CTGF/CCN2 Postconditioning Increases Tolerance of Murine Hearts towards Ischemia-Reperfusion Injury.

BACKGROUND AND PURPOSE: Previous studies of ischemia-reperfusion injury (IRI) in hearts from mice with cardiac-restricted overexpression of CCN2 have shown that CCN2 increases tolerance towards IRI. The objectives of this study were to investigate to what extent post-ischemic administration of recombinant human CCN2 (rhCCN2) would limit infarct size and improve functional recovery and what signaling pathways are involved. EXPERIMENTAL APPROACH: Isolated mice hearts were perfused ad modum Langendorff, subjected to no-flow, global ischemia, and subsequently, exposed to mammalian cell derived, full-length (38-40kDa) rhCCN2 (250 nM) or vehicle during the first 15 min of a 60 min reperfusion period. KEY RESULTS: Post-ischemic administration of rhCCN2 resulted in attenuation of infarct size from 58 ± 4% to 34 ± 2% (p < 0.001) which was abrogated by concomitant administration of the PI3 kinase inhibitor LY294002 (45 ± 3% vs. 50 ± 3%, ns). In congruence with reduction of infarct size rhCCN2 also improved recovery of left ventricular developed pressure (p < 0.05). Western blot analyses of extracts of ex vivo-perfused murine hearts also revealed that rhCCN2 evoked concentration-dependent increase of cardiac phospho-GSK3ß (serine-9) contents. CONCLUSIONS AND IMPLICATIONS: We demonstrate that post-ischemic administration of rhCCN2 increases the tolerance of ex vivo-perfused murine hearts to IRI. Mechanistically, this postconditioning effect of rhCCN2 appeared to be mediated by activation of the reperfusion injury salvage kinase pathway as demonstrated by sensitivity to PI3 kinase inhibition and increased CCN2-induced phosphorylation of GSK3ß (Ser-9). Thus, the rationale for testing rhCCN2-mediated post-ischemic conditioning of the heart in more complex models is established.

CCN2 exerts direct cytoprotective actions in adult cardiac myocytes by activation of the PI3-kinase/Akt/GSK-3ß signaling pathway.

We recently reported that transgenic mice with cardiac-restricted overexpression of CCN2/CTGF have substantially increased tolerance towards ischemia/reperfusion injury. The purpose of this study was to investigate to what extent fully differentiated cardiac myocytes are direct targets of CCN2, and to resolve the signaling mechanisms that convey the cardioprotective actions of CCN2. Akt and GSK-3ß were identified as putative intermediaries of intracellular signaling stimulated by recombinant human CCN2 (rhCCN2). Concentration-effect experiments revealed CCN2-stimulated phosphorylation of Akt (Ser473) and downstream GSK-3ß (Ser9) with EC50 ~250 nmol/L. CCN2-stimulated phosphorylation of Akt and GSK-3ß was sensitive to inhibition of PI3-kinase (LY294002). Phosphorylation of GSK-3ß was also sensitive to Akt-inhibition (API-2), demonstrating CCN2-engendered activation of a PI3-kinase/Akt/GSK-3ß-signaling pathway. A C-terminal peptide fragment of CCN2 (11.2 kD) displayed partial agonist activity, while two short peptides derived from the Thrombospondin- and the IGFBP- homology domains of CCN2, respectively, additively inhibited rhCCN2-stimulated Akt-phosphorylation. The viability of cardiac myocytes subjected to hypoxia/reoxygenation injury or doxorubicin-induced oxidative stress was assessed by assays of adenylate kinase and lactate dehydrogenase released from dying cells. Cardiac myocytes exposed to CCN2 displayed increased tolerance towards hypoxia/reoxygenation and doxorubicin-induced oxidative stress, an effect that was abrogated by inhibition of PI3-kinase. The cytoprotective actions of CCN2 reflected in the transcriptome of CCN2-stimulated cardiac myocytes (anti-apoptosis, stress, and wound-response gene programs). In conclusion, this study discloses the novel findings that cardiac myocytes are CCN2 target cells in which CCN2 increases tolerance towards hypoxia and oxidative stress via PI3-kinase-dependent Akt/GSK-3ß signaling.

Collagen isoform shift during the early phase of reverse left ventricular remodelling after relief of pressure overload.

AIMS: Aortic stenosis induces pressure overload and myocardial remodelling with concentric hypertrophy and alterations in extracellular matrix (ECM). Aortic valve replacement leads to reverse remodelling, a process of which knowledge is scarce. The aims of the present study were to examine alterations in myocardial gene expression and subsequently identify molecular alterations important for the early phase of reverse remodelling. METHODS AND RESULTS: After 4 weeks of ascending aortic banding, mice were subjected to a debanding operation (DB) and followed for 3, 7, or 14 days. Cardiac function was assessed by echocardiography/tissue Doppler ultrasonography. Myocardial gene expression was examined using Affymetrix microarray and the topGO software and verified by real-time polymerase chain reaction. Quantitative measurements of collagen subtypes were performed. Aortic banding increased left ventricular mass by 60%, with normalization to sham level 14 days after DB. Extracellular matrix genes were the most regulated after DB. Three days after DB, collagen I was transiently increased, whereas collagens III and VIII increased later at 7 days. CONCLUSION: The ECM genes were the most altered during reverse remodelling. There was a change in isoform constitution as collagen type I increased transiently at 3 days followed by a later increase in types III and VIII at 7 days after DB. This might be important for the biomechanical properties of the heart and recovery of cardiac function.

Activation of Notch signaling in cardiomyocytes during post-infarction remodeling.

OBJECTIVE: Notch signaling is crucial for cell-to-cell interaction during cardiovascular development and may influence differentiation, proliferation, and apoptotic events. We investigated whether Notch signaling is activated during myocardial remodeling in heart failure (HF). DESIGN: Myocardial gene expression and localization of Notch receptors (Notch1-4) and ligands (Jagged1-2, and Delta-like (Dll)-1 and 4) were investigated in rats with HF after induction of myocardial infarction and in humans with HF. RESULTS: All Notch receptors and ligands investigated and Notch intracellular domain (NICD) were present in rat and human myocardial tissue and in cardiomyocytes with differences in their relative expression levels and altered expression levels in failing vs. non-failing myocardium. In isolated rat cardiomyocytes, Notch3 and Notch4 appeared to be the major Notch receptors, and Notch3 and Notch4 mRNA levels and NICD-3 and -4 in cardiomyocytes were upregulated in chronic HF (p < 0.05), indicating increased Notch3 and Notch4 signaling. CONCLUSION: The Notch signaling system is present in the cardiomyocytes and activated in HF, indicating a role of Notch signaling during myocardial remodeling in HF.

Induction of pulmonary connective tissue growth factor in heart failure is associated with pulmonary parenchymal and vascular remodeling.

OBJECTIVES: Pulmonary remodeling is a well recognized consequence of heart failure (HF). However, the cellular and molecular mechanisms orchestrating the structural alterations of the lungs in HF are poorly understood. We have previously reported induction of the profibrotic peptide connective tissue growth factor (CTGF) in myocardial tissue of rats with HF, suggesting a role of CTGF during myocardial remodeling. The aim of the present study was to explore the potential role of CTGF in pulmonary remodeling in HF. METHODS: Pulmonary tissue samples were obtained from rats with myocardial infarction (MI) subsequent to ligation of the left coronary artery. Real-time quantitative RT-PCR was employed to investigate mRNA levels. The cellular distribution of CTGF was analysed by immunohistochemistry. RESULTS: Seven days after induction of myocardial infarction (MI) and HF in rats we found 2.3-fold and 1.9-fold increase of pulmonary transforming growth factor-beta1 and procollagen alpha1(I) mRNA levels, respectively, and typical morphological characteristics of pulmonary remodeling including interstitial fibrosis and medial thickening of pulmonary arteries. Pulmonary CTGF mRNA levels were substantially elevated in HF rats compared to sham-operated rats (4-fold; P<0.05) and corresponded with similar increase (3-fold; P<0.05) of pulmonary CTGF protein contents. Immunohistochemical analysis revealed increased pulmonary anti-CTGF immunoreactivity in HF, with immunostaining predominantly localized to alveolar macrophages and interstitial fibroblasts. Isolated alveolar macrophages from HF rats demonstrated substantial induction of CTGF mRNA expression (16-fold; p<0.05). Interestingly, platelets caused robust induction of CTGF mRNA expression in alveolar macrophages upon co-culture in vitro. CONCLUSION: Pulmonary CTGF was substantially increased in parallel with pulmonary remodeling in rats with HF. Our data indicate that alveolar macrophages are a major source of increased pulmonary CTGF in HF and that CTGF may be a player in the profibrotic mechanisms associated with HF.