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Department of Pharmacology and Cancer Biology (J.M.C., A.R.M.) and Department of Medicine (L.M., H.A.R., A.R.M.), Duke University Medical Center, Durham, North Carolina 27710
Address all correspondence and requests for reprints to: A. R. Means, Department of Pharmacology and Cancer Biology, Box 3813, Durham, North Carolina 27710. E-mail: means001{at}mc.duke.edu.
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ABSTRACT |
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genes. Included among these isoforms are those that localize to both cytoplasm and nucleus. Collectively, the increased levels of CaMKII isoforms result in a constitutive increase in the Ca2+/calmodulin-independent activity of CaMKII in the ventricles. We conclude that CaMKII is the multifunctional CaMK most likely to mediate Ca2+- dependent protein phosphorylation events in response to TAC-induced cardiac hypertrophy. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Several studies have reported that the hypertrophic response in primary cultures of cardiomyocytes was decreased by agents such as KN-62 (3, 4) that selectively inhibit the three members of the multifunctional calmodulin-dependent kinase (CaMK) family CaMK I, CaMK II, and CaMK IV. Conversely, hypertrophy-inducing stimuli in vitro and in failing human hearts have been reported to increase Ca2+/CaMK activity, although the identity of the multifunctional CaMK responsible remains unclear (3, 5). For example, a correlation exists between the expression of the hypertrophy marker atrial natriuretic factor (ANF) and activation of CaMKII in both cardiomyocyte cultures and in vivo (6, 7). In support of a role for CaMKII, cardiac hypertrophy occurs in transgenic mice that overexpress wild-type (WT) CaMKII (8). On the other hand, primary cultures of cardiac myocytes also develop hypertrophy when they are forced to overexpress constitutively active forms of either CaMKI or CaMKIV (9). Finally, cardiac hypertrophy occurs in transgenic mice that overexpress a constitutively active form of CaMKIV in the ventricles (9). These observations suggest that the development of cardiac hypertrophy can be caused by increased expression of any of the three multifunctional CaMKs.
Cardiomyocytes have been reported to express CaMKI (10), CaMKII (11, 12, 13, 14, 15, 16, 17), and CaMKIV (3, 18), although there is controversy about the presence of CaMKIV in the heart (19, 20, 21). Since the major multifunctional CaMKs are all activated by increases in cytoplasmic Ca2+, exhibit overlapping substrate specificity in vitro (22, 23), and are similarly inhibited by KN-62 (24), it has been difficult to assign specific roles for these kinases in cardiac cells utilizing existing techniques. Our study was designed to clarify roles for the three CaMKs in cardiac hypertrophy by addressing the changes in these enzymes induced in mouse ventricular myocytes by transverse aortic constriction (TAC). TAC was chosen because this experimental technique increases blood pressure overload and leads to development of cardiac hypertrophy that is similar to the response induced by naturally occurring pressure overload in many human patients (25). We conclude that specific isoforms of CaMKII are most likely to play a primary role in development of TAC-induced cardiac hypertrophy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, TAC significantly increased total CaMK activity in ventricular extracts compared with that present in sham-operated mice by 33% (P = 0.011). Thus, TAC does result in an increase in total CaMK activity, a finding common to the other hypertrophic stimuli that have been tested. We concluded that TAC was a suitable stimulus to use in future studies designed to identify the specific CaMK responsible for the increase in total CaMK activity in hypertrophied mouse ventricles.
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was determined in the presence of an excess of calcium and CaM. Thus, this activity is best correlated to the mass of CaMK present in the extract. Of the three multifunctional CaMKs, only CaMKII and CaMKIV become independent of Ca2+/calmodulin (CaM) once activated and demonstrate protein kinase activity when assayed in the presence of EGTA (26, 27). To examine whether the increase in total CaMK activity might include effects on CaMKII or CaMKIV, autonomous activity was measured in ventricular extracts, using the same conditions as above except for the presence of EGTA instead of Ca2+. Figure 1B shows that there is an increase of autonomous activity in TAC extracts compared with controls although the extent of the increase is quite variable. We conclude that TAC is likely to increase the total amount of CaMK in ventricular myocytes and, based on the increase in autonomous activity, that CaMKII or CaMKIV may be affected by the hypertrophic state.
TAC Does Not Increase CaMKI Levels in the Ventricles
The only multifunctional CaMK that fails to develop autonomous activity is CaMKI. To examine whether this enzyme is involved in the response to TAC we first confirmed that CaMKI was present in mouse ventricles (Fig. 2), an observation that agrees with previous reports (10). Since CaMKI remains Ca2+/CaM-dependent, involvement of this enzyme in the hypertrophic response should either be reflected by an increase in its mass or its efficacy in phosphorylating autocamtide-2 relative to the other CaMKs. Regarding the first possibility, examination of CaMKI levels by semiquantitative Western blot shows that, compared with sham-operated mice, TAC does not change CaMKI protein levels (Fig. 2). To examine the second possibility, we determined the relative affinities of CaMKI and CaMKII for phosphorylation of autocamtide-2 and found that the Km for CaMKI activated by CaMKK (CaMK kinase) was actually higher than that for CaMKII (1.0 µM vs. 0.5 µM, respectively). We conclude from these experiments that changes in CaMKI are unlikely to account for the increased CaMK activity in response to TAC.
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) or by in situ hybridization (results not shown). In addition, TAC did not induce CaMKIV expression in the ventricles, suggesting that it is extremely unlikely that CaMKIV plays any role in TAC-induced hypertrophy. However, overexpression of constitutively active CaMKIV in cardiomyocytes in vitro, as well as in transgenic mice, induces cardiomyocyte hypertrophy (9). Therefore, we were concerned that a level of CaMKIV below the level of detection by the two techniques we used might still be important for cardiac hypertrophy. To address this possibility we used a strain of mice null for the Camk4 gene (Camk4-/-), subjected them to TAC, and monitored the hypertrophic response of the ventricles relative to that of WT mice. Seven days after TAC the postsurgical survival rates for WT and Camk4-/- mice were similar (75% and 67% for WT and Camk4-/-, respectively). At the same time point, the transstenotic pressure gradient induced by TAC was also similar in WT and Camk4-/- mice (Table 1), indicating that pressure overload occurs in WT and Camk4-/- mice to the same extent. Moreover, at the 7-d time point TAC resulted in a significant increase in normalized heart and left ventricle mass in Camk4-/- mice, as shown in Table 1, and the increase was very similar to that observed in WT mice (41% and 46%, respectively, in both mouse strains). A two-factor ANOVA analysis (the TAC factor and the genotype factor) indicates that there is no statistical difference for the TAC-genotype interaction. Microscopic observation of left ventricles confirmed the presence of hypertrophy at the myocyte level in both Camk4-/- and WT mice (results not shown). Finally, cardiac function was monitored by echocardiography, and the functional parameters fractional shortening, mean velocity of circumferential shortening, and heart rate in Camk4-/- mice were indistinguishable from WT before or after TAC (data not shown), indicating that CaMKIV does not play a role in the functional adaptation of the ventricles after pressure overload. Therefore, CaMKIV is not required for myocyte hypertrophy or functional adaptation during hypertrophy.
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-actin (sk -A), by Northern blot analysis 7 d after either sham operation or TAC. As shown in Fig. 3, ANF mRNA was expressed in ventricles of embryos (d 17 post coitum), as well as in ventricles of adult WT hypertrophied hearts, but only at very low levels in ventricles of adult WT or Camk4-/- sham-operated mice. TAC increased the expression of ANF in Camk4-/- mice to the same extent as in WT. Similarly, TAC increased the expression of sk -A in Camk4-/- mice to the same level as in WT, which was much higher than in sham-operated Camk4-/- or WT mice (Fig. 3; skeletal muscle RNA is also shown as a positive control). Thus, CaMKIV is not necessary for expression of mRNA from ventricular hypertrophy marker genes in response to TAC.
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TAC Increases CaMKII Levels in the Ventricles
By process of elimination we suspected CaMKII to be the multifunctional CaMK involved in the hypertrophic response to TAC. To begin this investigation we first determined the CaMKII isoforms present in ventricular extracts. CaMKII proteins are encoded by four genes, , ß, , and . Whereas the - and ß-genes are neuron specific, the - and -genes are expressed in most somatic cells. Therefore, we used antibodies specific for the various - and -isoforms of CaMKII to evaluate the pattern of expression in the heart before and after TAC by Western blot. The results shown in Fig. 4A reveal that CaMKII is normally undetectable in ventricles whereas CaMKII is expressed as a doublet of isoforms with the top band being the most abundant. As is clearly evident from Fig. 4A, TAC induces the expression of two CaMKII -isoforms, as well as increases the amount of one of the CaMKII -isoforms, specifically the lower band of the doublet. In fact TAC increases the total levels of CaMKII by 70%, based on quantification of the Western blots by densitometry (the average of three independent experiments). These results provide evidence that TAC increases the total amount of CaMKII protein in the ventricles and also alters the isoforms of the enzyme that are expressed.
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, shows that TAC increases CaMK activity by 39% compared with WT (P = 0.004), indicating that CaMKII is primarily responsible for the increase in total CaMK activity induced by TAC shown in Fig. 1A.
Figure 1B showed a trend toward increased but variable autonomous CaMK activity 7 d after TAC. To determine whether this increase was also due to CaMKII, we used Western blot analysis (middle panel of Fig. 4A) to examine the degree of autophosphorylation of T287, which is the modification of CaMKII responsible for its autonomous activity (26). This autoposphorylated residue of CaMKII can be specifically detected with an antibody against phosphorylated T286 (equivalent to T287 in the and isoforms). Quantification of the Western blots after using the antiphospho-T287 CaMKII antibody revealed that TAC significantly increased phospho-CaMKII in ventricular extracts by 51% compared with sham-operated ventricles (P 
0.012). This result confirms that TAC leads to an increase in the amount of active CaMKII in vivo. Interestingly, the results also show that TAC changes the pattern of autonomously active CaMKII isoforms from a major phosphorylated lower CaMKII band to a major phosphorylated upper CaMKII band (Fig. 4A, middle and right panels). In addition TAC also results in activation of the CaMKII upper band (Fig. 4A, middle and left panels). Thus, TAC not only induces an increase in the total amount and activity of CaMKII, but also influences which isoforms are expressed as well as those that are autonomously active at ambient Ca2+ concentration.
The identity of the different isoforms of CaMKII that undergo changes in the ventricles after TAC cannot be determined by Western blot, since both CaMKII and genes express several mRNA splice variants in the heart, and there are no antibodies available that specifically detect protein derived from the individual splice variants. However, there are specific RT-PCR primers for each of the eight CaMKII mRNA splice variants (17), as well as primers that will produce different size products for several of the CaMKII splice variants. To carry out these experiments we attempted to use equivalent amounts of input mRNA, and included RT-PCR of GAPDH to allow us to correct for any small differences in starting material, therefore providing semiquantitative comparisons. The RT-PCR results showed that overall, the amount of CaMKII mRNA (the sum of the individual splicing variants) increased 2-fold after TAC (Fig. 5A), which correlated well with the increase in CaMKII protein. However, TAC resulted in differential effects on the specific splice variants. For example, the sham-operated ventricles expressed very little of the 3 and 4 variants, but these variants showed the largest fold increases of any after TAC (10- and 60-fold, respectively). On the other hand, four other variants showed TAC-induced changes as 2, 9, 7, and 8 each increased by 2- to 3-fold, whereas the levels of 6 and 10, as measured by quantitation of the band intensity in three separate experiments, failed to show any change in level. The entire experiment to quantify the amounts of each mRNA splice variant produced from the gene was repeated three times and the results were similar in each case.
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by RT-PCR, in this case using a pair of primers common to all the splice variants followed by gel analysis of the size of the products. Figure 5B shows that the mRNAs encoding the splice variants of CaMKII were virtually undetectable in normal hearts (we could only observe a very faint band corresponding to C). However, TAC induced two major bands of mRNA corresponding to C (predicted to be a 536-bp product) and B (predicted to be a 604-bp product). These results indicate that TAC up-regulates several CaMKII splice variants as well as two splice variants. This up-regulation of mRNAs is reflected at the protein level, and some of the CaMKII and isoforms become selectively activated in the ventricular myocyte as evidenced by their increased autophosphorylation. Together the TAC-induced changes in CaMKII mRNAs and proteins lead to selective changes in mass and autonomous activity of both CaMKII and isoforms. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Our analysis of CaMKI reveals that it cannot account for the increase in total CaMK activity that is manifest 7 d after TAC-induced cardiac hypertrophy. However, although CaMKI is not increased in mass or activity after TAC, we cannot exclude the possibility that CaMKI is transiently activated at some time during the 7-d period as both CaMKI and its activator CaMKK are present in mammalian heart (28). Unfortunately, the tools to properly address CaMKI function in the heart [e.g. specific antibodies to phospho-T177 (which is the target of the activator CaMKK) to monitor changes in the state of CaMKI activation (29) or a mouse model with a CaMKI deficiency in the heart] are not available. However, the fact that 1) CaMKI is expressed in all tissues examined (10, 30) and 2) CaMKI activity has been estimated to be only 1/5 to 1/40 of CaMKII activity in the heart (28) suggest that CaMKI may have a general function common in all tissues.
CaMKIV seemed a better candidate to be involved in cardiac hypertrophy because it localizes to the nucleus in most cells in which it is expressed and is involved in the regulation of transcriptional responses (31). One transcription factor critical for initiation of gene expression in cardiomyocytes is MEF-2 (32), and constitutively active forms of CaMKIV can increase transcription from genes containing MEF-2-response elements when expressed ectopically in cells (9, 33). In addition, overexpression of a constitutively active fragment of CaMKIV in ventricular myocytes of transgenic mice is sufficient to induce a hypertrophic response (9). However, we show here that CaMKIV is not required for cardiac function or hypertrophy by analyzing mice null for the unique Camk4 gene. In fact, at least based on the methods we have used, CaMKIV does not appear to be expressed in ventricles of adult mice, an observation entirely consistent with our previous study of mouse embryogenesis that failed to detect CaMKIV mRNA in cardiac primordia or heart at any time during development (34).
What then might explain the observation that targeted overexpression of truncated active CaMKIV results in ventricular hypertrophy? A number of possibilities can be envisioned. First, the truncated form of CaMKIV may be mislocalized and is certainly unregulated in the cell (35). Second, all evidence implicating CaMKIV is based on overexpression of the constitutively active fragment, which may result in altered substrate specificity. For example, CaMKIV can regulate the activity of both CREB and MEF-2 (31). Perhaps these proteins are normally physiologically relevant substrates for other protein kinases (that could be unrelated to CaMKs), which do become activated in response to hypertrophic stimuli. Third, overexpression of an active version of CaMKIV might intersect with and interrupt another signaling pathway. The CaMK cascade (31), comprised of a CaMKIV kinase and CaMKIV, can influence the MAPK (36), protein kinase B (AKT) (37), and protein kinase A (38) pathways when various components of these pathways are overexpressed in cells.
On a positive note, our results clearly show that CaMKII and CaMKII levels are specifically increased during the development of TAC-induced hypertrophy. Moreover, both of these CaMKII genes produce primary transcripts that are alternatively spliced to produce a number of protein isoforms, and a subset of these alternatively spliced mRNAs are specifically up-regulated during hypertrophy. Indeed some of the changes in mRNA splice variants we identify here are similar to those found previously in other hypertrophy models. For instance, 9 is increased in spontaneously hypertensive rats, 2 and 3 are increased in rats overexpressing the human renin gene, and 4 is increased in all the above models as well as in rats overexpressing the mouse renin gene (39). Interestingly 4, which is the only splice variant of the gene reported to be increased in all models of hypertrophy studied, is typically expressed in skeletal muscle (11) and is considered a fetal isoform of CaMKII in the heart (40). Therefore, its induction by TAC may reflect reactivation of the fetal gene program that occurs during cardiac hypertrophy.
Unfortunately, establishing the identity of the protein isoforms produced from the CaMKII mRNA splice variants is quite difficult. For example, note that although eight different CaMKII mRNA splice variants are expressed after TAC (Fig. 5A), only two bands corresponding to CaMKII are visible at the protein level (Fig. 4A). In fact, the 6, 7, 8, and 10 isoforms would be expected to be shorter than the 2 protein, but they are not visible on Western blots using antibodies to either CaMKII or phospho-CaMKII, the later capable of recognizing all phospho-CaMKII isoforms. This suggests that not all of the splice variants may be translated into proteins. On the other hand, the 2 splice variant may produce the CaMKII isoform that increases in mass but decreases its phosphorylation state after TAC (Fig. 4A) since it has been speculated to migrate as the lower band detected with the - specific antibody (17). Why autophosphorylation of this CaMKII isoform would decrease after TAC even though it is increased in mass is not clear. Possibilities include the following: 1) it could have been activated and inactivated before the 7-d time point after TAC that we examined; 2) the subcellular localization might preclude activation at ambient Ca2+ concentrations; 3) it could be associated with one of the protein phosphatases so that inactivation rapidly follows activation; or 4) it will become activated only in response to specific extrinsic signals that raise the intracellular concentration of Ca2+ in close proximity to its cellular location.
We postulate that the CaMKII isoform most relevant to hypertrophy may be the one that exhibits the highest degree of autophosphorylation after TAC, which is the upper band detected by the CaMKII -specific antibody in Fig. 4A. This band has been speculated to be a mix of 3 (also known as B) and 9 (17), and 3 is the isoform most highly increased in humans with dilated cardiomyopathy (41). Relative to cardiac hypertrophy, 3 has two additional characteristics that make it particularly interesting. First, in addition to 7 and 11a (42), which have not been proven to exist at the protein level, 3 is the only mRNA splice variant of CaMKII whose protein product contains a nuclear localization signal, and nuclear localization is required for the CaMKII-mediated activation of the well known hypertrophy marker gene ANF (6). Second, cardiac-specific overexpression of a full-length version of 3 induces cardiac hypertrophy in transgenic mice (8). Collectively, these data suggest that CaMKII 3 may play an important nuclear role in the development of cardiac hypertrophy.
TAC of the mouse also induces the expression of CaMKII isoforms in the ventricles, whose expression during hypertrophy has not, to our knowledge, been previously addressed. Our results reveal that only two mRNA splice variants of CaMKII are increased, namely CaMKII B and C. The correspondence between these mRNA variants and the protein bands identified by Western blot analysis can only be speculative, but based on the RT-PCR product length and protein size, the upper and more autophosphorylated protein should correspond to the B isoform, which contains 23 more residues than C. Neither B nor C contains a nuclear localization sequence, which suggests that CaMKII may also be required in the cytoplasm or in association with membranes during TAC-induced hypertrophy. Perhaps this putative nonnuclear function is to enhance contractility of the heart by modulating excitation-contraction coupling (43).
Our study also demonstrates that TAC results in an increase in autonomously active CaMKII in the ventricles. This conclusion is based on studies employing two independent methods to monitor autonomous activity, i.e. quantification of the CaMKII activity in ventricular extracts measured in the presence of EGTA and Western blot analysis of the degree of T287 phosphorylation, a modification that has been previously shown to be both necessary and sufficient for generation of autonomous activity (26). The Ca2+/CaM-independent activity assay carried out with ventricular extracts has the advantage of being quantitative but the disadvantage of being affected by the presence of other proteins, such as protein phosphatases, in the extract. Indeed, CaMKII has been identified in complex with either PP1 or PP2A in vivo (8, 44); therefore, inclusion of phosphatase inhibitors is critical during the kinase reaction. Although in our experiments a cocktail of protein phosphatase inhibitors was used, phosphatase activity during tissue and extract preparation could also help explain the high variability among different extracts. The second approach has the disadvantage of being only semiquantitative but the advantage that the samples are processed directly into sodium dodecyl sulfate sample buffer so that phosphatase activity is not a concern. An additional advantage of this technique is that it can distinguish differential activation of the various isoforms of CaMKII. For example, TAC results in decreased phosphorylation of the lower band but increased phosphorylation of the upper band of CaMKII (Fig. 4A).
Although how the fine tuning of the autonomous activity of CaMKII isoforms plays a role in TAC-induced hypertrophy remains an open question, we can pose several possibilities. First, the subcellular localization of the different isoforms, such as 3, 7, and 11a that contain a nuclear localization sequence (42), may be an important factor. Second, regulation of the various isoforms may be slightly but significantly different, as has been shown to be the case for isoforms produced from alternatively spliced Drosophila CaMKII mRNAs. In this regard, the amino acid composition of the variable domain of Drosophila CaMKII can alter both the activation by calmodulin and substrate specificity of CaMKII (45). Third, the different isoforms may form complexes with other regulatory or structural proteins, such as phosphatases (44), which would add another level of regulation.
In conclusion, our study shows that CaMKII is the only one of the three multifunctional CaMKs induced in the heart in response to TAC-mediated cardiac hypertrophy. Moreover, we demonstrate for the first time that the total amount of CaMKII protein, the expression of CaMKII isoforms, and regulation of the degree of autonomous activity among the different isoforms change in hypertrophied ventricles as compared with sham-operated controls. Specifically, selective mRNA splice variants that arise from the CaMKII and genes and produce a variety of CaMKII protein isoforms are induced after TAC in the mouse. Thus, CaMKII is regulated by TAC at different levels, which control the amount of mRNA, the manner in which the pre-mRNA is spliced, the amount of enzyme in the ventricles, and the autonomous activity of the enzyme isoforms. We believe these results strongly implicate CaMKII as the multifunctional CaMK most likely to be involved in cardiac hypertrophy and form the framework for further investigation of how changes in CaMKII and cardiac hypertrophy are related.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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TAC
TAC was performed as described previously (25). Since cardiac hypertrophy is maximal by 7 d after TAC (25, 47), mice were anesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg ip) at this time point, the systolic pressure gradient for each mouse was determined by measuring the difference between the right carotid and left axillary arterial systolic pressure (25, 47, 48), and the ventricles were processed as indicated.
Enzyme Assays
Ventricular homogenates were assayed for CaMK activity in 50 µl of a reaction mixture as described previously (7) using 100 µM of the substrate peptide autocamtide-2 or 5 µM of the substrate autocamtide-3 (Calbiochem, La Jolla, CA), as indicated.
Western Immunoblot Analysis
Tissue was homogenized and processed for Western blot analysis as described previously (7). The following antibodies were used: anti-CaMKIV, anti-CaMKII , and anti-CaMKII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), antiphospho-CaMKII (Affinity BioReagents, Inc., Golden, CO), or anti-CaMKI (generated by C. R. Kahl, laboratory of A.R. Means).
RNA Analysis
Ten micrograms of total RNA from microdissected left ventricles, leg skeletal muscle, or embryonic ventricle (17 d post coitum) were analyzed by Northern blot. A riboprobe specific for ANF (49) or probes specific for sk -A or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as described previously (7).
RT-PCR was performed using specific primers for GAPDH and each CaMKII isoform as described previously (17). The specific primers for the CaMKII gene (forward: 5'-AACCAGATGCTGACCATAAACCC-3' and reverse complement: 5'-GGATAGGCTTGCTGTTCTTGGAC-3') were based on the sequences described previously for rat CaMKII (50) (GenBank accession no. J04063) and the products were identified by size based on the sequences described for the CaMKII isoforms by H. Singer (15). All the RT-PCR reactions were done using polyA-RNA purified from tissue by the mRNA isolation system (Promega Corp., Madison, WI).
Statistical Analysis
Data are expressed as mean ± SE. Student’s t test was used to evaluate CaMK activity before and after TAC, except when indicated that the two-factor ANOVA was used. Two factor ANOVA was also used to evaluate the hypertrophic response after TAC. In this case the significance of the overall treatment (TAC) or genotype (Camk4) factor is expressed as the main effect in the table, and the interaction between the two factors is expressed as TAC-genotype interaction. The Statview software (Abacus Concepts, Inc., Berkeley, CA) was used for the analysis. Statistical significance was accepted when P < 0.05.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: ANF, Atrial natriuretic factor; CaM, calmodulin; CaMK, CaM-dependent protein kinase; CaMKK, CaMK kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; sk -A, skeletal muscle -actin; TAC, transverse aortic constriction; WT, wild-type.
Received for publication October 11, 2002. Accepted for publication November 11, 2002.
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T. Zhang, L. S. Maier, N. D. Dalton, S. Miyamoto, J. Ross Jr, D. M. Bers, and J. H. Brown The {delta}C Isoform of CaMKII Is Activated in Cardiac Hypertrophy and Induces Dilated Cardiomyopathy and Heart Failure Circ. Res., May 2, 2003; 92(8): 912 - 919. [Abstract] [Full Text] [PDF]
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