AKAP150 participates in calcineurin/NFAT activation during the down-regulation of voltage-gated K(+) currents in ventricular myocytes following myocardial infarction

Abstract

The Ca(2+)-responsive phosphatase calcineurin/protein phosphatase 2B dephosphorylates the transcription factor NFATc3. In the myocardium activation of NFATc3 down-regulates the expression of voltage-gated K(+) (Kv) channels after myocardial infarction (MI). This prolongs action potential duration and increases the probability of arrhythmias. Although recent studies infer that calcineurin is activated by local and transient Ca(2+) signals the molecular mechanism that underlies the process is unclear in ventricular myocytes. Here we test the hypothesis that sequestering of calcineurin to the sarcolemma of ventricular myocytes by the anchoring protein AKAP150 is required for acute activation of NFATc3 and the concomitant down-regulation of Kv channels following MI. Biochemical and cell based measurements resolve that approximately 0.2% of the total calcineurin activity in cardiomyocytes is associated with AKAP150. Electrophysiological analyses establish that formation of this AKAP150-calcineurin signaling dyad is essential for the activation of the phosphatase and the subsequent down-regulation of Kv channel currents following MI. Thus AKAP150-mediated targeting of calcineurin to sarcolemmal micro-domains in ventricular myocytes contributes to the local and acute gene remodeling events that lead to the down-regulation of Kv currents.

Keywords: A-kinase anchoring protein AKAP; Acute transcriptional response; Calcineurin; Myocardial infarction.

Figure 1. AKAP150 anchors low levels of calcineurin in the heart

A–C, Evaluation of expression levels of AKAP150 (A top panel and B) and calcineurin (A mid panel and C). D, Immunoprecipitation of AKAP150 followed by immunoblot detection of AKAP150 (top panel) and CaN_B (mid panel) in heart lysates from wildtype animals. E–F, Densitometry analysis of AKAP150 (E) and CaN_B (F) enrichment. G, Bar plot of phosphatase activity in AKAP150 enriched fractions.

Figure 2. Blunted phenylephrine (PE)-induced NFATc3 translocation in AKAP150^−/− cardiomyocytes

A, Western blot and B, immunofluorescence evaluation of AKAP150 expression and distribution, respectively, in adult cardiomyocytes from Wildtype (WT) and AKAP150^−/− mice. C, Representative images of neonatal cardiomyocytes from WT and AKAP150^−/− mice expressing an NFATc3-tagged with GFP that were cultured under control conditions and in the presence of 100 µM PE. D, Amalgamated data of the percentage of neonatal cardiomyocytes showing NFATc3-GFP nuclear accumulation relative to total number of cells from WT and AKAP150^−/− mice that were treated under control conditions (WT: n = 100; AKAP150^−/−: n = 95) and in the presence of 100 µM PE (WT: n = 226; AKAP150^−/−: n = 160). E, Inhibition of PP2B activity decreases PE-induced NFATc3-GFP nuclear accumulation in WT cardiomyocytes. Bar plot shows percent of WT neonatal cardiomyocytes with NFATc3-GFP nuclear accumulation relative to total number of cells in control (n = 20), PE (n = 35) and PE + CsA (n = 71). *P < 0.05.

Figure 3. Expression of AKAP79 in AKAP150^−/− and AKAP150ΔPIX neonatal cardiomyocytes rescues NFATc3 activation

A, Bar plot of NFAT-GFP nuclear accumulation in response to PE stimulation after over-expression of AKAP79-mCherry in AKAP150ΔPIX neonatal cardiomyocytes (n = 190). B, Representative images showing AKAP150^−/− (top) and AKAP150ΔPIX (bottom) cardiomyocytes co-expressing NFATc3-GFP and AKAP79-mCherry. C, Amalgamated data of NFATc3-GFP nuclear accumulation in response to PE stimulation in AKAP150^−/− and AKAP150ΔPIX neonatal cardiomyocytes over-expressing AKAP79-mCherry (n = 140 cells per condition). *P < 0.05.

Figure 4. Ablation of AKAP150 prevents K_V remodeling in adult cardiomyocytes after MI

A, Western Blot analysis of AKAP150 and PP2B protein expression in WT (n = 3) control and MI (n = 3) hearts. B, Bar plot of cellular PP2B activity (PO released / µg protein) in WT control (n = 3) and MI hearts (n = 3). C, Summary data for transcript levels of K_V1.5, K_V4.2, K_V4.3 and K_V2.1 subunits in WT and AKAP150^−/− control (n = 3) and MI (n =3) hearts. D, Representative blots of immunoreactive bands for K_V1.5, K_V4.2, K_V4.3 and K_V2.1 subunits in control (n = 3) and MI (n =3) hearts from WT and AKAP150^−/− mice and (E) corresponding densitometry data. Representative whole-cell K_V currents and current-voltage relationship of I_trans and I_sust in WT (F and G) and AKAP150^−/− (H and I) control and MI cells (WT: n = 10 and 8 cells for control and MI respectively) from 3 hearts; AKAP150^−/−: n = 8 cells for both control and MI group, from 3 hearts). *P < 0.05.