Isoform- and tissue-specific regulation of the Ca²⁺-sensitive transcription factor NFAT in cardiac myocytes and heart failure

Abstract

Nuclear factors of activated T cells (NFATs) are Ca(2+)-sensitive transcription factors that have been implicated in hypertrophy, heart failure (HF), and arrhythmias. Cytosolic NFAT is activated by dephosphorylation by the Ca(2+)-sensitive phosphatase calcineurin, resulting in translocation to the nucleus, which is opposed by kinase activity, rephosphorylation, and nuclear export. Four different NFAT isoforms are expressed in the heart. The activation and regulation of NFAT in adult cardiac myocytes, which may depend on the NFAT isoform and cell type, are not fully understood. This study compared basal localization, import, and export of NFATc1 and NFATc3 in adult atrial and ventricular myocytes to identify isoform- and tissue-specific regulatory mechanisms of NFAT activation under physiological conditions and in HF. NFAT-green fluorescent protein fusion proteins and NFAT immunocytochemistry were used to analyze NFAT regulation in adult cat and rabbit myocytes. NFATc1 displayed basal nuclear localization in atrial and ventricular myocytes, an effect that was attenuated by reducing intracellular Ca(2+) concentration and inhibiting calcineurin, and enhanced by the inhibition of nuclear export. In contrast, NFATc3 was localized to the cytoplasm but could be driven to the nucleus by angiotensin II and endothelin-1 stimulation in atrial, but not ventricular, cells. Inhibition of nuclear export (by leptomycin B) facilitated nuclear localization in both cell types. Ventricular myocytes from HF rabbits showed increased basal nuclear localization of endogenous NFATc3 and reduced responsiveness of NFAT translocation to phenylephrine stimulation. In control myocytes, Ca(2+) overload, leading to spontaneous Ca(2+) waves, induced substantial translocation of NFATc3 to the nucleus. We conclude that the activation of NFAT in adult cardiomyocytes is isoform and tissue specific and is tightly controlled by nuclear export. NFAT is activated in myocytes from HF animals and may be secondary to Ca(2+) overload.

Activation of NFATc1 is directly mediated by IP3 in adult cardiac myocytes

“…The average NFAT Nuc /NFAT Cyt ratios were 0.34 Ϯ 0.03 (n ϭ 66) for atrial myocytes and 0.59 Ϯ 0.05 (n ϭ 23) for ventricular myocytes. The basal nuclear localization of NFATc1 is consistent with our recent data from adult cat myocytes (28). The cytoplasmic distribution of NFATc3 is due to the enhanced regulation of this isoform by nuclear export processes (26,29).…”

“…The cells were infected with adenoviruses (Ad) for NFATc1-GFP or NFATc3-GFP (28) and were imaged 48 h after infection. NFAT-GFP fusion proteins are widely used to study NFAT in living cells because they behave similarly to endogenous proteins (4,9,14,18,26,28,29).…”

“…The rephosphorylation by cellular kinases (e.g., p38, JNK2, and GSK3) inactivates NFAT and initiates export back to the cytoplasm (25,33). In addition to these enzymatic activities, the precise regulation of NFAT in muscle cells also depends on NFAT isoform, cell and tissue type (28,29 release from endogenous stores through IP 3 receptors (IP 3 Rs). Neurohumoral agonists [e.g., angiotensin II (ANG II), endothelin-1 (ET-1), and phenylephrine (Phe)] stimulate G q protein-coupled receptors, a pathway that has been suggested to activate NFAT in cardiac myocytes (19,21).…”

Caveolin-3 Overexpression Attenuates Cardiac Hypertrophy via Inhibition of T-type Ca2+ Current Modulated by Protein Kinase Cα in Cardiomyocytes

“…Cav-3 Overexpression Attenuates the Activation of Calcineurin/ NFAT Signaling in Neonatal Ventricular Myocytes-The activation of the Ca 2ϩ -dependent calcineurin-NFAT signaling pathway is involved in Ang-II-induced pathological cardiac hypertrophy (39,40). We hypothesized that the enhanced I Ca, T due to reduced Cav-3 expression will activate the Ca 2ϩ -dependent calcineurin/ NFAT signaling following Ang-II stimulation, and this effect can be reversed with stable Cav-3 expression.…”

Cardiac Hypertrophy Is Inhibited by a Local Pool of cAMP Regulated by Phosphodiesterase 2

“…Although NFAT has not been reported to be part of a macromolecular complex organized by AKAPs, there is some evidence that this transcription factor also resides in distinct cytosolic domains and that its regulation occurs locally. 47 Based on the above observations, a possible scenario is therefore that PDE2 controls a pool of cAMP that activates an AKAP-bound PKA type II by guest on May 11, 2018 http://circres.ahajournals.org/ Downloaded from to phosphorylate a local NFAT, possibly counterbalancing its dephosphorylation by a local Ca 2+ -calmodulin-dependent phosphatase. Further studies are required to explore this hypothesis.…”