Abstract
The calcium-activated phosphatase calcineurin has been implicated as a critical intracellular signal transducer of cardiomyocyte hypertrophy. Although previous data suggested the nuclear factor of activated T-cells (NFAT) as its sole transcriptional effector, the absolute requirement of NFAT as a mediator of calcineurin signaling has not been examined in the heart. We therefore investigated the expression and activation profile of NFAT genes in the heart. Four members (NFATc1-c4) are expressed in cardiomyocytes, elicit nuclear translocation upon calcineurin activation, and are able to drive transactivation of cardiac promoter luciferase constructs. To define the necessary function of NFAT factors as hypertrophic transducers, a dominant negative NFAT construct was created, encompassing part of the N-terminal region of NFATc4 containing a conserved calcineurin-binding motif. Cotransfection of this construct dose-dependently abrogated promoter activation, irrespective of the NFAT isoform used, whereas a control construct with the calcineurin-binding motif mutated displayed no such effects. Adenoviral gene transfer of dominant negative NFAT rendered cardiomyocytes resistant toward all aspects of calcineurin or agonist-induced cardiomyocyte hypertrophy, whereas adenoviral gene transfer of the control construct had no discernable effect on these parameters. These results indicate that multiple NFAT isoforms are expressed in cardiomyocytes where they function as necessary transducers of calcineurin in facilitating cardiomyocyte hypertrophy.
Highlights
Heart failure is a leading cause of morbidity and mortality in industrial countries, affecting over 10 million Americans and Western Europeans, with a 5-year mortality approaching 50%
All blots were probed for 18 S to verify quality and equal loading of RNA. These data indicate that transcripts for all four calcineurin-regulated nuclear factor of activated T-cells (NFAT) factors are present in cardiomyocytes, with those for NFATc3 and NFATc4 being present at the highest levels
All cells examined were positive for sarcomeric actin using phalloidin staining, thereby confirming their identity as cardiomyocytes. These results indicate that NFATc1, -c2, and -c3 are sensitive to endogenous calcineurin activation as demonstrated previously for NFATc4 [10] and point toward a potential contribution for all NFAT members in calcineurin signaling in the ventricular cardiomyocyte
Summary
Heart failure is a leading cause of morbidity and mortality in industrial countries, affecting over 10 million Americans and Western Europeans, with a 5-year mortality approaching 50%. Previous data suggested the nuclear factor of activated T-cells (NFAT) as its sole transcriptional effector, the absolute requirement of NFAT as a mediator of calcineurin signaling has not been examined in the heart.
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