeutic intervention. JQ1, a small molecule inhibitor of bromodomain and extraterminal (BET) acetyl-lysine reader proteins, was identified in a high throughput screen designed to discover novel small molecule regulators of cardiomyocyte hypertrophy. JQ1 dose-dependently blocked agonist-dependent hypertrophy of cultured neonatal rat ventricular myocytes (NRVMs) and reversed the prototypical gene program associated with pathological cardiac hypertrophy. JQ1 also blocked left ventricular hypertrophy (LVH) and improved cardiac function in adult mice subjected to transverse aortic constriction (TAC). The BET family consists of BRD2, BRD3, BRD4 and BRDT. BRD4 protein expression was increased during cardiac hypertrophy, and hypertrophic stimuli promoted recruitment of BRD4 to the transcriptional start site (TSS) of the gene encoding atrial natriuretic factor (ANF). Binding of BRD4 to the ANF TSS was associated with increased phosphorylation of local RNA polymerase II. These findings define a novel function for BET proteins as signal-responsive regulators of cardiac hypertrophy, and suggest that small molecule inhibitors of these epigenetic reader proteins have potential as therapeutics for heart failure.

mt-tRNAs in the polymerase gamma mutant heart.

Introduction: Mice harboring a D257A mutation in the proofreading domain of the mitochondrial DNA polymerase, Polymerase Gamma (POLG), experience severe metabolic dysfunction and display hallmarks of accelerated aging. We previously reported a mitochondrial unfolded protein response (UPTmt) - like (UPRmt-like) gene and protein expression pattern in the right ventricular tissue of POLG mutant mice. Aim: We sought to determine if POLG mutation altered the expression of genes encoded by the mitochondria in a way that might also reduce proteotoxic stress. Methods and Results: The expression of genes encoded by the mitochondrial DNA was interrogated via RNA-seq and northern blot analysis. A striking, location-dependent effect was seen in the expression of mitochondrial-encoded tRNAs in the POLG mutant as assayed by RNA-seq. These expression changes were negatively correlated with the tRNA partner amino acid's amyloidogenic potential. Direct measurement by northern blot was conducted on candidate mt-tRNAs identified from the RNA-seq. This analysis confirmed reduced expression of MT-TY in the POLG mutant but failed to show increased expression of MT-TP, which was dramatically increased in the RNA-seq data. Conclusion: We conclude that reduced expression of amyloid-associated mt-tRNAs is another indication of adaptive response to severe mitochondrial dysfunction in the POLG mutant. Incongruence between RNA-seq and northern blot measurement of MT-TP expression points towards the existence of mt-tRNA post-transcriptional modification regulation in the POLG mutant that alters either polyA capture or cDNA synthesis in RNA-seq library generation. Together, these data suggest that 1) evolution has distributed mt-tRNAs across the circular mitochondrial genome to allow chromosomal location-dependent mt-tRNA regulation (either by expression or PTM) and 2) this regulation is cognizant of the tRNA partner amino acid's amyloidogenic properties.

Estrogen Receptor-ß Agonists Modulate T-Lymphocyte Activation and Ameliorate Left Ventricular Remodeling During Chronic Heart Failure.

BACKGROUND: CD4+ T cells temporally transition from protective to pathological during ischemic heart failure (HF; 8 weeks postmyocardial infarction). Cellular mechanisms mediating this shift are unknown. METHODS: RNA-sequencing of cardiac CD4+ T cells and flow cytometric analysis of immune cells was conducted. RESULTS: RNA-sequencing of CD4+ T cells from the failing hearts of male mice indicated activation of ER (estrogen receptor)-α signaling. Flow cytometric analysis showed that ERα in CD4+ T cells decreases significantly at 3-day postmyocardial infarction but increases during HF. To antagonize ERα, we tested a novel ERß agonist (OSU-ERb-012) to inhibit T cells and blunt left ventricular remodeling. Proliferation assays showed that OSU-ERb-012 dose-dependently inhibited proliferation and proinflammatory cytokine expression in anti-CD3/CD28 stimulated splenic T cells isolated from both the sexes. For in vivo efficacy, 10- to 12-week-old male and ovariectomized female mice were randomized at 4 weeks postmyocardial infarction and treated with either vehicle or drug (60 mg/kg per day; oral). While vehicle-treated HF mice displayed progressive left ventricular dilatation with significantly increased end-systolic and end-diastolic volumes from 4 to 8 weeks postmyocardial infarction, treatment with OSU-ERb-012 significantly blunted these changes and stopped left ventricular remodeling in both the sexes. Reduction in tibia-normalized heart and left ventricular weights, cardiomyocyte hypertrophy and interstitial fibrosis further supported these results. Additionally, OSU-ERb-012 treatment selectively inhibited cardiac, splenic, and circulating CD4+ T cells without affecting other myeloid and lymphoid cells in the HF mice. CONCLUSIONS: Our studies indicate that ERß agonists and OSU-ERb-012, in particular, could be used as selective immunomodulatory drugs to inhibit CD4+ T cells during chronic HF.

Molecular signature of cardiac remodeling associated with Polymerase Gamma mutation.

AIMS: Metabolic function/dysfunction is central to aging biology. This is well illustrated by the Polymerase Gamma (POLG) mutant mouse where a key residue of the mitochondrial DNA polymerase is mutated (D257A), causing loss of mitochondrial DNA stability and dramatically accelerated aging processes. Given known cardiac phenotypes in the POLG mutant, we sought to characterize the course of cardiac dysfunction in the POLG mutant to guide future intervention studies. MATERIALS AND METHODS: Cardiac echocardiography and terminal hemodynamic analyses were used to define the course of dysfunction in the right and left cardiac ventricles in the POLG mutant. We also conducted RNA-seq analysis on cardiac right ventricles to identify mechanisms engaged by severe metabolic dysfunction and compared this analysis to several publically available datasets. KEY FINDINGS: Interesting sex differences were noted as female POLG mutants died earlier than male POLG mutants and LV chamber diameters were impacted earlier in females than males. Moreover, male mutants showed LV wall thinning while female mutant LV walls were thicker. Both males and females displayed significant RV hypertrophy. POLG mutants displayed a gene expression pattern associated with inflammation, fibrosis, and heart failure. Finally, comparative omics analyses of publically available data provide additional mechanistic and therapeutic insights. SIGNIFICANCE: Aging-associated cardiac dysfunction is a growing clinical problem. This work uncovers sex-specific cardiac responses to severe metabolic dysfunction that are reminiscent of patterns seen in human heart failure and provides insights to the molecular mechanisms engaged downstream of severe metabolic dysfunction that warrant further investigation.

A novel protein kinase C target site in protein kinase D is phosphorylated in response to signals for cardiac hypertrophy.

Protein kinase D (PKD) regulates cardiac myocyte growth and contractility through phosphorylation of proteins such as class IIa histone deacetylases (HDACs) and troponin I (TnI). In response to agonists that activate G-protein-coupled receptors (GPCRs), PKD is phosphorylated by protein kinase C (PKC) on two serine residues (Ser-738 and Ser-742 in human PKD1) within an activation loop of the catalytic domain, resulting in stimulation of PKD activity. Here, we identify a novel PKC target site located adjacent to the auto-inhibitory pleckstrin homology (PH) domain in PKD. This site (Ser-412 in human PKD1) is conserved in each of the three PKD family members and is efficiently phosphorylated by multiple PKC isozymes in vitro. Employing a novel anti-phospho-Ser-412-specific antibody, we demonstrate that this site in PKD is rapidly phosphorylated in primary cardiac myocytes exposed to hypertrophic agonists, including norepinephrine (NE) and endothelin-1 (ET-1). Differential sensitivity of this event to pharmacological inhibitors of PKC, and data from in vitro enzymatic assays, suggest a predominant role for PKCδ in the control of PKD Ser-412 phosphorylation. Together, these data suggest a novel, signal-dependent mechanism for controlling PKD function in cardiac myocytes.

Matrix-Degrading Enzyme Expression and Aortic Fibrosis During Continuous-Flow Left Ventricular Mechanical Support.

BACKGROUND: The effects of nonphysiological flow generated by continuous-flow (CF) left ventricular assist devices (LVADs) on the aorta remain poorly understood. OBJECTIVES: The authors sought to quantify indexes of fibrosis and determine the molecular signature of post-CF-LVAD vascular remodeling. METHODS: Paired aortic tissue was collected at CF-LVAD implant and subsequently at transplant from 22 patients. Aortic wall morphometry and fibrillar collagen content (a measure of fibrosis) was quantified. In addition, whole-transcriptome profiling by RNA sequencing and follow-up immunohistochemistry were performed to evaluate CF-LVAD-mediated changes in aortic mRNA and protein expression. RESULTS: The mean age was 52 ± 12 years, with a mean duration of CF-LVAD of 224 ± 193 days (range 45-798 days). There was a significant increase in the thickness of the collagen-rich adventitial layer from 218 ± 110 µm pre-LVAD to 410 ± 209 µm post-LVAD (P < 0.01). Furthermore, there was an increase in intimal and medial mean fibrillar collagen intensity from 22 ± 11 a.u. pre-LVAD to 41 ± 24 a.u. post-LVAD (P < 0.0001). The magnitude of this increase in fibrosis was greater among patients with longer durations of CF-LVAD support. CF-LVAD led to profound down-regulation in expression of extracellular matrix-degrading enzymes, such as matrix metalloproteinase-19 and ADAMTS4, whereas no evidence of fibroblast activation was noted. CONCLUSIONS: There is aortic remodeling and fibrosis after CF-LVAD that correlates with the duration of support. This fibrosis is due, at least in part, to suppression of extracellular matrix-degrading enzyme expression. Further research is needed to examine the contribution of nonphysiological flow patterns on vascular function and whether modulation of pulsatility may improve vascular remodeling and long-term outcomes.