PI3K as Mediator of Apoptosis and Contractile Dysfunction in TGFß1-Stimulated Cardiomyocytes.

BACKGROUND: TGFß1 is a growth factor that plays a major role in the remodeling process of the heart by inducing cardiomyocyte dysfunction and apoptosis, as well as fibrosis thereby restricting heart function. TGFß1 mediates its effect via the TGFß receptor I (ALK5) and the activation of SMAD transcription factors, but TGFß1 is also known as activator of phosphoinositide-3-kinase (PI3K) via the non-SMAD signaling pathway. The aim of this study was to investigate whether PI3K is also involved in TGFß1-induced cardiomyocytes apoptosis and contractile dysfunction. METHODS AND RESULTS: Incubation of isolated ventricular cardiomyocytes with TGFß1 resulted in impaired contractile function. Pre-incubation of cells with the PI3K inhibitor Ly294002 or the ALK5 inhibitor SB431542 attenuated the decreased cell shortening in TGFß1-stimulated cells. Additionally, TGFß-induced apoptosis was significantly reduced by the PI3K inhibitor Ly294002. Administration of a PI3Kγ-specific inhibitor AS605240 abolished the TGFß effect on apoptosis and cell shortening. This was also confirmed in cardiomyocytes from PI3Kγ KO mice. Induction of SMAD binding activity and the TGFß target gene collagen 1 could be blocked by the PI3K inhibitor Ly294002, but not by the specific PI3Kγ inhibitor AS605240. CONCLUSIONS: TGFß1-induced SMAD activation, cardiomyocyte apoptosis, and impaired cell shortening are mediated via both, the ALK5 receptor and PI3K, in adult cardiomyocytes. PI3Kγ specifically contributes to apoptosis induction and impairment of contractile function independent of SMAD signaling.

TGFß receptor activation enhances cardiac apoptosis via SMAD activation and concomitant NO release.

UNLABELLED: Transforming growth factor ß (TGFß) expression is induced in the myocardium during transition from compensated hypertrophy to heart failure. In cardiomyocytes, stimulation with TGFß results in restricted contractile function and enhanced apoptosis. Nitric oxide (NO) also induces apoptosis and influences cardiac function. Therefore, we wanted to know whether NO is causally involved in TGFß-induced apoptosis. In isolated ventricular cardiomyocytes of adult rat incubation with TGFß(1) increased NO release which was inhibited by NOS inhibitor ETU but not with iNOS inhibitor (1400 W) or nNOS inhibitor (TFA). In addition, TGFß-induced apoptosis was blocked with ETU and ODQ, but not with 1400 W or TFA. The consequent assumption that endothelial NOS is involved in TGFß-induced NO formation and apoptosis was supported by increased phosphorylation of eNOS at serine 1177 and by the fact that TGFß did not increase NO release in eNOS KO mice. Furthermore, TGFß-induced apoptosis, NO formation, SMAD binding activity and SMAD2 phosphorylation were blocked by a TGFß receptor antagonist, but only apoptosis and NO formation could be blocked with ETU. Expression of SMAD7 was increased after TGFß stimulation and blocked with TGFß receptor antagonist but not after blocking NO synthase with ETU. CONCLUSION: In cardiomyocytes TGFß-induced apoptosis is mediated via TGFß receptor activation that concomitantly activates SMAD transcription factors and the eNOS/NO/sGC pathway. Both of these pathways are needed for apoptosis induction by TGFß. This reveals a new pathway of cardiac NO release and identifies NO as a possible contributor to heart failure progression mediated by TGFß.