LEOPARD-type SHP2 mutant Gln510Glu attenuates cardiomyocyte differentiation and promotes cardiac hypertrophy via dysregulation of Akt/GSK-3β/β-catenin signaling

Abstract

LEOPARD syndrome (LS) is an autosomal dominant inherited multisystemic disorder. Most cases involve mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase Src homology 2-containing protein phosphatase 2 (SHP2). LS frequently causes severe hypertrophic cardiomyopathy (HCM), even from the fetal period. However, the molecular pathogenesis has not been clearly elucidated. Here, we analyzed the roles of the LS-type SHP2 mutant Gln510Glu (Q510E), which showed the most severe type of HCM in LS, in cardiomyocyte differentiation, and in morphological changes. We generated mutant P19CL6 cell lines, the most convenient cardiomyocyte differentiation model, which continuously expressed SHP2-Q510E, SHP2-D61N (Noonan-type mutant), wild-type SHP2, and green fluorescent protein (native SHP2 expression only). SHP2-Q510E mutant P19CL6 cells showed significant attenuation of myofibrillogenesis, with increased proliferative activity. Mature cardiomyocytes from the SHP2-Q510E mutant were significantly larger than those of controls and the other mutants. However, expression of cardiac-specific transcriptional factors (Gata4, Tbx5, and Nkx2.5) did not differ significantly between the LS-type SHP2-Q510E mutants and the other mutants and controls. Our results indicate that SHP2-Q510E mutants can differentiate into cardiac progenitors but are inhibited from undergoing terminal differentiation into mature cardiomyocytes. In contrast, Akt and glycogen synthase kinase (GSK)-3β phosphorylation were upregulated, and nuclear β-catenin at the late stage of differentiation was highly accumulated in SHP2-Q510E mutant P19CL6 cells. Supplementation with the phosphoinositide 3-kinase/Akt inhibitor LY-294002 during the late stage of differentiation was found to partially restore myofibrillogenesis while suppressing the increase in size of individual mature cardiomyocytes derived from the SHP2-Q510E mutants. Our findings suggest that dysregulation of the Akt/GSK-3β/β-catenin pathway can contribute to the pathogenesis of HCM in LS patients, not only through hypertrophic changes in individual cardiac cells but also via the expansion of cardiac progenitors.

New Approaches to Prevent LEOPARD Syndrome-associated Cardiac Hypertrophy by Specifically Targeting Shp2-dependent Signaling

“…Importantly, our novel findings demonstrate that pharmacological inhibition of particular signaling steps upstream of mTOR could become feasible therapeutic approaches in the clinical arena. Our results complement the recent findings of two other groups showing that inhibition of phosphoinositide 3 kinase with LY294002 or wortmannin is also effective against the prohypertrophic effects of LS mutations in Shp2 (8,9). This is not surprising because PI3K is an important activator of Akt and, in turn, is regulated by FAK (28,29).…”

Noonan and LEOPARD syndrome Shp2 variants induce heart displacement defects in zebrafish

“…Hyperactivation of AKT signaling in response to LS-Shp2 is responsible for HCM in mouse hearts (Ishida et al, 2011;Marin et al, 2011;Schramm et al, 2012). We also observed elevated pAkt levels in response to LS-Shp2 in zebrafish embryos (supplementary material Fig.…”

Elevated Ca2+transients and increased myofibrillar power generation cause cardiac hypercontractility in a model of Noonan syndrome with multiple lentigines

“…Using various in vitro and in vivo models, we and others (8,17,30,43) have previously found that NSML mutations in SHP2 result in increased stimulation of Akt and mammalian target of rapamycin. This leads to enhanced growth signaling and thereby cardiomyocyte hypertrophy.…”