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(Circulation Research. 2003;92:904.)
© 2003 American Heart Association, Inc.

Cellular Biology

Transgenic CaMKII_C Overexpression Uniquely Alters Cardiac Myocyte Ca²⁺ Handling

Reduced SR Ca²⁺ Load and Activated SR Ca²⁺ Release

Lars S. Maier^*, Tong Zhang^*, Lu Chen, Jaime DeSantiago, Joan Heller Brown, Donald M. Bers

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

Correspondence to Donald M. Bers, PhD, Department of Physiology, Stritch School of Medicine, Loyola University Chicago, 2160 South First Ave, Maywood, IL 60153. E-mail dbers{at}lumc.edu

	Abstract

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Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is the predominant cardiac isoform, and the _C splice variant is cytoplasmic. We overexpressed CaMKII_C in mouse heart and observed dilated heart failure and altered myocyte Ca²⁺ regulation in 3-month-old CaMKII_C transgenic mice (TG) versus wild-type littermates (WT). Heart/body weight ratio and cardiomyocyte size were increased about 2-fold in TG versus WT. At 1 Hz, twitch shortening, [Ca²⁺]_i transient amplitude, and diastolic [Ca²⁺]_i were all reduced by 50% in TG versus WT. This is explained by >50% reduction in SR Ca²⁺ content in TG versus WT. Peak Ca²⁺ current (I_Ca) was slightly increased, and action potential duration was prolonged in TG versus WT. Despite lower SR Ca²⁺ load and diastolic [Ca²⁺]_i, fractional SR Ca²⁺ release was increased and resting spontaneous SR Ca²⁺ release events (Ca²⁺ sparks) were doubled in frequency in TG versus WT (with prolonged width and duration, but lower amplitude). Enhanced Ca²⁺ spark frequency was also seen in TG at 4 weeks (before heart failure onset). Acute CaMKII inhibition normalized Ca²⁺ spark frequency and I_Ca, consistent with direct CaMKII activation of ryanodine receptors (and I_Ca) in TG. The rate of [Ca²⁺]_i decline during caffeine exposure was faster in TG, indicating enhanced Na⁺-Ca²⁺ exchange function (consistent with protein expression measurements). Enhanced diastolic SR Ca²⁺ leak (via sparks), reduced SR Ca²⁺-ATPase expression, and increased Na⁺-Ca²⁺ exchanger explain the reduced diastolic [Ca²⁺]_i and SR Ca²⁺ content in TG. We conclude that CaMKII_C overexpression causes acute modulation of excitation-contraction coupling, which contributes to heart failure.

Key Words: calcium • Ca²⁺/calmodulin-dependent protein kinase II • sarcoplasmic reticulum • ryanodine receptor • heart

	Introduction

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Calcium/calmodulin-dependent protein kinase II (CaMKII) can phosphorylate and alter the function of many substrates.^1,2 CaMKII was initially identified in the nervous system, but is found in most tissues, including heart.^3,4 CaMKII is the predominant isoform in heart.^3,5 CaMKII phosphorylates several Ca²⁺ transport proteins, including ryanodine receptors (RyRs)^6,7 and phospholamban (PLB).^8,9 CaMKII is involved in L-type Ca²⁺ current (I_Ca) facilitation^10,11 and frequency-dependent acceleration of relaxation (FDAR; which depends on SR Ca²⁺ uptake).^12–16

Ramirez et al¹⁷ found that the nuclear CaMKII isoform (_B) caused transcriptional activation and expression of atrial natriuretic peptide (ANF, a hypertrophic signaling marker) in neonatal rat ventricular myocytes. Transgenic overexpression of CaMKII_B or expression of CaMKIV (also nuclear) induces cardiac hypertrophy.^18,19 However, in vivo expression of cytoplasmic CaMKII (_C) has not been examined.

In human heart failure (HF), CaMK activity is increased 2- to 3-fold, which could be compensatory because it correlated positively with cardiac index and ejection fraction in patients.^20,21 Because phosphatase activity is also enhanced in human HF,²² the net phosphorylation state of individual targets is unclear. For example, whereas many protein kinase A (PKA) targets are relatively dephosphorylated in HF, RyRs can be hyperphosphorylated due to reduced local phosphatase bound to RyRs.²³

To investigate CaMKII effects on cellular Ca²⁺ regulation, we overexpressed cytoplasmic CaMKII_C in mouse heart.²⁴ The transgenic line studied here exhibits 3-fold increase in CaMKII activity, profound dilated hypertrophy, and ventricular dysfunction.²⁴ We compared Ca²⁺ regulation in cardiomyocytes isolated from 3-month-old CaMKII_C transgenic mice (TG) versus wild-type littermates (WT). We found major reductions in diastolic [Ca²⁺]_i, twitch [Ca²⁺]_i, SR Ca²⁺ content, and SR Ca²⁺-ATPase (SERCA2), PLB, and RyR expression. However, the frequency of Ca²⁺ sparks (indicative of diastolic spontaneous SR Ca²⁺ release)²⁵ was greatly enhanced, demonstrating increased diastolic SR Ca²⁺ leak. There was also enhanced Na⁺-Ca²⁺ exchange (NCX) function and expression. Reduced contractile function in TG mice is explained by a combination of weaker SR Ca²⁺ pumping, greater NCX function, and higher SR Ca²⁺ leak.

	Materials and Methods

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Generating CaMKII_C Transgenic Mice and Myocyte Isolation
Transgenic mice were generated as in Zhang et al.²⁴ In the present study, Ca²⁺ handling was assessed using (unless noted) 3-month-old CaMKII_C TG mice (n=8) exhibiting a 3-fold increase in CaMKII activity (TGM line²⁴), and age-matched WT littermates (n=8). Ventricular myocytes were isolated as reported¹⁴ and kept in 100 µmol/L Ca²⁺ MEM solution for use within 4 hours. Myocyte volume was calculated from lengthxwidthx40% of width.²⁵ 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.

Shortening and Ca²⁺ Measurements Using Inverted Microscopes
Shortening and [Ca²⁺]_i were measured as reported previously.¹⁵ Diastolic [Ca²⁺]_i was measured in a subset of myocytes (n=8 for both TG and WT) loaded with 10 µmol/L indo-1-AM (Molecular Probes). These values were used to calculate [Ca²⁺]_i in fluo experiments. Fluorescence was excited at 365±25 nm and monitored at 405±10 nm (F₄₀₅) and 485±10 nm (F₄₈₅). Fluorescence ratio (R=F₄₀₅/F₄₈₅) was translated as [Ca²⁺]_i=K_dß(R-R_min)/(R_max-R), where ß is the ratio of maximum to minimum F₄₈₅ (1.9) and K_d was 844 nmol/L.²⁶ R_min is R at [Ca²⁺]_i<<K_d (0.75±0.03) and R_max is R at saturating [Ca²⁺]_i (2.5±0.2) with no difference between groups (n=8 for TG and WT). Nor was there altered compartmentalization of indo-1 (ie, not released by 10 µmol/L digitonin; 44±5% in WT and 42±5% in TG). Average resting [Ca²⁺]_i ([Ca²⁺]_i-rest) without stimulation for >3 minutes was 144±30 nmol/L in WT and 68±24 nmol/L in TG (P<0.05). Other myocytes were loaded with 10 µmol/L fluo-3-AM (Molecular Probes; n=19 and 10 for TG and WT).¹⁵ Fluo-3 was excited at 480±5 nm and fluorescence measured at 535±20 nm.

Ca²⁺ Measurements Using Confocal Microscopy
Ca²⁺ signals were recorded after incubating myocytes with 10 µmol/L fluo-4-AM (Molecular Probes; n=11 and 13 for TG and WT) on a laser scanning confocal microscope (Biorad Radiance 2000 MP).²⁷ Fluo-4 was excited at (488 nm) and measured at >515 nm. For fluo-3 and fluo-4, [Ca²⁺]_i was calibrated by [Ca²⁺]_i= K_d(F/F₀)/(K_d/[Ca²⁺]_i-rest+1-F/F₀) with K_d=1100 nmol/L²⁸ and [Ca²⁺]_i-rest=144 and 68 nmol/L for WT and TG, respectively, as measured in indo-1 experiments. Significant WT versus TG differences remained even assuming unaltered [Ca²⁺]_i-rest=100 nmol/L. However, measured [Ca²⁺]_i-rest was used. Results were comparable among indo-1, fluo-3, and fluo-4, so results were pooled.

Ca²⁺ sparks were characterized by an algorithm (IDL 5.3)²⁹ using a threshold of 3.8xSD with human verification. We measured Ca²⁺ spark peak (F/F₀), duration (full-duration-half-maximum, FDHM), width, or spatial size (full-width-half-maximum, FWHM). Ca²⁺ spark frequency (CaSpF) was obtained after 1 Hz stimulation and normalized to cell volume and scan rate, as sparks (pL^-1 s^-1), assuming voxel length and width of 0.2 µm, and depth of 1 µm.

Solutions and Experimental Protocol
Normal Tyrode’s solution (NT) contained (in mmol/L) 140 NaCl, 6 KCl, 10 HEPES, 10 glucose, 1 MgCl₂, and 1 CaCl₂ (23°C). SR Ca²⁺ load was evaluated by Ca²⁺ transient amplitudes induced by rapid caffeine (10 mmol/L) application, and the exponential rate constant of [Ca²⁺]_i decline indicates NCX function (k_NCX). This is because SR Ca²⁺ uptake is prevented and other Ca²⁺ pathways (mitochondrial uniporter and sarcolemmal Ca²⁺ ATPase) are negligible.²⁵

I_Ca and Action Potential Measurements
I_Ca was recorded as reported previously.¹³ Pipettes (1 to 2 M) were filled with (in mmol/L) 105 CsCl, 20 HEPES, 5 BAPTA, 1 di-bromo-BAPTA, 1.49 CaCl₂, and 5 MgATP; [Ca²⁺]=100 nmol/L (pH 7.2). Myocytes were superfused with NT in which KCl was replaced by 4 mmol/L CsCl. Holding potential was -90 mV. Five prepulses to 0 mV assured steady state. Test pulses (200 ms) were preceded by 50 ms at -50 mV to inactivate Na⁺ current. I_Ca facilitation was measured with 10 steps to 0 mV from rest (with no prepulses, but the same Na⁺ current inactivating pulse). In some experiments, 1 µmol/L KN-93 was added (10 minutes) to inhibit CaMKII.

Action potentials (APs) were recorded using high resistance electrodes (>10 M) filled with (in mmol/L) 120 K-aspartate, 7 NaCl, 8 KCl, 10 HEPES, 1 MgCl₂, and 5 MgATP (pH 7.2). Myocytes superfused with NT were stimulated by 1- to 3-ms pulses of 1 to 2 nA.

Western Blots
Cardiac homogenates from the same age and TG line were subjected to Western blot analysis as described previously.¹⁸ Antibodies used were anti-SERCA2, anti-PLB, anti-RyR (Affinity Bioreagents), and anti-NCX (monoclonal R3F1, a gift from K.D. Philipson, UCLA, Los Angeles, Calif).

Statistics
Results are mean±SEM with significance (P<0.05) determined using unpaired Student’s t test or 2-way repeated measurements ANOVA. Time constants of [Ca²⁺]_i decline, _Ca were determined by monoexponential least-squares fit.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cardiac Hypertrophy and Increased Myocyte Size
Cardiac overexpression of CaMKII_C induces hypertrophy, ventricular dilation, and mortality, related to gene dosage (see Zhang et al).²⁴ The heart/body weight ratio was more than 2-fold greater in TG (n=4) versus WT mice (n=4; 17.0±3.3 versus 7.4±0.8 mg/g; P<0.05). In addition, isolated TG myocytes (n=58) were almost twice as large as WT (n=60) myocytes (52.3±0.1 versus 29.1±0.1 pL; P<0.05).

[Ca²⁺]_i Transients and SR Ca²⁺ Content
Figure 1A shows steady-state twitch Ca²⁺ transients (1 Hz) in WT and TG myocytes. Mean twitch Ca²⁺ transient amplitude ([Ca²⁺]_i) in TG was decreased by 50% versus WT (Table, Figure 1B). This reduction probably results from decreased SR Ca²⁺ content in TG, because caffeine-induced Ca²⁺ transients were also strongly depressed (Figure 1, Table). In addition, diastolic [Ca²⁺]_i was lower (96±13 in TG versus 200±21 nmol/L in WT; P<0.05). Twitch contraction amplitude was also decreased by 50% in TG versus WT (Table). Notably, the ratio of twitch/caffeine [Ca²⁺]_i (an index of SR fractional Ca²⁺ release, or amount of Ca²⁺ released during a twitch versus that Ca²⁺ stored in the SR) was significantly increased in TG versus WT (Figure 1B, Table). Thus, whereas SR Ca²⁺ content is reduced, the fraction of SR Ca²⁺ released during a twitch is increased.

View larger version (26K): [in this window] [in a new window]   Figure 1. Decreased twitch [Ca2+]i transients and SR Ca2+ content in TG. A, Superimposed twitch and caffeine-induced Ca2+ transients at 1 Hz from TG and WT myocytes. B, Average [Ca2+]i for twitches, caffeine-induced Ca2+ transients, and fractional release (ratio of twitch/caffeine [Ca2+]i) for WT (n=31/22) and TG (n=38/33). *P<0.05 of TG vs WT.

View this table: [in this window] [in a new window]   Table 1. Twitch and Ca2+ Transient Characteristics

We also translated [Ca²⁺]_i to changes in total cytosolic [Ca²⁺] ([Ca²⁺]_Tot), assuming unchanged cytosolic Ca²⁺ buffering (and as in other myocytes; [Ca²⁺]_Tot=244/{1+673/[Ca²⁺]_i}²⁵). Twitch [Ca²⁺]_Tot was 39±3 and 29±3 µmol/L cytosol for WT and TG, respectively (P=0.03). SR Ca²⁺ content was 142±9 versus 80±6 µmol/L cytosol for WT versus TG (P<0.001). This indicates that fractional release increased from 29±3% for WT to 41±4% for TG (P=0.03). This is interesting because the intrinsic effect of reduced SR Ca²⁺ content would be to reduce fractional release and consequently twitch [Ca²⁺]_i.³⁰

L-Type Ca²⁺ Currents and Action Potentials
Higher I_Ca could also increase fractional release, so we measured I_Ca. Figure 2A shows that the voltage-dependence of I_Ca was not different, although mean peak I_Ca at 0 mV was slightly increased in TG (-4.1±0.4 A/F) versus WT (-3.5±0.4 A/F; P=0.08). However, after CaMKII inhibition by KN-93, peak I_Ca was almost identical between TG and WT. Thus, the trend toward higher I_Ca in TG could be a direct effect of the overexpressed CaMKII_C.

View larger version (31K): [in this window] [in a new window]   Figure 2. Ca2+ currents and action potentials (APs). A, ICa-V relationships from TG (n=23) and WT (n=12) myocytes. Inset with superimposed ICa shows slower decline in TG. CaMKII inhibition (1 µmol/L KN-93) decreased peak current at 0 mV. B, ICa facilitation during 10 pulses to 0 mV (after rest) was similar in TG and was reversed by KN-93. C, Superimposed APs and mean data for AP durations at 50 and 90% repolarization. *P<0.05 of TG vs WT.

The 17% higher peak I_Ca in TG could partly explain the higher fractional release. However, the I_Ca dependence of fractional release (for a given SR Ca²⁺ load) is linear or saturating,³⁰ so this effect should be <17%. In contrast, a small reduction in SR Ca²⁺ load can dramatically reduce fractional release.³⁰ Thus, the large reduction in SR Ca²⁺ load and small increase in I_Ca in TG mice would still be expected to reduce, rather than increase, fractional release. This makes the significant increase in fractional release all the more remarkable. Given the lower number of RyRs in TG (Figure 4), this may reflect enhanced RyR Ca²⁺ sensitivity.

View larger version (39K): [in this window] [in a new window]   Figure 4. Western blot analysis of protein expression in cardiac homogenates at 3 months of age for WT (n=6 to 8) and TG (n=6 to 8). A, SR Ca2+-ATPase (SERCA2). B, Phospholamban (PLB). C, Ryanodine receptor (RyR2). D, Na+-Ca2+ exchanger (NCX). *P<0.05 of TG vs WT; ***P<0.001 of TG vs WT.

CaMKII is known to play a role in I_Ca facilitation.^10,11 Figure 2B shows that after a 1-minute rest, the increase in I_Ca amplitude (during 10 pulses to 0 mV) is reversed by the CaMKII inhibitor KN-93. I_Ca facilitation was still present in TG myocytes, but it may start from a partially activated baseline. This is consistent with the basal I_Ca elevation being KN-93-sensitive, and also the observation that the t_1/2 of control I_Ca decline was slower in TG (25±1 ms) versus WT (19±2 ms) mice (see Figure 2A, inset). This is a hallmark of CaMKII-dependent I_Ca facilitation.¹⁰

Figure 2C shows typical APs. Mean AP duration at 50% and 90% repolarization were significantly increased in TG versus WT. No changes were found in resting potential (-79±2 mV in WT versus -78±2 mV in TG) or AP amplitude (106±11 mV in WT versus 102±9 mV in TG; P=N.S.). The AP prolongation could be partly due to enhanced I_Ca, but other currents could also contribute.

The slightly increased I_Ca and APD would by themselves be expected to increase twitch Ca²⁺ transients. Thus, smaller Ca²⁺ transients in TG are due mainly to reduced SR Ca²⁺ content (that occurs despite greater Ca²⁺ influx).

Relaxation of Twitch and Caffeine-Induced [Ca²⁺]_i Transients
Figure 3 shows time courses of relaxation and [Ca²⁺]_i decline during twitches and caffeine-induced contractures. Time to peak shortening and _Ca and half-relaxation time (RT_50%) for twitches were slightly increased in TG versus WT (Table). When SR Ca²⁺ uptake was inhibited by caffeine, _Ca and RT_50% (Figure 3B) reflect mainly NCX function in reducing [Ca²⁺]_i and relaxation.²⁵ From the faster [Ca²⁺]_i decline and relaxation in TG versus WT in caffeine (Table), we infer a 30% increase in NCX function in TG.

View larger version (27K): [in this window] [in a new window]   Figure 3. Rates of [Ca2+]i decline. A, Normalized twitch [Ca2+]i decline, mean time constants (Ca), and half-times of relaxation (RT50%) in TG and WT. B, Corresponding values for caffeine-induced contractures. *P<0.05 of TG vs WT.

SERCA2 contributes strongly to twitch relaxation and [Ca²⁺]_i decline.²⁵ We used a simplified analysis of the competition between SERCA2 and NCX, using the rate constants for [Ca²⁺]_i decline in caffeine to infer NCX function (k_NCX), and during the twitch (k_Tw) to infer function of NCX+SERCA2.²⁵ Then the SR Ca²⁺ transport rate is k_SR=k_Tw-k_NCX, and the relative contribution by the SR Ca²⁺-ATPase is k_SR/k_Tw. Thus, for WT, k_Tw=3.41 s^-1, k_NCX=0.41 s^-1, k_SR=3.00 s^-1, and SERCA contributes 88% (3/3.41), similar to our more detailed mouse analysis.¹⁴ In TG the rate of [Ca²⁺]_i decline attributable to SERCA2 was decreased by 12% (k_SR=2.64 s^-1), whereas that for NCX increased by 27% (k_NCX=0.52 s^-1; k_SR/k_Tw was still 84%). Using cell relaxation, this analysis is indirect, but indicates 27% functional deficit in SERCA2 and a 92% increase in NCX function.

Expression of SERCA2, PLB, RyR, and NCX
Figure 4 shows reduced expression of SERCA2 and PLB protein (by 32 and 17%; P<0.05), and mRNA²⁴ (by 67 and 41%; P<0.05). The SERCA2/PLB ratio was also 17±6% lower in TG (P<0.05), suggesting greater Ca²⁺-pump inhibition. RyR protein expression was decreased by 58% (P<0.05), whereas NCX expression was increased more than 2-fold (P<0.05). The SERCA2 and NCX data are consistent with the above functional observations.

Ca²⁺ Sparks
With lower SR Ca²⁺ content and diastolic [Ca²⁺]_i in the TG myocytes, lower diastolic CaSpF was expected. However, Figures 5 and 6A show that CaSpF after 1 Hz stimulation was dramatically increased in TG versus WT myocytes (185±30 versus 80±7 sparks pL^-1 s^-1; P<0.05). On the other hand, Ca²⁺ spark amplitude was reduced in TG (1.67±0.01 versus 2.07±0.02 F/F₀). Spatial spread was increased (1.80±0.03 versus 1.35±0.03 µm), and duration was prolonged (35.7±0.8 versus 21.3±1.0 ms). Usually CaSpF and amplitude are altered in the same direction. In addition to higher FDHM in TG, the single event distribution was shifted toward longer durations, suggesting longer RyR openings (Figure 6B). Similar differences were obtained after 2 Hz stimulation or rest (not shown). The overall rate of diastolic SR Ca²⁺ leak should be related to the product CaSpFxamplitudexFDHMxFWHM, which was 4.3 times higher in TG versus WT.

View larger version (67K): [in this window] [in a new window]   Figure 5. Representative Ca2+ sparks in TG vs WT. A, Representative longitudinal line scan images, with line plots of [Ca2+]i at sites indicated by white bars. B, 3-D surface plot of signal averaged Ca2+ sparks from the cells in A (6 sparks for WT, 7 sparks for TG).

View larger version (29K): [in this window] [in a new window]   Figure 6. Ca2+ spark characteristics. Average data for (A) Ca2+ sparks frequency (CaSpF), amplitude, and width. B, Average spark duration and distribution of duration, fit with f=axexp(-0.5x{ln[x/x0]/b}2). C, CaSpF in young TG mice is decreased by blocking CaMKII (1 µmol/L KN-93). *P<0.05 of TG vs WT.

The Ca²⁺ spark data are surprising in two ways. First, reduced SR Ca²⁺ content or [Ca²⁺]_i as in the TG mice would be expected to decrease CaSpF.^28,31 This suggests that there might be a relatively direct enhancement of spontaneous SR Ca²⁺ release (eg, increased RyR Ca²⁺ sensitivity) in TG mice. The lower Ca²⁺ spark amplitude in TG is consistent with the lower SR Ca²⁺ content (lower Ca²⁺ driving force) and fewer expressed RyRs. The second surprise is that despite the lower Ca²⁺ spark amplitude, the temporal and spatial spread was greater (Figure 6A). This may reflect longer RyR open time and SR Ca²⁺ release (again, despite lower SR Ca²⁺ content), consistent with altered RyR gating in TG.

To test whether the increased CaSpF is an acute CaMKII-dependent effect, we used the specific CaMKII inhibitor KN-93. KN-93 dramatically reduced CaSpF in TG mice (eliminating the WT versus TG difference, not shown). Figure 6C shows that in TG mice aged 4 weeks (before heart failure onset²⁴), CaSpF was already increased versus 4-week WT littermates. SR Ca²⁺ load was also 47% lower in these 4-week TG versus WT. KN-93 did not lower SR Ca²⁺ content in either 4-week WT or TG. Nor did it alter CaSpF in WT myocytes, but acute CaMKII inhibition reduced CaSpF in TG to the same level as WT (Figure 6C). This strengthens the conclusion that CaMKII overexpression causes acute CaMKII-dependent RyR phosphorylation (before the onset of failure), which alters Ca²⁺ sparks.

Frequency-Dependent Acceleration of Relaxation
CaMKII has been implicated mechanistically in FDAR, which occurs in both normal and failing hearts.² We studied FDAR at 0.2 to 4 Hz stimulation frequencies (Figure 7). As in the 1 Hz data, twitch [Ca²⁺]_i and shortening were depressed in TG versus WT (P<0.05 at most frequencies), but the slightly negative staircase was similar in both (Figure 7A). There was prominent FDAR in WT and TG cells, apparent for both _Ca (P<0.05) and RT_50% (P<0.05; Figure 7B). Thus, FDAR was not abolished by overexpression of cytosolic CaMKII. On the other hand, comparing the extent of FDAR at the extremes of the frequencies tested (as ratio of _Ca at 4 versus 0.2 Hz), a significant enhancement of FDAR was evident for TG versus WT (to 36 versus 45%; inset Figure 7B).

View larger version (34K): [in this window] [in a new window]   Figure 7. Frequency-dependent acceleration of relaxation (FDAR). A, [Ca2+]i- and shortening-frequency relationships in TG (n=27) vs WT (n=22). B, Mean values for [Ca2+]i decline (Ca) and relaxation (RT50%). Inset, Ratio of Ca at 4 Hz/0.2 Hz as a measure of FDAR. *P<0.05 vs 0.2 Hz; #P<0.05 of TG vs WT.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
CaMKII is critically involved in numerous cardiac Ca²⁺-dependent processes, including excitation-contraction (E-C) coupling and hypertrophic signaling.^1,2 Transient expression of CaMKII_B (the nuclear isoform) induced hypertrophy and ANF production in neonatal rat ventricular myocytes.¹⁷ Transgenic mice overexpressing CaMKII_B also showed cardiac hypertrophy and ventricular dilation,¹⁸ as did mice expressing CaMKIV.¹⁹

In the present study, transgenic overexpression of cytosolic CaMKII_C leads to dramatic dilated cardiac hypertrophy²⁴ and uniquely altered myocyte Ca²⁺ handling. We found the following: (1) twitch shortening and [Ca²⁺]_i are both reduced in TG versus WT; (2) SR Ca²⁺ content and diastolic [Ca²⁺]_i are lower in TG versus WT (consistent with reduced SERCA2 expression, increased SR Ca²⁺ leak and increased NCX expression); (3) despite lower SR Ca²⁺ load, diastolic CaSpF and twitch fractional release are increased in TG versus WT (with prolonged spark duration), suggesting altered RyR function in TG mice; and (4) caffeine-induced Ca²⁺ transients decline more rapidly, indicating enhanced NCX function in TG versus WT (consistent with the increased NCX protein expression). We suggest that CaMKII_C overexpression acutely activates RyR (and I_Ca), while also contributing to the contractile dysfunction of HF.

Acute Effects of CaMKII on E-C Coupling
The RyR can be phosphorylated by CaMKII,⁶ and most (but not all) lipid bilayer studies indicate that this increases RyR open probability.^6,7 In intact myocytes, Li et al¹³ demonstrated that endogenous CaMKII increased SR Ca²⁺ release for a given SR Ca²⁺ content and I_Ca trigger (ie, increased fractional release). The effect was Ca²⁺- and CaMKII-specific because smaller conditioning Ca²⁺ transients (unlikely to activate CaMKII) failed to produce this effect. These findings are consistent with observations that protein phosphatase inhibitors enhance, and phosphatases reduce, SR Ca²⁺ release for a given I_Ca and SR Ca²⁺ load.^32,33 Wu et al³⁴ found opposite results, suggesting that CaMKII inhibits SR Ca²⁺ release (although SR Ca²⁺ content was not determined in the same experiments). Our results support the hypothesis that CaMKII increases RyR activity (diastolic and systolic).

CaMKII also phosphorylates PLB at Thr-17, which increases SERCA2 rate.⁹ The function of CaMKII is controversial because levels of PLB Thr-17 phosphorylation in vivo are not appreciably elevated by enhanced Ca²⁺ transients alone, unless pH is reduced, phosphatases are inhibited, or ß-adrenergic receptors are activated (which phosphorylates PLB at Ser-16).^9,35,36 On the other hand, Hagemann et al³⁷ showed frequency-dependent increases in PLB Thr-17 phosphorylation and function in the absence of PKA activation or Ser-16 phosphorylation.

In conclusion, overexpression of CaMKII might be expected to increase SR Ca²⁺ uptake, sensitize SR Ca²⁺ release, and enhance twitch Ca²⁺ transients. Although we observed enhanced CaSpF and enhanced fractional SR Ca²⁺ release (consistent with sensitized RyRs), the SR content and twitch Ca²⁺ transients were depressed. This results from progressive NCX and SERCA/PLB expression changes that exacerbate the SR Ca²⁺ load reduction and cause HF.

Impaired Contractility and [Ca²⁺]_i Transients
Severe hypertrophy and HF are typically characterized by reduced [Ca²⁺]_i transients and by some combination of reduced SERCA2 function and/or enhanced NCX function.^25,38 This slows [Ca²⁺]_i decline and reduces SR Ca²⁺ content.^25,38–40 Both, reduced SERCA2 function and enhanced NCX function lower SR Ca²⁺ load. However, with respect to diastolic function these NCX and SERCA2 effects can be offsetting, such that relaxation may be little changed.^40,41 The TG mice in this study show this HF phenotype.

The effect of decreased SERCA2 expression on twitch [Ca²⁺]_i decline might be partly counterbalanced by the reduced PLB expression and increased CaMKII-dependent phosphorylation of PLB at Thr-17.²⁴ This may explain why the rate of SR-dependent [Ca²⁺]_i decline (k_SR) was only reduced by 12% in TG versus WT. It is worth noting that a 32% decrease in SERCA2 expression reduces V_max, whereas the PLB-dependent phosphorylation shifts K_m (increasing Ca²⁺ transport at low [Ca²⁺]_i, but not at higher [Ca²⁺]_i). Presumably the higher PLB Thr-17 phosphorylation in TG mice is a direct effect of CaMKII_C overexpression.²⁴ In addition, NCX function (and expression) is increased, which can unload the SR and lower diastolic [Ca²⁺]_i. Finally, there is a large 4-fold increase in diastolic SR Ca²⁺ leak in TG based on Ca²⁺ spark measurements. Thus, there may be 3 factors contributing to reduce SR Ca²⁺ content: (1) reduced SERCA2 function; (2) enhanced Ca²⁺ extrusion via NCX; and (3) increased diastolic SR Ca²⁺ leak. The last of these is most likely to be a direct acute effect of CaMKII, mediated by RyR phosphorylation (there is no evidence for CaMKII in NCX function).²⁵ A direct effect of CaMKII to increase diastolic SR Ca²⁺ release could exacerbate the depressed SR Ca²⁺ content and systolic dysfunction.

Diastolic SR Ca²⁺ Release and Ca²⁺ Sparks
In the TG mice, Ca²⁺ sparks have smaller amplitude, but longer duration and increased frequency. The net result is a substantial (4-fold) increase in resting SR Ca²⁺ leak. The increased CaSpF in TG (Figures 5 and 6) is surprising, considering the reduced SR Ca²⁺ content and diastolic [Ca²⁺]_i. Both of these factors normally depress CaSpF.^28,31 The increased CaSpF is most easily explained by enhanced RyR opening at resting [Ca²⁺]_i (eg, increased RyR Ca²⁺ sensitivity). This might also explain the apparent E-C coupling enhancement (higher fractional SR Ca²⁺ release in Figure 1B), in the face of reduced SR Ca²⁺ load (which itself would reduce fractional SR Ca²⁺ release³⁰).

The likely explanation for smaller Ca²⁺ spark amplitude is the lower SR Ca²⁺ content and the reduced number of expressed RyRs (eg, with fewer RyRs participating in the average Ca²⁺ spark). Shorter release time could also contribute, but is inconsistent with the FWHM and FDHM data, which suggest a longer release duration.

We hypothesize that the increased Ca²⁺ spark frequency and duration in TG mice are due to RyR modulation by CaMKII-mediated phosphorylation (before the onset of HF).²⁴ Increased PKA-dependent RyR phosphorylation has also been implicated in HF, due to lower RyR-phosphatase association.²³ However, in our TG mice we do not find altered PKA or phosphatase associated with the RyR.²⁴ We infer that the increased RyR phosphorylation results from overexpressed CaMKII (which readily phosphorylates RyRs⁶ and coimmunoprecipitates with the RyR²⁴). The inhibition of enhanced CaSpF by KN-93 is also consistent with this direct CaMKII effect on RyRs.

I_Ca Facilitation
The small increase in peak I_Ca seen in TG versus WT (and slowing of inactivation) may be a direct effect of CaMKII overexpression and phosphorylation (as in Ca²⁺-dependent I_Ca facilitation),^10,11 because it was acutely reversed by KN-93. However, I_Ca facilitation still occurred in TG, indicating that the extent of basal CaMKII activation was not saturating for this CaMKII-dependent modulation.

Frequency-Dependent Acceleration of Relaxation
FDAR is an important intrinsic mechanism that contributes to faster relaxation (and diastolic filling) at higher heart rates. FDAR is also reflected in the rate of [Ca²⁺]_i decline and is attributable to altered SR Ca²⁺ uptake.¹² Schouten¹⁶ proposed that FDAR might be due to enhanced SR Ca²⁺ uptake secondary to CaMKII mediated PLB phosphorylation. Whereas PLB is a logical CaMKII target, FDAR is still prominent in PLB-knockout mice and still sensitive to CaMKII inhibition by KN-93.^14,15 Thus, whereas PLB might contribute to FDAR, it cannot be the sole mechanism. Some groups have not seen marked FDAR inhibition by CaMKII inhibitors KN-93 and KN-62.^42–44 In the present study, FDAR was clearly demonstrable in both WT and TG mice, with FDAR only slightly (albeit significantly) enhanced in TG mice. This might imply that the CaMKII levels in TG mice (with 3-fold increase in activity²⁴) do not greatly alter FDAR. CaMKII may indeed be involved in FDAR, but endogenous CaMKII in WT may not be rate limiting. Thus, our results are consistent with a role of CaMKII in FDAR, but do not provide proof for CaMKII involvement.

In summary, TG CaMKII_C overexpression causes dilated HF, with profound myocyte Ca²⁺ handling changes. Direct CaMKII-dependent RyR phosphorylation probably explains the increased diastolic SR Ca²⁺ leak (which reduces SR Ca²⁺ content) and also the enhanced fractional SR Ca²⁺ release during E-C coupling (even with less SR Ca²⁺). Enhanced Ca²⁺ extrusion via NCX and reduced SERCA2 function may further depress SR Ca²⁺ content and systolic function, contributing to HF.

	Acknowledgments

 
We acknowledge the support from NIH: HL-30077 and HL-64724 (D.M.B.); HL-46345 and HL-28143 (J.H.B.). Dr Maier is in the Emmy-Noether-Program of the DFG (MA 1982/1-1&1/2). Dr Zhang is an American Heart Association postdoctoral fellow.

	Footnotes

 
*Both authors contributed equally to this study.

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

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