Search

We aimed to study the cardiac expression of bone morphogenetic protein 2, its receptor 1 b, and connective tissue growth factor, factors implicated in cardiac embryogenesis, following ischemia/hypoxia, heart failure, and in remodeling hearts from humans and mice. Biopsies from the left ventricle of patients with end-stage heart failure due to dilated cardiomyopathy or coronary artery disease were compared with donor hearts and biopsies from patients with normal heart function undergoing coronary artery bypass grafting. Mouse model of post-infarction remodeling was made by permanent ligation of the left coronary artery. Hearts were analyzed by real-time polymerase chain reaction and Western blotting after 24 hours and after 2 and 4 weeks. Patients with dilated cardiomyopathy and mice post-infarction had increased cardiac expression of connective tissue growth factor. Bone morphogenetic protein 2 was increased in human hearts failing due to coronary artery disease and in mice post-infarction. Gene expression of bone morphogenetic protein receptor 1 beta was reduced in hearts of patients with failure, but increased two weeks following permanent ligation of the left coronary artery in mice. In conclusion, connective tissue growth factor is upregulated in hearts of humans with dilated cardiomyopathy, bone morphogenetic protein 2 is upregulated in remodeling due to myocardial infarction while its receptor 1 b in human failing hearts is downregulated. A potential explanation might be an attempt to engage regenerative processes, which should be addressed by further, mechanistic studies.

Aims Heart failure is associated with activation of fetal gene programs. Bone morphogenetic proteins (BMPs) regulate embryonic development through interaction with BMP receptors (BMPRs) on the cell surface. We investigated if the expression of BMP4 and its receptors BMPR1a and BMPR2 were activated in post-infarction remodeling and heart failure. Main methods Left ventricular biopsies were taken from explanted hearts of patients with end-stage heart failure due to dilated cardiomyopathy (CMP; n = 15) or ischemic heart disease (CAD; n = 9), and compared with homograft control preparations from organ donors deceased due to non-cardiac causes (n = 7). Other samples were taken from patients undergoing coronary artery bypass grafting (CABG; n = 11). Mice were subjected to induced infarction by permanent coronary artery ligation or sham operation, and hearts were sampled serially thereafter (n = 7 at each time point). Key findings Human and mouse hearts expressed BMP4 and both receptor subtypes. CABG and CMP patients had increased expression of mRNA encoding for BMP4, but unchanged protein. Mouse hearts had increased BMP4 precursor protein 24 h after infarction. BMPR1a protein decreased in CAD patients and initially in postinfarcted mouse hearts, but increased again in the latter after two weeks. Human recombinant BMP4 promoted survival after H 2 O 2 injury in HL-1 cells, and also protected adult mouse cardiomyocytes against hypoxia–reoxygenation injury. Significance Adult hearts express BMP4, the mRNA increasingly so in patients with coronary artery disease with good cardiac function. BMPRs are downregulated in cardiac remodeling and failure. Recombinant BMP4 has protective effects on cultured cardiomyocytes.

Coronary atherosclerosis can lead to myocardial infarction, and secondarily to post-infarct remodelling and heart failure. Retinoic acid (RA) influences cell proliferation. We hypothesized that RA could influence gene expression and proliferation of cardiovascular cells. Left ventricular biopsies from patients with end-stage heart failure due to coronary artery disease (CAD) or dilated cardiomyopathy were investigated for the content of RA metabolites using liquid chromatography mass spectrometry (LC-MS/MS), and compared with healthy donors. All-trans retinoic acid (ATRA) was increased in the hearts of CAD patients. Gene expression (quantitative PCR) of RA target genes was not influenced in failing hearts, but was increased in the hearts of patients with CAD undergoing open heart surgery. The expression of RA target genes was increased in atherosclerotic lesions from carotid arteries compared to healthy arteries. Stimulation of cardiomyocytes, cardiofibroblasts, smooth muscle cells and endothelial cells with ATRA increased the gene expression of the key enzymes. Cardiofibroblast and smooth muscle cell proliferation were reduced by ATRA, which increased endothelial cell proliferation. Coronary artery disease leads to increased expression of RA target genes. ATRA accumulated in the failing human heart. All investigated cell types present in the heart had induced expression of RA target genes when stimulated with ATRA, which also influenced cell proliferation.

Myocardial connective tissue growth factor (CTGF/CCN2) is induced in heart failure, a condition associated with diminution of β-adrenergic receptor (β-AR) responsiveness. Accordingly, we aimed to investigate whether CTGF could play a mechanistic role in regulation of β-AR responsiveness. Concentration-response curves of isoproterenol-stimulated cAMP generation in cardiomyocytes from transgenic mice with cardiac-restricted overexpression of CTGF (Tg-CTGF) or cardiomyocytes pretreated with recombinant human CTGF (rec-hCTGF) revealed marked reduction of both β₁-AR and β₂-AR responsiveness. Consistently, ventricular muscle strips from Tg-CTGF mice stimulated with isoproterenol displayed attenuation of maximal inotropic responses. However, no differences of maximal inotropic responses of myocardial fibers from Tg-CTGF mice and nontransgenic littermate control (NLC) mice were discerned when stimulated with supramaximal concentrations of dibutyryl-cAMP, indicating preserved downstream responsiveness to cAMP. Congruent with a mechanism of desensitization of β-ARs, mRNA and protein levels of G protein-coupled receptor kinase 5 (GRK5) were found isoform-selective upregulated in both cardiomyocytes from Tg-CTGF mice and cardiomyocytes exposed to rec-hCTGF. Corroborating a mechanism of GRK5 in CTGF-mediated control of β-AR sensitivity, Chinese hamster ovary cells pretreated with rec-hCTGF displayed increased agonist- and biased ligand-stimulated β-arrestin binding to β-ARs. Despite increased sensitivity of cardiomyocytes from GRK5-knockout (KO) mice to β-adrenergic agonists, pretreatment of GRK5-KO cardiomyocytes with rec-hCTGF, as opposed to cardiomyocytes from wild-type mice, did not alter β-AR responsiveness. Finally, Tg-CTGF mice subjected to chronic exposure (14 days) to isoproterenol revealed blunted myocardial hypertrophy and preserved cardiac function versus NLC mice. In conclusion, this study uncovers a novel mechanism controlling β-AR responsiveness in cardiomyocytes involving CTGF-mediated regulation of GRK5.

Lactate is transported by stereo-specific, pH-dependent monocarboxylate transporters (MCTs), of which MCT1 is expressed in abundance in rodent and human hearts. This study investigated the expression and functional role of MCT1 in mouse hearts during acute myocardial ischemia.Mice hearts were isolated and Langendorff-perfused with induced global ischemia (40 min) and reperfusion with function and infarct size as end-points. Hearts were collected serially for protein extraction and immunoblotting with an MCT1 antibody, and for determining subcellular localization with immunogold EM. Immortalized cardiomyocytes (HL-1 cells) were injured with hydrogen peroxide, and cell death in the presence or absence of the competitive inhibitor of MCT1, d-lactate, was evaluated by Trypan blue exclusion.MCT1 expression increased after 15 min reperfusion (p0.05), but was not significantly increased after 60 min. MCT1 was localized in mitochondria, plasma membrane, and intercalated disks. At 15 min reperfusion, gold particle count was increased in the intercalated disks (p0.05). d-lactate administration to isolated hearts either for 5 min before ischemia or at the first 5 min of reperfusion increased infarct size (p0.01). A significant impairment of left ventricular performance was found when d-lactate was given before ischemia. MCT1 expression was not influenced by d-lactate. When HL-1 cells were treated with d-lactate before injury was induced with hydrogen peroxide, cell death was increased (p0.05).Inhibition of MCT1 increases cell death. Increased MCT1 expression after ischemia and reperfusion is likely to restore cardiac pH through lactate export.

Heart rupture and heart failure are deleterious complications of myocardial infarction. The ShcA gene encodes for three protein isoforms, p46-, p52- and p66ShcA. p66ShcA induces oxidative stress. We studied the role of p66ShcA post-infarction. Expression of p66ShcA was analyzed in myocardium of patients with stable angina (n = 11), in explanted hearts with end-stage ischemic heart failure (n = 9) and compared to non-failing hearts not suitable for donation (n = 7). p66ShcA was increased in the patients with stable angina, but not in the patients with end-stage heart failure. Mice (n = 105) were subjected to coronary artery ligation. p66ShcA expression and phosphorylation were evaluated over a 6-week period. p66ShcA expression increased transiently during the first weeks post-infarction. p66ShcA knockout mice (KO) were compared to wild type (n = 82 in total). KO had improved survival and reduced occurrence of heart rupture post-infarction. Expression of cardiac matrix metalloproteinase 2 (MMP-2) was reduced; fibroblast activation and collagen accumulation were facilitated, while oxidative stress was attenuated in KO early post-infarction. 6 weeks post-infarction, reactive fibrosis and left ventricular dilatation were diminished in KO. p66ShcA regulation of MMP-2 was demonstrated in cultured fibroblasts: lack or overexpression of p66ShcA in vitro altered expression of MMP-2. Myocardial infarction induced cardiac p66ShcA. Deletion of p66ShcA improved early survival, myocardial healing and reduced cardiac fibrosis. Upon myocardial infarction p66ShcA regulates MMP-2 activation. The role of p66ShcA in human cardiac disease deserves further study as a potential target for reducing adverse cardiac remodeling post-infarction.

We tested if remote gene delivery of hypoxia-inducible factor 1 alpha (HIF-1 alpha) protected hearts against induced ischemia, hypothesizing that gene delivery into skeletal muscle may lead to secretion of proteins with actions elsewhere. Murine quadriceps muscles were transfected with DNA encoding for human HIF-1 alpha, which resulted in a local, but lasting expression (mRNA and protein, where the latter had nuclear localization). Subjection of isolated hearts to global ischemia and reperfusion 1, 4, and 8 weeks after gene delivery resulted in infarct size reduction (p < 0.05). Supporting that this was due to paracrine effects, HL-1 cells treated with conditioned media from cells transfected with HIF-1 alpha or serum from HIF-1 alpha-treated mice were protected against H(2)O(2)-induced cell death (p < 0.05, respectively). The latter protection was reduced when a heme oxygenase activity blocker was used. Taqman low-density array of 47 HIF-1 alpha-regulated genes at the treatment site showed nine specific upregulations (p < 0.05). Of the corresponding proteins, PDGF-B and adrenomedullin were upregulated in the heart. HIF-1 alpha treatment induced an increased vascularization of the heart and skeletal muscle. In conclusion, remote delivery of DNA for HIF-1 alpha was cardioprotective, represented by consistent infarct size reduction, which may be due to release of paracrine factors from the transfected muscle.

The present study investigates whether the cardioprotection achieved by gene delivery of hypoxia-inducible factor-1 alpha (HIF-1 alpha) depends on the downstream factor haem oxygenase (HMOX)-1.Immortalized cardiomyocytes (HL-1 cells) were transfected with HIF-1 alpha or HMOX-1 and injured with hydrogen peroxide (H(2)O(2)), and death was evaluated by trypan blue staining. Quadriceps muscles of mice were treated with DNA for HIF-1 alpha and HMOX-1, or sham-treated and electroporated, and 3 days later, hearts were isolated and subjected to global ischaemia and reperfusion. Some HIF-1 alpha- and sham-treated mice received the HMOX blocker zinc deuteroporphyrin 2,4-bis-glycol (ZnBG) (n = 6-8 in each group). HL-1 cells were stimulated with bilirubin or the carbon monoxide donor CORM-2 before injury with H(2)O(2). HL-1 cells which were transfected with HIF-1 alpha or HMOX-1 had an increased survival to H(2)O(2)-induced injury compared with empty vector (n = 10-12 per group; P0.01 for both). When HMOX-1-luciferase reporter mice were treated with HIF-1 alpha in the quadriceps muscle, increased luciferase activity was found locally, but nowhere else. Mice pre-treated with HIF-1 alpha or HMOX-1 had a reduced infarct size, improved post-ischaemic function, and increased serum bilirubin (P0.05). ZnBG inhibited all these effects afforded by HIF-1 alpha. Stimulation of HL-1 cells with bilirubin and CORM-2 reduced cell death evoked by H(2)O(2) (P0.05 for both, n = 11-15 in each group).HIF-1 alpha and HMOX-1 provided protection against H(2)O(2)-induced damage in HL-1 cells. Remote gene delivery of HIF-1 alpha afforded cardioprotective effects. These were dependent on HMOX activity, as an HMOX blocker abolished the effects, and they were mimicked by pre-treatment with HMOX-1. Downstream to HMOX-1, bilirubin as well as carbon monoxide may be organ effectors.

Purpose: Bone morphogenetic protein-2 (BMP2) is known for its wide role in embryonic heart development, encompassing the differentiation of cardiomyocytes, vascularization, valvulogenesis and connective tissue formation. However, BMP2 function in the adult heart is unknown. Recent work indicates that other BMP family members may be involved in myocardial remodeling. We hypothesized that BMP2 is expressed during ischemic cardiac injury and in heart failure. Furthermore we wanted to study which cells in the heart express BMP2 and what stimuli may induce this expression. Methods: Left ventricular biopsies were sampled from explanted hearts of patients with end-stage dilated cardiomyopathy and ischemic heart disease, from hearts of patients undergoing coronary artery bypass grafting and from donor hearts. Mice were subjected to infarction by permanent coronary artery ligation or sham operated, and hearts were sampled serially thereafter. Other hearts were dissected into ventricles and atria, as well as used for cell isolation and culture. Human umbilical vein endothelial cells (HUVEC) were subjected to mechanical stretch, hypoxia/reoxygenation and oxidative stress by means of hydrogen peroxide. Expression of BMP2 and some of its downstream targets was assessed using qPCR and Western blot. Results: BMP2 mRNA was upregulated in biopsies of coronary artery bypass grafting patients and in infarcted mouse hearts, but not in end-stage heart failure. Examination of its cellular source in untreated heart revealed that BMP2 protein expression was confined to cardiac myocytes, rather than to cardiac fibroblasts or endothelial cells, with the left ventricle displaying higher BMP2 expression than the atria. HUVEC had similar levels of BMP2 expression as cardiomyocytes. To investigate the potential mechanism of BMP2 activation, we subjected HUVEC to different modalities of cellular stress. Mechanical stress, but not hypoxia/reoxygenation or hydrogen peroxide induced BMP2 expression, as well expression of its downstream target genes sprouty-1 and beta-catenin. Conversely, oxidative stress strongly inhibited expression of sprouty-1 and BMP signaling co-factor thrombospondin-1. Conclusion: BMP2 is induced in biopsies from CABG patients and in postinfarction mouse hearts. Activation of BMP2 and its targets was also observed in HUVECs subjected to mechanical stress, but not in hypoxia/reoxygenation or oxidative stress. The significance of BMP2 activation in these situations is not clarified.