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(Circulation Research. 2006;98:235.)
© 2006 American Heart Association, Inc.

Cellular Biology

Increased Sarcoplasmic Reticulum Calcium Leak but Unaltered Contractility by Acute CaMKII Overexpression in Isolated Rabbit Cardiac Myocytes

Michael Kohlhaas, Tong Zhang, Tim Seidler, Darya Zibrova, Nataliya Dybkova, Astrid Steen, Stefan Wagner, Lu Chen, Joan Heller Brown, Donald M. Bers, Lars S. Maier

From the Abteilung Kardiologie & Pneumologie/Herzzentrum (M.K., T.S., D.Z., N.D., A.S., S.W., L.S.M.), Georg-August-Universität Göttingen, Germany; Department of Pharmacology (T.Z., J.H.B.), University of California San Diego; and Department of Physiology (L.C., D.M.B.), Stritch School of Medicine, Loyola University Chicago, Ill.

Correspondence to Lars S. Maier, MD, Abt. Kardiologie & Pneumologie/Herzzentrum, Georg-August-Universität Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany. E-mail lmaier{at}med.uni-goettingen.de

	Abstract

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The predominant cardiac Ca²⁺/calmodulin-dependent protein kinase (CaMK) is CaMKII. Here we acutely overexpress CaMKII_C using adenovirus-mediated gene transfer in adult rabbit ventricular myocytes. This circumvents confounding adaptive effects in CaMKII_C transgenic mice. CaMKII_C protein expression and activation state (autophosphorylation) were increased 5- to 6-fold. Basal twitch contraction amplitude and kinetics (1 Hz) were not changed in CaMKII_C versus LacZ expressing myocytes. However, the contraction–frequency relationship was more negative, frequency-dependent acceleration of relaxation was enhanced (_0.5Hz/_3Hz=2.14±0.10 versus 1.87±0.10), and peak Ca²⁺ current (I_Ca) was increased by 31% (–7.1±0.5 versus –5.4±0.5 pA/pF, P<0.05). Ca²⁺ transient amplitude was not significantly reduced (–27%, P=0.22), despite dramatically reduced sarcoplasmic reticulum (SR) Ca²⁺ content (41%; P<0.05). Thus fractional SR Ca²⁺ release was increased by 60% (P<0.05). Diastolic SR Ca²⁺ leak assessed by Ca²⁺ spark frequency (normalized to SR Ca²⁺ load) was increased by 88% in CaMKII_C versus LacZ myocytes (P<0.05; in an multiplicity-of-infection–dependent manner), an effect blocked by CaMKII inhibitors KN-93 and autocamtide-2–related inhibitory peptide. This enhanced SR Ca²⁺ leak may explain reduced SR Ca²⁺ content, despite measured levels of SR Ca²⁺-ATPase and Na⁺/Ca²⁺ exchange expression and function being unaltered. Ryanodine receptor (RyR) phosphorylation in CaMKII_C myocytes was increased at both Ser2809 and Ser2815, but FKBP12.6 coimmunoprecipitation with RyR was unaltered. This shows for the first time that acute CaMKII_C overexpression alters RyR function, leading to enhanced SR Ca²⁺ leak and reduced SR Ca²⁺ content but without reducing twitch contraction and Ca²⁺ transients. We conclude that this is attributable to concomitant enhancement of fractional SR Ca²⁺ release in CaMKII_C myocytes (ie, CaMKII-dependent enhancement of RyR Ca²⁺ sensitivity during diastole and systole) and increased I_Ca.

Key Words: calcium • CaMKII • excitation–contraction coupling • sarcoplasmic reticulum

	Introduction

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Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that phosphorylates numerous target proteins.^1,2 The major cardiac isoform is CaMKII, and the splice variant CaMKII_C is primarily cytosolic, whereas CaMKII_B is nuclear because of a nuclear localization sequence.³

During excitation–contraction coupling (ECC), Ca²⁺ entry, mainly via voltage dependent L-type Ca²⁺ channels (I_Ca), triggers sarcoplasmic reticulum (SR) Ca²⁺ release via ryanodine receptors (RyRs), via Ca²⁺-induced Ca²⁺ release.^4,5 The resultant increase in intracellular [Ca²⁺] ([Ca²⁺]_i), causes Ca²⁺ binding to troponin C, which activates myofilaments, leading to contraction. For relaxation to occur, Ca²⁺ must be removed from the cytoplasm. SR Ca-ATPase (SERCA) and Na⁺/Ca²⁺-exchanger (NCX) are the main mechanisms for Ca²⁺ removal.^4,5 CaMKII can modulate ECC by phosphorylating several important Ca²⁺-dependent regulatory proteins in heart, including Ca²⁺ transport proteins, such as RyR and phospholamban (PLB), and possibly L-type Ca²⁺ channels.^2,5

CaMKII is directly associated with RyR and overexpression of CaMKII in transgenic mouse cardiomyocytes increases SR Ca²⁺ release as shown by increased frequency of spontaneous SR Ca²⁺ release events (Ca²⁺ sparks).^6,7 Blocking CaMKII (using KN-93) decreases Ca²⁺ spark frequency dramatically, providing evidence for a direct relationship between CaMKII activity and the increased spark frequency.⁷ These results in myocytes from CaMKII transgenic mouse hearts were confirmed by Currie et al,⁸ who showed that the specific CaMKII peptide inhibitor autocamtide-2–related inhibitory peptide (AIP) depresses Ca²⁺ spark frequency in rabbit hearts because of decreased endogenous CaMKII-dependent RyR phosphorylation. Wehrens et al⁹ showed using site-directed mutagenesis that CaMKII-dependent phosphorylation of RyR was at Ser2815, rather than at Ser2809 (which they find is a PKA target and phosphorylation causes FKBP12.6 dissociation). Using single-channel measurements in lipid bilayers, Wehrens et al also showed that CaMKII-dependent RyR phosphorylation increased RyR open probability (P_o), without alteration of FKBP12.6 association.⁹

CaMKII may also be involved in the pathogenesis of hypertrophy and heart failure.² In human heart failure, CaMKII expression are increased.^10,11 In neonatal ventricular myocytes, overexpression of CaMKII_B caused transcriptional activation of atrial natriuretic peptide gene expression (a hypertrophic signaling marker).¹² Furthermore, overexpression of the cytoplasmic _C isoform in mouse heart results in profound contractile dysfunction and heart failure.^6,7 In our previous studies using these animals, we described major alterations in intracellular Ca²⁺ handling with marked reductions in Ca²⁺ transients, SR Ca²⁺ content, and SERCA, PLB, and RyR protein expression and enhanced NCX function and expression, all of which are typical for heart failure. Most remarkably, however, with respect to the RyR, the frequency of Ca²⁺ sparks (indicative of diastolic spontaneous SR Ca²⁺ release events or opening of RyR clusters) was greatly enhanced, demonstrating increased diastolic SR Ca²⁺ leak despite reduced SR Ca²⁺ load and diastolic [Ca²⁺]_i⁷ (which by themselves would normally reduce SR Ca²⁺ leak).¹³ We showed that this was most likely attributable to increased CaMKII-dependent RyR phosphorylation increasing RyR openings, because Ca²⁺ spark frequency could be reduced back to normal levels by blocking CaMKII. Backphosphorylation and subsequent studies using phospho-CaMKII antibodies indeed showed increased RyR phosphorylation in transgenic versus wild type.^6,7

Although these results show that CaMKII_C overexpression can cause heart failure and altered cellular Ca²⁺ transport, it was unclear how direct effects of acute CaMKII-dependent protein phosphorylation alter Ca²⁺ handling functionally and with respect to protein expression, especially in the context of possible developmental changes or adaptive responses associated with heart failure induction as reported previously.^6,7 Therefore, we have acutely overexpressed CaMKII_C in ventricular rabbit myocytes and compared these with LacZ-expressing control cells to investigate intracellular Ca²⁺ handling. We demonstrate that acute CaMKII_C overexpression enhances SR Ca²⁺ leak and reduces SR Ca²⁺ content. However, in acute CaMKII_C overexpression, we do not see alterations in the protein expression levels or function of NCX and SERCA (in striking contrast to the failing transgenic mice), and twitch contractions and Ca²⁺ transients are unaltered. This is attributed to an increased fractional SR Ca²⁺ release (and I_Ca), which may result from the same CaMKII_C-dependent enhancement of RyR Ca²⁺ sensitivity that enhances diastolic SR Ca²⁺ leak.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generating Adenoviral Vectors and Cardiac Myocyte Isolation
Adenoviral vectors were generated as published previously.^14,15 For adenoviral transfection, ventricular myocytes from rabbit hearts (female Chinchilla Bastards; 1.3- to 2.0-kg weight) were isolated using standard procedures^16,17 with collagenase B (0.5 mg/mL, Boehringer-Mannheim, Mannheim, Germany) and protease (0.02 mg/mL, Sigma, St Louis, Mo). Cells were plated at a density &4.2x10³ rod-shaped cells/cm² on culture dishes (55 mm) and incubated for 24 hours in supplemented M199 tissue culture medium (Sigma-Aldrich Chemie, Taufkirchen, Germany). All procedures involving animals were performed in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996). Initially, myocytes were transfected with multiplicities of infections (MOIs) of 1, 10, and 100 to test for CaMKII_C protein overexpression and phosphorylation levels. For functional experiments, myocytes were then transfected with either CaMKII_C, or LacZ using a MOI of 10 or 100 for 24 hours at 37°C in a humidified incubator (5% CO₂, 95% O₂). Myocyte volume was calculated from myocytes lengthxwidthx40% of width.⁷

Shortening and Ca²⁺ Measurements Using Inverted Microscopes
Shortening and [Ca²⁺]_i measurements were performed as reported previously.^7,17 Briefly, myocytes were loaded with the Ca²⁺-sensitive dye indo-1/acetoxymethyl ester to measure diastolic [Ca²⁺]. Because no difference in diastolic Ca²⁺ was found, additional experiments used fluo-3/acetoxymethyl ester (10 µmol/L, respectively; Molecular Probes). Excitation wavelengths (360±5 nm for indo and 480±15 nm for fluo) using a 75-W xenon arc lamp on the stage of a Nikon Eclipse TE200-U inverted microscope. Emitted fluorescence was measured using photomultipliers (at 405±15 nm and 485±12.5 nm for indo and 535±20 nm for fluo; IonOptix Corp, Milton, Mass). From the raw fluorescence, indo-1 ratio was calculated (405 nm/485 nm), and for fluo-3, F/F₀ was calculated by dividing by the baseline fluorescence F₀, after subtraction of the background fluorescence (IonWizard, IonOptix Corp). Myocytes were field-stimulated (voltage 25% above threshold) at 1 Hz and 37°C until steady state.

L-Type Ca²⁺ Current Measurements
I_Ca was recorded by voltage-clamp as reported previously.⁷ Briefly, low-resistance (&2 to 3 M) electrodes were pulled and filled with K-free internal solution containing (in mmol/L) 105 CsCl, 20 HEPES, 5 BAPTA, 1 di-bromo-BAPTA, 1.49 CaCl₂, and 5 MgATP resulting in a free [Ca²⁺] of 100 nmol/L (pH 7.2). Myocytes were superfused with K-free external solution containing (mmol/L) 140 NaCl, 4 CsCl, 5 HEPES, 10 glucose, 1 MgCl₂, and 2 CaCl₂ (pH 7.4). Current–voltage relationships were established as follows: holding potential was –90 mV; 5 prepulses were applied to 0 mV to ensure equal SR Ca²⁺ loading; brief Na⁺ current-inactivating pulses (50 ms, –50 mV) preceded test potentials steps (between –40 mV and +40 mV, 200 ms, in 10 mV steps). I_Ca facilitation was assessed by repetitive depolarizations to 0 mV after a pause of 1 minute, and amplitudes and kinetics were measured and analyzed (EPC10, Heka Electronics Inc, Lambrecht, Germany).

Confocal Microscopy
Ca²⁺ signals were recorded in fluo-4 loaded myocytes on a laser scanning confocal microscope (Bio-Rad Radiance 2000MP).⁷ Fluo-4 was excited via an argon laser (488 nm) and emitted fluorescence (F) was collected through a 515 nm long-pass emission filter. [Ca²⁺]_i was calibrated by the equation [Ca²⁺]_i=K_d(F/F₀)/(K_d/[Ca²⁺]_i-rest+1–F/F₀) with K_d=1100 nmol/L and [Ca²⁺]_i-rest=100 nmol/L.⁷

Ca²⁺ sparks were analyzed by a program (IDL 5.3)⁷ that detects Ca²⁺ sparks as areas of increased fluorescence with respect to the SD of background fluorescence. We used a Ca²⁺ spark threshold of 3.8xSD, with human verification. Peaks of Ca²⁺ sparks were normalized as F/F₀, and duration was taken from the full-duration half-maximum (FDHM). Width or spatial size was taken as full-width half-maximum (FWHM). Ca²⁺ spark frequency (CaSpF) was obtained by averaging the number of sparks in images recorded after 1 Hz stimulation and normalized to cell volume and scan rate as sparks (pL^–1s^–1), assuming voxel length and width of 0.2 µm and depth of 1 µm.

Solutions and Experimental Protocol
Normal Tyrode’s solution contained (mmol/L) 140 NaCl, 6 KCl, 10 HEPES, 10 glucose, 1 MgCl₂, and 2 CaCl₂ (37°C). SR Ca²⁺ load was evaluated by Ca²⁺ transient amplitudes induced by rapid caffeine (10 mmol/L) application. NCX function was assessed measuring Ca²⁺ decay 50% relaxation (RT_50%) after caffeine application. During this procedure, Ca²⁺ uptake by SERCA is prevented and other Ca²⁺ elimination pathways (eg, mitochondrial uniporter, sarcolemmal Ca²⁺ ATPase, contributing <1% each) can be neglected.⁵ In a subset of experiments, KN-93 (1 µmol/L) or the membrane permeant AIP (20 µmol/L) was added to the external bath solution or the patch-pipette to inhibit CaMKII. Enough time was allowed for KN-93 and AIP to inhibit CaMKII. In another subset of experiments, tetracaine was used to inhibit SR Ca²⁺ leak (1 mmol/L).

For shortening-frequency measurements, stimulation frequency was varied stepwise (from 0.5 to 3 Hz), waiting at intermediate frequencies until steady state was reached. For postrest measurements, a rest interval of 30 s was applied measuring steady-state and postrest twitch contraction amplitude. Of note, rabbit myocytes are known to show rest decay of twitches.⁵

Protein Expression, Phosphorylation Levels, and Immunocytochemistry
Western blot analysis was performed as described previously^6,7 using an anti-CaMKII antibody (Santa Cruz), as well as antibodies for SERCA, NCX (Affinity BioReagents), and PLB (Upstate). For phosphorylation levels of CaMKII (Affinity BioReagents), PLB-Thr17, and PLB-Ser16 (Cyclacel) in transfected myocytes, phospho-specific antibodies were used. RyR expression and phosphorylation levels were investigated using antibodies kindly provided by Dr A. Marks (Columbia University, New York).¹⁸ For immunohistochemical experiments (epifluorescence), diaminobenzidine staining was performed (picture plus, Zymed) using a hemagglutinin (HA) antibody (Roche) against, which was coexpressed with CaMKII. In parallel, a fluorescent Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc) against anti-HA was used for confocal images.

Coimmunoprecipitation and Immunoblotting
Coimmunoprecipitation studies were performed to test for FKBP12.6/RyR2 interaction. CaMKII_C and LacZ transfected myocytes (5x10⁵) were lysed in 250 µL of lysis buffer containing (in mmol/L) 50 HEPES, pH 7.4, 500 KCl, 1% Triton X-100, and 5 EDTA and supplemented with protease inhibitors (0.2 mmol/L pefabloc SC, 100 nmol/L aprotinin, 1 µmol/L leupeptin, 1 µmol/L pepstatin A, 1 mmol/L benzamidine, 1 µmol/L of calpain inhibitor I, and 1 µmol/L of calpain inhibitor II). After centrifugation for 5 minutes (10 000g, 4°C), cell lysates (1 mg) were suspended in PBS (1 mL). Eight micrograms of anti-RyR antibody (34°C clone, Affinity BioReagents) was added to the samples. After 4 hours of incubation at 4°C, protein G-sepharose beads were added to the samples, incubated for a further 4 hours at 4°C, and washed 3 times with PBS. Afterward, the beads were resuspended in SDS loading buffer and heated at 95°C for 5 minutes, and the eluates recovered by centrifugation were subjected to 4% to 20% linear gradient SDS-PAGE.

Immunoblotting was performed as described previously. Primary antibodies used were rabbit polyclonal anti-FKBP12.6 (1:500, SA-169; raised by Eurogentec, Hestal, Belgium), mouse monoclonal anti-RyR (1:500, C3–33 clone; Affinity BioReagents), and rabbit polyclonal anti–phospho-RyR (1:5000, RyR2-P2809 and RyR2-P2815; generous gifts of Dr A. Marks, Columbia University, New York). Secondary antibody used were donkey anti-rabbit whole Ig (1:10000, Amersham) and donkey anti-mouse affinity-purified IgG (1:500, Affinity BioReagents). Immunoreactive bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce).

Statistics
Results are expressed as mean±SEM. Significance (P<0.05) was determined using unpaired Student’s t test or 2-way repeated measurements ANOVA followed by Student–Newman–Keuls test as appropriate. Time constants of [Ca²⁺]_i decline, _Ca, were monoexponential least-square fits. Time constants ₁ and ₂ of I_Ca inactivation were fitted biexponentially.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CaMKII Overexpression in Ventricular Rabbit Myocytes
Figure 1A shows a typical CaMKII Western blot with rabbit myocytes infected at MOI100 (24 hours). Average overexpression of CaMKII was &6-fold that of LacZ controls (P<0.05; n=5). In addition, myocytes showed MOI-dependent (1, 10, 100) increases in CaMKII protein expression, and also in CaMKII phosphorylation status, with maximum phosphorylation increases of &5-fold at MOI100 (P<0.05). Figure 1B shows that expressed CaMKII_C is localized in the cytosol (no nuclear staining). Myocyte volume (Figure 1C) was not significantly increased in CaMKII_C (41.6±2.5 pL; n=66) versus LacZ control (35.9±1.8 pL; n=154). Similarly, in voltage-clamped myocytes, membrane capacity was not different between LacZ (112.0±5.1 pF) and CaMKII_C (113.5±6.9 pF). These results also suggest no alteration in surface to volume ratio.

View larger version (29K): [in this window] [in a new window]   Figure 1. CaMKIIC overexpression in rabbit ventricular myocytes. A, Western blots (MOI100, 24 hours) showing expression levels of CaMKII (n=5) vs LacZ control (n=5) and MOI-dependent increase in phosphorylation status. B, Immunohistochemical staining and confocal images showing cytosolic (but not nuclear) expression of CaMKIIC vs no staining in LacZ. C, Myocyte volume in CaMKIIC (n=66) vs LacZ (n=154). *P<0.05 vs LacZ.

Twitch Shortening and Ca²⁺ Transients
Twitch fractional shortening at 1 Hz (Figure 2A) was not altered in CaMKII_C versus LacZ (3.6±0.2% versus 3.7±0.3% resting cell length; P=0.79). Ca²⁺ transients were also not significantly decreased (P=0.22; Figure 2B), and there was no change in diastolic [Ca²⁺]_i as measured by indo-1 (diastolic F₄₀₅/F₄₈₅ was 0.46±0.02 versus 0.48±0.01; P=0.26). Twitch relaxation and [Ca²⁺]_i decline were not changed, indicating unaltered basal SR Ca²⁺ ATPase function (particularly because NCX function was also unaltered; see below). However, KN-93 significantly prolonged relaxation (RT_80%) at 1 Hz to 225±25 ms versus 187±16 ms (P<0.05). On increasing stimulation frequency from 0.5 to 3 Hz, there tended to be a greater decrease in contraction amplitude in CaMKII_C-transfected myocytes (18%; n=46) versus LacZ myocytes (7%; n=29; P=NS). Frequency-dependent acceleration of relaxation (FDAR) (Figure 2D) was apparent in both CaMKII_C and LacZ cells but was significantly enhanced in CaMKII_C versus LacZ myocytes, consistent with a role for CaMKII in FDAR. The FDAR index _0.5Hz/_3Hz=2.14±0.10 in CaMKII_C versus 1.87±0.10 in LacZ (P<0.05). KN-93 pretreatment partially inhibited FDAR in both myocyte types (not shown).

View larger version (20K): [in this window] [in a new window]   Figure 2. Twitches and intracellular Ca2+ transients. A, Original traces showing twitch shortening at 1 Hz, as well as mean data for fractional shortening (resting cell length [RCL]) and RT50% for LacZ (n=29) and CaMKIIC (n=46). B, Original Ca2+ transients at 1 Hz and average data for Ca2+ transient amplitude ([Ca2+]) and RT50% for LacZ (n=8) and CaMKIIC (n=11). C, Influence of increasing stimulation frequency (0.5 to 3 Hz) on fractional shortening in CaMKIIC-transfected myocytes (n=39) vs LacZ (n=28). D, FDAR index (0.5Hz/ 3Hz) was used to show differences in relaxation behavior between LacZ and CaMKIIC. *P<0.05 vs LacZ.

Ca²⁺ Currents
Figure 3A shows I_Ca–voltage relationships, where peak I_Ca was increased in CaMKII_C versus LacZ by 31% at 0 mV (–7.1±0.5 versus –5.4±0.5 pA/pF; P<0.05). These effects can be significantly reversed by CaMKII inhibition with AIP (–5.8±0.5 pA/pF; P<0.05 versus CaMKII_C). The I_Ca inactivation time constants ₁ and ₂ were significantly prolonged in CaMKII_C versus LacZ myocytes and could be completely reversed by AIP (P<0.05 versus CaMKII_C; Figure 3B). Repeated depolarization to 0 mV after a 1 minute rest causes a CaMKII-dependent I_Ca facilitation (Figure 3C) that was enhanced in CaMKII_C versus LacZ myocytes (P<0.05). The CaMKII inhibitor KN-93 abolished facilitation in CaMKII_C myocytes (P<0.05) and LacZ myocytes (not shown).

View larger version (19K): [in this window] [in a new window]   Figure 3. L-type Ca2+ current (ICa). ICa was significantly increased in CaMKIIC vs LacZ. A, Current–voltage relations. CaMKIIC (n=11), LacZ (n=8), CaMKIIC+AIP (n=13). *P<0.05 vs LacZ, #P<0.05 vs CaMKIIC. B, Inactivation is slowed by CaMKIIC but reaccelerated in the presence of AIP. Original traces normalized to peak ICa and mean data for fast (1) and slow inactivation (2) calculated by biexponential fits. *P<0.05 vs LacZ, #P<0.05 vs CaMKIIC. C, Increased facilitation in CaMKIIC (n=11) vs LacZ (n=11). KN-93 (n=8) reverses facilitation.

SR Ca²⁺ Content and NCX Function
Because no significant changes in twitch shortening or Ca²⁺ transients were found, SR Ca²⁺ load might be expected to be unchanged. However, SR Ca²⁺ content measured by caffeine-induced Ca²⁺ transients was dramatically reduced (by 41%) in CaMKII_C versus LacZ (310±79 versus 521±120 nmol/L; P<0.05; Figure 4A). To assess NCX function, we measured the half-time of [Ca²⁺]_i decline during caffeine-induced Ca²⁺ transients. No change was detectable for NCX function in CaMKII_C versus LacZ (Figure 4B).

View larger version (25K): [in this window] [in a new window]   Figure 4. SR Ca2+ content and NCX function. A, Original traces showing SR Ca content measured by caffeine-induced Ca2+ transients in CaMKIIC (n=11) vs LacZ (n=8). B, As a measure for NCX function, Ca2+ decay during caffeine contractures was analyzed. C, Postrest contractions in CaMKIIC (n=47) vs LacZ (n=61). *P<0.05 vs LacZ. D, Western blots showing unchanged SERCA and NCX protein expression at different MOIs.

During rest, Ca²⁺ that leaks from the SR is partly taken up by the SR and partly extruded by NCX. In rabbit cardiac myocytes, Ca²⁺ is predominantly transported out of the cell via NCX, which leads to a gradual decrease in SR Ca²⁺ content and Ca²⁺ release during postrest twitches (rest decay). This differs from rest potentiation that is typical in rat or mouse myocytes.⁵ Figure 4C shows that rest decay was more pronounced in CaMKII_C (23±4%; P<0.05) versus LacZ (10±4%). This could reflect enhanced SR Ca²⁺ leak or NCX function in CaMKII_C myocytes. Given the unaltered NCX and SERCA function in CaMKII_C cells, enhanced SR Ca²⁺ leak seems likely.

To test whether SERCA or NCX expression differs between LacZ and CaMKII_C myocytes, Western blots were performed at different MOIs of 10 and 100 (normalized to GAPDH). SERCA (n=3) and NCX (n=3) protein expression was unaltered.

SR Ca²⁺ Leak and Ryanodine Receptor Phosphorylation
To more directly assess SR Ca²⁺ leak, we measured Ca²⁺ spark frequency. Because SR Ca²⁺ content is a major determinant of Ca²⁺ spark frequency and SR Ca²⁺ content is very low in CaMKII_C myocytes, spark frequency was normalized to SR Ca²⁺ content (measured at the same time). Normalized Ca²⁺ spark frequency was increased by 88% in CaMKII_C versus LacZ (1.9±0.1 versus 1.0±0.1; P<0.05; Figure 5A), indicating enhanced SR Ca²⁺ leak at a given SR Ca²⁺ content. Similarly, fractional SR Ca²⁺ release during a normal twitch was significantly increased in CaMKII_C cells (0.41±0.06 versus 0.26±0.03; P<0.05; Figure 5B). Although part of this 58% increase in fractional release may be attributable to the 23% increase in peak I_Ca, it may also reflect enhanced RyR sensitivity to Ca²⁺ (especially because the lower SR Ca²⁺ content by itself would tend to greatly reduce fractional SR Ca²⁺ release).^19,20

View larger version (34K): [in this window] [in a new window]   Figure 5. SR Ca2+ leak and RyR phosphorylation. A, Mean data for Ca2+ spark frequency normalized to SR Ca content. B, Fractional release as a ratio for twitch Ca2+ transient/caffeine transient. C, RyR phosphorylation at Ser2809 (normalized to RyR expression) at different MOI and MOI100 compared with Ser2815. D, Coimmunoprecipitation of FKBP12.6 and RyR2 from isolated myocytes overexpressing CaMKIIC showing unaltered in FKBP12.6/ RyR binding. The complexes were immunoprecipitated and blotted with anti-RyR (top), anti-FKBP12.6 (middle), and anti-phospho-RyR2 (S2809) (bottom) antibodies. + indicates recombinant FKBP12.6 loaded as molecular weight standard. Incubation of samples with control antibody or with protein G-sepharose beads alone did not lead to specific immunoprecipitation (lanes 6 and 7, respectively); treatment of cell lysates with 20 µmol/L FK506 before immunoprecipitation completely abolished RyR2-FKBP12.6 association (lane 8), confirming specificity of interaction.

The enhanced SR Ca²⁺ leak and fractional release were associated with significantly increased RyR phosphorylation at Ser2809 and Ser2815 (71±26 and 171±70% respectively, P<0.05; Figure 5C). To investigate whether FKBP12.6 association with RyR2 was altered, coimmunoprecipitation of equal amounts of myocyte lysates with anti-RyR antibody. At equal amount of RyR2 precipitated in each sample, no decrease in FKBP12.6 was observed in cells overexpressing CaMKII_C (Figure 5D), whereas RyR2 phosphorylation was increased. Thus, the increased phosphorylation of RyR elicited by CaMKII affects RyR function but does not dissociate FKBP12.6 from RyR2, in agreement with others.⁹

Reversal of SR Ca²⁺ Leak and CaMKII-Dependent PLB Phosphorylation
The effects of CaMKII_C overexpression are MOI dependent. Figure 6A and 6B show a dose-dependent increase in SR Ca²⁺ spark frequency and amplitude. Figure 6C shows that Ca²⁺ spark frequency can be significantly decreased by the CaMKII_C inhibitors AIP or KN-93 or by inhibiting RyR gating by tetracaine. Figure 6D shows an alternative SR Ca²⁺ leak measurement,¹³ where abrupt RyR block by tetracaine (in Ca²⁺-free, Na⁺-free solution) causes [Ca²⁺]_i to decline and SR Ca²⁺ content to rise (F/F₀ decreased from 1.05±0.08 to 0.75±0.06; P<0.05).

View larger version (26K): [in this window] [in a new window]   Figure 6. Reversal of SR Ca2+ leak and CaMKII-dependent PLB phosphorylation. A, Mean data for Ca2+ spark frequency for LacZ (n=169) and CaMKIIC overexpression using MOI10 (n=75) and 100 (n=112). *P<0.05 vs LacZ, #P<0.05 vs MOI100. B, Mean data for Ca2+ spark amplitude for LacZ and CaMKIIC overexpression using MOI10 and 100. *P<0.05 vs LacZ, #P<0.05 vs MOI100. C, Mean data for Ca2+ spark frequency for CaMKIIC (MOI100) overexpression (n=112) and in the presence of CaMKIIC inhibition using AIP (n=48) and KN-93 (n=8) but also tetracaine (n=6). *P<0.05 vs CaMKIIC. D, Original experiments to inhibit SR Ca2+ leak using tetracaine showing decreased diastolic F/F0 and increase SR Ca2+ content. E, Western blots (n=4) showing specific PLB-Thr17 phosphorylation but decreased PLB-Ser16 phosphorylation in the presence of unchanged PLB protein expression. *P<0.05 vs LacZ being 0%.

In addition, acute CaMKII_C overexpression at MOI100 resulted in increased PLB-Thr17 phosphorylation (+125±42%, P<0.05) but a slight decrease in PLB-Ser16 phosphorylation (–44±26%; P=NS; Figure 6E). In contrast, PLB protein expression is unchanged (as SERCA and NCX in Figure 4D).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study shows for the first time that acute overexpression of CaMKII_C (24 hours) results in increased Ca²⁺ leak from the SR and decreased SR Ca²⁺ load most likely because of RyR phosphorylation. We conclude that the effects of CaMKII_C overexpression on RyR function and SR Ca²⁺ leak as observed in transgenic mice previously are mimicked by adenoviral overexpression in myocytes. Most importantly, however, in contrast to chronic CaMKII_C overexpression in mice, these acute alterations in ECC together with increased I_Ca do not lead to decreased twitch contractions or Ca²⁺ transients. Therefore, CaMKII_C mediated phosphorylation directly increases diastolic RyR opening and enhances ECC efficacy.

CaMKII_C Overexpression
Using adenovirus-mediated gene transfer, we elevated CaMKII_C expression in rabbit cardiac myocytes, and overexpression was specifically in the cytosolic (versus nuclear) compartment, consistent with CaMKII_C lacking the 11 amino acid nuclear localization sequence in the _B splice variant.³ Whereas our previous results in transgenic CaMKII_C mice show clear hypertrophy on the whole heart and myocyte level,⁷ there was no significant increase in myocyte size after 24 hours of overexpression of CaMKII_C. In CaMKII_C transgenic mice, the more prolonged overexpression of CaMKII_C or its possible multimerization with the nuclear CaMKII_B in vivo could contribute to the otherwise unknown hypertrophic mechanism. Notably, in both transgenic CaMKII_C mice and the present acute CaMKII_C overexpressing rabbit myocytes, a major functional finding was increased SR Ca²⁺ leak associated with enhanced RyR phosphorylation and reduced SR Ca²⁺ content. It is possible that the increased diastolic Ca²⁺ leak from the SR may activate Ca²⁺-dependent hypertrophic signaling pathways.^3,21,22

L-Type Ca²⁺ Current
In the present study, CaMKII_C overexpression resulted in significantly enhanced peak I_Ca and also prolonged I_Ca inactivation parameters ₁ and ₂. Because CaMKII can activate I_Ca^23–25 and we see enhanced I_Ca facilitation, the 31% increase in peak I_Ca may reflect a relatively direct CaMKII-dependent regulatory effect on I_Ca. This interpretation is supported by the observation that acute CaMKII inhibition by KN-93 or AIP blocks both amplitude and inactivation effects. Indeed, both higher I_Ca amplitude and slowed inactivation are hallmarks of CaMKII-dependent I_Ca facilitation,^5,23 consistent with a common fundamental mechanism. The increased I_Ca, together with the increases in fractional Ca²⁺ release from the SR, results in unchanged twitch contraction (at least at low stimulation rates), even in the face of decreased SR Ca²⁺ content.

SR Ca²⁺ Content and Contractions
The SR is central in cardiac ECC and CaMKII can accelerate SERCA function via PLB phosphorylation.^2–5 Surprisingly, we did not detect altered SERCA function at baseline contraction frequency in CaMKII_C versus LacZ myocytes. However, we do see modestly enhanced FDAR in the CaMKII_C versus LacZ myocytes (as in transgenic CaMKII_C mice).⁷ FDAR is thought to reflect CaMKII-dependent enhancement of SR Ca²⁺ uptake (even though it does not require PLB).²⁶ Thus, acute CaMKII appears to have only modest effects on SERCA function here. These modest effects on the rate of [Ca²⁺]_i decline here and in our previous study⁷ may be because the absolute extent of CaMKII-dependent PLB phosphorylation may be small²⁷ and the increased phosphorylation at PLB-Thr17 may be counterbalanced by less at Ser16, as seen in the present study.

In transgenic CaMKII_C-overexpressing mouse hearts,⁷ SR Ca²⁺ content was also reduced, but that could have been attributable to the enhanced SR Ca²⁺ leak (and RyR phosphorylation), increased NCX function, or reduced SERCA function that are associated with the heart failure phenotype, as in other heart failure models.^28–31 With acute CaMKII_C expression here, a more modest reduction in myocyte SR Ca²⁺ content occurs with enhanced SR Ca²⁺ leak but unaltered NCX and SERCA function and protein expression. This argues strongly in favor of enhanced SR Ca²⁺ leak causing reduced SR Ca²⁺ content here. However, the more severe reduction in SR Ca²⁺ content in heart failure (whether induced by transgenic CaMKII overexpression or otherwise) is attributable not only to enhanced SR Ca²⁺ leak but also to enhanced NCX function and reduced SERCA function.^28–31

In isolated single-channel RyR recordings, CaMKII has been shown to increase cardiac RyR open probability.^8,32 At an intermediate level of isolation, CaMKII greatly enhanced Ca²⁺ spark frequency in permeabilized PLB-KO mouse myocytes (without enhanced SR Ca²⁺ content).³³ In addition, CaMKII is associated with the RyR in the cell,^7–9 and can phosphorylate the RyR.^7–9,32,34 The cardiac RyR has been reported to be phosphorylated by CaMKII at both Ser2809 and Ser2815 sites.^9,32,34 Mark and colleagues have reported that these sites are segregated (CaMKII only at 2815 and PKA only at 2809) and that Ser2809 phosphorylation causes dissociation of FKBP12.6 from RyR and consequent RyR opening, whereas CaMKII-dependent phosphorylation activates RyR without causing FKBP12.6 dissociation.^9,18 Likewise, here we do not see FKBP12.6 dissociation from the RyR in myocytes overexpressing CaMKII_C, despite some increase in RyR phosphorylation at Ser2809. Thus RyR phosphorylation appears to cause enhanced SR Ca²⁺ leak and reduced SR Ca²⁺ content in CaMKII_C versus LacZ myocytes.

A remarkable finding here is that twitch contractions and Ca²⁺ transients are almost unaffected by the dramatically reduced SR Ca²⁺ content. This may be attributable in part to the enhanced I_Ca (as above), but a major factor is probably the sort of autoregulation described previously by Trafford et al³⁵ in the presence of low caffeine concentration (which causes diastolic SR Ca²⁺ leak and sensitizes the RyR to Ca²⁺). They showed that altered RyR gating only produces transient changes in Ca²⁺ transients (but sustained changes in SR Ca²⁺ content and fractional release). That is, abruptly CaMKII may enhance SR Ca²⁺ release, but this causes more Ca²⁺ extrusion (via NCX) and reduces SR Ca²⁺ content. With the lower SR Ca²⁺ content the enhanced fractional release only results (in the steady state) in the same amount of SR Ca²⁺ release. This may be what is happening here with acute CaMKII_C overexpression. Indeed, Shannon et al³⁶ recently simulated Ca²⁺ homeostasis mathematically showing that enhanced RyR Ca²⁺ sensitivity (as by caffeine or phosphorylation) increased SR Ca²⁺ leak and reduced SR Ca²⁺ content but enhanced SR fractional release without decreasing the size of the steady-state [Ca²⁺]_i transient (as seen here).

Thus, enhanced RyR Ca²⁺ sensitivity by itself may contribute substantially to SR Ca²⁺ unloading on CaMKII overexpression (or in heart failure), without itself being appreciably negatively inotropic. Other factors must be largely responsible for the systolic dysfunction seen in CaMKII_C transgenic mice or other heart failure models (eg, reduced SERCA function and enhanced NCX function).^7,29,31 Indeed, in heart failure, CaMKII is overexpressed,^11,37 SR Ca²⁺ leak is enhanced,^31,37 and block of CaMKII in heart failure can greatly enhance SR Ca²⁺ content without improving systolic function.³⁷ We conclude that CaMKII-dependent enhancement of RyR Ca²⁺ sensitivity (and thus leak) does not contribute appreciably to systolic dysfunction, but the enhanced diastolic SR Ca²⁺ leak could possibly increase the propensity for triggered arrhythmias.³⁸

	Acknowledgments

 
D.M.B. is funded by NIH grants HL-30077 and HL-64724 and J.H.B. by HL-46345 and HL-28143. T.Z. is the recipient of a Scientist Development Grant from the American Heart Association. L.S.M. is funded by the Deutsche Forschungsgemeinschaft (DFG) through an Emmy Noether grant (MA 1982/4–1), by a Young Investigator Award of the GlaxoSmithKline Research Foundation, and by a grant from the Medical Faculty of the University of Göttingen (Anschubfinanzierung).

	Footnotes

 
Original received July 18, 2005; resubmission received October 31, 2005; accepted December 8, 2005.

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