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, 105 (10), 1395-406

CaM Kinase Signaling Induces Cardiac Hypertrophy and Activates the MEF2 Transcription Factor in Vivo

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CaM Kinase Signaling Induces Cardiac Hypertrophy and Activates the MEF2 Transcription Factor in Vivo

R Passier et al. J Clin Invest.

Abstract

Hypertrophic growth is an adaptive response of the heart to diverse pathological stimuli and is characterized by cardiomyocyte enlargement, sarcomere assembly, and activation of a fetal program of cardiac gene expression. A variety of Ca(2+)-dependent signal transduction pathways have been implicated in cardiac hypertrophy, but whether these pathways are independent or interdependent and whether there is specificity among them are unclear. Previously, we showed that activation of the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin or its target transcription factor NFAT3 was sufficient to evoke myocardial hypertrophy in vivo. Here, we show that activated Ca(2+)/calmodulin-dependent protein kinases-I and -IV (CaMKI and CaMKIV) also induce hypertrophic responses in cardiomyocytes in vitro and that CaMKIV overexpressing mice develop cardiac hypertrophy with increased left ventricular end-diastolic diameter and decreased fractional shortening. Crossing this transgenic line with mice expressing a constitutively activated form of NFAT3 revealed synergy between these signaling pathways. We further show that CaMKIV activates the transcription factor MEF2 through a posttranslational mechanism in the hypertrophic heart in vivo. Activated calcineurin is a less efficient activator of MEF2-dependent transcription, suggesting that the calcineurin/NFAT and CaMK/MEF2 pathways act in parallel. These findings identify MEF2 as a downstream target for CaMK signaling in the hypertrophic heart and suggest that the CaMK and calcineurin pathways preferentially target different transcription factors to induce cardiac hypertrophy.

Figures

Figure 1
Figure 1
CaMKI and -IV activate hypertrophy-responsive cardiac promoters through a calcineurin-independent mechanism. Transient transfection of cardiomyocytes with ANF-luciferase promoter (a) or α-skeletal actin-luciferase (b) and expression vectors encoding activated calcineurin (CN), CaMKIV, or CaMKI in the presence or absence of cyclosporin A (CsA), as indicated. Data are presented as mean ± SEM. All transfections were performed in triplicate.
Figure 2
Figure 2
Western analysis of immunoprecipitated CaMKIV proteins. Brain and heart extracts (500 μg) from wild-type and CaMKIV transgenic (CaMKIV-Tg) mice were either immunoprecipitated with anti-CaMKIV mAb (lanes 2–4) or with anti-Flag mAb (lanes 5 and 6). Immunoprecipitates were separated by SDS-PAGE electrophoresis and subjected to Western analysis using the anti-CaMKIV antibody. As a positive control, 10 μL of Jurkat cells lysate was used (lane 1). ATruncated CaMKIV protein. IgH, immunoglobulin heavy chains.
Figure 3
Figure 3
Cardiac hypertrophy in CaMKIV transgenic mice. (a) Heart weight/body weight ratios (×1,000) from wild-type (WT) and CaMKIV transgenic mice (n = 6 for each group) were measured at 4, 8, 12, and 24 weeks (wk) of age. (b) Myocyte area per nucleus was measured in WT and CaMKIV transgenic hearts at 6, 8, and 20 weeks (n = 6 for each group). (c) Northern hynbridization analysis of ANF and αMHC mRNA levels in WT (n = 5) and CaMKIV transgenic mice (n = 6) at 3 months of age, divided by GAPDH mRNA levels. Data are presented as mean ± SEM. AP < 0.05 versus WT animals.
Figure 4
Figure 4
Hearts from wild-type and CaMKIV transgenic mice. Whole hearts from wild-type (a) and CaMKIV transgenic (b) mice. Hearts from wild-type (c) and CaMKIV transgenic (d) mice, cut at the midsagittal level and parallel to the base. The same sections of wild-type (e) and CaMKIV transgenic (f) hearts are presented at a higher magnification (×40), showing cardiomyocyte enlargement in the transgenic hearts. All hearts were collected from 6-month-old mice. lv, left ventricle; rv, right ventricle.
Figure 5
Figure 5
Transthoracic echocardiography in wild-type and CaMKIV transgenic mice. Representative M-mode images (bottom) and ECG (top) of wild-type (a) and CaMKIV transgenic (b) mice at 6 months of age. IVS, interventricular septum; LV, left ventricle; PW, posterior wall.
Figure 6
Figure 6
Intercrosses between the CaMKIV and NFATΔ317 transgenic mice. (a) Histological sections of wild-type, CaMKIV, NFATΔ317, and CaMKIV + NFATΔ317 transgenic mice at 6 weeks of age. All sections were cut at the midsagittal level and parallel to the base. (b) Heart weight/body weight ratio (×1,000) of wild-type (WT), CaMKIV, NFATΔ317, and CaMKIV + NFATΔ317 at 6–8 weeks of age (n = 5 for each group). AP < 0.05 versus WT animals.
Figure 7
Figure 7
CaM kinase-dependent activation of MEF2 in vivo. (a) Induction of MEF2 activity by CaMKIV in the intact heart. MEF2 indicator mice were bred with mice harboring an αMHC-CaMKIV or αMHC-calcineurin (CN) transgenes, as described in the text. Littermates positive for the lacZ transgene and lacking (left) or containing the CaMKIV or calcineurin transgene were sacrificed at 8 weeks of age, and hearts were stained for lacZ expression. LacZ expression was not detected above background levels in control hearts, whereas lacZ expression was detected throughout the CaMKIV transgenic heart. In αMHC-calcineurin transgenics, lacZ staining was observed sporadically in subsets of hypertrophic cardiomyocytes. This was revealed more clearly in histological cross section (lower panels). (b) β-Galactosidase assays were performed on cardiac extracts from wild-type, αMHC-CaMKIV, and αMHC-calcineurin transgenic mice harboring the MEF2-lacZ transgene, as described in Methods. (c) Extracts were prepared from hearts of wild-type, αMHC-CaMKIV, and αMHC-calcineurin transgenic littermates and used for gel mobility shift assays with a 32P-labeled MEF2 site as probe. Anti-MEF2A antibody was added to assays as indicated. Comparable amounts of MEF2 DNA binding activity were detected in both extracts, and all activity was supershifted with anti-MEF2A antibody. Nonimmune serum had no effect on the MEF2-DNA protein complex (data not shown). Only the region of the gel containing the shifted probe is shown.
Figure 8
Figure 8
Calmodulin-dependent transcriptional pathways for cardiac hypertrophy. Activated calcineurin has been shown to act through NFAT3, which associates with GATA4, to induce hypertrophy (12). Activated CaMK stimulates transcriptional activity of MEF2 through a posttranslational mechanism. Calcineurin only weakly activates MEF2 in the heart (indicated by a broken line).

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