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

The predominant cardiac Ca2+/calmodulin-dependent protein kinase (CaMK) is CaMKII&dgr;. Here we acutely overexpress CaMKII&dgr;C using adenovirus-mediated gene transfer in adult rabbit ventricular myocytes. This circumvents confounding adaptive effects in CaMKII&dgr;C transgenic mice. CaMKII&dgr;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&dgr;C versus LacZ expressing myocytes. However, the contraction–frequency relationship was more negative, frequency-dependent acceleration of relaxation was enhanced (&tgr;0.5Hz/&tgr;3Hz=2.14±0.10 versus 1.87±0.10), and peak Ca2+ current (ICa) 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&dgr;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&dgr;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&dgr;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&dgr;C myocytes (ie, CaMKII-dependent enhancement of RyR Ca2+ sensitivity during diastole and systole) and increased ICa.

C a 2ϩ /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), Ca 2ϩ entry, mainly via voltage dependent L-type Ca 2ϩ channels (I Ca ), triggers sarcoplasmic reticulum (SR) Ca 2ϩ release via ryanodine receptors (RyRs), via Ca 2ϩ -induced Ca 2ϩ release. 4,5 The resultant increase in intracellular [Ca 2ϩ ] ([Ca 2ϩ ] i ), causes Ca 2ϩ binding to troponin C, which activates myofilaments, leading to contraction. For relaxation to occur, Ca 2ϩ must be removed from the cytoplasm. SR Ca-ATPase (SERCA) and Na ϩ /Ca 2ϩ -exchanger (NCX) are the main mechanisms for Ca 2ϩ removal. 4,5 CaMKII can modulate ECC by phosphorylating several important Ca 2ϩ -dependent regulatory proteins in heart, including Ca 2ϩ transport proteins, such as RyR and phospholamban (PLB), and possibly L-type Ca 2ϩ channels. 2,5 CaMKII is directly associated with RyR and overexpression of CaMKII in transgenic mouse cardiomyocytes increases SR Ca 2ϩ release as shown by increased frequency of spontaneous SR Ca 2ϩ release events (Ca 2ϩ sparks). 6,7 Blocking CaMKII (using KN-93) decreases Ca 2ϩ 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 Ca 2ϩ spark frequency in rabbit hearts because of decreased endogenous CaMKIIdependent RyR phosphorylation. Wehrens et al 9 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 Ca 2ϩ handling with marked reductions in Ca 2ϩ transients, SR Ca 2ϩ 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 Ca 2ϩ sparks (indicative of diastolic spontaneous SR Ca 2ϩ release events or opening of RyR clusters) was greatly enhanced, demonstrating increased diastolic SR Ca 2ϩ leak despite reduced SR Ca 2ϩ load and diastolic [Ca 2ϩ ] i 7 (which by themselves would normally reduce SR Ca 2ϩ leak). 13 We showed that this was most likely attributable to increased CaMKII-dependent RyR phosphorylation increasing RyR openings, because Ca 2ϩ 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 Ca 2ϩ transport, it was unclear how direct effects of acute CaMKIIdependent protein phosphorylation alter Ca 2ϩ 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 Ca 2ϩ handling. We demonstrate that acute CaMKII␦ C overexpression enhances SR Ca 2ϩ leak and reduces SR Ca 2ϩ 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 Ca 2ϩ transients are unaltered. This is attributed to an increased fractional SR Ca 2ϩ release (and I Ca ), which may result from the same CaMKII␦ C -dependent enhancement of RyR Ca 2ϩ sensitivity that enhances diastolic SR Ca 2ϩ leak.

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 Ϸ4.2ϫ10 3 rod-shaped cells/cm 2 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% CO 2 , 95% O 2 ). Myocyte volume was calculated from myocytes lengthϫwidthϫ40% of width. 7

Shortening and Ca 2؉ Measurements Using Inverted Microscopes
Shortening and [Ca 2ϩ ] i measurements were performed as reported previously. 7,17 Briefly, myocytes were loaded with the Ca 2ϩ -sensitive dye indo-1/acetoxymethyl ester to measure diastolic [Ca 2ϩ ]. Because no difference in diastolic Ca 2ϩ was found, additional experiments used fluo-3/acetoxymethyl ester (10 mol/L, respectively; Molecular Probes). Excitation wavelengths (360Ϯ5 nm for indo and 480Ϯ15 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 405Ϯ15 nm and 485Ϯ12.5 nm for indo and 535Ϯ20 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/F 0 was calculated by dividing by the baseline fluorescence F 0 , 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.

Confocal Microscopy
Ca 2ϩ 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. [Ca 2ϩ ] i was calibrated by the equation [Ca 2ϩ ] i ϭK d (F/F 0 )/(K d /[Ca 2ϩ ] i-restϩ1ϪF/F 0 ) with K d ϭ1100 nmol/L and [Ca 2ϩ ] i-rest ϭ100 nmol/L. 7 Ca 2ϩ sparks were analyzed by a program (IDL 5.3) 7 that detects Ca 2ϩ sparks as areas of increased fluorescence with respect to the SD of background fluorescence. We used a Ca 2ϩ spark threshold of 3.8ϫSD, with human verification. Peaks of Ca 2ϩ sparks were normalized as F/F 0 , and duration was taken from the full-duration half-maximum (FDHM). Width or spatial size was taken as fullwidth half-maximum (FWHM). Ca 2ϩ 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 Ϫ1 s Ϫ1 ), assuming voxel length and width of 0.2 m and depth of 1 m.

Solutions and Experimental Protocol
Normal Tyrode's solution contained (mmol/L) 140 NaCl, 6 KCl, 10 HEPES, 10 glucose, 1 MgCl 2 , and 2 CaCl 2 (37°C). SR Ca 2ϩ load was evaluated by Ca 2ϩ transient amplitudes induced by rapid caffeine (10 mmol/L) application. NCX function was assessed measuring Ca 2ϩ decay 50% relaxation (RT 50% ) after caffeine application. During this procedure, Ca 2ϩ uptake by SERCA is prevented and other Ca 2ϩ elimination pathways (eg, mitochondrial uniporter, sarcolemmal Ca 2ϩ ATPase, contributing Ͻ1% each) can be neglected. 5 In a subset of experiments, KN-93 (1 mol/L) or the membrane permeant AIP (20 mol/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 Ca 2ϩ 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, phosphospecific 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 (5ϫ10 5 ) were lysed in 250 L 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 mol/L leupeptin, 1 mol/L pepstatin A, 1 mmol/L benzamidine, 1 mol/L of calpain inhibitor I, and 1 mol/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.

Statistics
Results are expressed as meanϮSEM. Significance (PϽ0.05) 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 [Ca 2ϩ ] i decline, Ca , were monoexponential least-square fits. Time constants 1 and 2 of I Ca inactivation were fitted biexponentially. Figure 1A shows a typical CaMKII Western blot with rabbit myocytes infected at MOI100 (24 hours). Average overexpression of CaMKII␦ was Ϸ6-fold that of LacZ controls (PϽ0.05; nϭ5). In addition, myocytes showed MOIdependent (1, 10, 100) increases in CaMKII protein expression, and also in CaMKII phosphorylation status, with maximum phosphorylation increases of Ϸ5-fold at MOI100 (PϽ0.05). 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 (41.6Ϯ2.5 pL; nϭ66) versus LacZ control (35.9Ϯ1.8 pL; nϭ154). Similarly, in voltage-clamped myocytes, membrane capacity was not different between LacZ (112.0Ϯ5.1 pF) and CaMKII␦ C (113.5Ϯ6.9 pF). These results also suggest no alteration in surface to volume ratio.

SR Ca 2؉ Content and NCX Function
Because no significant changes in twitch shortening or Ca 2ϩ transients were found, SR Ca 2ϩ load might be expected to be unchanged. However, SR Ca 2ϩ content measured by caffeineinduced Ca 2ϩ transients was dramatically reduced (by 41%) in CaMKII␦ C versus LacZ (310Ϯ79 versus 521Ϯ120 nmol/L; PϽ0.05; Figure 4A). To assess NCX function, we measured the half-time of [Ca 2ϩ ] i decline during caffeine-induced Ca 2ϩ transients. No change was detectable for NCX function in CaMKII␦ C versus LacZ ( Figure 4B). During rest, Ca 2ϩ that leaks from the SR is partly taken up by the SR and partly extruded by NCX. In rabbit cardiac myocytes, Ca 2ϩ is predominantly transported out of the cell via NCX, which leads to a gradual decrease in SR Ca 2ϩ content and Ca 2ϩ 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 (23Ϯ4%; PϽ0.05) versus LacZ (10Ϯ4%). This could reflect enhanced SR Ca 2ϩ leak or NCX function in CaMKII␦ C myocytes. Given the unaltered NCX and SERCA function in CaMKII␦ C cells, enhanced SR Ca 2ϩ leak seems likely.

Circulation Research February 3, 2006 SR Ca 2؉ Leak and Ryanodine Receptor Phosphorylation
To more directly assess SR Ca 2ϩ leak, we measured Ca 2ϩ spark frequency. Because SR Ca 2ϩ content is a major determinant of Ca 2ϩ spark frequency and SR Ca 2ϩ content is very low in CaMKII␦ C myocytes, spark frequency was normalized to SR Ca 2ϩ content (measured at the same time). Normalized Ca 2ϩ spark frequency was increased by 88% in CaMKII␦ C versus LacZ (1.9Ϯ0.1 versus 1.0Ϯ0.1; PϽ0.05; Figure 5A), indicating enhanced SR Ca 2ϩ leak at a given SR Ca 2ϩ content. Similarly, fractional SR Ca 2ϩ release during a normal twitch was significantly increased in CaMKII␦ C cells (0.41Ϯ0.06 versus 0.26Ϯ0.03; PϽ0.05; 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 Ca 2ϩ (especially because the lower SR Ca 2ϩ content by itself would tend to greatly reduce fractional SR Ca 2ϩ release). 19,20 The enhanced SR Ca 2ϩ leak and fractional release were associated with significantly increased RyR phosphorylation at Ser2809 and Ser2815 (71Ϯ26 and 171Ϯ70% respectively, PϽ0.05; 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 elic-ited by CaMKII affects RyR function but does not dissociate FKBP12.6 from RyR2, in agreement with others. 9

Reversal of SR Ca 2؉ 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 Ca 2ϩ spark frequency and amplitude. Figure 6C shows that Ca 2ϩ 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 Ca 2ϩ leak measurement, 13 where abrupt RyR block by tetracaine (in Ca 2ϩ -free, Na ϩ -free solution) causes [Ca 2ϩ ] i to decline and SR Ca 2ϩ content to rise (F/F 0 decreased from 1.05Ϯ0.08 to 0.75Ϯ0.06; PϽ0.05).

Discussion
The present study shows for the first time that acute overexpression of CaMKII␦ C (24 hours) results in increased Ca 2ϩ leak from the SR and decreased SR Ca 2ϩ load most likely because of RyR phosphorylation. We conclude that the effects of CaMKII␦ C overexpression on RyR function and SR Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ leak associated with enhanced RyR phosphorylation and reduced SR Ca 2ϩ content. It is possible that the increased diastolic Ca 2ϩ leak from the SR may activate Ca 2ϩ -dependent hypertrophic signaling pathways. 3,21,22

L-Type Ca 2؉ Current
In the present study, CaMKII␦ C overexpression resulted in significantly enhanced peak I Ca and also prolonged I Ca inac- tivation 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 Ca 2ϩ release from the SR, results in unchanged twitch contraction (at least at low stimulation rates), even in the face of decreased SR Ca 2ϩ content.

SR Ca 2؉ 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 CaMKIIdependent enhancement of SR Ca 2ϩ 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 [Ca 2ϩ ] i decline here and in our previous study 7 may be because the absolute extent of CaMKIIdependent PLB phosphorylation may be small 27 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 Ca 2ϩ content was also reduced, but that could have been attributable to the enhanced SR Ca 2ϩ 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 Ca 2ϩ content occurs with enhanced SR Ca 2ϩ leak but unaltered NCX and SERCA function and protein expression. This argues strongly in favor of enhanced SR Ca 2ϩ leak causing reduced SR Ca 2ϩ content here. However, the more severe reduction in SR Ca 2ϩ content in heart failure (whether induced by transgenic CaMKII overexpression or otherwise) is attributable not only to enhanced SR Ca 2ϩ 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 Ca 2ϩ spark frequency in permeabilized PLB-KO mouse myocytes (without enhanced SR Ca 2ϩ content). 33 In addition, CaMKII is associated with the RyR in the cell, 7-9 and can phosphorylate the RyR. [7][8][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 Ca 2ϩ leak and reduced SR Ca 2ϩ content in CaMKII␦ C versus LacZ myocytes. Figure 6. Reversal of SR Ca 2ϩ leak and CaMKII-dependent PLB phosphorylation. A, Mean data for Ca 2ϩ spark frequency for LacZ (nϭ169) and CaMKII␦ C overexpression using MOI10 (nϭ75) and 100 (nϭ112). *PϽ0.05 vs LacZ, #PϽ0.05 vs MOI100. B, Mean data for Ca 2ϩ spark amplitude for LacZ and CaMKII␦ C overexpression using MOI10 and 100. *PϽ0.05 vs LacZ, #PϽ0.05 vs MOI100. C, Mean data for Ca 2ϩ spark frequency for CaMKII␦ C (MOI100) overexpression (nϭ112) and in the presence of CaMKII␦ C inhibition using AIP (nϭ48) and KN-93 (nϭ8) but also tetracaine (nϭ6). *PϽ0.05 vs CaMKII␦ C . D, Original experiments to inhibit SR Ca 2ϩ leak using tetracaine showing decreased diastolic F/F 0 and increase SR Ca 2ϩ content. E, Western blots (nϭ4) showing specific PLB-Thr17 phosphorylation but decreased PLB-Ser16 phosphorylation in the presence of unchanged PLB protein expression. *PϽ0.05 vs LacZ being 0%.
A remarkable finding here is that twitch contractions and Ca 2ϩ transients are almost unaffected by the dramatically reduced SR Ca 2ϩ 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 al 35 in the presence of low caffeine concentration (which causes diastolic SR Ca 2ϩ leak and sensitizes the RyR to Ca 2ϩ ). They showed that altered RyR gating only produces transient changes in Ca 2ϩ transients (but sustained changes in SR Ca 2ϩ content and fractional release). That is, abruptly CaMKII may enhance SR Ca 2ϩ release, but this causes more Ca 2ϩ extrusion (via NCX) and reduces SR Ca 2ϩ content. With the lower SR Ca 2ϩ content the enhanced fractional release only results (in the steady state) in the same amount of SR Ca 2ϩ release. This may be what is happening here with acute CaMKII␦ C overexpression. Indeed, Shannon et al 36 recently simulated Ca 2ϩ homeostasis mathematically showing that enhanced RyR Ca 2ϩ sensitivity (as by caffeine or phosphorylation) increased SR Ca 2ϩ leak and reduced SR Ca 2ϩ content but enhanced SR fractional release without decreasing the size of the steadystate [Ca 2ϩ ] i transient (as seen here).
Thus, enhanced RyR Ca 2ϩ sensitivity by itself may contribute substantially to SR Ca 2ϩ 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 Ca 2ϩ leak is enhanced, 31,37 and block of CaMKII in heart failure can greatly enhance SR Ca 2ϩ content without improving systolic function. 37 We conclude that CaMKII-dependent enhancement of RyR Ca 2ϩ sensitivity (and thus leak) does not contribute appreciably to systolic dysfunction, but the enhanced diastolic SR Ca 2ϩ leak could possibly increase the propensity for triggered arrhythmias. 38