Increased regulatory activity of the calcineurin/NFAT pathway in human heart failure

Abstract

Cardiac hypertrophy may initiate progression to a compromised cardiac function. While the clinical consequences of hypertrophy are well understood, only little is known about the underlying molecular pathways. As reported from animal experiments, the Ca(2+)-calmodulin activated phosphatase calcineurin and its downstream transcriptional effector NFAT have been implicated as transducers of the hypertrophic response. To study whether the calcineurin pathway is activated in human heart failure, we investigated samples of human left ventricular myocardium from patients with dilated (idiopathic) cardiomyopathy (DCM, NYHA IV, n=8) in comparison with non-failing controls (NF, n=8). We not only analyzed the pathway by measuring the calcineurin activity, but also by determination of the protein expression of the calcineurin B subunit and additional key markers of the calcineurin signaling cascade (NFAT-3, GATA-4). Calcineurin enzymatic activity was increased by 80% in human dilated cardiomyopathy compared with non-failing human hearts (135.424+/-11.69 and 83.484+/-1.81 nmol Pi/min per microl). This was in line with increased protein expression of calcineurin B in DCM (71.18+9.11 vs. 46.41+/-11.23 densitometric units (DU)/microg protein). In order to verify the activated calcineurin pathway as described in animal models, we compared the protein expression of NFAT-3 in homogenates within nuclear extracts. In nuclear extracts the protein level of NFAT-3 was increased in dilated cardiomyopathy compared with non-failing myocardium (104.01+/-8.85 vs. 71.47+/-8.79 DU/microg protein). In contrast, in homogenates the expression of NFAT-3 was higher in the non-failing tissue indicating subcellular redistribution (19.56+/-3.36 vs. 25.84+/-3.16 DU/microg protein). The protein expression of GATA-4 was increased in DCM (43.14+/-2.89 vs. 29.87+/-2.17 DU/microg protein). In human heart failure (DCM) the calcineurin signaling pathway is activated not only by an increased activity of calcineurin and expression of GATA-4, but also by the shift from dephosphorylated NFAT-3 to the nucleus indicating subcellular redistribution and regulatory activation.