Published online before print February 27, 2003, doi: 10.1161/01.RES.0000064566.91495.0C
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© 2003 American Heart Association, Inc.
Cellular Biology |
Calmodulin Regulation of Excitation-Contraction Coupling in Cardiac Myocytes
From the National Laboratory of Biomembrane and Membrane Biotechnology (D.Y., C.W., H.C.), College of Life Sciences, Peking University, Beijing, China; Laboratory of Cardiovascular Sciences (D.Y., L.-S.S., W.-Z.Z., K.C., W.W., R.-P.X., H.C.), National Institute on Aging, National Institutes of Health, Baltimore, Md; the Department of Physiology (Y.W.), School of Medicine, University of Maryland, Baltimore, Md; Cardiovascular Research Group (S.R.W.C.), Departments of Physiology & Biophysics and Biochemistry & Molecular Biology, University of Calgary, Calgary, Alberta, Canada.
Correspondence to Heping Cheng, PhD, Laboratory of Cardiovascular Sciences, NIA, NIH, Baltimore, MD 21224.E-mail chengp{at}grc.nia.nih.gov
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Abstract |
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Calmodulin (CaM) as a ubiquitous Ca2+ sensor interacts with multiple key molecules involved in excitation-contraction (EC) coupling. In the present study, we report that adenoviral expression of a mutant CaM lacking all of its four Ca2+-binding sites, CaM(1-4), at a level 6.5-fold over endogenous CaM markedly increases the amplitude and abbreviates the decay time of Ca2+ transients and contraction in cultured rat ventricular myocytes. To determine the underlying mechanisms, we examined the properties of L-type Ca2+ channels, Ca2+/CaM-dependent protein kinase II (CaMKII), and phospholamban (PLB) in the sarcoplasmic reticulum (SR). We found that CaM(1-4) expression markedly augmented L-type Ca2+ current amplitude and slowed its inactivation. Surprisingly, overexpression of CaM(1-4) increased CaMKII activity and phosphorylation of PLB-Thr-17. Moreover, CaM(1-4) elevated diastolic Ca2+ and caffeine-labile Ca2+ content of the SR. Inhibition of CaMKII by KN-93 or a myristoylated autocamtide-2 related inhibitory peptide prevented the aforementioned PLB phosphorylation and reversed the positive inotropic and relaxant effects, indicating that CaMKII is essential to CaM(1-4) actions. These results demonstrate that CaM modulates Ca2+ influx, SR Ca2+ release, and Ca2+ recycling during cardiac EC coupling. A novel finding of this study is that expression of a Ca2+-insensitive CaM mutant can lead to activation of CaMKII in cardiac myocytes.
Key Words: calmodulin • Ca2+/calmodulin-dependent protein kinase II • L-type Ca2+ channels • phospholamban
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Introduction |
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Intracellular Ca2+ acts as a universal and versatile second messenger that regulates vital biological processes, including cell contraction, synaptic transmission, hormone secretion, cell growth, and cell death.1,2 The specificity and versatility of Ca2+ signaling are in part endowed by the spatial and temporal patterning of both the amplitude and frequency of Ca2+ signals. During eukaryotic evolution, nature has also evolved a sophisticated means to orchestrate the intricate Ca2+ signaling, ie, the use of small Ca2+-binding proteins, such as calmodulin (CaM), as Ca2+ transducer to sort the Ca2+ signals toward different cellular effectors. The multifunctional CaM contains 148 highly conserved amino acid residues, with two pairs of Ca2+ binding sites of affinities in its N- and C-terminal lobes, and an effector-binding motif connecting these two domains.3 CaM binds to different effectors in either Ca2+-free (apoCaM)4 or Ca2+-bound forms,3 mediating the Ca2+-dependent regulation of diverse cellular processes.
CaM is known to interact with multiple proteins involved in excitation-contraction (EC) coupling and Ca2+ homeostasis.5,6 Studies in heterologous expression system have shown that CaM preassociates with the C-terminus of Ca2+ channels located in the plasma membrane and bifurcately mediates Ca2+-dependent inactivation7–9 and facilitation7,9 of the channel. Cardiac sarcoplasmic reticulum (SR) vesicles bind approximately four CaM molecules per type 2 ryanodine receptor (RyR2) tetramer in the absence of Ca2+ or 7.5 CaM molecules per tetramer in the presence of 100 µmol/L Ca2+.10 Similarly, skeletal muscle isoform of RyR (RyR1) can bind both Ca2+-free and Ca2+-bound CaM, at a stoichiometry of 4 CaM per RyR1 tetramer,11 exerting bifunctional regulation of Ca2+-dependent channel activation and inactivation.12 Furthermore, Ca2+-bound CaM can activate many types of protein kinases (eg, Ca2+/CaM-dependent kinase II, CaMKII)13 or phosphatases (eg, calcineurin)14 to regulate the phosphorylation status, thereby affecting the functioning of a wide spectrum of effector proteins, including L-type Ca2+ channel (LCC),15,16 Na+ channel,17 and the SR Ca2+-ATPase regulator protein phospholamban (PLB).18,19
To delineate potential roles of CaM in regulating cardiac EC coupling and Ca2+ signaling, we used a molecular approach to alter the Ca2+-sensing ability of CaM in cultured rat ventricular myocytes, and characterized its functional consequences, including LCC Ca2+ current (ICa), intracellular Ca2+ transients, cell contraction, protein phosphorylation, and kinase activity. Our results indicate that expression of a mutant CaM that is deficient in Ca2+ binding results in prominent contractile and Ca2+ phenotypes. Our data also suggest a previously unappreciated mode of CaMKII activation.
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Materials and Methods |
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Construction of Viral Vectors
A synthetic gene encoding the wild-type (WT) bovine CaM20 was used as a template for the construction of the mutant CaM with residues Asp21, 57, 94, and 130 all changed to Ala [CaM(1-4)] by site-directed mutagenesis. These Asp residues are in the first positions of the four Ca2+ coordinating sites of CaM, and the Ala replacements result in a loss of Ca2+-dependent shift in electrophoretic mobility in nondenaturing gel in the absence (2 mmol/L EGTA) or presence of 5 mmol/L Ca2+.21 The generation and amplification of adenoviruses harboring the CaM(1-4) target-gene were performed in HEK293 cells.
Cell Culture and Adenoviral Infection
Single cardiac myocytes were isolated from the hearts of 2 to 3 month old Sprague-Dawley rats using a standard enzymatic technique, then cultured and infected with adenoviral vectors at a multiplicity of infection (moi) of 100, as described previously.22,23 Briefly, myocytes were plated at a density of 0.5 to 1x104/cm2 in dishes precoated with 10 µg/mL laminin (Upstate Biotechnology). The culture medium23 (M199, SIGMA) contains (in mmol/L) creatine 5, L-carnitine 2, taurine 5, insulin-transferrin-selenium-X 0.1%, penicillin, and streptomycin 1%, and HEPES 25 (adjusted with NaOH, pH 7.4at 37°C). Cells were used 48 hours after infection, unless specified otherwise.
Immunostaining
After fixation and blocking, cells were incubated with anti-CaM antibody (1:100, Upstate Biotechnology) at 4°C overnight and then stained with Cy5-conjugated secondary antibody (1:500, Jackson ImmunoResearch Laboratories). Confocal images were obtained with a Zeiss LSM 510 (Carl Zeiss Inc, Germany). In the negative control, cells were incubated with only the secondary antibodies, and displayed negligible fluorescence under otherwise identical conditions (data not shown).
Western Blotting
For quantifying the expression of WT and mutant CaM, cell lysate (30 µg protein) were loaded in a Ca2+-free loading buffer containing 20 mmol/L EDTA and immunoblotted using anti-CaM antibody (1:1000, Upstate Biotechnology) and HRP-conjugated secondary antibody (Bio-Rad). To detect PLB phosphorylation, the phosphorylation site-specific P-Thr-17PLB antibody (1:10000, PhosphoProtein Research, UK) and HRP-conjugated secondary antibody (Bio-Rad) were employed. After incubation with a peroxidase-conjugated antibody (Dianova), films were exposed to the chemiluminescence (ECL, Amersham Pharmacia Biotech) reaction and quantified with a video documentation system (Bio-Rad).
Ca2+ Transients and Contraction Measurement
Myocytes were field-stimulated at 0.5Hz with perfusion solution containing (in mmol/L) NaCl 137, KCl 4.9, CaCl2 1, MgSO4 1.2, NaH2PO4 1.2, glucose 15, and HEPES 20 (pH 7.4 adjusted with NaOH). In indicator-unloaded myocytes, cell length was monitored by an optical edge tracking method24 at a 3-ms time resolution. In a subset of experiments, myocytes were loaded with the Ca2+ indicator fluo-4/AM (20 µmol/L and 30 minutes; or 5 µmol/L and 15 minutes for freshly isolated cells) (Molecular Probes) and line scan images of Ca2+ transients were obtained with a Zeiss LSM 410. For ratiometric Ca2+ measurement, cells were loaded with 25 µmol/L indo-1/AM for 30 minutes, and cytosolic free Ca2+ concentration was indexed by the emission ratio of r410/490.
Measurement of ICa
An Axopatch 200B patch-clamp amplifier (Axon Instruments) was used for the recording of ICa. The pipette (1.5 to 3 M
) filling solution contains (in mmol/L) CsCl 110, MgCl2 1.5, MgATP 5, NaCl 10, TEA-Cl 10, EGTA 4, CaCl2 2, and HEPES 20 (pH 7.2 adjust with CsOH). The extracellular solution contains (in mmol/L) NaCl 137, CsCl 5.4, MgCl2 1.2, NaH2PO4 1, CaCl2 1, glucose 20, tetrodotoxin 0.01, and HEPES 20 (pH 7.4 adjust with NaOH). ICa was activated by 400 ms-depolarizations from holding potential of -60 mV to test potentials ranging from -40 to +60 mV. Serial resistance was compensated by 70% to 75%. The magnitude of ICa was indexed by the difference between the peak inward current and the baseline. All measurements were performed at room temperature (23°C to 25°C).
Coimmunoprecipitation
Adenoviral infected myocytes or HEK293 cells were lysated in a buffer containing (in mmol/L) NaCl 150, EDTA 10, Na3VO4 1, NP-40 1%, Tris-Cl 20, and 1x protease inhibitor cocktail (pH 7.4). Cell lysate (300 µg protein) was immunoprecipitated with an anti-CaMKII antibody (1:100, Santa Cruz Biotechnology). The precipitated immunoreactive complexes were then subjected to Western blotting using anti-CaM antibody described above.
Assay of CaMKII Activity
To measure CaMKII activity, cell lysate (200 µg protein) was first immunoprecipitated with anti-CaMKII antibody (1:100) in a Ca2+-free medium containing (in mmol/L) Tris-HCl 20, NaCl 150, Na2-EDTA 1, EGTA 1, Triton 1%, sodium pyrophosphate 2.5, ß-glycerophate 1, Na3VO4 1, and leupeptin 1 µg/mL (pH 7.4). The precipitated proteins were then incubated with a specific peptide substrate (KKALRRQETVDAL) to evaluate the kinase activity according to the manufacturer’s recommendations (Upstate Biotechnology Inc), as previously described.25
Statistics
Data were reported as mean±SEM. Student’s t test, paired t test, 
2 test, or ANOVA with repeated measurement was applied, when appropriate, to determine statistical significance of the differences. A value of P<0.05 was considered statistically significant.
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Expression and Subcellular Localization of CaM(1-4) in Cardiac Myocytes
Using Western blotting with an antibody recognizing the C-terminus of CaM, we detected a single band corresponding to endogenous CaM in cells expressing ß-galactosidase (ß-gal) as control, but two clearly separated bands in cells infected with Adv-CaM(1-4): the upper band corresponding to endogenous CaM, and the lower one corresponding to CaM(1-4) with a slightly greater electrophoretic mobility in Ca2+-free medium.21 CaM(1-4) was expressed in a time-dependent manner. In contrast, the amount of endogenous CaM was maintained at a constant level, regardless of CaM(1-4) expression (Figure 1A). At moi 100, the densitometry ratio of CaM measured in CaM(1-4) versus ß-gal group was 1.11±0.12 (n=6, P=0.16) or 1.20±0.22 (n=8, P=0.21); the ratio of mutant to endogenous CaM was 2.0±0.2 (n=6, P<0.01) or 6.5±1.0 (n=8, P<0.01) after 1- or 2-day culture, respectively (Figure 1B). Interestingly, infection with adv-CaM(WT) under otherwise identical conditions increased the abundance of CaM in HEK293 cells, but not in cardiac myocytes, suggesting a tight posttranscriptional regulation of this signaling molecule in the heart (authors’ unpublished results, 2003).
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The subcellular localization of CaM and its mutant was then visualized by confocal immunocytochemistry. In cells infected with adv-ß-gal, specific immunofluorescent signal from endogenous CaM was dim and grossly homogenous (Figure 1C). After two-day culture, nearly all myocytes exhibited intense immunostaining, with the signal concentrated beneath the surface membrane (Figure 1C). This pattern indicates that CaM(1-4) associates with both cytosolic and, preferentially, membrane-delimited effector proteins. Because no nuclei could be discerned against the background, CaM(1-4) was neither excluded nor enriched in the nuclear region, consistent with the idea that CaM can enter the nucleus.26
CaM(1-4) Expression Induced Positive Inotropic and Relaxant Effects
We opted to use cell contraction as the endpoint readout to examine the overall effect of CaM(1-4) expression on cardiac EC coupling. The Table shows that, as compared with freshly isolated myocytes, cultured and adv-ß-gal infected rat cardiac myocytes for up to 48 hours did not significantly alter the contractile parameters examined, whereas culture for one day resulted in slowing of relaxation in mouse cardiac myocytes.22 Figure 2A shows twitch amplitude (TA) in response to 0.5 Hz electrical stimulation applied after a 2-minute rest in indicator-unloaded myocytes infected with adv-ß-gal or adv-CaM(1-4). In control cells, there was a steep negative staircase of cell shortening, which is similar to that seen in fresh cells.27 However, only a mild beat-dependent change in cell shortening occurred in myocytes expressing CaM(1-4). At the steady state, TA and the maximal velocity of shortening were increased by 1.1- and 1.4-fold, respectively, relative to that in ß-gal group (Figure 2A, Table). The positive inotropic effect of CaM(1-4) was accompanied by a marked relaxant effect: the maximal rate of relaxation was increased by 163%, and the 90% relaxation time (T90) was diminished by 33% (Table). Thus, a high-level expression of CaM(1-4) exerts both positive inotropic and relaxant effects in cultured cardiac myocytes, indicating that CaM plays an important role in regulation of cardiac contractility.
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Effects of CaM(1-4) Expression on Intracellular Ca2+ Transients
To dissect cellular mechanisms underlying the contractile responses, we next measured intracellular Ca2+ in the presence and absence of CaM(1-4). Ratiometric Ca2+ measurement using indo-1 revealed no significant change at the resting Ca2+ level between CaM(1-4) (r410/490=0.93±0.02, n=35 cells from 5 hearts) and ß-gal group (0.92±0.02, n=35 from 5 hearts). However, electrical pacing at 0.5 Hz raised the diastolic Ca2+ level in CaM(1-4)–expressing cells (1.12±0.06, n=11 cells from 3 hearts; P<0.01 versus at rest), but not in ß-gal control (0.94±0.02, n=15 cells from 3 hearts; P>0.05 versus at rest), indicating a pacing-dependent accumulation of intracellular Ca2+ in the presence of CaM(1-4).
As compared with ß-gal group or fresh myocytes, the amplitude of Ca2+ transients in CaM(1-4) group increased markedly both in the first beat after rest and at the steady state (Figures 2B and 2C; Table). Concomitantly, CaM(1-4) expression was accompanied by an enhanced cell shortening with flattened negative staircase in these indicator-loaded myocytes (Figures 2C and 2D). The presence of exogenous Ca2+ buffer (ie, the Ca2+ indicator) had only a marginal effect in reducing TA (Figures 2A and 2D) except for the first beat after in ß-gal group, unmasking a trend for an increased first TA after rest in the CaM(1-4) group (15.0±1.4%, n=13; versus 10.9±2.3%, n=12 in ß-gal group; P=0.14). This suggests that the lack of CaM(1-4) effect on the first TA after rest in indicator-unloaded myocytes (Figure 2A) is due to a saturation of cell contractility. As was the case with cell-shortening, CaM(1-4) expression resulted in an accelerated relaxation of Ca2+ transients, even for the first beat after rest (Figure 2C). On average, the T90 of Ca2+ transient was reduced by 62% (Table). The accelerated decay of Ca2+ transients indicates a more powerful SR Ca2+ reuptake, whereas the augmented systolic Ca2+ indicates a greater SR Ca2+ release. It is worthy to note that these alterations in Ca2+ transients did not cause any instability in Ca2+ regulation, as CaM(1-4) did not increase the propensity of spontaneous propagating Ca2+ waves (4 out of 16 cells versus 3 out of 16 cells in ß-gal and CaM(1-4) group, respectively; P>0.05, 2 test).
Modulation of ICa by CaM(1-4)
It has been shown in a cell line7 or guinea pig cardiac myocytes28 that preassociation of Ca2+-insensitive CaM mutant to LCCs alters ICa kinetics by eliminating Ca2+-dependent inactivation of LCC, whereas ICa amplitude is either unchanged28 or even decreased (due to reduced LCC expression).7 Because ICa serves as the physiological trigger of SR Ca2+ release, we next examined whether expression of CaM(1-4) alters properties of ICa in rat heart cells. Figure 3A shows two families of ICa traces at 6 different membrane voltages from typical myocytes expressing ß-gal or CaM(1-4), with 1 mmol/L Ca2+ as the charge carrier. Two prominent changes in ICa were associated with CaM(1-4) expression: the slowing of ICa inactivation and the augmentation of peak ICa. Quantitatively, the 63% decay time (T63) of ICa was more than doubled at 0 mV; and its voltage dependence was transformed from a roughly "U"-shaped curve in ß-gal group to a sigmoid curve in CaM(1-4) group (Figure 3C). These observations are in agreement with previous observations7,28 and support the notion that preassociation of apoCaM to the channel mediates Ca2+-dependent inactivation of ICa.7–9 Furthermore, we detected a robust, 2-fold increase in ICa density (at 0 mV: -7.3±0.4 pA/pF, n=12 in ß-gal group; -14.9±1.0 pA/pF, n=12 in CaM(1-4) group; P<0.001), with the current-voltage relationship unchanged (Figure 3B). Thus, the greater and more persistent ICa can explain, at least in part, the augmented Ca2+ transients and contraction consequential to CaM(1-4) expression.
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Altered SR Ca2+ Content in the Presence of CaM(1-4)
Apart from ICa, SR Ca2+ content constitutes another important determinant of cardiac EC coupling. We therefore measured caffeine-labile SR Ca2+ load both at rest and after electrical pacing (0.5 Hz for 1 minute), with typical time courses and average data shown in Figure 4. CaM(1-4) expression was associated with an elevated SR Ca2+ content (index by 
r410/490 of indo-1 fluorescence) at rest, which was further increased after pacing (Figure 4B), to a level almost double that of paced ß-gal cells (Figure 4B). Moreover, we analyzed the half-decay time of caffeine-induced Ca2+ transients, which reflects the rate of Ca2+ clearance by Na+-Ca2+ exchange (and, to a minor extent, by sarcolemmal Ca2+ ATPase), and found no significant difference between groups (Figure 4C). Thus, an imbalance between sarcolemmal Ca2+ entry and extrusion, together with the enhanced SR Ca2+ recycling, can account for the elevation of SR Ca2+ as well as the aforementioned increase in diastolic Ca2+ on resuming electrical pacing in CaM(1-4) group.
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CaM(1-4) Expression Enhanced CaMKII Activity and PLB Phosphorylation at Thr-17
As a key physiological regulator of the SR Ca2+ ATPase, PLB can be phosphorylated by cAMP-dependent protein kinase A and CaMKII at Ser-16 and Thr-17, respectively.29 Phosphorylation of either site is sufficient to remove PLB-mediated inhibition of the Ca2+ pump, resulting in accelerated SR Ca2+ recycling and hastened cardiac relaxation.19 We hypothesized that the relaxant effect of CaM(1-4) is due to altered phosphorylation of PLB at Thr-17, which is caused by a direct or indirect activation of CaMKII by CaM(1-4). To test this possibility, we first assessed PLB phosphorylation using a site-specific antibody recognizing phosphorylated PLB at Thr-17. PLB phosphorylation at Thr-17 was increased 5.7±1.6-fold (n=5, P<0.01) in the presence of CaM(1-4) (Figure 5A), and this was reversed by incubating cells with a myristoylated autocamtide-2–related inhibitory peptide (AIP, 10 µmol/L), a highly specific membrane-permeable peptide inhibitor of CaMKII,30 or KN-93 (1 µmol/L), a synthetic CaMKII inhibitor (Figure 5A). Further, we performed CaMKII activity assay, which involved immunoprecipitation of CaMKII in Ca2+-free medium and measurement of 32P incorporation into a CaMKII-specific substrate peptide. Expression of CaM(1-4) led to a 60% elevation of total CaMKII activity, and the CaMKII activation was prevented by AIP added 24 hours before harvesting cells (Figure 5B).
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These observations indicate that overexpression of Ca2+-insensitive CaM mutant elevates CaMKII activity. In light of a present model in which apoCaM can dynamically associate with CaMKII through the C-terminal lobe,31 we hypothesized that the presence of excessive CaM(1-4) favors a Ca2+-independent association and partial activation of CaMKII. To test this possibility, CaMKII was precipitated in a Ca2+-free medium using anti-CaMKII antibody, then subjected to Western blot analysis using anti-CaM antibody. Our results revealed that a significant amount of CaM(1-4) was pulled down together with CaMKII in both rat cardiac myocytes and HEK293 cells (Figure 5C), indicating a Ca2+-independent association of CaM(1-4) with CaMKII.
CaMKII Activation Is Essential to CaM(1-4)–Mediated Contractile and Ca2+ Responses
To further determine roles of CaMKII activation in mediating CaM(1-4) actions, we examined contractile effects of CaM(1-4) in the presence of CaMKII inhibition in indicator-unloaded cells. In cells expressing ß-gal, inhibition of CaMKII by AIP (10 µmol/L) exerted no appreciable effects on either contraction amplitude or kinetics (Figure 6A), in agreement with previous reports that there is little basal CaMKII-mediated contractile modulation at 0.5 Hz pacing frequency.19 In sharp contrast, AIP rapidly and reversibly reduced the contraction amplitudes in cells expressing CaM(1-4) (Figure 6A). On average, TA was diminished to about 25% (Figure 6C) of that before AIP application (Figure 6B), whereas CaM(1-4)–mediated abbreviation of T90 was also abolished (Figure 6D). In indicator-loaded myocytes, AIP (10 µmol/L, 10 minutes) similarly inhibited Ca2+ transients (F/F0=3.06±0.29 and 2.18±0.22 before and after AIP, n=7; P<0.01). Thus, we conclude that CaM(1-4)–induced contractile and Ca2+ effects are largely mediated by CaMKII-dependent signaling pathway.
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Effects of Ca2+-Insensitive CaM on Cardiac EC Coupling
Cardiac EC coupling, in essence, is a cascade of Ca2+-mediated signal transduction, whereas many of the Ca2+ signaling molecules are themselves under feedback regulation by the intracellular Ca2+ with CaM as the Ca2+ sensor. The present study sought to determine whether and how CaM regulates EC coupling in intact cardiac myocytes by creating an experimental setting with a high-level expression of Ca2+-insensitive mutant CaM, CaM(1-4). When CaM(1-4) predominates over endogenous CaM, as seen in the Adv-CaM(1-4)–infected rat ventricular myocytes cultured for two days, CaM(1-4) should replace effector-bound apoCaM, resulting in a dominant-negative suppression for the subset of CaM signals mediated by Ca2+ binding to apoCaM preassociated with specific effectors.3,4,7 Because expression of endogenous CaM is unaltered (Figure 1), the subset of CaM signals mediated by interaction between Ca2+/CaM complexes and their effectors should remain intact (see next section).
In light of dichotomous CaM modulation of LCCs and RyRs, it is rather surprising that CaM(1-4) expression in cardiac myocytes is predominantly positively inotropic and lusitropic (Figure 2). The magnitudes of CaM(1-4)–elicited Ca2+ and contractile responses are comparable with those induced by an optimal ß-adrenergic receptor stimulation,32,33 the primary physiological regulatory mechanism of cardiac function. This finding suggests that, albeit non-essential to the EC coupling cascade per se, CaM acts as a powerful (negative) regulator of this process. Targeting the CaM signaling pathway might afford a therapeutic strategy to improve cardiac contraction and relaxation simultaneously.
Several molecular and cellular mechanisms may underlie the CaM(1-4)–mediated positive inotropic and lusitropic effects. Because ICa serves both as the trigger (the peak currents) and the loading Ca2+ (the sustained currents),34 augmentation of ICa density and suppression of ICa inactivation (Figure 3) provide the key explanation not only for the enhanced Ca2+ transient amplitude, but also for the pacing-dependent elevation of diastolic Ca2+ and SR Ca2+ content (Figure 4). Possible prolongation of action potential by the greater ICa in the presence of CaM(1-4)28 may have additional contribution to the contractile and Ca2+ responses. Furthermore, the unexpected PLB phosphorylation (Figures 5A and 5B) predicts an enhanced SR Ca2+ uptake, and should account for the accelerated relaxation of Ca2+ transients and contraction. Thirdly, the enhanced SR Ca2+ recycling, in conjunction with the enhanced ICa, would counteract negative regulators, such as the use-dependent inactivation35 or adaptation of RyR2,36 and give rise to the flattened negative staircase of cell contraction. Thus, a combination of increased ICa and elevated SR Ca2+-ATPase activity would readily explain major phenotypes of this acute gene manipulation. Finally, the observation that contractions in the presence of AIP were smaller in CaM(1-4) than ß-gal group also points to the possibility that CaM(1-4) expression exhibits a component of negative inotropy (eg, desensitization of contractile myofilaments), in analogy to ß-adrenergic modulation of cardiac EC coupling.
Possible Mechanism Underlying CaMKII Activation
The prevalent view is that CaMKII activation and the ensuing trans-subunit autophosphorylation require the binding of Ca2+/CaM to CaMKII complex.13,31 It is thus unexpected that the expression of mutant CaM devoid of Ca2+ sensing ability leads to an enhanced CaMKII activity and CaMKII-mediated protein phosphorylation (Figure 5). Three generic possibilities can be entertained in light of the present findings. First, CaM(1-4) might alter Ca2+ homeostasis and dynamics, enhancing Ca2+/CaM signaling and thereby CaMKII activity. Because neither resting Ca2+ nor endogenous CaM was up-regulated in CaM(1-4)–expressing cells, it alone does not appear to be able to explain the augmented basal PLB-Thr-17 phosphorylation and CaMKII activity measured in unpaced cells, the increased SR Ca2+ content at rest, and the relaxant effect for the first postrest beat. A second working hypothesis is that excessive CaM(1-4) displaces apoCaM from their effectors (eg, LCC), resulting in an increase in apoCaM concentration in the cytosol, while the concentration of total CaM remains unchanged. This not only precludes CaM activation at prebound sites (eg, LCC), but also increases the Ca2+/CaM available to activate CaMKII, and enhances phosphorylation of CaMKII target proteins (eg, PLB-Thr-17). An untested premise of this hypothesis, however, is that much of endogenous CaM must exist as apoCaM-effector complexes under physiological conditions. Thirdly, CaM(1-4) might somehow directly interact with CaMKII and partially activate the kinase via a previously unappreciated mode of action. In this respect, it has been proposed that CaM can interact with CaMKII through the C-terminal lobe in a Ca2+-independent manner.31 It has also been shown that autophosphorylated CaMKII exhibits a high affinity for CaM, trapping the CaM molecule that activates this enzyme.31 In the present study, coimmunoprecipitation data provide evidence for a physical association between CaM(1-4) and CaMKII. It is thus tempting to speculate that the greater occupancy of CaMKII by CaM(1-4) promotes partial CaMKII activation, conferring the major phenotypes observed. Regardless of the exact mechanism, the fact that CaMKII is substantially activated by CaM(1-4) expression cautions that mechanisms underlying LCC phenotypes of Ca2+-insensitive CaM mutant may be more complex than previously thought,7–9 particularly because LCC is a known substrate of CaMKII.3,31
In summary, we have provided the first molecular characterization of CaM regulation of EC coupling in intact cardiac myocytes. The present study demonstrates that expression of Ca2+-insensitive CaM mutant results in markedly increased ICa, SR Ca2+ content, Ca2+ transients, and cell contraction. To our surprise, these were accompanied by a profound relaxation effect due to an elevated CaMKII activity, PLB-Thr-17 phosphorylation, and Ca2+ recycling. Future investigation is warranted to elucidate the specific mechanism underlying CaMKII activation by CaM(1-4) and to appraise specific modes of CaM action on major components of EC coupling, including LCCs, RyRs, and sarcolemmal and SR Ca2+ pumps. The successful genetic manipulation of CaM in cultured cardiac myocytes, as demonstrated in this study, provides a useful experimental system to address these tantalizing issues on CaM regulation of EC coupling in the heart.
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Acknowledgments |
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This work was supported by NIH extramural (Y.W.) and intramural research programs (R.-P.X., H.C.); National Natural Science Foundation of China (D.Y., project 30200088); Major State Basic Research Development Program of China (H.C.); and research grants from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Alberta, N.W.T., and Nunavut (S.R.W.C.). We thank Dr Hans Vogel for providing the synthetic gene encoding the WT bovine calmodulin, Dr Edward G. Lakatta for critical comments, Bruce Ziman for cell isolation, and Xiaoli Li for technical assistance.
Received August 27, 2002; revision received February 11, 2003; accepted February 20, 2003.
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