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.

Connective tissue growth factor/CCN2 attenuates ß-adrenergic receptor responsiveness and cardiotoxicity by induction of G protein-coupled receptor kinase-5 in cardiomyocytes.

Myocardial connective tissue growth factor (CTGF/CCN2) is induced in heart failure, a condition associated with diminution of ß-adrenergic receptor (ß-AR) responsiveness. Accordingly, we aimed to investigate whether CTGF could play a mechanistic role in regulation of ß-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 ß1-AR and ß2-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 ß-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 ß-AR sensitivity, Chinese hamster ovary cells pretreated with rec-hCTGF displayed increased agonist- and biased ligand-stimulated ß-arrestin binding to ß-ARs. Despite increased sensitivity of cardiomyocytes from GRK5-knockout (KO) mice to ß-adrenergic agonists, pretreatment of GRK5-KO cardiomyocytes with rec-hCTGF, as opposed to cardiomyocytes from wild-type mice, did not alter ß-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 ß-AR responsiveness in cardiomyocytes involving CTGF-mediated regulation of GRK5.

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.