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(Circulation Research. 2003;92:912.)
© 2003 American Heart Association, Inc.

Integrative Physiology

The _C Isoform of CaMKII Is Activated in Cardiac Hypertrophy and Induces Dilated Cardiomyopathy and Heart Failure

Tong Zhang, Lars S. Maier, Nancy D. Dalton, Shigeki Miyamoto, John Ross, Jr, Donald M. Bers, Joan Heller Brown

From the Departments of Pharmacology (T.Z., S.M., J.H.B.) and Medicine (N.D.D., J.R.), University of California, San Diego, La Jolla, Calif; and the Department of Physiology (L.S.M., D.M.B.), Loyola University, Chicago, Ill. Present address for L.S.M. is the Department of Cardiology, Georg-August Universitaet Goettingen, Germany.

Correspondence to Joan Heller Brown, Department of Pharmacology, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0636. E-mail jhbrown{at}ucsd.edu

	Abstract

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Recent studies have demonstrated that transgenic (TG) expression of either Ca²⁺/calmodulin-dependent protein kinase IV (CaMKIV) or CaMKII_B, both of which localize to the nucleus, induces cardiac hypertrophy. However, CaMKIV is not present in heart, and cardiomyocytes express not only the nuclear CaMKII_B but also a cytoplasmic isoform, CaMKII_C. In the present study, we demonstrate that expression of the _C isoform of CaMKII is selectively increased and its phosphorylation elevated as early as 2 days and continuously for up to 7 days after pressure overload. To determine whether enhanced activity of this cytoplasmic _C isoform of CaMKII can lead to phosphorylation of Ca²⁺ regulatory proteins and induce hypertrophy, we generated TG mice that expressed the _C isoform of CaMKII. Immunocytochemical staining demonstrated that the expressed transgene is confined to the cytoplasm of cardiomyocytes isolated from these mice. These mice develop a dilated cardiomyopathy with up to a 65% decrease in fractional shortening and die prematurely. Isolated myocytes are enlarged and exhibit reduced contractility and altered Ca²⁺ handling. Phosphorylation of the ryanodine receptor (RyR) at a CaMKII site is increased even before development of heart failure, and CaMKII is found associated with the RyR in immunoprecipitates from the CaMKII TG mice. Phosphorylation of phospholamban is also increased specifically at the CaMKII but not at the PKA phosphorylation site. These findings are the first to demonstrate that CaMKII_C can mediate phosphorylation of Ca²⁺ regulatory proteins in vivo and provide evidence for the involvement of CaMKII_C activation in the pathogenesis of dilated cardiomyopathy and heart failure.

Key Words: Ca²⁺/calmodulin-dependent protein kinase II • transgenic mice • dilated cardiomyopathy • heart failure

	Introduction

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Multifunctional Ca²⁺/calmodulin-dependent protein kinases (CaM kinases or CaMKs) are transducers of Ca²⁺ signals that phosphorylate a wide range of substrates and thereby affect Ca²⁺-mediated cellular responses.¹ The family includes CaMKI and CaMKIV, monomeric enzymes activated by CaM kinase kinase,^2,3 and CaMKII, a multimer of 6 to 12 subunits activated by autophosphorylation.¹ The CaMKII subunits , ß, , and show different tissue distributions,¹ with the isoform predominating in the heart.^4–7 Splice variants of the isoform, characterized by the presence of a second variable domain,^4,7 include _B, which contains a nuclear localization signal (NLS), and _C, which does not. CaMKII composed of _B subunits localizes to the nucleus, whereas CaMKII_C localizes to the cytoplasm.^4,8,9

CaMKII has been implicated in several key aspects of acute cellular Ca²⁺ regulation related to cardiac excitation-contraction (E-C) coupling. CaMKII phosphorylates sarcoplasmic reticulum (SR) proteins including the ryanodine receptors (RyR2) and phospholamban (PLB).^10–14 Phosphorylation of RyR has been suggested to alter the channel open probability,^14,15 whereas phosphorylation of PLB has been suggested to regulate SR Ca²⁺ uptake.¹⁴ It is also likely that CaMKII phosphorylates the L-type Ca²⁺ channel complex or an associated regulatory protein and thus mediates Ca²⁺ current (I_Ca) facilitation^16–18 and the development of early after-depolarizations and arrhythmias.¹⁹ Thus, CaMKII has significant acute effects on E-C coupling and cellular Ca²⁺ regulation. Nothing is known about the CaMKII isoforms regulating these responses.

Contractile dysfunction develops with hypertrophy, characterizes heart failure, and is associated with changes in cardiomyocyte Ca²⁺ homeostasis.²⁰ CaMKII expression and activity are altered in the myocardium of rat models of hypertensive cardiac hypertrophy^21,22 and heart failure,²³ and in cardiac tissue from patients with dilated cardiomyopathy.^24,25 Whether these changes are causal or secondary to the underlying pathologies and whether they lead to altered phosphorylation of Ca²⁺ regulatory proteins in vivo are not known.

Several transgenic mouse models have confirmed a role for CaMK in the development of cardiac hypertrophy, as originally suggested by studies in isolated neonatal rat ventricular myocytes.^9,26–28 Hypertrophy develops in transgenic mice that overexpress CaMKIV,²⁷ but this isoform is not detectable in the heart,^4,29 and CaMKIV knockout mice still develop hypertrophy after transverse aortic constriction (TAC).²⁹ Transgenic mice overexpressing calmodulin developed severe cardiac hypertrophy,³⁰ later shown to be associated with an increase in activated CaMKII³¹; the isoform of CaMKII involved in hypertrophy could not be determined from these studies. We recently reported that transgenic mice that overexpress CaMKII_B, which is highly concentrated in cardiomyocyte nuclei, develop hypertrophy and dilated cardiomyopathy.³² To determine whether in vivo expression of the cytoplasmic CaMKII_C can phosphorylate cytoplasmic Ca²⁺ regulatory proteins and induce hypertrophy or heart failure, we generated transgenic (TG) mice that expressed the _C isoform of CaMKII under the control of the cardiac-specific -myosin heavy chain (MHC) promoter. Our findings implicate CaMKII_C in the pathogenesis of dilated cardiomyopathy and heart failure and suggest that this occurs at least in part via alterations in Ca²⁺ handling proteins.³³

	Materials and Methods

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Transverse Aortic Constriction (TAC) Surgery
Transverse thoracic aortic constriction was performed as described previously^34,35 on 8-week-old adult C57BL/6 male mice (Jackson Laboratory, Bar Harbor, Maine). At different time points (1 to 7 days) after surgery (n=3 to 5 for each time point), animals were euthanized and the hearts were removed. Left ventricles were weighed and quickly frozen in liquid nitrogen for protein and total RNA extraction.

Generation of CaMKII_C Transgenic Mice and Southern Blot Analysis
Hemagglutinin (HA)-tagged rat wild-type CaMKII_C cDNA (a gift from H. Schulman, Stanford University, Stanford, Calif) was subcloned into the SalI site of pBluescript-based TG vector (a gift from J. Robbins, University of Cincinnati, Cincinnati, Ohio) between the 5.5-kb murine -MHC promoter and a human growth hormone (HGH) polyadenylation sequences. Purified linear transgene fragments were injected into pronuclei of fertilized mouse oocytes. The resultant pups were screened for the presence of the transgene by PCR as described previously,³² using a CaMKII specific primer (5'-TTGAAGGGTGCCATCTTGACA-3') and a TG vector specific primer (5'-GGTCATGCATGCCTGGAATC-3'). To determine the transgene copy number, Southern blot analysis was performed with EcoRI-digested genomic DNA and a ³²P-labeled 1.7 kb EcoRI-SalI -MHC fragment as a probe. Founder mice were bred with C57BL/6 or Black Swiss wild-type (WT) mice to generate transgenic and WT offspring. All procedures were performed in accordance with Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee.

Western Blot and RNA Analysis
Cardiac homogenates were prepared and Western blot analysis was performed as described previously.³² Total RNA was prepared from ventricular tissue using Trizol reagent (GIBCO BRL) and dot blot analysis was performed as described previously.³² Semiquantitative RT-PCR was performed as described previously.³⁶

Immunoprecipitation and Back-Phosphorylation Assays
Cardiac homogenates were prepared, and immunoprecipitation and back-phosphorylation assays were performed as previously described using protein kinase A (PKA)³⁷ or CaMKII (a gift from H. Schulman and A. Hudmon, Stanford University, Calif).

Transthoracic Echocardiography
Transthoracic echocardiography was performed using an Agilent Technologies Sonos 5500 system with a 15 MHz transducer as described.³² Briefly, mice were anesthetized by intraperitoneal injection of 2.5% Avertin (15 µL/g body weight). M-mode and Doppler tracings were recorded at a sweep speed of 150 mm/sec. Measurements were obtained by an examiner blinded to the genotype of the animals.

Histological and Morphometric Analysis
Hearts from TG mice and WT controls were collected and fixed in 4% paraformaldehyde buffered with PBS, routinely dehydrated, and paraffin embedded. Hearts were sectioned at 5 µm and stained with hematoxylin and eosin, and Masson’s Trichrome.

Cardiac Myocyte Isolation and Immunocytochemical Staining
Single mouse ventricular myocytes were isolated from WT and CaMKII_C TG mice as reported.^32,33 Myocytes were measured to determine cell volume (lengthxwidthx40% of width).³⁸ Myocytes were field stimulated at 0.5 Hz (23°C) and twitch shortening was recorded. Immunocytochemical staining was performed as described previously.³²

Statistical Analysis
All data are reported as mean±SEM. Statistical significance of difference between WT and CaMKII_C TG mice was determined using unpaired Student’s t test. A value of P<0.05 was considered statistically significant.

	Results

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Expression and Activation of CaMKII_C Isoform After TAC
To determine whether CaMKII was regulated in pressure-overload–induced hypertrophy, CaMKII expression and phosphorylation were examined by Western blot analysis using left ventricular samples obtained at various times after TAC. A selective increase (1.6-fold) in the lower band of CaMKII was observed as early as 1 day and continuously for 4 days (2.3-fold) and 7 days (2-fold) after TAC (Figure 1A). This lower band has the same mobility as genetically encoded heterologously expressed CaMKII_C (Figure 1A) and corresponds to the expected molecular mass of about 56 kDa.³⁶ Although there are no commercially available antibodies that distinguish among the _B, _C and other splice variants of CaMKII, immunoreactive protein bands at 56 kDa and 58 kDa have been suggested to correspond to CaMKII_C and to CaMKII_B (plus ₉, which differs by only 3 amino acids), respectively.³⁶ To confirm that CaMKII_C was increased and determine whether this occurred at the transcriptional level, we performed semiquantitative RT-PCR using primers specific for the CaMKII_C isoform. These experiments revealed that mRNA levels for CaMKII_C were increased 1 to 7 days after TAC (Figure 1B). In addition to examining CaMKII expression, the activation state of CaMKII was monitored by its autophosphorylation, which confers Ca²⁺-independent activity. Western blots using phospho-CaMKII antibody demonstrated that 2 to 7 days of TAC increased phosphorylation of the _B, _C, and other detectable CaMKII isoforms by 2.3- to 3.8-fold (Figure 1C).

View larger version (32K): [in this window] [in a new window]   Figure 1. Expression and activation of CaMKIIC isoform after TAC. A, Western blot analysis of total CaMKII in left ventricular (LV) homogenates obtained at indicated times after TAC. Cardiomyocytes transfected with CaMKIIB and C (right) served as positive controls and molecular markers. Top band (58 kDa) represents CaMKIIB plus 9, and the bottom band (56 kDa) corresponds to CaMKIIC. *P<0.05 vs control. B, Semiquantitative RT-PCR using primers specific for CaMKIIC isoform (24 cycles) and GAPDH (19 cycles) using total RNA isolated from the same LV samples. C, Western blot analysis of phospho-CaMKII in LV homogenates obtained at various times after TAC. Three bands seen for each sample represent CaMKII subunit (uppermost), CaMKIIB plus 9 (58 kDa), and CaMKIIC (56 kDa). Quantitation is based on the sum of all of the bands. *P<0.05 vs control.

Generation and Identification of CaMKII_C Transgenic Mice
TG mice expressing HA-tagged rat wild-type CaMKII_C under the control of the cardiac-specific -MHC promoter were generated as described in Materials and Methods. By Southern blot analysis, 3 independent TG founder lines carrying 3, 5, and 15 copies of the transgene were identified. They were designated as TGL (low copy number), TGM (medium copy number), and TGH (high copy number), respectively (Figure 2A). The founder mice from the TGH line died at 5 weeks of age with marked cardiac enlargement. The other two lines showed germline transmission of the transgene. The transgene was expressed only in the heart and not in other organs, as determined with the anti-HA antibody (data not shown). The expressed transgene was also demonstrated by anti-HA immunostaining to be confined to the cytoplasm in cardiomyocytes isolated from these mice (Figure 2B). This contrasts with the nuclear localization of the _B isoform³² shown here for comparative purpose. Although CaMKII protein levels in TGL and TGM hearts were increased 12- and 17-fold over wild-type (WT) controls (Figure 2C), the amount of activated CaMKII was only increased 1.7- and 3-fold in TGL and TGM hearts (Figure 2D). The relatively small increase in CaMKII activity in the TG lines probably reflects the fact that the enzyme is not constitutively activated and that the availability of Ca²⁺/CaM, necessary for activation of the overexpressed CaMKII, is limited. Importantly, the extent of increase in active CaMKII in the TG lines was similar to that elicited by TAC.

View larger version (26K): [in this window] [in a new window]   Figure 2. Expression and activation of CaMKII in CaMKIIC transgenic mice. A, Transgene copy number based on Southern blots using genomic DNA isolated from mouse tails (digested with EcoRI). Probe (a 32P-labeled 1.7-kb EcoRI-SalI -MHC fragment) was hybridized to a 2.3-kb endogenous fragment (En) and a 3.9-kb transgenic fragment (TG). Transgene copy number was determined from the ratio of the 3.9-kb/2.3-kb multiplied by 2. B, Immunocytochemical staining of ventricular myocytes isolated from WT and CaMKII TG mice. Myocytes were cultured on laminin-coated slides overnight. Transgene was detected by indirect immunofluorescence staining using rabbit anti-HA antibody (1:100 dilution) followed by FITC-conjugated goat anti-rabbit IgG antibody (1:100 dilution). CaMKIIB localization to the nucleus in CaMKIIB TG mice (see Reference 32) is shown here for comparative purpose. C, Quantitation of the fold increase in CaMKII protein expression in TGL and TGM lines. Different amounts of ventricular protein (numbers) from WT control, TG (+) and their littermates (-) were immunoblotted with an anti-CaMKII antibody. Standard curve from the WT control was used to calculate fold increases in protein expression in TGL and TGM lines. D, Phosphorylated CaMKII in ventricular homogenates was measured by Western blot analysis (n=5 for each group). **P<0.01 vs WT.

Cardiac Overexpression of CaMKII_C Induces Cardiac Hypertrophy and Dilated Cardiomyopathy
There was significant enlargement of hearts from CaMKII_C TGM mice by 8 to 10 weeks (Figure 3A) and from TGL mice by 12 to 16 weeks. Histological analysis showed ventricular dilation (Figure 3B), cardiomyocyte enlargement (Figure 3C), and mild fibrosis (Figure 3D) in CaMKII_C TG mice. Quantitative analysis of cardiomyocyte cell volume from 12-week-old TGM mice gave values of 54.7±0.1 pL for TGM (n=96) versus 28.6±0.1 pL for WT littermates (n=94; P<0.001).

View larger version (123K): [in this window] [in a new window]   Figure 3. Cardiac pathology in CaMKIIC TG mice. Images from WT and TGM mice aged 6 and 13 weeks are shown for (A) whole hearts; (B) histological sections (bar=2 mm); (C) ventricular cross-sections at high-power magnification (indicating cardiomyocyte hypertrophy in CaMKIIC; bar=0.05 mm); and (D) Masson’s trichrome-stained sections (showing mild fibrosis in TGM mice; bar=0.2 mm). WT shown is aged 6 weeks.

As indicated above, the highest expressing founder died at 5 weeks with marked cardiac enlargement. Half of the TGM mice and one quarter of the TGL mice died prematurely, most by 16 weeks of age (Figure 4). Most of the TG mice also displayed other symptoms of heart failure, including pulmonary congestion (26% increase in lung/body weight ratio in TGM line at 12 weeks of age), pleural effusions, atrial dilation and thrombi, and severe edema (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 4. Survival curve for CaMKIIC TG mice. Numbers of mice were as follows: WT, n=29; TGL, n=32; and TGM, n=25.

CaMKII_C overexpression also induced a genetic program associated with hypertrophy and heart failure (Figure 5). Hypertrophic marker genes ANF, ß-MHC, and -skeletal actin mRNA levels were significantly increased in TG ventricles, especially in the TGM line. There were also significant decreases in -MHC, SERCA, and PLB mRNA levels in TG mice, characteristic of failing myocardium.

View larger version (38K): [in this window] [in a new window]   Figure 5. Ventricular gene expression in CaMKIIC TG mice. Ventricular RNA from WT, TGL, and TGM mice (n=8 for WT, n=6 for TGL and TGM) at 12 to 14 weeks of age was subjected to dot blot analysis using gene transcript–specific antisense oligonucleotide probes as indicated. GAPDH was used as the normalizing control. SK.Actin indicates -skeletal actin; CA. Actin, -cardiac actin. *P<0.05 vs WT. **P<0.01 vs WT.

M-mode echocardiography (Figure 6A) confirmed 50% increases in left ventricular mass to body weight ratio by 8 weeks for TGM and 16 weeks for the TGL line (Figure 6B). Ventricular dilation and cardiac dysfunction developed over time in proportion to the extent of transgene expression. Left ventricular end diastolic diameter (LVEDD) was increased by 35% to 45%, left ventricular posterior wall thickness (LVPW) decreased by 26% to 29% and fractional shortening decreased by 50% to 60% at 8 weeks for TGM and at 16 weeks for TGL (Figure 6B). None of these parameters were significantly altered at 4 weeks in TGM or up to 11 weeks in TGL mice, indicating that heart failure had not yet developed.

View larger version (46K): [in this window] [in a new window]   Figure 6. Dilated cardiomyopathy and dysfunction in CaMKIIC TG mice at both whole heart and single cell levels. A, Representative M-mode images of transthoracic echocardiography in TGM mice at 8 weeks of age. B, Averaged echocardiographic parameters for WT and CaMKIIC mice from both TGL and TGM lines at different ages. Data are presented for left ventricular (LV) end-diastolic diameter (EDD), posterior wall thickness (PW), and mass-to-body weight ratio (LVM/BW), and for calculated percent fractional shortening (FS).32 *P<0.05 vs WT. C, Decreased contractile function in ventricular myocytes isolated from 12-week-old TGM and WT controls presented as percent change of resting cell length (RCL) stimulated at 0.5 Hz. Representative trace and mean values are shown. *P<0.05 vs WT.

To assess contractile function at the single cell level, ventricular myocytes were isolated and stimulated at 0.5 Hz. Contractile function was significantly decreased (Figure 6C) in 12-week-old TGM (2.25±0.34% RCL; n=27) versus WT mice (4.19±0.90% RCL; n=22; P<0.05) and average half-relaxation time (RT_50%) was slightly increased in TGM (231±31 ms; n=27) versus WT mice (164±21 ms; n=22; P=0.13).

Cardiac Overexpression of CaMKII_C Results in Changes in the Phosphorylation of Ca²⁺ Handling Proteins
To assess the possible involvement of phosphorylation of Ca²⁺ cycling proteins in the phenotypic changes observed in the CaMKII_C TG mice, we first compared PLB phosphorylation state in homogenates from 12- to 14-week-old TGM and WT littermates. Western blots using antibodies specific for phosphorylated PLB showed a 2.3-fold increase in phosphorylation of Thr17 (the CaMKII site) in hearts from TGM versus WT (Figure 7A). Phosphorylation of PLB at the CaMKII site was also increased 2-fold in 4- to 5-week-old TGM mice (Figure 7B). Significantly, phosphorylation of the PKA site (Ser16) was unchanged in either the older or the younger TGM mice (Figures 7A and 7B).

View larger version (20K): [in this window] [in a new window]   Figure 7. Phosphorylation of PLB in CaMKIIC TG mice. Thr17 and Ser16 phosphorylated PLB was measured by Western blots using specific anti-phospho antibodies. Ventricular homogenates were from 12- to 14-week-old WT and TGM mice (A) or 4 to 5-week-old WT and TGM mice (B). Data were normalized to total PLB examined by Western blots (data not shown here). n=6 to 8 mice per group; *P<0.05 vs WT.

To examine RyR2 phosphorylation status, we used back-phosphorylation assays with purified PKA and ³²P-ATP.³⁷ PKA and CaMKII have been reported to phosphorylate the same site (Ser2809) on the RyR2.¹⁰ As shown in Figure 8A, PKA phosphorylated the immunoprecipitated RyR from 12- to 14-week-old WT mice in a PKI-dependent manner. The extent of back-phosphorylation was significantly less in CaMKII_C TGM mice, indicating that in vivo phosphorylation is higher in the TGM mice. Using the reciprocal of the PKA-dependent back-phosphorylation as an index of in vivo phosphorylation, endogenous RyR2 phosphorylation is 2.5-fold higher in TGM than WT hearts (Figure 8B).

View larger version (79K): [in this window] [in a new window]   Figure 8. Phosphorylation of RyR2 in CaMKIIC TG mice. A, RyR2 was immunoprecipitated (IP’d) from 12- to 14-week-old TGM and WT control mouse hearts and back-phosphorylated with PKA as described in Materials and Methods. PKA inhibitor (PKI, 0.5 µmol/L) was used to define PKA-independent phosphorylation. Phosphorylation of RyR2 was quantified using a Phosphoimager. Bottom panels demonstrate that equal amounts of RyR2 were IP’d from WT and TG hearts and that CaMKII is co-IP’d with the RyR2. B, Relative in vivo RyR2 phosphorylation was calculated as the inverse of the PKA-dependent increase in 32P incorporation (after normalization to the amount of IP’d RyR2). n=4 mice per group; *P<0.05 vs WT. C, RyR2 was IP’d from 4- to 5-week-old TGM and WT control mouse hearts and back-phosphorylated with PKA or CaMKII. PKI was used to define PKA-independent phosphorylation, and KN-93 (10 µmol/L) was used to define CaMKII-independent phosphorylation. D, Equal amounts of RyR2 were IP’d from WT and TG hearts; the amount of CaMKII associated with RyR2 in TG hearts was significantly increased but there was no change in PKA or the phosphatases PP1 or PP2A associated with RyR2 in TG hearts.

To demonstrate that the RyR2 phosphorylation changes observed in the CaMKII transgenic mice are not secondary to development of heart failure, we performed biochemical studies examining RyR2 phosphorylation in 4- to 5-week-old TGM mice. At this age, most mice showed no signs of hypertrophy or heart failure (see Figure 6B) and there was no significant increase in myocyte size (21.3±1.3 versus 27.7±4.6 pL; P=0.14). Also, twitch Ca²⁺ transient amplitude ([Ca²⁺]_i) was not yet significantly depressed, and mean [Ca²⁺]_i (1 Hz) was only 20% lower (192±36 versus 156±13 nmol/L; P=0.47) versus 50% lower in TGM at 13 weeks.³³ The in vivo phosphorylation of RyR2, determined by back-phosphorylation, was significantly (2.1±0.3-fold; P<0.05) increased in these 4- to 5-week-old TGM animals (Figure 8C), an increase equivalent to that seen in 12- to 14-week-old mice. We also performed the RyR2 back-phosphorylation assay using purified CaMKII rather than PKA. RyR2 phosphorylation at the CaMKII site was also significantly increased (2.2±0.3-fold; P<0.05) in 4- to 5-week-old TGM mice (Figure 8C).

CaMKII was found to coimmunoprecipate with the RyR2 (Figures 8A and 8D). The amount of CaMKII in the immunoprecipitates was significantly greater in TG versus WT hearts at both 12 to 14 (Figure 8A) and 4 to 5 weeks (Figure 8D). The association of CaMKII with the RyR2 is consistent with a physical interaction between this protein kinase and its substrate. The catalytic subunit of PKA and the phosphatases PP1 and PP2A were also present in the RyR2 immunoprecipitates, but not different in WT versus TG mouse hearts (Figure 8D). These data provide further evidence that the increase in RyR2 phosphorylation, which precedes development of failure in the 4- to 5-week-old CaMKII_C TG hearts, can be attributed to the increased activity of CaMKII.

	Discussion

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CaMKII is involved in the dynamic modulation of cellular Ca²⁺ regulation and has been implicated in the development of cardiac hypertrophy and heart failure.¹⁴ Published data from CaMK-expressing TG mice demonstrate that forced expression of CaMK can induce cardiac hypertrophy and lead to heart failure.^27,32 However, the CaMK genes expressed in these mice are neither the endogenous isoforms of the enzyme nor the isoforms likely to regulate cytoplasmic Ca²⁺ handling, because they localize to the nucleus. Data presented in this study provide several important advances in our understanding of the role of CaMKII in the heart. First, we demonstrate that the cytoplasmic cardiac isoform of CaMKII is upregulated at the expression level and is in the active state (based on autophosphorylation) after pressure overload induced by TAC. Second, we demonstrate that two cytoplasmic CaMKII substrates (PLB and RyR) are phosphorylated in vivo when CaMKII is overexpressed and its activity increased to an extent seen under pathophysiological conditions. Moreover, CaMKII is found to associate physically with the RyR in the heart. Finally, our data indicate that heart failure can result from activation of the cytoplasmic form of CaMKII and this may be due to altered Ca²⁺ handling.

Differential Regulation of CaMKII Isoforms in Cardiac Hypertrophy
The isoform of CaMKII that predominates in the heart is the isoform.^4–7 Neither the nor the ß isoforms are expressed and there is only a low level of expression of the isoforms.³⁹ Both _B and _C splice variants of CaMKII are present in the adult mammalian myocardium^36,40 and expressed in distinct cellular compartments.^4,8,9 The possibility that these isoforms of CaMKII are differentially regulated and play distinct roles in cardiac physiology is intriguing. One previous report examined changes in CaMKII expression and showed that in a spontaneously hypertensive rat (SHR) model of cardiac hypertrophy the transcript levels of _B and _C were unchanged, whereas the embryonic ₄ and the minor ₉ variants were upregulated.²¹ On the other hand, the expression of the _B isoform appeared to be increased at both the transcript and protein levels in failing human myocardium.²⁵ There is, however, little further information regarding selective regulation of the cardiac isoforms in hypertrophy and failure.

We suggest that the CaMKII isoforms are differentially regulated in pressure-overload–induced hypertrophy, because the expression of CaMKII_C is selectively increased as early as 1 day after TAC. Studies using RT-PCR confirm that CaMKII_C is regulated at the transcriptional level in response to TAC. In addition, activation of both CaMKII_B and CaMKII_C, as indexed by autophosphorylation, increases as early as 2 days after TAC. Activation of CaMKII_B by TAC is relevant to our previous work indicating its role in hypertrophy.^9,32 The increased expression, as well as activation of the CaMKII_C isoform, suggests that it could also play a critical role in both the acute and longer responses to pressure overload.

Phosphorylation State of RyR2 and PLB in CaMKII_C TG Mice
Both PKA and CaMKII can phosphorylate the RyR2 and PLB in the heart.^10,37 It is therefore of interest that mice overexpressing CaMKII_C develop a dilated cardiomyopathy similar to that shown for PKA transgenic mice.⁴¹ There is growing evidence that an altered function of the RyR2 contributes to cardiac dysfunction in heart failure.^15,42 Data from Marks’ laboratory has demonstrated that in failing human hearts, RyR2 is hyperphosphorylated and that consequent dissociation of FKBP12.6 from RyR results in its increased sensitivity to Ca²⁺ and aberrant RyR channel function.³⁷ As demonstrated in the present study, CaMKII as well as PKA associates with the RyR2 and a greater amount of CaMKII is associated with the RyR in CaMKII_C TG mouse hearts, where the RyR2 is highly phosphorylated. These data suggest that CaMKII, like PKA, is part of a macromolecular signaling complex and that it contributes to phosphorylation of the RyR2. Importantly, phosphorylation of RyR2 is found to be increased in young transgenic mice, preceding the development of heart failure. The observation that there is increased CaMKII associated with the RyR2 in these TG mice, whereas PKA and phosphatases are unchanged, provides further evidence that the increased phosphorylation of the RyR2 is due to CaMKII.

We also demonstrate that phosphorylation of the Thr17 site on PLB is increased by CaMKII_C expression in TG hearts. This increase in PLB phosphorylation would be expected to enhance SERCA activity and Ca²⁺ uptake and thus improve contractile function. However, the total amount of SERCA is reduced in CaMKII_C TG hearts at both the mRNA (Figure 5) and protein level.³³ The reduction in SERCA and decrease in SERCA/PLB ratio³³ may dictate the overall phenotype such that SR Ca²⁺ uptake is actually diminished. The observation that there is no increase in phosphorylation of PLB at Ser16, the PKA site, is noteworthy, however, because it suggests that PKA activity is not upregulated (nor phosphatases locally downregulated) in these TG mice.

CaMKII Signaling in Dilated Cardiomyopathy and Heart Failure
It has been previously reported that CaMK activity is increased 3-fold in cardiomyopathic human hearts²⁴ and that the expression of CaMKII is increased 2-fold in failing human myocardium,²⁵ implicating CaMKII in the development of heart failure. We recently reported that transgenic mice overexpressing the nuclear CaMKII_B isoform developed hypertrophy and dilation.³² Notably, the phenotype of the CaMKII_B transgenic mice was relatively mild by comparison to the mice generated in this study. In light of the different subcellular localization of CaMKII_B and _C isoforms, we predicted that CaMKII_C would have specificity for phosphorylating cytoplasmic substrates. Indeed, not only are the RyR2 and PLB phosphorylated in CaMKII_C TG mice, but associated Ca²⁺ handling defects are evident in cardiomyocytes isolated from these mice.³³ A significant result of the increased phosphorylation of RyR2 is increased Ca²⁺ spark frequency and duration, in the face of decreased diastolic Ca²⁺ and SR Ca²⁺ content.³³ The resulting increase in diastolic SR Ca²⁺ leak, by decreasing SR Ca²⁺ content, could contribute to the diminished contractile function, seen in the CaMKII_C TG mice.

In conclusion, we demonstrate here that CaMKII_C can phosphorylate RyR2 and PLB when expressed in vivo at levels leading to 2- to 3-fold increases in its activity. Similar increases in CaMKII activity occur with TAC or in heart failure. Data presented in this study and in the accompanying article³³ suggest that altered phosphorylation of Ca²⁺ cycling proteins is a major component of the observed decrease in contractile function in CaMKII_C TG mice. The early occurrence of increased CaMKII activity after TAC, and of RyR and PLB phosphorylation in the CaMKII_C TG mice suggest that CaMKII_C plays an important role in the pathogenesis of dilated cardiomyopathy and heart failure. These results have major implications for considering CaMKII and its isoforms in exploring new treatment strategies for heart failure.

	Acknowledgments

 
This work was supported by NIH grant HL-46345 (to J.H.B. and J.R.), HL-28143 (to J.H.B.), and HL-30077 and HL-64724 (to D.M.B.). Dr T. Zhang is supported by an American Heart Association postdoctoral fellowship. Dr L.S. Maier is in the Emmy-Noether-Program of the DFG MA 1982/1-1&1/2. We thank Dr H. Schulman for providing HA-tagged rat wild-type CaMKII_C cDNA as well as valued advice.

Received September 5, 2002; revision received January 21, 2003; accepted March 20, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
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