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Substantial evidence suggests that the progressive loss of cardiomyocytes caused by apoptosis significantly contributes to the development of heart failure. beta-Adrenergic receptor activation and subsequent persistent phosphodiesterase 3A (PDE3A) downregulation and concomitant inducible cAMP early repressor (ICER) upregulation (PDE3A/ICER feedback loop) has been proposed to play a key role in the pathogenesis of cardiomyocyte apoptosis. In contrast, insulin-like growth factor-1 can activate cell survival pathways, providing protection against cell death and restoring muscle function. In this study, we found that insulin-like growth factor-1 activates extracellular signal-regulated kinase 5 (ERK5) and inhibits PDE3A/ICER feedback loop. Insulin-like growth factor-1 normalized isoproterenol-mediated PDE3A downregulation and ICER upregulation via ERK5/MEF2 activation, and also inhibited isoproterenol-induced myocyte apoptosis. To determine the physiological relevance of ERK5 activation in regulating PDE3A/ICER feedback loop, we investigated the PDE3A/ICER expression and cardiomyocyte apoptosis in transgenic mice with cardiac specific expression of a constitutively active form of mitogen-activated protein (MAP)/extracellular signal-regulated protein kinase (ERK) kinase 5alpha (MEK5alpha) (CA-MEK5alpha-Tg). In wild-type mice, pressure overload- or doxorubicin-induced significant reduction of PDE3A expression and subsequent ICER induction. Cardiac specific expression of CA-MEK5alpha rescued pressure overload- or doxorubicin-mediated PDE3A downregulation and ICER upregulation and inhibited myocyte apoptosis as well as subsequent cardiac dysfunction in vivo. These data suggest that preventing the feedback loop of PDE3A/ICER by ERK5 activation could inhibit progression of myocyte apoptosis as well as cardiac dysfunction. These data suggest a new therapeutic paradigm for end stage of heart failure by inhibiting the PDE3A/ICER feedback loop via activating ERK5.

Rationale: Cardiac dysfunction is accelerated in DM patients after MI. Previously, we reported the critical role of ERK5 and CHIP association on CHIP Ub E3 ligase activity, which inhibits inducible cAMP early repressor (ICER)-mediated apoptosis and left ventricle (LV) dysfunction after MI in DM (DM + MI). Yet the regulatory mechanism of ERK5-CHIP has not been established. Objective: Since we found that p90RSK activation was increased in DM heart, we investigated whether p90RSK activation may inhibit ERK5-mediated CHIP activation, and subsequent ICER induction and apoptosis. Methods and Results: The inhibition of p90RSK activation prevented the reduction of ERK5-CHIP binding, CHIP activity, as well as ICER induction and cardiac apoptosis both in vitro after angiotensin II (ang II) stimulation and in vivo after DM + MI. p90RSK and CHIP share a same binding site with ERK5 C-terminal domain (aa571–807), and overexpression of both p90RSK and ERK5 (aa571–807) fragment, but not kinase dead mutant of p90RSK, inhibited ERK5-CHIP association, suggesting the critical role of p90RSK activation on ERK5-CHIP interaction, and competitive nature of p90RSK and CHIP against ERK5 association. Furthermore. LC-MS/MS analysis identified ERK5-S496 as being directly phosphorylated by p90RSK, and ERK5 S496A mutant significantly impaired ang II-mediated inhibition of CHIP Ub ligase activity, suggesting the critical role of Ser-496 phoaphorylation of ERK5 on CHIP activity. Therefore, p90RSK activation is critical for both p90RSK-ERK5 association as well as ERK5-Ser496 phosphorylation, and following disruption of ERK5-CHIP interaction and subsequent inhibition of CHIP Ub ligase activity. The reduction of CHIP Ub ligase activity and LV dysfunction were accelerated both in cardio-specific ERK5 knock out and wild type p90RSK transgenic mice (WT-p90RSK-Tg). Furthermore, double transgenic mice of WT-p90RSK and constitutively active form of MEK5α (specific ERK5 activator) inhibited single WT-p90RSK-Tg-medaited reduction of CHIP Ub ligase activity, LV dysfunction, and improved mortality after MI. Conclusions: These data strongly suggested that p90RSK activation accelerated cardiac dysfunction and apoptosis after DM + MI via inhibiting ERK5-CHIP module.

Shear stress–induced extracellular signal-regulated kinase (ERK)5 activation and the consequent regulation of Kruppel-like factor 2 and endothelial nitric oxide synthase expression represents one of the antiinflammatory and vascular tone regulatory mechanisms maintaining normal endothelial function. Endothelial dysfunction is a major initiator of atherosclerosis, a vascular pathology often associated with diabetes. Small ubiquitin-like modifier (SUMO) covalently attaches to certain residues of specific target transcription factors and could inhibit its activity. We investigated whether H 2 O 2 and AGE (advanced glycation end products), 2 well-known mediators of diabetes, negatively regulated ERK5 transcriptional activity and laminar flow–induced endothelial nitric oxide synthase expression through ERK5 SUMOylation. H 2 O 2 and AGE induced endogenous ERK5 SUMOylation. In addition, ERK5 SUMOylation was increased in the aortas from diabetic mice. ERK5 transcriptional activity, but not kinase activity, was inhibited by expression of Ubc9 (SUMO E2 conjugase) or PIAS1 (E3 ligase), suggesting the involvement of ERK5 SUMOylation on its transcriptional activity. Point-mutation analyses showed that ERK5 is covalently modified by SUMO at 2 conserved sites, Lys6 and Lys22, and that the SUMOylation defective mutant of ERK5, dominant negative form of Ubc9 (DN-Ubc9), and small interfering RNA PIAS1 reversed H 2 O 2 and AGE–mediated reduction of shear stress–mediated ERK5/myocyte enhancer factor 2 transcriptional activity, as well as promoter activity of Kruppel-like factor 2. Finally, PIAS1 knockdown reversed the inhibitory effect of H 2 O 2 in shear stress–induced Kruppel-like factor 2 and endothelial nitric oxide synthase expression. These data clearly defined SUMOylation-dependent ERK5 transcriptional repression independent of kinase activity and suggested this process as among the molecular mechanisms of diabetes-mediated endothelial dysfunction.