FHL2 binds calcineurin and represses pathological cardiac growth.

Stress-induced hypertrophic growth of the heart predisposes the heart 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 ß-adrenergic signaling and is required for isoproterenol-induced myocardial hypertrophy. Based on these facts, we hypothesized that FHL2 suppresses stress-induced activation of calcineurin. FHL2 is upregulated in mouse hearts exposed to isoproterenol, a ß-adrenergic agonist, and isoproterenol-induced increases in the NFAT target genes RCAN1.4 and BNP were amplified significantly in FHL2 knockout (FHL2(-/-)) mice compared with levels in 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 interleukin 2 (IL-2). Consistent with the in vivo data, small interfering RNA (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 cyclosporine, confirming the calcineurin dependence of the response. Overexpression of FHL2 inhibited activation of both NFAT reporter constructs. Furthermore, NRVMs overexpressing 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 colocalization of FHL2 and calcineurin predominantly at the sarcomere and activation of calcineurin by endothelin-1-facilitated interaction between FHL2 and calcineurin. FHL2 is an endogenous, agonist-dependent suppressor of calcineurin.

Metabolic stress-induced activation of FoxO1 triggers diabetic cardiomyopathy in mice.

The leading cause of death in diabetic patients is cardiovascular disease; diabetic cardiomyopathy is typified by alterations in cardiac morphology and function, independent of hypertension or coronary disease. However, the molecular mechanism that links diabetes to cardiomyopathy is incompletely understood. Insulin resistance is a hallmark feature of diabetes, and the FoxO family of transcription factors, which regulate cell size, viability, and metabolism, are established targets of insulin and growth factor signaling. Here, we set out to evaluate a possible role of FoxO proteins in diabetic cardiomyopathy. We found that FoxO proteins were persistently activated in cardiac tissue in mice with diabetes induced either genetically or by high-fat diet (HFD). FoxO activity was critically linked with development of cardiomyopathy: cardiomyocyte-specific deletion of FoxO1 rescued HFD-induced declines in cardiac function and preserved cardiomyocyte insulin responsiveness. FoxO1-depleted cells displayed a shift in their metabolic substrate usage, from free fatty acids to glucose, associated with decreased accumulation of lipids in the heart. Furthermore, we found that FoxO1-dependent downregulation of IRS1 resulted in blunted Akt signaling and insulin resistance. Together, these data suggest that activation of FoxO1 is an important mediator of diabetic cardiomyopathy and is a promising therapeutic target for the disease.

STIM1-dependent store-operated Ca²âº entry is required for pathological cardiac hypertrophy.

Alterations in intracellular Ca(2+) homeostasis are an important trigger of pathological cardiac remodeling; however, mechanisms governing context-dependent changes in Ca(2+) influx are poorly understood. Store-operated Ca(2+) entry (SOCE) is a major mechanism regulating Ca(2+) trafficking in numerous cell types, yet its prevalence in adult heart and possible role in physiology and disease are each unknown. The Ca(2+)-binding protein, stromal interaction molecule 1 (STIM1), is a Ca(2+) sensor in the sarcoplasmic reticulum (SR), capable of triggering SOCE by interacting with plasma membrane Ca(2+) channels. We report that SOCE is abundant and robust in neonatal cardiomyocytes; however, SOCE is absent from adult cardiomyocytes. Levels of STIM1 transcript and protein correlate with the amplitude of SOCE, and manipulation of STIM1 protein levels (via shRNA) or activity (via expression of constitutively active or dominant-negative mutants) reveals a critical role for STIM1 in activating SOCE in cardiac myocytes. In neonatal hearts a recently identified STIM1 splice variant (STIM1L) is predominant but diminishes with maturation, only to reemerge with agonist- or afterload-induced cardiac stress. To test for pathophysiological relevance, we evaluated both in vitro and in vivo models of cardiac hypertrophy, finding that STIM1 expression is re-activated by pathological stress to trigger significant SOCE-dependent Ca(2+) influx. STIM1 amplifies agonist-induced hypertrophy via activation of the calcineurin-NFAT pathway. Importantly, inhibition of STIM1 suppresses agonist-triggered hypertrophy, pointing to a requirement for SOCE in this remodeling response. Stress-triggered STIM1 re-expression, and consequent SOCE activation, are critical elements in the upstream, Ca(2+)-dependent control of pathological cardiac hypertrophy.