Transcriptomic Signatures of End-Stage Human Dilated Cardiomyopathy Hearts with and without Left Ventricular Assist Device Support.

Left ventricular assist device (LVAD) use in patients with dilated cardiomyopathy (DCM) can lead to a differential response in the LV and right ventricle (RV), and RV failure remains the most common complication post-LVAD insertion. We assessed transcriptomic signatures in end-stage DCM, and evaluated changes in gene expression (mRNA) and regulation (microRNA/miRNA) following LVAD. LV and RV free-wall tissues were collected from end-stage DCM hearts with (n = 8) and without LVAD (n = 8). Non-failing control tissues were collected from donated hearts (n = 6). Gene expression (for mRNAs/miRNAs) was determined using microarrays. Our results demonstrate that immune response, oxygen homeostasis, and cellular physiological processes were the most enriched pathways among differentially expressed genes in both ventricles of end-stage DCM hearts. LV genes involved in circadian rhythm, muscle contraction, cellular hypertrophy, and extracellular matrix (ECM) remodelling were differentially expressed. In the RV, genes related to the apelin signalling pathway were affected. Following LVAD use, immune response genes improved in both ventricles; oxygen homeostasis and ECM remodelling genes improved in the LV and, four miRNAs normalized. We conclude that LVAD reduced the expression and induced additional transcriptomic changes of various mRNAs and miRNAs as an integral component of the reverse ventricular remodelling in a chamber-specific manner.

Structural Valve Deterioration Is Linked to Increased Immune Infiltrate and Chemokine Expression.

We aim to investigate whether structural valve deterioration (SVD) of bioprosthetic xenogenic tissue heart valves (XTHVs) is associated with increased immune cell infiltration and whether co-expression of several chemokines correlates with this increase in immune infiltrate. Explanted XTHVs from patients undergoing redo valve replacement for SVD were obtained. Immunohistochemical, microscopic, and gene expression analysis approaches were used. XTHVs (n = 37) were obtained from 32 patients (mean 67.7 years) after a mean time of 11.6 years post-implantation. Significantly increased immune cellular infiltration was observed in the explanted SVD valves for all immune cell types examined, including T cells, macrophages, B cells, neutrophils, and plasma cells, compared to non-SVD controls. Furthermore, a significantly increased chemokine gradient in explanted SVD valves accompanied immune cell infiltration. These data suggest the development of SVD is associated with a significantly increased burden of immune cellular infiltrate correlated to the induction of a chemokine gradient around the XHTV, representing chronic immune rejection.Graphical abstract Proposed interaction between innate and adaptive immunity leading to the development of structural valve deterioration in xenogenic tissue heart valves.

Elevated Angiotensin 1-7/Angiotensin II Ratio Predicts Favorable Outcomes in Patients With Heart Failure.

BACKGROUND: ACE2 (angiotensin-converting enzyme 2) and Ang 1-7 (angiotensin 1-7) are endogenous negative regulators of the renin-angiotensin system exerting cardioprotective effects in models of heart failure. Recombinant human ACE2 markedly increased plasma Ang 1-7 and lowered Ang II levels in phase II clinical trials. We hypothesize that the dynamic state of this renin-angiotensin system protective arm could influence long-term outcomes in patients with heart failure. METHODS: One hundred ten patients with heart failure were prospectively enrolled from our outpatient clinic and the emergency department. Comprehensive circulating and equilibrium levels of plasma angiotensin peptide profiles were assessed using novel liquid chromatography-mass spectrometry/mass spectroscopy techniques. Plasma aldosterone, B-type natriuretic peptide, active renin concentration, and clinical profiles were captured at baseline. During a median follow-up of 5.1 years (interquartile range, 4.7-5.7 years), composite clinical outcomes were assessed using all-cause in-patient hospitalizations and mortality. RESULTS: Circulating and equilibrium angiotensin peptide levels strongly correlated in our patient cohort. Adjusting for covariates, elevated equilibrium (hazard ratio, 0.38 [95% CI, 0.18-0.81] P=0.012), and circulating (hazard ratio, 0.38 [95% CI, 0.18-0.80] P=0.011) Ang 1-7/Ang II ratios were associated with improved survival. Lower hospitalization duration was also associated with elevated equilibrium (P<0.001) and circulating (P=0.023) Ang 1-7/Ang II ratios. Importantly, individual Ang 1-7 and Ang II peptide levels failed to predict all-cause mortality or hospitalization duration in our patient cohort. CONCLUSIONS: We extensively profiled plasma angiotensin peptides in patients with heart failure and identified elevated Ang 1-7/Ang II ratio, as an independent and incremental predictor of beneficial outcomes, higher survival rate, and decreased hospitalization duration. These findings provide important clinical evidence supporting strategies aiming to promote the beneficial Ang 1-7/Mas axis concurrent with renin-angiotensin system blockade therapies inhibiting the detrimental Ang II/AT1 receptor axis.

Apelin protects against abdominal aortic aneurysm and the therapeutic role of neutral endopeptidase resistant apelin analogs.

Abdominal aortic aneurysm (AAA) remains the second most frequent vascular disease with high mortality but has no approved medical therapy. We investigated the direct role of apelin (APLN) in AAA and identified a unique approach to enhance APLN action as a therapeutic intervention for this disease. Loss of APLN potentiated angiotensin II (Ang II)-induced AAA formation, aortic rupture, and reduced survival. Formation of AAA was driven by increased smooth muscle cell (SMC) apoptosis and oxidative stress in Apln-/y aorta and in APLN-deficient cultured murine and human aortic SMCs. Ang II-induced myogenic response and hypertension were greater in Apln-/y mice, however, an equivalent hypertension induced by phenylephrine, an α-adrenergic agonist, did not cause AAA or rupture in Apln-/y mice. We further identified Ang converting enzyme 2 (ACE2), the major negative regulator of the renin-Ang system (RAS), as an important target of APLN action in the vasculature. Using a combination of genetic, pharmacological, and modeling approaches, we identified neutral endopeptidase (NEP) that is up-regulated in human AAA tissue as a major enzyme that metabolizes and inactivates APLN-17 peptide. We designed and synthesized a potent APLN-17 analog, APLN-NMeLeu9-A2, that is resistant to NEP cleavage. This stable APLN analog ameliorated Ang II-mediated adverse aortic remodeling and AAA formation in an established model of AAA, high-fat diet (HFD) in Ldlr-/- mice. Our findings define a critical role of APLN in AAA formation through induction of ACE2 and protection of vascular SMCs, whereas stable APLN analogs provide an effective therapy for vascular diseases.

PI3Kα Pathway Inhibition With Doxorubicin Treatment Results in Distinct Biventricular Atrophy and Remodeling With Right Ventricular Dysfunction.

Background Cancer therapies inhibiting PI 3Kα (phosphoinositide 3-kinase-α)-dependent growth factor signaling, including trastuzumab inhibition of HER 2 (Human Epidermal Growth Factor Receptor 2), can cause adverse effects on the heart. Direct inhibition of PI 3Kα is now in clinical trials, but the effects of PI 3Kα pathway inhibition on heart atrophy, remodeling, and function in the context of cancer therapy are not well understood. Method and Results Pharmacological PI 3Kα inhibition and heart-specific genetic deletion of p110α, the catalytic subunit of PI 3Kα, was characterized in conjunction with anthracycline (doxorubicin) treatment in female murine models. Biventricular changes in heart morphological characteristics and function were analyzed, with molecular characterization of signaling pathways. Both PI 3Kα inhibition and anthracycline therapy promoted heart atrophy and a combined effect of distinct right ventricular dilation, dysfunction, and cardiomyocyte remodeling in the absence of pulmonary arterial hypertension. Congruent findings of right ventricular dilation and dysfunction were seen with pharmacological and genetic suppression of PI 3Kα signaling when combined with doxorubicin treatment. Increased p38 mitogen-activated protein kinase activation was mechanistically linked to heart atrophy and correlated with right ventricular dysfunction in explanted failing human hearts. Conclusions PI 3Kα pathway inhibition promotes heart atrophy in mice. The right ventricle is specifically at risk for dilation and dysfunction in the setting of PI 3K inhibition in conjunction with chemotherapy. Inhibition of p38 mitogen-activated protein kinase is a proposed therapeutic target to minimize this mode of cardiotoxicity.

PI3Kα-regulated gelsolin activity is a critical determinant of cardiac cytoskeletal remodeling and heart disease.

Biomechanical stress and cytoskeletal remodeling are key determinants of cellular homeostasis and tissue responses to mechanical stimuli and injury. Here we document the increased activity of gelsolin, an actin filament severing and capping protein, in failing human hearts. Deletion of gelsolin prevents biomechanical stress-induced adverse cytoskeletal remodeling and heart failure in mice. We show that phosphatidylinositol (3,4,5)-triphosphate (PIP3) lipid suppresses gelsolin actin-severing and capping activities. Accordingly, loss of PI3Kα, the key PIP3-producing enzyme in the heart, increases gelsolin-mediated actin-severing activities in the myocardium in vivo, resulting in dilated cardiomyopathy in response to pressure-overload. Mechanical stretching of adult PI3Kα-deficient cardiomyocytes disrupts the actin cytoskeleton, which is prevented by reconstituting cells with PIP3. The actin severing and capping activities of recombinant gelsolin are effectively suppressed by PIP3. Our data identify the role of gelsolin-driven cytoskeletal remodeling in heart failure in which PI3Kα/PIP3 act as negative regulators of gelsolin activity.

TIMP3 deficiency exacerbates iron overload-mediated cardiomyopathy and liver disease.

Chronic iron overload results in heart and liver diseases and is a common cause of morbidity and mortality in patients with genetic hemochromatosis and secondary iron overload. We investigated the role of tissue inhibitor of metalloproteinase 3 (TIMP3) in iron overload-mediated tissue injury by subjecting male mice lacking Timp3 ( Timp3-/-) and wild-type (WT) mice to 12 wk of chronic iron overload. Whereas WT mice with iron overload developed diastolic dysfunction, iron-overloaded Timp3-/- mice showed worsened cardiac dysfunction coupled with systolic dysfunction. In the heart, loss of Timp3 was associated with increased myocardial fibrosis, greater Timp1, matrix metalloproteinase ( Mmp) 2, and Mmp9 expression, increased active MMP-2 levels, and gelatinase activity. Iron overload in Timp3-/- mice showed twofold higher iron accumulation in the liver compared with WT mice because of constituently lower levels of ferroportin. Loss of Timp3 enhanced the hepatic inflammatory response to iron overload, leading to greater neutrophil and macrophage infiltration and increased hepatic fibrosis. Expression of inflammation-related MMPs (MMP-12 and MMP-13) and inflammatory cytokines (IL-1ß and monocyte chemoattractant protein-1) was elevated to a greater extent in iron-overloaded Timp3-/- livers. Gelatin zymography demonstrated equivalent increases in MMP-2 and MMP-9 levels in WT and Timp3-/- iron-overloaded livers. Loss of Timp3 enhanced the susceptibility to iron overload-mediated heart and liver injury, suggesting that Timp3 is a key protective molecule against iron-mediated pathology. NEW & NOTEWORTHY In mice, loss of tissue inhibitor of metalloproteinase 3 ( Timp3) was associated with systolic and diastolic dysfunctions, twofold higher hepatic iron accumulation (attributable to constituently lower levels of ferroportin), and increased hepatic inflammation. Loss of Timp3 enhanced the susceptibility to iron overload-mediated injury, suggesting that Timp3 plays a key protective role against iron-mediated pathology.

Advanced iron-overload cardiomyopathy in a genetic murine model is rescued by resveratrol therapy.

Iron-overload cardiomyopathy is prevalent on a worldwide basis and is a major comorbidity in patients with genetic hemochromatosis and secondary iron overload. Therapies are limited in part due to lack of a valid preclinical model, which recapitulates advanced iron-overload cardiomyopathy. Male hemojuvelin (HJV) knockout (HJVKO) mice, which lack HJV, a bone morphogenetic co-receptor protein required for hepcidin expression and systemic iron homeostasis, were fed a high-iron diet starting at 4 weeks of age for a duration of 1 year. Aged HJVKO mice in response to iron overload showed increased myocardial iron deposition and mortality coupled with oxidative stress and myocardial fibrosis culminating in advanced iron-overload cardiomyopathy. In a parallel group, iron-overloaded HJVKO mice received resveratrol (240 mg/day) at 9 months of age until 1 year of age. Echocardiography and invasive pressure-volume (PV) loop analyses revealed a complete normalization of iron-overload mediated diastolic and systolic dysfunction in response to resveratrol therapy. In addition, myocardial sarcoplasmic reticulum Ca2+ ATPase (SERCa2a) levels were reduced in iron-overloaded hearts and resveratrol therapy restored SERCa2a levels and suppressed up-regulation of the sodium-calcium exchanger (NCX1). Further, iron-mediated oxidative stress and myocardial fibrosis were suppressed by resveratrol treatment with concomitant activation of the p-Akt and p-AMP-activated protein kinase (AMPK) signaling pathways. A combination of ageing and high-iron diet in male HJVKO mice results in a valid preclinical model that recapitulates iron-overload cardiomyopathy in humans. Resveratrol therapy resulted in normalization of cardiac function demonstrating that resveratrol represents a feasible therapeutic intervention to reduce the burden of iron-overload cardiomyopathy.

Roles of Angiotensin Peptides and Recombinant Human ACE2 in Heart Failure.

BACKGROUND: The renin-angiotensin system (RAS) is activated in heart failure (HF) and inhibition of RAS is a mainstay therapy for HF. Angiotensin-converting enzyme 2 (ACE2) and its product, angiotensin 1-7 (Ang-[1-7]), are important negative regulators of the RAS. OBJECTIVES: A comprehensive examination of angiotensin peptide levels and therapeutic effects of recombinant human ACE2 (rhACE2) on peptide metabolism was evaluated in human plasma and explanted heart tissue from patients with HF. METHODS: Using prospective cohorts with chronic (n = 59) and acute (n = 42) HF, plasma angiotensin analysis was performed using a unique liquid chromatography-mass spectrometry/mass spectroscopy method quantifying circulating and equilibrium levels. Angiotensin II (Ang II) metabolism was examined in human explanted hearts with dilated cardiomyopathy (n = 25). RESULTS: The dynamic range of the RAS was large, with equilibrium angiotensin levels being 8- to 10-fold higher compared with circulating angiotensin levels. In chronic HF patients receiving ACE inhibition, plasma Ang II was suppressed and plasma Ang-(1-7) was elevated, whereas acute HF and patients receiving angiotensin receptor blocker had higher plasma Ang II with lower Ang-(1-7) levels. Suppressed Ang-(1-7)/Ang II ratio was associated with worsening HF symptoms and longer hospitalization. Recombinant human ACE2 effectively metabolized Ang-(1-10) and Ang II into Ang-(1-9) and Ang-(1-7), respectively. Myocardial Ang II levels in explanted human hearts with dilated cardiomyopathy were elevated despite ACE inhibition with elevated chymase activity, and Ang II was effectively converted to Ang-(1-7) by rhACE2. CONCLUSIONS: Plasma angiotensin peptides represent a dynamic network that is altered in HF and in response to rhACE2. An increased plasma Ang-(1-7) level is linked to ACE inhibitor use, whereas acute HF reduced Ang-(1-7) levels and suppressed the Ang-(1-7)/Ang II ratio. Increased chymase activity elevated Ang II levels in failing human hearts. Use of rhACE2 effectively normalized elevated Ang II while increasing Ang-(1-7) and Ang-(1-9) levels.

Females Are Protected From Iron-Overload Cardiomyopathy Independent of Iron Metabolism: Key Role of Oxidative Stress.

BACKGROUND: Sex-related differences in cardiac function and iron metabolism exist in humans and experimental animals. Male patients and preclinical animal models are more susceptible to cardiomyopathies and heart failure. However, whether similar differences are seen in iron-overload cardiomyopathy is poorly understood. METHODS AND RESULTS: Male and female wild-type and hemojuvelin-null mice were injected and fed with a high-iron diet, respectively, to develop secondary iron overload and genetic hemochromatosis. Female mice were completely protected from iron-overload cardiomyopathy, whereas iron overload resulted in marked diastolic dysfunction in male iron-overloaded mice based on echocardiographic and invasive pressure-volume analyses. Female mice demonstrated a marked suppression of iron-mediated oxidative stress and a lack of myocardial fibrosis despite an equivalent degree of myocardial iron deposition. Ovariectomized female mice with iron overload exhibited essential pathophysiological features of iron-overload cardiomyopathy showing distinct diastolic and systolic dysfunction, severe myocardial fibrosis, increased myocardial oxidative stress, and increased expression of cardiac disease markers. Ovariectomy prevented iron-induced upregulation of ferritin, decreased myocardial SERCA2a levels, and increased NCX1 levels. 17ß-Estradiol therapy rescued the iron-overload cardiomyopathy in male wild-type mice. The responses in wild-type and hemojuvelin-null female mice were remarkably similar, highlighting a conserved mechanism of sex-dependent protection from iron-overload-mediated cardiac injury. CONCLUSIONS: Male and female mice respond differently to iron-overload-mediated effects on heart structure and function, and females are markedly protected from iron-overload cardiomyopathy. Ovariectomy in female mice exacerbated iron-induced myocardial injury and precipitated severe cardiac dysfunction during iron-overload conditions, whereas 17ß-estradiol therapy was protective in male iron-overloaded mice.

ACE2/Ang 1-7 axis: A critical regulator of epicardial adipose tissue inflammation and cardiac dysfunction in obesity.

Obesity is characterized by an excessive fat accumulation in adipose tissues leading to weight gain and is increasing in prevalence and is strongly associated with metabolic and cardiovascular disorders. The renin-angiotensin system (RAS) has emerged as a key pathogenic mechanism for these disorders; activated RAS and angiotensin (Ang) II production results in worsening of cardiovascular diseases and angiotensin converting enzyme 2 (ACE2) negatively regulates RAS by metabolizing Ang II into Ang 1-7. ACE2 is expressed in the adipocytes and its expression is upregulated in response to high fat diet induced obesity in mice. Loss of ACE2 results in heart failure with preserved ejection fraction which is mediated in part by epicardial adipose tissue inflammation. Angiotensin 1-7 reduces the obesity associated cardiac dysfunction predominantly via its role in adiponectin expression and attenuation of epicardial adipose tissue inflammation. Human heart disease is also linked with inflammed epicardial adipose tissue. Here, we discuss the important interpretation of the novel of ACE2/Ang 1-7 pathway in obesity associated cardiac dysfunction.

A Disintegrin and Metalloprotease-17 Regulates Pressure Overload-Induced Myocardial Hypertrophy and Dysfunction Through Proteolytic Processing of Integrin ß1.

A disintegrin and metalloprotease-17 (ADAM17) belongs to a family of transmembrane enzymes, and it can mediate ectodomain shedding of several membrane-bound molecules. ADAM17 levels are elevated in patients with hypertrophic and dilated cardiomyopathy; however, its direct role in hypertrophic cardiomyopathy is unknown. Cardiomyocyte-specific ADAM17 knockdown mice (ADAM17(flox/flox)/αMHC-Cre; ADAM17(f/f)/Cre) and littermates with intact ADAM17 levels (ADAM17(f/f)) were subjected to cardiac pressure-overload by transverse aortic constriction. Cardiac function/architecture was assessed by echocardiography at 2 and 5 weeks post transverse aortic constriction. ADAM17 knockdown enhanced myocardial hypertrophy, fibrosis, more severe left ventricular dilation, and systolic dysfunction at 5 weeks post transverse aortic constriction. Pressure overload-induced upregulation of integrin ß1 was much greater with ADAM17 knockdown, concomitant with the greater activation of the focal adhesion kinase pathway, suggesting that integrin ß1 could be a substrate for ADAM17. ADAM17 knockdown did not alter other cardiomyocyte integrins, integrin α5 or α7, and HB-EGF (heparin-bound epidermal growth factor), another potential substrate for ADAM17, remained unaltered after pressure overload. ADAM17-mediated cleavage of integrin ß1 was confirmed by an in vitro assay. Intriguingly, ADAM17 knockdown did not affect the myocardial hypertrophy induced by a subpressor dose of angiotensin II, which occurs independent from the integrin ß1-mediated pathway. ADAM17-knockdown enhanced the hypertrophic response to cyclic mechanical stretching in neonatal rat cardiomyocytes. This study reports a novel cardioprotective function for ADAM17 in pressure overload cardiomyopathy, where loss of ADAM17 promotes hypertrophy by reducing the cleavage of cardiac integrin ß1, a novel substrate for ADAM17. This function of ADAM17 is selective for pressure overload-induced myocardial hypertrophy and dysfunction, and not agonist-induced hypertrophy.

Differential impact of mechanical unloading on structural and nonstructural components of the extracellular matrix in advanced human heart failure.

Adverse remodeling of the extracellular matrix (ECM) is a significant characteristic of heart failure. Reverse remodeling of the fibrillar ECM secondary to mechanical unloading of the left ventricle (LV) by left ventricular assist device (LVAD) has been subject of intense investigation; however, little is known about the impacts on nonfibrillar ECM and matricellular proteins that also contribute to disease progression. Explanted failing hearts were procured from patients with nonischemic dilated cardiomyopathy (DCM) with or without LVAD support, and compared to nonfailing control hearts. LV free wall specimens were formalin-fixed, flash-frozen or optimum cutting temperature-mount frozen. Histologic and biochemical assessment of fibrillar ECM showed that LVAD support was associated with lower levels of insoluble collagen, collagen type I mRNA, and collagen I/III ratio compared with no-LVAD hearts. A disintegrin and Metalloproteinase with Thrombospondin Motifs-2 (ADAM-TS2), a procollagen endopeptidase, was reduced in no-LVAD but not in LVAD hearts. The rise in ECM proteolytic activities was significantly lower in LVAD hearts. Matrix metalloproteinases (MMP1, MMP2, MMP8, MMP13, and MT1-MMP/MMP14) were comparable between DCM hearts. Tissue inhibitor of metalloproteinase (TIMP)3 and TIMP4 messenger RNA and protein showed the greatest reduction in no-LVAD hearts. Basement membrane proteins exhibited less severe disarray of laminin and fibronectin-1 in LVAD-supported hearts. The rise in matricellular protein, osteopontin, was suppressed in LVAD hearts, whereas secreted protein, acidic, cysteine-rich (SPARC) levels was unaffected by LVAD. Mechanical unloading of the failing DCM hearts can restore the fibrillar ECM and the basement membrane, contributing toward improved clinical outcomes. However, persistent elevation of matricellular proteins such as SPARC could contribute to the relapse of failing hearts on removal of LVAD support.

ACE2 Deficiency Worsens Epicardial Adipose Tissue Inflammation and Cardiac Dysfunction in Response to Diet-Induced Obesity.

Obesity is increasing in prevalence and is strongly associated with metabolic and cardiovascular disorders. The renin-angiotensin system (RAS) has emerged as a key pathogenic mechanism for these disorders; angiotensin (Ang)-converting enzyme 2 (ACE2) negatively regulates RAS by metabolizing Ang II into Ang 1-7. We studied the role of ACE2 in obesity-mediated cardiac dysfunction. ACE2 null (ACE2KO) and wild-type (WT) mice were fed a high-fat diet (HFD) or a control diet and studied at 6 months of age. Loss of ACE2 resulted in decreased weight gain but increased glucose intolerance, epicardial adipose tissue (EAT) inflammation, and polarization of macrophages into a proinflammatory phenotype in response to HFD. Similarly, human EAT in patients with obesity and heart failure displayed a proinflammatory macrophage phenotype. Exacerbated EAT inflammation in ACE2KO-HFD mice was associated with decreased myocardial adiponectin, decreased phosphorylation of AMPK, increased cardiac steatosis and lipotoxicity, and myocardial insulin resistance, which worsened heart function. Ang 1-7 (24 µg/kg/h) administered to ACE2KO-HFD mice resulted in ameliorated EAT inflammation and reduced cardiac steatosis and lipotoxicity, resulting in normalization of heart failure. In conclusion, ACE2 plays a novel role in heart disease associated with obesity wherein ACE2 negatively regulates obesity-induced EAT inflammation and cardiac insulin resistance.

Iron-overload injury and cardiomyopathy in acquired and genetic models is attenuated by resveratrol therapy.

Iron-overload cardiomyopathy is a prevalent cause of heart failure on a world-wide basis and is a major cause of mortality and morbidity in patients with secondary iron-overload and genetic hemochromatosis. We investigated the therapeutic effects of resveratrol in acquired and genetic models of iron-overload cardiomyopathy. Murine iron-overload models showed cardiac iron-overload, increased oxidative stress, altered Ca(2+) homeostasis and myocardial fibrosis resulting in heart disease. Iron-overload increased nuclear and acetylated levels of FOXO1 with corresponding inverse changes in SIRT1 levels in the heart corrected by resveratrol therapy. Resveratrol, reduced the pathological remodeling and improved cardiac function in murine models of acquired and genetic iron-overload at varying stages of iron-overload. Echocardiography and hemodynamic analysis revealed a complete normalization of iron-overload mediated diastolic and systolic dysfunction in response to resveratrol therapy. Myocardial SERCA2a levels were reduced in iron-overloaded hearts and resveratrol therapy restored SERCA2a levels and corrected altered Ca(2+) homeostasis. Iron-mediated pro-oxidant and pro-fibrotic effects in human and murine cardiomyocytes and cardiofibroblasts were suppressed by resveratrol which correlated with reduction in iron-induced myocardial oxidative stress and myocardial fibrosis. Resveratrol represents a clinically and economically feasible therapeutic intervention to reduce the global burden from iron-overload cardiomyopathy at early and chronic stages of iron-overload.

Determinants of ventricular arrhythmias in human explanted hearts with dilated cardiomyopathy.

BACKGROUND: The molecular and cellular determinants of ventricular tachycardia (VT) in patients with nonischaemic dilated cardiomyopathy (NIDCM) remain poorly defined. MATERIALS AND METHODS: We examined 20 NIDCM hearts where VT was reported in 10 cases and VT was absent in 10 cases, using a double-blinded case-control study design, and assessed the molecular and cellular features of the adverse myocardial remodelling. RESULTS: Explanted hearts from patients with VT showed greater hypertrophic changes based on cardiomyocyte cross-sectional area and expression of disease markers, and increased myocardial fibrosis which extended into the left ventricular and right ventricular outflow tract regions. The VT group also showed increased oxidative stress with reduction in reduced glutathione levels. Connexin 43 levels in the intercalated discs showed increased levels in the VT group with reduced phosphorylation. Microarray mRNA analysis of gene expression in the left ventricle (LV) free wall revealed several families of genes which were differentially upregulated or downregulated in hearts with documented VT compared to hearts without VT. Notably, we identified reduced expression of the Ca(2+) -activated K(+) channel (KCNN2) and increased expression of the transient receptor potential cation channel 7 (TRPM7) and intracellular chloride channel 3. Western blot analysis on LV membrane fractions showed reduced KCNN2 and increased TRPM7 levels in hearts with VT. CONCLUSIONS: In explanted human hearts with NIDCM, VT is associated with greater hypertrophy, oxidative stress and myocardial fibrosis, differential gene expression, and altered ion channel levels indicative of a distinctive adverse myocardial remodelling process associated with clinically significant VT.

Cardiomyocyte A Disintegrin And Metalloproteinase 17 (ADAM17) Is Essential in Post-Myocardial Infarction Repair by Regulating Angiogenesis.

BACKGROUND: A disintegrin and metalloproteinase 17 (ADAM17) is a membrane-bound enzyme that mediates shedding of many membrane-bound molecules, thereby regulating multiple cellular responses. We investigated the role of cardiomyocyte ADAM17 in myocardial infarction (MI). METHODS AND RESULTS: Cardiomyocyte-specific ADAM17 knockdown mice (ADAM17(flox/flox)/α-MHC-Cre; f/f/Cre) and parallel controls (ADAM17(flox/flox); f/f) were subjected to MI by ligation of the left anterior descending artery. Post MI, f/f/Cre mice showed compromised survival, higher rates of cardiac rupture, more severe left ventricular dilation, and suppressed ejection fraction compared with parallel f/f-MI mice. Ex vivo ischemic injury (isolated hearts) resulted in comparable recovery in both genotypes. Myocardial vascular density (fluorescent-labeled lectin perfusion and CD31 immunofluorescence staining) was significantly lower in the infarct areas of f/f/Cre-MI compared with f/f-MI mice. Activation of vascular endothelial growth factor receptor 2 (VEGFR2), its mRNA, and total protein levels were reduced in infarcted myocardium in ADAM17 knockdown mice. Transcriptional regulation of VEGFR2 by ADAM17 was confirmed in cocultured cardiomyocyte-fibroblast as ischemia-induced VEGFR2 expression was blocked by ADAM17-siRNA. Meanwhile, ADAM17-siRNA did not alter VEGFA bioavailability in the conditioned media. ADAM17 knockdown mice (f/f/Cre-MI) exhibited reduced nuclear factor-κB activation (DNA binding) in the infarcted myocardium, which could underlie the suppressed VEGFR2 expression in these hearts. Post MI, inflammatory response was not altered by ADAM17 downregulation. CONCLUSIONS: This study highlights the key role of cardiomyocyte ADAM17 in post-MI recovery by regulating VEGFR2 transcription and angiogenesis, thereby limiting left ventricular dilation and dysfunction. Therefore, ADAM17 upregulation, within the physiological range, could provide protective effects in ischemic cardiomyopathy.

Antagonism of angiotensin 1-7 prevents the therapeutic effects of recombinant human ACE2.

UNLABELLED: Activation of the angiotensin 1-7/Mas receptor (MasR) axis counteracts angiotensin II (Ang II)-mediated cardiovascular disease. Recombinant human angiotensin-converting enzyme 2 (rhACE2) generates Ang 1-7 from Ang II. We hypothesized that the therapeutic effects of rhACE2 are dependent on Ang 1-7 action. Wild type male C57BL/6 mice (10-12 weeks old) were infused with Ang II (1.5 mg/kg/d) and treated with rhACE2 (2 mg/kg/d). The Ang 1-7 antagonist, A779 (200 ng/kg/min), was administered to a parallel group of mice. rhACE2 prevented Ang II-induced hypertrophy and diastolic dysfunction while A779 prevented these beneficial effects and precipitated systolic dysfunction. rhACE2 effectively antagonized Ang II-mediated myocardial fibrosis which was dependent on the action of Ang 1-7. Myocardial oxidative stress and matrix metalloproteinase 2 activity was further increased by Ang 1-7 inhibition even in the presence of rhACE2. Activation of Akt and endothelial nitric oxide synthase (eNOS) by rhACE2 were suppressed by the antagonism of Ang 1-7 while the activation of pathological signaling pathways was maintained. Blocking Ang 1-7 action prevents the therapeutic effects of rhACE2 in the setting of elevated Ang II culminating in systolic dysfunction. These results highlight a key cardioprotective role of Ang 1-7, and increased Ang 1-7 action represents a potential therapeutic strategy for cardiovascular diseases. KEY MESSAGES: Activation of the renin-angiotensin system (RAS) plays a key pathogenic role in cardiovascular disease. ACE2, a monocarboxypeptidase, negatively regulates pathological effects of Ang II. Antagonizing Ang 1-7 prevents the therapeutic effects of recombinant human ACE2. Our results highlight a key protective role of Ang 1-7 in cardiovascular disease.