Background: Stress-induced hypertrophic growth of the heart predisposes to arrhythmia, contractile dysfunction, and clinical heart failure. FHL2 (four-and-a-half LIM domain protein 2) is expressed predominantly in the heart, and inactivation of the gene coding for FHL2 leads to exaggerated responsiveness to adrenergic stress. Activation of calcineurin occurs downstream of beta-adrenergic signaling and is required for isoproterenol-induced myocardial hypertrophy. Based on these facts, we hypothesized that FHL2 suppresses stress-induced activation of calcineurin.

Methods and Results: FHL2 is up-regulated in mouse hearts exposed to isoproterenol, a beta-adrenergic agonist, and isoproterenol-induced increases in NFAT target genes RCAN1.4 and BNP were amplified significantly in FHL2 knockout (FHL2-/-) mice as compared with wild-type (WT) mice. To determine whether the effect of FHL2 on NFAT target gene transcript levels occurred at the level of transcription, HEK 293 cells and neonatal rat ventricular myocytes (NRVMs) were transfected with a luciferase reporter construct harboring the NFAT-dependent promoters of either RCAN1 or IL-2. Consistent with the in vivo data, siRNA knockdown of FHL2 led to increased activation of these promoters 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 inhibited activation of both NFAT reporter constructs. Furthermore, NRVMs over-expressing FHL2 exhibited reduced hypertrophic growth in response to constitutively active calcineurin as measured by cell cross-sectional area and fetal gene expression. Finally, immunostaining in isolated adult cardiomyocytes revealed co-localization of FHL2 and calcineurin predominantly at the sarcomere, and activation of calcineurin by endothelin-1 facilitated interaction between FHL2 and calcineurin.

Conclusions: FHL2 in an endogenous, agonist-dependent suppressor of calcineurin.

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