CaM kinase signaling induces cardiac hypertrophy and activates the MEF2 transcription factor in vivo (original) (raw)
Activation of hypertrophic-responsive gene promoters by CaMKI and -IV. To begin to investigate the potential role of CaMK signaling in cardiac hypertrophy, we tested whether activated forms of CaMKI and CaMKIV, lacking the COOH-terminal regulatory region required for Ca2+/calmodulin-dependent regulation, could activate the promoters of the ANF and α-skeletal actin genes linked to luciferase in transiently transfected cardiomyocytes. Consistent with the known responsiveness of these promoters to hypertrophic signals, activity of both promoters was upregulated by CaMKI and CaMKIV (Figure 1). These promoters were also activated to comparable levels by calcineurin (Figure 1). Expression of CaMKI and IV together did not result in additional activation of the ANF or α-skeletal actin promoters above that seen with either kinase alone. In contrast, the maximal effects of calcineurin and CaMKI or -IV were additive. These results suggested that these CaMKs acted through a common pathway to activate the hypertrophic response and that this pathway was separate from the calcineurin pathway.
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.
To investigate further the possible involvement of calcineurin in hypertrophic signaling by CaMKI and -IV, we tested the effects of CsA on induction of the ANF and α-skeletal actin promoters by these kinases (Figure 1). CsA completely blocked hypertrophic signaling by calcineurin and, unexpectedly, partially affected activation by CaMKI and -IV. In the presence of both calcineurin and these CaMKs, CsA reduced expression of the ANF and α-skeletal actin promoters approximately to the level observed with CsA and CaMKI or -IV alone, which was substantially higher than the level seen with CsA and calcineurin. We conclude that the calcineurin and CaMK pathways are cooperative and that calcineurin may be required for maximal responsiveness to CaMK, but calcineurin activation cannot account for the complete response to CaMKI and -IV.
Creation of αMHC-CaMKIV transgenic. In light of the ability of CaMKI and -IV to induce hypertrophic-responsive promoters in primary cardiomyocytes, we extended our studies to investigate whether CaMK signaling could also induce cardiac hypertrophy in vivo, by generating mice that expressed activated CaMKIV in the heart, under control of the αMHC promoter. Five founders carrying the CaMKIV transgene were obtained. One of the founders died at 3 weeks of age with an estimated copy number of 50. Three of the surviving lines had a single copy of the transgene, and one line had three copies. Founder transgenic mice were bred to FVB mice to generate F1 offspring. Transgene expression was determined by Northern analysis using a probe specific to the coding region of human CaMKIV. All transgenic lines expressed the CaMKIV transgene in the heart (data not shown).
The level of expression of CaMKIV protein in hearts of αMHC-CaMKIV transgenic mice was determined by immunoprecipitation and Western blot analysis. As a positive control, parallel assays were performed on extracts from brain, the tissue with highest levels of CaMKIV expression (39). As seen in Figure 2, immunoprecipitations of brain extracts with CaMKIV antibody yielded the predicted 61-kDa CaMKIV protein. To determine whether the CaMKIV transgenic mice expressed the truncated CaMKIV protein, heart extracts from transgenic mice were immunoprecipitated with anti-CaMKIV or anti-Flag mAb and immunoblotted with anti-CaMKIV. The CaMKIV antibody used in these experiments recognizes the NH2-terminus of CaMKIV and therefore detects the endogenous as well as the truncated CaMKIV protein. Both anti-Flag and anti-CaMKIV immunoprecipitations of the transgenic heart extracts demonstrated clearly the expected 40-kDa CaMKIV truncated protein. The exogenous CaMKIV protein was expressed at a level approximating the level of CaMKIV expression in brain, which has been reported to be 50- to 100-times higher than the level in heart (39). The immunoglobulin heavy chain band migrates slightly below the predicted position of endogenous CaMKIV. This precluded reliable detection of the low level of endogenous CaMKIV protein in cardiac extracts.
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.
Cardiac hypertrophy in vivo in response to activated CaMKIV expression. Examination of the hearts of αMHC-CaMKIV transgenic mice beginning at 1 month of age revealed moderate enlargement. At 8, 12, and 24 weeks of age, the heart weight/body weight ratios of the transgenics were significantly increased by 28%, 38% and 25%, respectively (Figure 3a and Figure 4).There was no difference between body weight from wild-type and transgenic mice, indicating that the increases of heart weight/body weight ratios were due to an increase in heart weight. In all four transgenic lines, an increase of heart weight/body weight ratio was observed, although the rate of progression of cardiac disease was most severe in the line with three copies of the transgene. The transgenic line with an estimated 50 copies of the transgene, which died at 3 weeks of age, showed extreme dilated cardiomyopathy (data not shown). The early lethality in this animal and the fact that viable transgenic lines had only one to three copies of the transgene may indicate that activated CaMKIV is a highly potent hypertrophic stimulus that can only be tolerated at relatively low levels. Cardiomyocyte areas were not increased in 6-week-old CaMKIV transgenic hearts, moderately increased at 8 weeks, and significantly increased at 20 weeks of age (Figure 3b and Figure 4f), indicating a slow progression of cardiac hypertrophy, as suggested by the heart weight/body weight ratios.
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. A_P_ < 0.05 versus WT animals.
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.
We examined expression of the hypertrophic-responsive cardiac genes, ANF and αMHC, in CaMKIV transgenic mice by Northern analysis of RNA from heart. As shown in Figure 3c, ANF transcripts were dramatically upregulated (24-fold), whereas αMHC was downregulated (12-fold) in hypertrophic transgenic hearts. GAPDH transcripts were measured to correct for differences in RNA amounts between the samples (Figure 3c).
Transthoracic echocardiography in CaMKIV transgenic mice. On the basis of histological sections from CaMKIV transgenic hearts at 2 months of age, we observed an increase in wall thickness without significant increases of the inner ventricular radius, consistent with parallel sarcomere replication in concentric hypertrophy (40). However, at 6 months of age, cardiac wall thickening was frequently accompanied by ventricular dilation, suggesting progression from concentric hypertrophy to a dilated hypertrophic phenotype. To correlate abnormalities further in cardiac structure (as observed in histological section) and function, we measured wall thickness, ventricular diameter, and cardiac function by transthoracic echocardiography in 3- and 6-month-old transgenic mice and wild-type littermates. At 3 months, IVSD, LVEDD, and LVPWD in transgenic mice were increased by 18%, 20%, and 29%, respectively. Furthermore, in transgenic mice, LVESD was increased by 27%, and the calculated LVm was increased by 77%, whereas the heart rate (HR) was decreased by 23%. Although cardiac function, measured by FS, was not significantly different in wild-type and transgenic mice at 3 months of age, a trend toward decreased FS was observed in transgenic mice (Table 1).
Echocardiographic parameters for wild-type (WT) and CaMKIV transgenic mice
At 6 months of age, LVEDD and LVESD in transgenic mice were significantly increased by 21% and 64%, respectively (Figure 5 and Table 1). Accordingly, FS was decreased by 37% in transgenic mice. The calculated LVm was also increased by 43% in 6-month old transgenic mice (Table 1). These data demonstrate that early-onset hypertrophy at 3 months of age in CaMKIV transgenic mice, with no significant change in cardiac function, is accompanied by a moderate increase in left ventricular chamber dilation. By 6 months, cardiac dysfunction progresses to dilated cardiomyopathy with pronounced left ventricular chamber dilation, and significantly reduced FS.
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.
Synergy between CaMKIV and calcineurin/NFAT3 pathways in vivo. Previously, we showed that expression of a constitutively active mutant form of NFAT3, called NFATΔ317, in the heart resulted in hypertrophy (12). To begin to investigate the potential relationship between the CaMKIV and NFAT signaling pathways, we intercrossed mice expressing the αMHC-CaMKIV and αMHC-NFATΔ317 transgenes. At 6–8 weeks of age, hypertrophy in each line was relatively modest, whereas in the double transgenics, hypertrophy was greatly enhanced (Figure 6). Several attempts were also made to intercross αMHC-calcineurin and αMHC-CaMKIV transgenic mice. We were only able to obtain one double transgenic mouse, which displayed severe dilated cardiomyopathy at 3 weeks of age. None of the CaMKIV or the calcineurin transgenics displayed this cardiac phenotype at 3 weeks of age (data not shown).These results suggest that the CaMKIV and calcineurin/ NFAT3 pathways can synergize to control cardiac growth. Hypertrophy in response to NFATΔ317 is less pronounced than for activated calcineurin, which is likely to explain why NFATΔ317/CaMKIV double transgenics showed greater viability than calcineurin/CaMKIV mice.
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). A_P_ < 0.05 versus WT animals.
CaMK signaling specifically stimulates activity of the MEF2 transcription factor in vivo. MEF2 transcription factors regulate numerous cardiac genes and have been shown to act as end points in Ca2+-dependent signaling pathways (reviewed in ref. 29). To determine whether MEF2 might be a downstream target for CaMKIV signaling in the heart, we intercrossed the αMHC-CaMKIV transgenics with MEF2-indicator mice, which harbor a lacZ transgene under transcriptional control of three tandem copies of the MEF2 consensus binding site (34). This MEF2-dependent lacZ reporter gene is expressed throughout the embryonic heart (34), reflecting the important role of MEF2 factors in activation of muscle-specific gene expression during development (41).
Although MEF2 protein is expressed at high levels in the adult heart (42, 43), the MEF2-dependent lacZ transgene was not expressed above background levels in the heart after birth (Figure 7a), consistent with the notion that MEF2 factors require specific signaling events or cofactors for activation. Indeed, the MEF2-lacZ transgene was upregulated to extremely high levels of expression throughout the heart when it was introduced by breeding into the αMHC-CaMKIV transgenic line (Figure 7a). Quantitative β-galactosidase assays on cardiac extracts showed a greater than 100-fold increase in expression of the lacZ transgene in the heart in response to CaMKIV (Figure 7b), demonstrating that CaMK signaling is a potent inducer of MEF2 activity in cardiomyocytes in vivo.
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.
To assess the specificity of the response of the MEF2-lacZ reporter to CaMK signaling, we assayed its expression in αMHC-calcineurin transgenic mice, which show a much more profound hypertrophic response than the αMHC-CaMKIV transgenics (12). Despite the extreme hypertrophy in αMHC-calcineurin transgenics, the MEF2-lacZ transgene was activated only about eightfold in hearts from these mice, based on quantitative β-galactosidase assays (Figure 7b). In contrast to αMHC-CaMKIV transgenics, which showed extremely high lacZ staining throughout the heart, staining was observed only sporadically in cardiomyocytes from αMHC-calcineurin transgenics, as seen in histological cross-sections (Figure 7a).
In transgenic mice bearing the hsp-lacZ transgene linked to multimers of a mutant MEF2 site, there was no lacZ expression (data not shown). These results demonstrate the dependence of transgene expression on MEF2 binding in vivo.
CaMK signaling does not alter MEF2 DNA binding activity in vivo. We next investigated whether the dramatic increase in MEF2 transcriptional activity in response to CaMK signaling was accompanied by an increase in MEF2 DNA binding activity. Extracts were prepared from wild-type, αMHC-CaMKIV, and αMHC-calcineurin transgenic mice and tested for MEF2 DNA binding activity by gel mobility shift assays with a 32P-labeled MEF2 binding site as probe. The level of MEF2 DNA binding activity was comparable in cardiac extracts from wild-type, αMHC-CaMKIV, and αMHC-calcineurin (Figure 7c) mice. Thus, despite a greater than 100-fold increase in transcriptional activity of MEF2 in hearts from αMHC-CaMKIV transgenics, there appeared to be no difference in MEF2 DNA binding activity, suggesting that CaMK signaling activates preexisting MEF2 protein.
To confirm that all binding activity observed with this assay was attributable to MEF2, we performed antibody “supershift” assays. In the presence of anti-MEF2A antibody, the entire MEF2-DNA complex was supershifted to a slower-migrating ternary complex, indicating that all of the MEF2 binding activity is composed of either MEF2A homo- or heterodimers (Figure 7c). Previous studies have demonstrated that this antibody is specific for MEF2A and does not recognize other MEF2 isoforms (our unpublished observations). Western blot analysis of cardiac extracts with anti-MEF2A antibody also confirmed that there was no difference in the amount of MEF2 protein in extracts from wild-type and hypertrophic mice (data not shown).