of phosphoinositide 3-kinase signaling in developing heart: effect of acute ␤-adrenergic stimulation.

Our results demonstrate that the peripheral administration of Wt lin(-)c-kit(+) stem cells restores ventricular function and promotes angiogenic response following MI. These benefits were abrogated in MI mice receiving Akt-1(-/-) stem cells, suggesting the pivotal role of Akt-1 in mediating stem cells to protect MI hearts.

Diabetic nephropathy is associated with mesangial ECM (extracellular matrix) accumulation. We have shown that AT-1R [Ang II (angiotensin II) type I receptor] signalling induces ECM proteins via transactivation of PI3K (phosphoinositide 3-kinase) in mesangial cells. In the present study, we examined the mechanisms underlying the effect of high ambient glucose on cell proliferation and ECM expansion in a mesangial context. High glucose induced increases in PI3K activity, proliferation and ECM accumulation in mesangial cells. These effects were abrogated by losartan, an AT-1R antagonist, but not by [Sar1,Thr8]-Ang II (Sar is sarcosine), an inactive analogue of Ang II, or by a neutralizing antibody against Ang I/II. Overexpression of a constitutively active PI3Kalpha or AT-1R alone was sufficient to induce similar changes by high glucose. In contrast, overexpression of an inactive AT-1R lowered the basal levels and rendered the cells non-responsive to high glucose. Moreover, cells overexpressing wild-type AT-1R had enhanced sensitivity to acute Ang II stimulation. These cells, however, did not respond to conditioned medium obtained from mesangial cells cultured in high glucose. We further demonstrated that iAng (intracellular Ang II) can be induced by high glucose but only under certain conditions. Efficient suppression of iAng by short hairpin RNA against angiotensinogen, however, did not affect high glucose-induced effects on MES-13 cells. These results suggest that high ambient glucose induces activation of AT-1R in an Ang II-independent manner to transactivate PI3K, resulting in proliferation and ECM accumulation in mesangial cells.

Cardiomyocyte development switches from hyperplasmic to hypertrophic growth between postnatal days 3 and 4 in rats. The mechanisms responsible for this transition have been controversial. beta-Adrenergic receptor (betaAR) activation of mitogenic responses in vitro has been reported. We hypothesized that tonic activation of the betaAR signaling regulates cell division in neonatal cardiomyocytes via effects on signaling kinases known to be important in cell cycle regulation. The purpose of the current study was to elucidate the roles of betaAR in rat cardiomyocyte growth in vivo. We demonstrated that betaAR blockade induced a significant reduction in cardiomyocyte proliferation as measured by the BrdU labeling index. Blockade of betaAR did not affect p38 or p44/42 MAPK activities. We further demonstrated that betaAR blockade induced a prompt deactivation of the p70 ribosomal protein S6 kinase (p70 S6K). To confirm these results, we measured p70 S6K activity directly. Basal activity of p70 S6K in neonatal cardiomyocytes was fourfold higher than that of insulin-treated adult rat liver. The activity of p70 S6K was reduced by 60% within 1 min after betaAR blockade. We conclude that the betaAR are involved in regulation of neonatal cardiomyocyte proliferation and that this mitogenic control may be mediated via the p70 S6K pathway.

Recent studies have demonstrated that the beta2-adrenergic receptor (beta2AR)-Galphai signaling pathway exerts a cardiac antiapoptotic effect. The goals of this study were to determine the intracellular signaling factors involved in beta2AR-mediated protection against doxorubicin-induced apoptosis in H9c2 cardiomyocyte and explore the impact of high ambient glucose on the antiapoptotic effect. Under physiological glucose environment (100 mg/dl), beta2AR stimulation prevented doxorubicin-induced apoptosis, which was attenuated by cotreatment with wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, or transfection of a dominant-negative Akt. Inhibition of Src kinase with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine or cSrc small interfering RNA 32 also attenuated the antiapoptotic effect. Inhibition of platelet-derived growth factor receptor (PDGFR) with AG1296 reversed the beta2AR-induced antiapoptotic effect. Transfection of an active Src cDNA (Y529F) alone was sufficient to render the cells resistant to apoptosis, and the resistance was blocked by wortmannin. Transfection of an active PI3K minigene (iSH2-p110) alone also induced resistance to apoptosis, and the resistance was reversed by an Akt-inhibitor but not by AG1296. High ambient glucose (450 mg/dl) caused two major effects: 1) it significantly reduced betaAR-induced PDGFR phosphorylation, Src kinase activity, and activation of PI3K signaling pathway; and 2) it partially attenuated beta2AR-induced antiapoptotic effect. These data provide in vitro evidence supporting a signaling cascade by which beta2AR exerts a protective effect against doxorubicin-induced apoptosis via sequential involvement of Galphai, Gbetagamma, Src, PDGFR, PI3K, and Akt. High ambient glucose significantly attenuates beta2AR-mediated cardioprotection by suppressing factors involved in this cascade including PDGFR, Src, and PI3K/Akt.