Cardiomyocyte d-dopachrome tautomerase protects against heart failure.
ABSTRACT: The mechanisms contributing to heart failure remain incompletely understood. d-dopachrome tautomerase (DDT) is a member of the macrophage migration inhibitory factor family of cytokines and is highly expressed in cardiomyocytes. This study examined the role of cardiomyocyte DDT in the setting of heart failure. Patients with advanced heart failure undergoing transplantation demonstrated decreased cardiac DDT expression. To understand the effect of loss of cardiac DDT in experimental heart failure, cardiomyocyte-specific DDT-KO (DDT-cKO) and littermate control mice underwent surgical transverse aortic constriction (TAC) to induce cardiac pressure overload. DDT-cKO mice developed more rapid cardiac contractile dysfunction, greater cardiac dilatation, and pulmonary edema after TAC. Cardiomyocytes from DDT-cKO mice after TAC had impaired contractility, calcium transients, and reduced expression of the sarcoplasmic reticulum calcium ATPase. The DDT-cKO hearts also exhibited diminished angiogenesis with reduced capillary density and lower VEGF-A expression after TAC. In pharmacological studies, recombinant DDT (rDDT) activated endothelial cell ERK1/2 and Akt signaling and had proangiogenic effects in vitro. The DDT-cKO hearts also demonstrated more interstitial fibrosis with enhanced collagen and connective tissue growth factor expression after TAC. In cardiac fibroblasts, rDDT had an antifibrotic action by inhibiting TGF-β-induced Smad-2 activation. Thus, endogenous cardiomyocyte DDT has pleiotropic actions that are protective against heart failure.
Project description:A vital step in the development of heart failure is the transition from compensatory cardiac hypertrophy to decompensated dilated cardiomyopathy (DCM) during cardiac remodeling under mechanical or pathological stress. However, the molecular mechanisms underlying the development of DCM and heart failure remain incompletely understood. In the present study, we investigate whether Gab1, a scaffolding adaptor protein, protects against hemodynamic stress-induced DCM and heat failure. We first observed that the protein levels of Gab1 were markedly reduced in hearts from human patients with DCM and from mice with experimental viral myocarditis in which DCM developed. Next, we generated cardiac-specific Gab1 knockout mice (Gab1-cKO) and found that Gab-cKO mice developed DCM in hemodynamic stress-dependent and age-dependent manners. Under transverse aorta constriction (TAC), Gab1-cKO mice rapidly developed decompensated DCM and heart failure, whereas Gab1 wild-type littermates exhibited adaptive left ventricular hypertrophy without changes in cardiac function. Mechanistically, we showed that Gab1-cKO mouse hearts displayed severe mitochondrial damages and increased cardiomyocyte apoptosis. Loss of cardiac Gab1 in mice impaired Gab1 downstream MAPK signaling pathways in the heart under TAC. Gene profiles further revealed that ablation of Gab1 in heart disrupts the balance of anti- and pro-apoptotic genes in cardiomyocytes. These results demonstrate that cardiomyocyte Gab1 is a critical regulator of the compensatory cardiac response to aging and hemodynamic stress. These findings may provide new mechanistic insights and potential therapeutic target for DCM and heart failure.
Project description:The cellular response to stress involves the recruitment and coordination of molecular signaling pathways that prevent cell death. D-dopachrome tautomerase (DDT) is an enzyme that lacks physiologic substrates in mammalian cells, but shares partial sequence and structural homology with macrophage migration inhibitory factor (MIF). Here, we observed that DDT is highly expressed in murine cardiomyocytes and secreted by the heart after ischemic stress. Antibody-dependent neutralization of secreted DDT exacerbated both ischemia-induced cardiac contractile dysfunction and necrosis. We generated cardiomyocyte-specific DDT knockout mice (Myh6-Cre Ddtfl/fl), which demonstrated normal baseline cardiac size and function, but had an impaired physiologic response to ischemia-reperfusion. Hearts from Myh6-Cre Ddtfl/fl mice exhibited more necrosis and LV contractile dysfunction than control hearts after coronary artery ligation and reperfusion. Furthermore, treatment with DDT protected isolated hearts against injury and contractile dysfunction after ischemia-reperfusion. The protective effect of DDT required activation of the metabolic stress enzyme AMP-activated protein kinase (AMPK), which was mediated by a CD74/CaMKK2-dependent mechanism. Together, our data indicate that cardiomyocyte secretion of DDT has important autocrine/paracrine effects during ischemia-reperfusion that protect the heart against injury.
Project description:The Ras-related guanosine triphosphatase RhoA mediates pathological cardiac hypertrophy, but also promotes cell survival and is cardioprotective after ischemia/reperfusion injury. To understand how RhoA mediates these opposing roles in the myocardium, we generated mice with a cardiomyocyte-specific deletion of RhoA. Under normal conditions, the hearts from these mice showed functional, structural, and growth parameters similar to control mice. Additionally, the hearts of the cardiomyocyte-specific, RhoA-deficient mice subjected to transverse aortic constriction (TAC)-a procedure that induces pressure overload and, if prolonged, heart failure-exhibited a similar amount of hypertrophy as those of the wild-type mice subjected to TAC. Thus, neither normal cardiac homeostasis nor the initiation of compensatory hypertrophy required RhoA in cardiomyocytes. However, in response to chronic TAC, hearts from mice with cardiomyocyte-specific deletion of RhoA showed greater dilation, with thinner ventricular walls and larger chamber dimensions, and more impaired contractile function than those from control mice subjected to chronic TAC. These effects were associated with aberrant calcium signaling, as well as decreased activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and AKT. In addition, hearts from mice with cardiomyocyte-specific RhoA deficiency also showed less fibrosis in response to chronic TAC, with decreased transcriptional activation of genes involved in fibrosis, including myocardin response transcription factor (MRTF) and serum response factor (SRF), suggesting that the fibrotic response to stress in the heart depends on cardiomyocyte-specific RhoA signaling. Our data indicated that RhoA regulates multiple pathways in cardiomyocytes, mediating both cardioprotective (hypertrophy without dilation) and cardio-deleterious effects (fibrosis).
Project description:Pathological cardiomyocyte hypertrophy is associated with significantly increased risk of heart failure, one of the leading medical causes of mortality worldwide. MicroRNAs are known to be involved in pathological cardiac remodeling. However, whether miR-99a participates in the signaling cascade leading to cardiac hypertrophy is unknown. To evaluate the role of miR-99a in cardiac hypertrophy, we assessed the expression of miR-99a in hypertrophic cardiomyocytes induced by isoprenaline (ISO)/angiotensin-II (Ang II) and in mice model of cardiac hypertrophy induced by transverse aortic constriction (TAC). Expression of miR-99a was evaluated in these hypertrophic cells and hearts. We also found that miR-99a expression was highly correlated with cardiac function of mice with heart failure (8 weeks after TAC surgery). Overexpression of miR-99a attenuated cardiac hypertrophy in TAC mice and cellular hypertrophy in stimuli treated cardiomyocytes through down-regulation of expression of mammalian target of rapamycin (mTOR). These results indicate that miR-99a negatively regulates physiological hypertrophy through mTOR signaling pathway, which may provide a new therapeutic approach for pressure-overload heart failure.
Project description:Chronic pressure-overload (PO)- induced cardiomyopathy is one of the leading causes of left ventricular (LV) remodeling and heart failure. The role of the ? isoform of glycogen synthase kinase-3 (GSK-3?) in PO-induced cardiac remodeling is unclear and its downstream molecular targets are largely unknown. To investigate the potential roles of GSK-3?, cardiomyocyte-specific GSK-3? conditional knockout (cKO) and control mice underwent trans-aortic constriction (TAC) or sham surgeries. Cardiac function in the cKOs and littermate controls declined equally up to 2?weeks of TAC. At 4?week, cKO animals retained concentric LV remodeling and showed significantly less decline in contractile function both at systole and diastole, vs. controls which remained same until the end of the study (6?wk). Histological analysis confirmed preservation of LV chamber and protection against TAC-induced cellular hypertrophy in the cKO. Consistent with attenuated hypertrophy, significantly lower level of cardiomyocyte apoptosis was observed in the cKO. Mechanistically, GSK-3? was found to regulate mitochondrial permeability transition pore (mPTP) opening and GSK-3?-deficient mitochondria showed delayed mPTP opening in response to Ca2+ overload. Consistently, overexpression of GSK-3? in cardiomyocytes resulted in elevated Bax expression, increased apoptosis, as well as a reduction of maximum respiration capacity and cell viability. Taken together, we show for the first time that GSK-3? regulates mPTP opening under pathological conditions, likely through Bax overexpression. Genetic ablation of cardiomyocyte GSK-3? protects against chronic PO-induced cardiomyopathy and adverse LV remodeling, and preserves contractile function. Selective inhibition of GSK-3? using isoform-specific inhibitors could be a viable therapeutic strategy to limit PO-induced heart failure.
Project description:BACKGROUND:Inflammation is associated with cardiac remodeling and heart failure, but how it is initiated in response to nonischemic interventions in the absence of cell death is not known. We tested the hypothesis that activation of Ca2+/calmodulin-dependent protein kinase II ? (CaMKII?) in cardiomyocytes (CMs) in response to pressure overload elicits inflammatory responses leading to adverse remodeling. METHODS:Mice in which CaMKII? was selectively deleted from CMs (cardiac-specific knockout [CKO]) and floxed control mice were subjected to transverse aortic constriction (TAC). The effects of CM-specific CaMKII? deletion on inflammatory gene expression, inflammasome activation, macrophage accumulation, and fibrosis were assessed by quantitative polymerase chain reaction, histochemistry, and ventricular remodeling by echocardiography. RESULTS:TAC induced increases in cardiac mRNA levels for proinflammatory chemokines and cytokines in ?3 days, and these responses were significantly blunted when CM CaMKII? was deleted. Apoptotic and necrotic cell death were absent at this time. CMs isolated from TAC hearts mirrored these robust increases in gene expression, which were markedly attenuated in CKO. Priming and activation of the NOD-like receptor pyrin domain-containing protein 3 inflammasome, assessed by measuring interleukin-1? and NOD-like receptor pyrin domain-containing protein 3 mRNA levels, caspase-1 activity, and interleukin-18 cleavage, were increased at day 3 after TAC in control hearts and in CMs isolated from these hearts. These responses were dependent on CaMKII? and associated with activation of Nuclear Factor-kappa B and reactive oxygen species. Accumulation of macrophages observed at days 7 to 14 after TAC was diminished in CKO and, by blocking Monocyte Chemotactic Protein-1 signaling, deletion of CM Monocyte Chemotactic Protein-1 or inhibition of inflammasome activation. Fibrosis was also attenuated by these interventions and in the CKO heart. Ventricular dilation and contractile dysfunction observed at day 42 after TAC were diminished in the CKO. Inhibition of CaMKII, Nuclear Factor-kappa B, inflammasome, or Monocyte Chemotactic Protein-1 signaling in the first 1 or 2 weeks after TAC decreased remodeling, but inhibition of CaMKII after 2 weeks did not. CONCLUSIONS:Activation of CaMKII? in response to pressure overload triggers inflammatory gene expression and activation of the NOD-like receptor pyrin domain-containing protein 3 inflammasome in CMs. These responses provide signals for macrophage recruitment, fibrosis, and myocardial dysfunction in the heart. Our work suggests the importance of targeting early inflammatory responses induced by CM CaMKII? signaling to prevent progression to heart failure.
Project description:The aim of this study was to determine whether endogenous GLUT1 induction and the increased glucose utilization that accompanies pressure overload hypertrophy (POH) are required to maintain cardiac function during hemodynamic stress, and to test the hypothesis that lack of GLUT1 will accelerate the transition to heart failure. To determine the contribution of endogenous GLUT1 to the cardiac adaptation to POH, male mice with cardiomyocyte-restricted deletion of the GLUT1 gene (G1KO) and their littermate controls (Cont) were subjected to transverse aortic constriction (TAC). GLUT1 deficiency reduced glycolysis and glucose oxidation by 50%, which was associated with a reciprocal increase in fatty acid oxidation (FAO) relative to controls. Four weeks after TAC, glycolysis increased and FAO decreased by 50% in controls, but were unchanged in G1KO hearts relative to shams. G1KO and controls exhibited equivalent degrees of cardiac hypertrophy, fibrosis, and capillary density loss after TAC. Following TAC, in vivo left ventricular developed pressure was decreased in G1KO hearts relative to controls, but+dP/dt was equivalently reduced in Cont and G1KO mice. Mitochondrial function was equivalently impaired following TAC in both Cont and G1KO hearts. GLUT1 deficiency in cardiomyocytes alters myocardial substrate utilization, but does not substantially exacerbate pressure-overload induced contractile dysfunction or accelerate the progression to heart failure.
Project description:An important event in the pathogenesis of heart failure is the development of pathological cardiac hypertrophy. In cultured cardiomyocytes, the transcription factor Gata4 is required for agonist-induced hypertrophy. We hypothesized that, in the intact organism, Gata4 is an important regulator of postnatal heart function and of the hypertrophic response of the heart to pathological stress. To test this hypothesis, we studied mice heterozygous for deletion of the second exon of Gata4 (G4D). At baseline, G4D mice had mild systolic and diastolic dysfunction associated with reduced heart weight and decreased cardiomyocyte number. After transverse aortic constriction (TAC), G4D mice developed overt heart failure and eccentric cardiac hypertrophy, associated with significantly increased fibrosis and cardiomyocyte apoptosis. Inhibition of apoptosis by overexpression of the insulin-like growth factor 1 receptor prevented TAC-induced heart failure in G4D mice. Unlike WT-TAC controls, G4D-TAC cardiomyocytes hypertrophied by increasing in length more than width. Gene expression profiling revealed up-regulation of genes associated with apoptosis and fibrosis, including members of the TGF-beta pathway. Our data demonstrate that Gata4 is essential for cardiac function in the postnatal heart. After pressure overload, Gata4 regulates the pattern of cardiomyocyte hypertrophy and protects the heart from load-induced failure.
Project description:Although postcapillary pulmonary hypertension (PH) is an important prognostic factor for patients with heart failure (HF), its pathogenesis remains to be fully elucidated. To elucidate the different roles of Rho-kinase isoforms, ROCK1 and ROCK2, in cardiomyocytes in response to chronic pressure overload, we performed transverse aortic constriction (TAC) in cardiac-specific ROCK1-deficient (cROCK1-/-) and ROCK2-deficient (cROCK2-/-) mice. Cardiomyocyte-specific ROCK1 deficiency promoted pressure-overload-induced cardiac dysfunction and postcapillary PH, whereas cardiomyocyte-specific ROCK2 deficiency showed opposite results. Histological analysis showed that pressure-overload-induced cardiac hypertrophy and fibrosis were enhanced in cROCK1-/- mice compared with controls, whereas cardiac hypertrophy was attenuated in cROCK2-/- mice after TAC. Consistently, the levels of oxidative stress were up-regulated in cROCK1-/- hearts and down-regulated in cROCK2-/- hearts compared with controls after TAC. Furthermore, cyclophilin A (CyPA) and basigin (Bsg), both of which augment oxidative stress, enhanced cardiac dysfunction and postcapillary PH in cROCK1-/- mice, whereas their expressions were significantly lower in cROCK2-/- mice. In clinical studies, plasma levels of CyPA were significantly increased in HF patients and were higher in patients with postcapillary PH compared with those without it. Finally, high-throughput screening demonstrated that celastrol, an antioxidant and antiinflammatory agent, reduced the expressions of CyPA and Bsg in the heart and the lung, ameliorating cardiac dysfunction and postcapillary PH induced by TAC. Thus, by differentially affecting CyPA and Bsg expressions, ROCK1 protects and ROCK2 jeopardizes the heart from pressure-overload HF with postcapillary PH, for which celastrol may be a promising agent.
Project description:Adhesive intercellular connections at cardiomyocyte intercalated disks (IDs) support contractile force and maintain structural integrity of the heart muscle. Disturbances of the proteins at IDs deteriorate cardiac function and morphology. An adaptor protein afadin, one of the components of adherens junctions, is expressed ubiquitously including IDs. At present, the precise role of afadin in cardiac physiology or disease is unknown. To explore this, we generated conditional knockout (cKO) mice with cardiomyocyte-targeted deletion of afadin. Afadin cKO mice were born according to the expected Mendelian ratio and have no detectable changes in cardiac phenotype. On the other hand, chronic pressure overload induced by transverse aortic constriction (TAC) caused systolic dysfunction, enhanced fibrogenesis and apoptosis in afadin cKO mice. Afadin deletion increased macrophage infiltration and monocyte chemoattractant protein-1 expression, and suppressed transforming growth factor (TGF) ? receptor signaling early after TAC procedure. Afadin also associated with TGF? receptor I at IDs. Pharmacological antagonist of TGF? receptor I (SB431542) augmented mononuclear infiltration and fibrosis in the hearts of TAC-operated control mice. In conclusion, afadin is a critical molecule for cardiac protection against chronic pressure overload. The beneficial effects are likely to be a result from modulation of TGF? receptor signaling pathways by afadin.