Berdymammet Hojayev

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.

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 β-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 β-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.

I n the course of daily life, the heart reacts to alterations in workload demand with changes in contractility and beating rate; over time, robust changes in ventricular volume and mass take place. In the context of pathological triggers, however, cardiac remodeling is ultimately maladaptive and a discrete milestone in disease pathogenesis. The end result is heart failure, a syndrome with 50% 5-year mortality, rivaling the most lethal cancers.

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.