Background: Yap1 regulates cardiac development, yet the function of Yap1 in the adult heart remains unknown. Results: Yap1 promotes cardiomyocyte survival, hypertrophy, and proliferation and protects against chronic myocardial infarction (MI). Conclusion: Yap1 is critical for basal heart homeostasis, and Yap1 deficiency exacerbates myocardial injury. Significance: Increasing cardiomyocyte survival and proliferation may afford protection in vivo against MI injury.

SUMMARY High energy production in mitochondria is essential for maintaining cardiac contraction in the heart. Genes regulating mitochondrial function are commonly downregulated during heart failure. Here we show that both PPARα and Sirt1 are upregulated by pressure overload in the heart. Haploinsufficiency of either PPARα or Sirt1 attenuated pressure overload-induced cardiac hypertrophy and failure, whereas simultaneous upregulation of PPARα and Sirt1 exacerbated the cardiac dysfunction. PPARα and Sirt1 coordinately suppressed genes involved in mitochondrial function that are regulated by estrogen related receptors (ERRs). PPARα bound and recruited Sirt1 to the ERR response element (ERRE), thereby suppressing ERR target genes in an RXR-independent manner. Downregulation of ERR target genes was also observed during fasting, and this appeared to be an adaptive response of the heart. These results suggest that suppression of the ERR transcriptional pathway by PPARα/Sirt1, a physiological fasting response, is involved in the progression of heart failure by promoting mitochondrial dysfunction.

The signaling mechanisms facilitating cardiomyocyte (CM) differentiation from bone marrow (BM)-derived mesenchymal stem cells (MSCs) are not well understood. 5-Azacytidine (5-Aza), a DNA demethylating agent, induces expression of cardiac-specific genes, such as Nkx2.5 and ␣-MHC, in mouse BMderived MSCs. 5-Aza treatment caused significant up-regulation of glycogen synthase kinase (GSK)-3␤ and down-regulation of ␤-catenin, whereas it stimulated GSK-3␣ expression only modestly. The promoter region of GSK-3␤ was heavily methylated in control MSCs, but was demethylated by 5-Aza. Although overexpression of GSK-3␤ potently induced CM differentiation, that of GSK-3␣ induced markers of neuronal and chondrocyte differentiation. GSK-3 inhibitors, including LiCl, SB 216743, and BIO, abolished 5-Aza-induced up-regulation of CM-specific genes, suggesting that GSK-3 is necessary and sufficient for CM differentiation in MSCs. Although specific knockdown of endogenous GSK-3␤ abolished 5-Aza-induced expression of cardiac specific genes, surprisingly, that of GSK-3␣ facilitated CM differentiation in MSCs. Although GSK-3␤ is found in both the cytosol and nucleus in MSCs, GSK-3␣ is localized primarily in the nucleus. Nuclear-specific overexpression of GSK-3␤ failed to stimulate CM differentiation. Down-regulation of ␤-catenin mediates GSK-3␤-induced CM differentiation in MSCs, whereas up-regulation of c-Jun plays an important role in mediating CM differentiation induced by GSK-3␣ knockdown. These results suggest that GSK-3␣ and GSK-3␤ have distinct roles in regulating CM differentiation in BM-derived MSCs. GSK-3␤ in the cytosol induces CM differentiation of MSCs through down-regulation of ␤-catenin. In contrast, GSK-3␣ in the nucleus inhibits CM differentiation through down-regulation of c-Jun.

Rationale Glycogen synthase kinase-3β (GSK-3β) upregulates cardiac genes in bone marrow-derived mesenchymal stem cells (MSCs) in vitro. Ex vivo modification of signaling mechanisms in MSCs may improve the efficiency of cardiac cell based therapy (CRT). Objective To test the effect of GSK-3β upon the efficiency of CBT with MSCs after myocardial infarction (MI). Methods and Results MSCs overexpressing either GSK-3β (GSK-3β-MSCs) or LacZ (LacZ-MSCs) or saline was injected into the heart after coronary ligation. A significant improvement in the mortality and left ventricular (LV) function was observed at 12 weeks in GSK-3β-MSC-injected mice compared to in LacZ-MSC- or saline-injected mice. MI size and LV remodeling were reduced in GSK-3β-MSC-injected mice compared to in LacZ-MSC- or saline-injected ones. GSK-3β increased survival and increased cardiomyocyte (CM) differentiation of MSCs, as evidenced by activation of an Nkx2.5-LacZ reporter and upregulation of troponin T. Injection of GSK-3β-MSCs induced Ki67 positive myocytes and c-Kit positive cells, suggesting that GSK-3β-MSCs upregulate cardiac progenitor cells. GSK-3β-MSCs also increased capillary density and upregulated paracrine factors, including vascular endothelial growth factor A (Vegfa). Injection of GSK-3β-MSCs in which Vegfa had been knocked-down abolished the increase in survival and capillary density. However, the decrease in MI size and LV remodeling, and the improvement of LV function were still observed in MI mice injected with GSK-3β-MSCs without Vegfa. Conclusions GSK-3β significantly improves the efficiency of CBT with MSCs in the post-MI heart. GSK-3β not only increases survival of MSCs but also induces CM differentiation and angiogenesis through Vegfa-dependent and -independent mechanisms.

Rationale Overexpression of muscle atrophy F-box (MAFbx/atrogin-1), an E3 ubiquitin ligase, induces proteasomal degradation in cardiomyocytes. The role of endogenous MAFbx in regulating cardiac hypertrophy and failure remains unclear. Objective We investigated the role of MAFbx in regulating cardiac hypertrophy and function in response to pressure overload. Transverse aortic constriction (TAC) was applied to MAFbx KO and wild type (WT) mice. Methods and Results Expression of MAFbx in WT mice was significantly increased by TAC. TAC-induced increases in cardiac hypertrophy were significantly smaller in MAFbx KO than in WT mice. There was significantly less lung congestion and interstitial fibrosis in MAFbx KO than in WT mice. MAFbx KO also inhibited β-adrenergic cardiac hypertrophy. DNA microarray analysis revealed that activation of genes associated with the transcription factor binding site for the NF-κB family were inhibited in MAFbx KO mice compared with WT mice after TAC. Although the levels of IκB-α were significantly decreased after TAC in WT mice, they were increased in MAFbx KO mice. MAFbx regulates ubiquitination and proteasomal degradation of IκB-α in cardiomyocytes. In primary cultured rat cardiomyocytes, phenylephrine-induced activation of NF-κB and hypertrophy were significantly suppressed by MAFbx knock-down, but were partially rescued by overexpression of NF-κB p65. Conclusions MAFbx plays an essential role in mediating cardiac hypertrophy in response to pressure overload. Downregulation of MAFbx inhibits cardiac hypertrophy in part through stabilization of IκB-α and inactivation of NF-κB. Taken together, inhibition of MAFbx attenuates pathological hypertrophy, thereby protecting the heart from progression into heart failure.