FHL2 Inhibits Calcineurin and Represses Pathological Cardiac Hypertrophy
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Stress-induced cardiac hypertrophy is a hallmark feature of pathological remodeling which, left unchecked, predisposes hearts to arrhythmia and failure. FHL2 is a member of the four-and-a-half LIM domain (FHL) family of proteins expressed predominantly in the heart. Targeted disruption of FHL2 leads to an exaggerated response to beta-agonist (isoproterenol)-induced cardiac hypertrophy. Isoproterenol-induced hypertrophy relies on activation of the calcineurin-NFAT pathway, and inhibition of calcineurin is sufficient to block growth in response to isoproterenol. I also observed that FHL2 is up-regulated in mouse hearts after isoproterenol treatment. Based on this, we hypothesized that FHL2 negatively regulates the calcineurin-NFAT pathway and consequently, the hypertrophic growth response. To determine whether calcineurin signaling is enhanced in the absence of FHL2, wild type (WT) and FHL2 knockout (FHL2-/-) mice were treated with isoproterenol (32 mg/kg/day). We observed a significant increase in isoproterenol-induced expression of the NFAT target genes RCAN1.4 and BNP in FHL2-/- hearts as compared to WT. To determine whether the effect of FHL2 on the abundance of NFAT target gene transcripts was mediated by calcineurin-NFAT-dependent transcription, HEK 293 cells were transfected with luciferase reporter constructs containing the NFAT-driven promoters of either RCAN1 or IL-2. Consistent with the in vivo data, knockdown of FHL2 message using siRNA led to increases in both RCAN1 and IL-2 promoter activities elicited by constitutively active calcineurin or the calcium ionophore, ionomycin. Importantly, activation of the RCAN1 promoter by ionomycin, in control and FHL2 knockdown cells, was abolished by the calcineurin inhibitor cyclosporin A, confirming the calcineurin dependence of the response. Over-expression of FHL2 in HEK 293 cells inhibited the activation of both NFAT reporters triggered by either constitutively active calcineurin or ionomycin. Furthermore, neonatal rat ventricular myocytes over-expressing FHL2 exhibited reduced hypertrophic growth in response to constitutively active calcineurin (measured by cell cross-sectional area and fetal gene expression). Finally, immunostaining of adult cardiomyocytes revealed co-localization of FHL2 and calcineurin predominantly at the sarcomere, and activation of calcineurin by endothelin-1 treatment resulted in interaction between FHL2 and calcineurin as demonstrated by coimmunoprecipitation. These observations demonstrate that FHL2 represses calcineurin-NFAT signaling and thereby suppresses hypertrophic cardiac growth at least in part by interacting with calcineurin and inhibiting its activation.