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Am J Physiol Heart Circ Physiol 290: H599-H606, 2006. First published September 2, 2005; doi:10.1152/ajpheart.00214.2005
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Targeted inhibition of sarcoplasmic reticulum CaMKII activity results in alterations of Ca2+ homeostasis and cardiac contractility

Yong Ji,1 Wen Zhao,2 Bailing Li,1 Jaime Desantiago,3 Eckard Picht,3 Marcia A. Kaetzel,1 Jo El J. Schultz,2 Evangelia G. Kranias,2 Donald M. Bers,3 and John R. Dedman1

1Departments of Genome Science and 2Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio; and 3Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois

Submitted 7 March 2005 ; accepted in final form 29 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transgenic (TG) mice expressing a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitory peptide targeted to the cardiac myocyte longitudinal sarcoplasmic reticulum (LSR) display reduced phospholamban phosphorylation at Thr17 and develop dilated myopathy when stressed by gestation and parturition (Ji Y, Li B, Reed TD, Lorenz JN, Kaetzel MA, and Dedman JR. J Biol Chem 278: 25063–25071, 2003). In the present study, these animals (TG) are evaluated for the effect of inhibition of sarcoplasmic reticulum (SR) CaMKII activity on the contractile characteristics and Ca2+ cycling of myocytes. Analysis of isolated work-performing hearts demonstrated moderate decreases in the maximal rates of contraction and relaxation (±dP/dt) in TG mice. The response of the TG hearts to increases in load is reduced. The TG hearts respond to isoproterenol (Iso) in a dose-dependent manner; the contractile properties were reduced in parallel to wild-type hearts. Assessment of isolated cardiomyocytes from TG mice revealed 40–47% decrease in the maximal rates of myocyte shortening and relengthening under both basal and Iso-stimulated conditions. Although twitch Ca2+ transient amplitudes were not significantly altered, the rate of twitch intracellular Ca2+ concentration decline was reduced by ~47% in TG myocytes, indicating decreased SR Ca2+ uptake function. Caffeine-induced Ca2+ transients indicated unaltered SR Ca2+ content and Na+/Ca2+ exchange function. Phosphorylation assays revealed an ~30% decrease in the phosphorylation of ryanodine receptor Ser2809. Iso stimulation increased the phosphorylation of both phospholamban Ser16 and the ryanodine receptor Ser2809 but not phospholamban Thr17 in TG mice. This study demonstrates that inhibition of SR CaMKII activity at the LSR results in alterations in cardiac contractility and Ca2+ handling in TG hearts.

transgenic mice; calcium/calmodulin-dependent protein kinase II; cardiac contractility; {beta}-adrenergic stimulation; phospholamban; ryanodine receptor

THE CARDIAC sarcoplasmic reticulum (SR) contains pathways for the release of Ca2+ via ryanodine receptors (RyR), which triggers contraction, as well as for its uptake mediated by the SR Ca2+ ATPase (SERCA), which initiates relaxation. Ca2+/calmodulin-dependent protein kinase II (CaMKII), a critical transducer of Ca2+ signals (4), is localized at distinct compartments of cardiomyocytes, including SR, nucleus, and cytosol, and phosphorylates a wide range of substrates (21, 34). CaMKII is critical in modulating Ca2+ homeostasis in cardiomyocytes (21, 34). However, the precise cellular mechanisms underlying the role of CaMKII in cardiac function are undefined.

The phosphorylation of RyR at serine-2809 (Ser2809) by CaMKII (7, 27, 32) and cAMP-dependent protein kinase A (PKA) (23, 27) has been reported to increase the open probability of the SR Ca2+ release channel (RyR) in lipid bilayers or permeabilized cardiac myocytes (7, 23, 32). Regulation of SERCA is primarily mediated by phosphorylation of phospholamban (PLN) at two adjacent sites, serine-16 (Ser16) and threonine-17 (Thr17), catalyzed by PKA and CaMKII, respectively (17, 20, 31). PLN phosphorylation relieves its inhibition on SERCA, enhancing the relaxation rate and contractility, and contributes to the {beta}-adrenergic response of the heart (17, 31). However, the relative contribution of each PLN phosphorylation to the {beta}-adrenergic stimulation of the in vivo cardiac function remains to be defined.

We produced transgenic (TG) mice in which the expression of a potent CaMKII autocamitide inhibitory peptide, AIP, is targeted to the cardiac longitudinal SR (LSR) by using a truncated phospholamban transmembrane domain (12). The heterozygous hearts displayed a ~60% decrease in PLN Thr17 phosphorylation with little change in PLN Ser16 phosphorylation (12). Whereas the in vivo measurement of cardiac function showed no significant decreases in positive or negative first derivative of intraventricular pressure (±dP/dt) in the TG hearts, 7-mo-old females, which had given birth to three litters, develop dilated heart failure. This consequence demonstrates difficulty of the TG mice to respond to physiological cardiovascular stress (12). To better understand the role of SR CaMKII in the regulation of Ca2+ homeostasis and cardiac contractility, isolated work-performing heart preparations as well as isolated cardiomyocytes were analyzed to evaluate the contractility properties and Ca2+ transient dynamics. Our findings demonstrate that CaMKII located at the LSR plays an important role in regulating cardiac contractility.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
TG mice. The design strategy and production of LSR-AIP4 TG mice were described previously (12). In brief, a synthetic gene expression unit, consisting of sequences encoding a tetramer of a potent CaMKII autocamitide inhibitory peptide (AIP4), FLAG epitope, and SR localization signal, was produced to generate TG mice. The SR localization signal is a truncated PLN transmembrane domain (amino acids 23–52) with mutations at Leu31 (L31A) and Asn34 (N34A) to ablate the PLN inhibition of SERCA activity (15). All experiments were performed with homozygous mice that were inbred with heterozygous mice for at least three generations in line 46. Three- to four-month-old female mice were used for all experiments. High expression of CaMKII inhibitory peptide in the cardiac SR of TG hearts was confirmed with anti-Flag antibody (12). All animal procedures followed were approved by the University of Cincinnati Institutional Animal Care and Use Committee.

Isolated work-performing hearts. The experimental conditions for the work-performing mouse heart preparations were described previously (10). Briefly, the hearts were attached by the aorta to a 20-gauge cannula and temporarily retrogradely perfused with oxygenized modified Krebs-Henseleit solution (KHS). For the measurement of intraventricular pressure a polyethylene-50 catheter was inserted into the apex of the left ventricle. The pulmonary vein was connected to a second cannula, and antegrade perfusion was initiated with a basal workload of 250 mmHg·ml·min–1 (5 ml/min venous return and 50 mmHg mean aortic pressure). Volume loadings (3–7 ml/min) were carried out in all work-performing heart preparations. Afterload (aortic resistance, mean aortic pressure) was kept constant at 50 mmHg, and venous return was increased until the contractility (+dP/dt) was no longer elevated. The cardiac work at different venous return conditions was calculated (and expressed as mmHg·ml·min–1). Hearts were allowed to equilibrate for 30 min before isoproterenol (Iso) was infused. A cumulative Iso concentration-response curve was obtained with Isoprel (0.2 mg/ml; Abbott, Chicago, IL) infusion from 0.8 to 160 nmol/l; each dose was applied for 5 min.

Isolation of mouse left ventricular myocytes and measurements of Ca2+ transients. Isolation of mouse left ventricular myocytes was carried out as described previously (8, 35). Cell shortening and Ca2+ transients were measured separately from cardiomyocytes at room temperature. Briefly, mouse hearts were excised from anesthetized (pentobarbital sodium, 70 mg/kg ip) adult mice, mounted in a Langendorff perfusion apparatus, and perfused with Ca2+-free Tyrode solution at 37°C for 3 min. The normal Tyrode solution contained (in mM) 140 NaCl, 4 KCl, 1 MgCl2, 10 glucose, and 5 HEPES, pH 7.4. Perfusion was then switched to the same solution containing 75 U/ml type 1 collagenase (Worthington), and perfusion continued until the heart became flaccid (~10–15 min). The left ventricular tissue was excised, minced, pipette-dissociated, and filtered through a 240-µm screen. Ca2+ concentration was sequentially raised from 25 µM, 100 µM, 200 µM, and 1 mM, and cells were suspended in 1.0 or 1.8 mM Ca2+-Tyrode for experiments.

To measure intracellular Ca2+ concentration ([Ca2+]i), cells were incubated at 23°C with the acetoxymethyl ester form of either fura-2 (2 µM for 30 min) or fluo-3 (10 µM for 20 min) and resuspended in Tyrode solution containing 1.8 mM Ca2+ (for fura-2) and 1 mM Ca2+ (for fluo-3). The myocyte suspension was placed in a Plexiglas chamber on the stage of an inverted epifluorescence microscope (Nikon Diaphot 200) and perfused with Tyrode solution at 23°C. Myocytes were field-stimulated (0.5–1 Hz, square waves), and contractions were measured via video edge detector (Crescent Electronics). Fractional shortening (as percentage of resting cell length) and maximal rates of contraction and relaxation (±dL/dt) were calculated (19, 20). For fura-2 measurements, cells were alternately excited at 340 and 380 nm with 510 nm emission using a Delta Scan dual-beam spectrophotofluorometer (Photon Technology International) with Ca2+ transients shown as the ratio of 510 nm signals at 340/380 nm. For fluo-3, excitation was at 480 ± 5 nm and emission measured at 535 ± 20 nm (and reported normalized to the diastolic value, where F is the fluorescence intensity and F0 is the intensity at rest (22). Baseline and amplitude times for 50% decay of the Ca2+ signal (T50) or exponential time constant ({tau}) were measured. SR Ca2+ content was measured as previously described by rapid application of 10 mM caffeine to induce SR Ca2+ release after steady-state stimulation at 1 Hz (1). Caffeine was continuously applied for 15 s to allow cytosolic Ca2+ removal mainly via Na+/Ca2+ exchange (NCX). The amplitude of the caffeine-induced Ca2+ transients was used as an index of SR Ca2+ content, and [Ca2+]i decline fit to a mono-exponential decline (time constant {tau}) was used to assess NCX function (8, 22).

Immunodetection of site-specific phosphorylation of PLN and RyR. In vivo phosphorylation of PLN and RyR was determined by perfusing the isolated wild-type (WT) and TG hearts in a Langendorff setup. One set of hearts (8 WT and 8 TG) was freeze-clamped after 30 min perfusion with KHS without Iso, and the other set (n = 8) was treated with Iso (100 nM) for 5 min after 25 min equilibration. Hearts were homogenized in buffer containing (in mM) 10 imidazole, 300 sucrose, 1 dithiothreitol, 25 sodium fluoride, and protease inhibitors (13). To stop the reaction mediated by protease, protein kinase, and phosphatase, the hearts after perfusion were immediately freeze-clamped in liquid N2 and were homogenized with 60°C tissue solubilization buffer; the homogenates were then further heated at 95°C for 10 min (14). The aliquots of the samples were stored at –80°C until use.

In vitro phosphorylation was performed using cardiac homogenates from mouse hearts, as described previously (12). For endogenous CaMKII phosphorylation, 10 µl (25 µg) of the cardiac homogenates were added to 10 µl of reaction mixture containing (in mM) 20 imidazole (pH 7.0), 10 MgCl2, 10 NaF, 0.5 EGTA, and 0.1 ATP. Additionally, 0.5 mM CaCl2, 2 µM calmodulin, and 1 µM PKA inhibitor peptide 5–24 amide (Sigma) were included. The reaction was carried out at 30°C. PKA phosphorylation of the cardiac homogenates was carried out in the above described reaction mixture with 20 U of the PKA catalytic subunit without the PKA inhibitor. Reactions were terminated with 4 µl of 6x SDS sample buffer after a 5-min (CaMKII) and 2-min (PKA) incubation, which was associated with optimal phosphate incorporation (19).

Twenty micrograms of protein were subjected to 15% [for PLN and troponin I (TnI)] or 6% (for RyR) SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with polyclonal anti-phosphorylated PLN Ser16, anti-phosphorylated PLN Thr17 antibody, anti-phosphorylated RyR Ser2809 antibody (Badrilla, Leeds, UK), or anti-phosphorylated TnI Ser22/ Ser23 antibody (Cell Signalling) and a monoclonal anti-PLN, anti-RyR, or polyclonal anti-calsequestrin antibodies (Affinity Bioreagents). Binding of the primary antibody was detected by peroxidase-conjugated secondary antibodies. Enhanced chemiluminescence (ECL) was performed using the Super Signal Chemiluminescent Detection System (Amersham Biosciences). The signals were analyzed using Image Pro 4.0 (Media Cybernetics) (12).

Statistical analysis. Results are expressed as means ± SE. Significance was estimated by Student's t-test for paired and unpaired observations and one-way ANOVA for multiple comparisons. P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
There was no significant difference in the body weights in 3- to 4-mo-old WT and AIP4-LSR homozygous mice, but the heart weight (both ventricles only, no atria) was increased in TG mice, resulting in a 14.7 ± 1.3% increase in the heart weight/body weight ratio (mg/g) in TG (6.36 ± 0.21, n = 6) compared with WT (5.57 ± 0.25, n = 6, P = 0.02) mice. Isolated TG myocytes (122.9 ± 2.9 µm, n = 38) were slightly enlarged compared with WT myocytes (111.9 ± 3.8 µm, n = 39, P = 0.01).

Response to loading and Iso stimulation in isolated work-performing AIP4-LSR TG hearts. Isolated work-performing heart preparations were used to assess the effect of targeted inhibition of SR CaMKII on the cardiac function in the absence of neural-hormonal factors. In particular, cardiac function and response to acute physiological stresses, including increases in preload (Frank-Starling response) and {beta}-adrenergic stimulation, were evaluated. Under baseline, unstimulated load conditions (250 mmHg·ml·min–1), TG hearts showed moderate decreases in the maximal rate of contraction (+dP/dt) (by 16.5 ± 3.7%, P < 0.01, n = 6) (Fig. 1A) and relaxation (–dP/dt) (by 10.5 ± 3.3%, P < 0.05, n = 6) in TG (Fig. 1B). However, no significant alterations in the time to peak pressure per unit pressure (TPP/mmHg; Fig. 1C) and time to 50% relaxation per unit pressure (RT1/2/mmHg; Fig. 1D) were observed in the LSR-AIP4 TG hearts at baseline conditions.



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  		Fig. 1. Cumulative dose response to isoproterenol (Iso; 0.08–160 nmol/l) in isolated work-performing hearts. A: positive 1st derivative of intraventricular pressure (+dP/dt). B: negative 1st derivative of intraventricular pressure (–dP/dt). C: time to peak pressure normalized to the pressure amplitude (TTP/mmHg), where pressure amplitude is systolic – end-diastolic pressure. D: time to 50% relaxation normalized to the pressure amplitude (RT1/2/mmHg), where pressure amplitude is systolic – diastolic pressure divided by 2. Data are derived from 6 wild-type (WT) and 6 transgenic (TG) hearts. *P < 0.05, **P < 0.01 vs. WT.

 
{beta}-Adrenergic stimulation of the heart produces an increase in contractility and a decrease in relaxation times. To explore whether the decrease of PLN Thr17 phosphorylation (12) due to targeted inhibition of SR CaMKII influences the cardiac response to {beta}-adrenergic stimulation in TG hearts, isolated WT and TG hearts were perfused with cumulative doses of the {beta}-agonist Iso (0.08–160 nmol/l). As shown in Fig. 1, in response to Iso, the maximal rates of contraction and relaxation (±dP/dt) increased, and the time to 50% relaxation per unit pressure (RT1/2/mmHg) decreased in parallel in WT and TG hearts. Expressed as percentage of baseline values, +dP/dt in response to Iso (which occurred at 80 nmol/l or 160 nmol/l) was 237 ± 24% in WT vs. 221 ± 18% in TG (P = 0.30), and –dP/dt increased up to 231 ± 16% in WT and 211 ± 8.6% in TG (P = 0.15). TPP/mmHg decreased to 53 ± 5.7% (WT) vs. 69 ± 6.5% (TG) (P = 0.05), and RT1/2/mmHg was decreased to 31 ± 4.8% (WT) and 37 ± 3.3% (TG) (P = 0.19). Although the Iso dose-response curve was similar in TG vs. WT hearts, over a wide range of Iso concentration (0.08–160 nM), the absolute values for TG were statistically lower for ±dP/dt and were prolonged for RT1/2/mmHg compared with WT controls (Fig. 1, A, B, and D). TPP/mmHg was slightly lower in TG hearts (Fig. 1C).

The capacity of the ventricle to vary the force of contraction as a function of the load is generally referred to as the Frank-Starling mechanism. To determine to what extent the WT and TG hearts could be loaded with increasing volume loads, cardiac minute work (mean aortic pressure x cardiac output) was varied from 100 to 600 mmHg·ml·min–1, and the results were plotted as Frank-Starling function curves (Fig. 2). The WT and TG hearts exhibit a classic Frank-Starling response, a strong positive correlation of +dP/dt to increased left ventricular work. Compared with the baseline value obtained at 250 mmHg·ml·min–1, there was a 54.1 ± 9.1% (P < 0.01, n = 6) increase in WT and a 42.2 ± 2.6% (P < 0.01, n = 6) increase in TG at an increased workload of 550 mmHg·ml·min–1. At all workloads, the TG hearts displayed lower absolute values for +dP/dt than those obtained in WT hearts, indicating that the TG hearts perform at a lower intrinsic contractile state. Figure 2 shows that the slope of the work response was altered [7.1 ± 0.8 (mmHg/s)/(mmHg·ml·min–1) in TG vs. 9.6 ± 0.9 (mmHg/s)/(mmHg·ml·min–1) in WT, n = 6, P < 0.05], indicating reduced response to higher workloads in TG hearts. In summary, the overall contractility of AIP4-LSR TG hearts is depressed under both basal and stimulated conditions induced by Iso perfusion and increased workload.



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  		Fig. 2. Frank-Starling left ventricular function curves (+dP/dt vs. cardiac work) in WT and longitudinal sarcoplasmic reticulum/autocamitide inhibitory peptide (LSR-AIP4) TG mouse hearts. Each point represents the average of 6 hearts from age-matched WT and TG mice, which were examined under varying preload to cover the range of left ventricular minute work from 100 to 600 mmHg·ml·min–1. The relations between increases in +dP/dt and cardiac work are given by the regression lines; {blacksquare} and {circ} represent the values obtained in WT and TG hearts, respectively. # Significantly different from WT at all workload levels (P < 0.05).

 
Alterations in mechanics in AIP4-LSR cardiomyocytes. Left ventricular myocytes were paced at 0.5 Hz, and cell shortening was measured. As shown in Fig. 3, while the fractional shortening of LSR-AIP4 myocytes was not significantly altered (Fig. 3A), the maximal rates of myocyte shortening (+dL/dt) and relengthening (–dL/dt) were decreased by 40.8 ± 4.3% (P < 0.01, n = 18) and 45.6 ± 5.5% (P < 0.01, n = 18), respectively, which reflect the reduced basal contraction and relaxation kinetics in TG hearts (Fig. 3, B and C). PLN is a prominent mediator of the cardiac response to {beta}-agonist stimulation (17, 20, 31). To determine whether the inhibition of CaMKII site (Thr17) affects the myocyte response to Iso, the effect of Iso on cardiomyocyte contraction and relaxation was evaluated. In the presence of 100 nM Iso, there were comparable increases in fractional shortenings (100 ± 14% in WT vs. 115 ± 23% in TG, P = 0.30), +dL/dt (198 ± 16.9% of WT vs. 144 ± 29.3% of TG, P = 0.06), and –dL/dt (213 ± 21.3% in WT vs. 243 ± 44.3% in TG, P = 0.27) (Fig. 3). The equivalence of Iso response in WT and TG myocytes indicates that inhibition of CaMKII at the LSR in TG mice does not prevent the myocyte response to Iso. However, the absolute values of the maximal contraction and relaxation rates in response to Iso are 42.1 ± 6.2% (P < 0.01) and 46.8 ± 3.3% (P < 0.01) lower than that in WT, similar with the decreased percentile values under basal conditions.



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  		Fig. 3. Contractile parameters of isolated ventricular myocytes from WT and TG hearts. Twitch contractions obtained from WT and TG myocytes paced at 0.5 Hz in the presence and absence of 100 nM Iso. A: fractional shortening. B: maximal rates of shortening (+dL/dt). C: maximal rates of relengthening (–dL/dt). **P < 0.01 for WT and TG vs. values in absence of Iso; ## P < 0.01, TG vs. WT.

 
Ca2+ transients in response to Iso and caffeine. To determine the effect of inhibition of SR CaMKII on cardiomyocyte Ca2+ handling, Ca2+ transients were measured in isolated left ventricular myocytes loaded with fura-2 and paced at 0.5 Hz. Figure 4A shows that the amplitude of Ca2+ transients were not significantly altered in TG myocytes. However, the T50 of [Ca2+]i decline was increased by 47.1 ± 6.6% (P = 0.01, n = 18) in LSR-AIP4 myocytes, indicating depressed Ca2+ removal mechanisms (Fig. 4B). Iso (100 nM) incubation increased Ca2+ transient amplitude by 70.5 ± 12.8% (P < 0.01) in WT and by 73.7 ± 14.6% (P < 0.01) in TG myocytes, whereas Iso reduced T50 by 42.0 ± 5.7% (P < 0.01) in WT vs. 41.6 ± 4.8% (P < 0.01) in TG. These results indicate that while TG myocytes respond to Iso, the increase of T50 remains extended compared with WT controls.



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  		Fig. 4. Ca2+ transients in isolated WT and TG myocytes. Ca2+ transients obtained from WT and TG myocytes paced at 0.5 Hz in the presence and absence of 100 nM Iso. A: Ca2+ amplitude (ratios of 340/380 nm); B: time to 50% decline of Ca2+ transients (T50). [Ca]i, intracellular calcium concentration. *P < 0.05, **P < 0.01 for WT and TG vs. values in absence of Iso; ## P < 0.01, TG vs. WT.

 
Parallel experiments using fluo-3 (1 mM extracellular Ca2+ concentration and 1-Hz steady-state stimulation) also showed prolonged [Ca2+]i decline ({tau}) by 27.8 ± 2.9% (P < 0.05, n = 8 for WT, n = 10 for TG, Fig. 5, A and D). Mean amplitudes of Ca2+ transients were on average smaller in LSR-AIP4 mice (although not significant) (Fig. 5C). This may be consistent with the small decrease in whole heart basal contractility (Fig. 1A).



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  		Fig. 5. Twitch- and caffeine-induced Ca2+ transients in WT and TG myocytes. Isolated myocytes were paced at 1 Hz; traces were normalized for twitch-induced (A) and caffeine-induced Ca2+ transients (B). Pooled data for Ca2+ transient amplitude ({Delta}[Ca2+]i) and time constant ({tau}) of [Ca2+]i decline are shown (C and D). F/F0, fluorescence intensity/intensity at rest. *P < 0.05, TG vs. WT.

 
SR Ca2+ content assessed by caffeine-induced Ca2+ transients on termination of steady-state stimulation at 1 Hz (Fig. 5C) was not significantly altered in TG mice. In addition, the ratio of twitch- to caffeine-induced {Delta}[Ca2+]i (an index of SR fractional Ca2+ release) was not changed in TG vs. WT (Fig. 5C). During sustained caffeine application, the {tau} of [Ca]i decline is indicative of Ca2+ extrusion via NCX, and Fig. 5, B and D, shows that this was unaltered in LSR-AIP4 mice. With similar NCX activity in WT and TG, the prolonged twitch Ca2+ transient decline can therefore be attributed to reduced SERCA function.

Phosphorylation status of PLN and RyR in AIP4-LSR TG mice. To explore the biochemical basis of the functional and Ca2+-handling alterations in TG mice, the levels and phosphorylation status of PLN and RyR were measured. Iso (100 nM) perfusion significantly increased phosphorylation at the CaMKII site (Thr17) in WT but not in TG hearts (Fig. 6, A and B), indicating that the CaMKII-PLN Thr17 pathway was highly suppressed because of the LSR targeting of the CaMKII inhibitory peptide. In contrast, Iso increased phosphorylation of PLN Ser16 in both WT and TG mice (Fig. 6, C and D). The expression of PLN protein was not altered in TG using SR calsequestrin protein level as an internal control (data not shown).



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  		Fig. 6. Phosphorylation status of phospholamban (PLN) in WT and TG hearts. WT and TG hearts were isolated and perfused with a Langendorff setup. One set of hearts was freeze-clamped after 30 min equilibration without Iso, and the other set was treated with Iso (100 nM) for 5 min after 25 min equilibration. Representative immunoblots showing the PLN phosphorylation (p) at Thr17 (A and B) and Ser16 (C and D) in response to Iso. Data are derived from 8 WT and 8 TG mice. In each graph, the phosphorylation signals are normalized to the expression of PLN in each individual heart. **P < 0.01, WT and TG vs. values in absence of Iso; ## P < 0.01, TG vs. WT.

 
When normalized by RyR protein, which was not altered in TG hearts (Fig. 7A), the phosphorylated RyR Ser2809 was decreased by 30.5 ± 6.8% (P < 0.05) and 16.4 ± 7.1% (P < 0.05), respectively, without or with perfusion of 100 nM Iso in TG hearts (Fig. 7, A and B). RyR Ser2809 has been shown to be phosphorylated by both PKA and CaMKII (7, 23, 27, 32). To determine whether the response to Iso is mediated by the PKA and/or CaMKII pathways, in vitro phosphorylation was carried out by incubating cardiac homogenates with either exogenous PKA catalytic subunit or CaM/Ca2+ to activate endogenous CaMKII. As shown in Fig. 7, C and E, in the absence of PKA or CaM/Ca2+, there was a 28.6 ± 5.3% (P < 0.05) decrease in the RyR phosphorylation in TG hearts, which is comparable with the in vivo measurements. Addition of PKA catalytic subunit significantly increased RyR phosphorylation by 40.4 ± 6.8% in WT (P < 0.01) and more significantly by 92.4 ± 15.3% in TG (P < 0.01) (Fig. 7, C and D), indicating the capacity of PKA-mediated RyR phosphorylation is preserved in SR vesicles isolated from LSR-AIP4 mice. On the other hand, incubation with CaM/Ca2+ increased the RyR Ser2809 phosphorylation by 13.2 ± 2.1% (P < 0.01) in WT but did not increase the RyR phosphorylation in TG mice (Fig. 7, E and F). When compared with WT, TG presented a 34.7 ± 4.1% (P < 0.01) decrease in RyR phosphorylation in the presence of CaM/Ca2+. These results demonstrate that the regulation of RyR phosphorylation is inhibited in LSR-AIP4 TG hearts.



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  		Fig. 7. Phosphorylation status of ryanodine receptor (RyR) in WT and TG hearts. WT and TG hearts were isolated and perfused with Iso in the Langendorff setup as described in MATERIALS AND METHODS. Representative immunoblots show the RyR phosphorylation at Ser2809 in response to Iso (A and B). For the assay of in vitro phosphorylation, cardiac homogenates from WT and TG mice were phosphorylated by either catalytic subunit of protein kinase A (PKA; C and D) or endogenous Ca2+/calmodulin-dependent protein kinase II (CaM/Ca2+) (E and F). Twenty micrograms of boiled samples were used to detect phosphorylated RyR Ser2809. Data are derived from 4 WT and 4 TG mice in each group. In each graph, the phosphorylation signals are normalized to the expression of RyR in each individual heart. *P < 0.05, **P < 0.01, WT and TG vs. values in absence of Iso; # P < 0.05, ## P < 0.01, TG vs. WT.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our findings demonstrate that SR CaMKII plays an important role in the regulation of cardiac contractility. Basal rates of contraction and relaxation are decreased in work-performing hearts (±dP/dt) and cardiomyocytes (±dL/dt) isolated from LSR-AIP4 TG mice. The decrease in the rate of relaxation is correlated with a decrease in the rate of Ca2+ decline (Fig. 4B), which is a consequence of reduced SR Ca2+ sequestration (Fig. 5A) (12) due to the targeting of AIP4 to the LSR. Our recent study has shown that frequency-dependent acceleration of relaxation, determined by SERCA activity (1), is attenuated in myocytes isolated from LSR-AIP4 TG mice (26) but is present in PLN knockout myocytes (8). These results suggest that SERCA function may also be affected by SR CaMKII inhibition via mechanisms other than PLN.

Neither SR Ca2+ content nor the amplitude of the [Ca2+]i was significantly depressed in TG mice, although they all tended to be low (Fig. 4A and Fig. 5C), which corresponds well with the small change in fractional shortening (Fig. 3A). The potential decrease in SR Ca2+ content, due to a decrease in SR Ca2+ uptake activity, might be limited here because the reduced RyR2809 phosphorylation would reduce the diastolic leak of Ca2+ from the SR (22, 23). On the other hand, the reduced RyR2809 phosphorylation, as a result of the possibility that the LSR-targeted AIP4 can inhibit CaMKII at the junction of the SR (26), did not appear to apply to excitation-contraction coupling because our data show unaltered fractional SR Ca2+ release (Fig. 5C). However, the TG cardiomyocytes demonstrated a significant decrease (~40%) in the rate of contraction (+dL/dt). The reduced rate of contraction during relatively high activating [Ca2+]i suggests that the myofilament characteristics are altered as well (16). Tong et al. (29) have shown that CaMKII inhibition reduced both myosin binding protein C and TnI phosphorylation, which decreased the rates of force development and relaxation without changing the magnitude of the [Ca2+]i. However, PKA-induced phosphorylation of TnI, a major determining factor of the Ca2+ sensitivity of the myofilaments (16), is not changed in LSR-AIP4 TG mice (unpublished data). Further detailed studies are necessary to determine the phosphorylation status of other myofilament proteins (16, 24, 29).

Increasing the physiological demand on LSR-AIP4 hearts by increasing the workload or {beta}-adrenergic stimulation does not restore the impairment of the cardiac contraction and relaxation function to the level of WT mice (Figs. 1–4). The decreases in the absolute values of the contractile response to workload and Iso stimulation indicate reductions in contraction and relaxation reserve in TG hearts. Studies with isolated cardiomyocytes also revealed a 40–47% decrease in the maximal contraction and relaxation rates in response to Iso (Fig. 3), which is similar to the extent of reduction in basal conditions. These results demonstrate that the reduced contractile properties in response to {beta}-adrenergic stimulation are the extension of the decreases in basal contractility. However, the slope of the Frank-Starling curve is attenuated in TG hearts (Fig. 2), suggesting additional possible mechanisms. The length-dependent activation of cardiac myocytes is a major contributor to the Frank-Starling relation of the heart (9). Thus the reduced slope of the Frank-Starling curve predicts reduced length dependence of activation. Studies have shown that the length-force relation is determined by both myofilament response to Ca2+ and intracellular Ca2+ amplitude (3, 9, 11, 16). Although no significant alterations were found for the [Ca2+]i amplitude or PKA-mediated cardiac TnI phosphorylation, LSR CaMKII inhibition may affect phosphorylation of other myofibrillar proteins. Regardless of the mechanism, our results demonstrating reduced cardiac response to cardiovascular stress support our previous findings that female TG mice that are confronted with increased physiological cardiovascular demands develop dilated heart failure (12).

The relevance of CaMKII-mediated phosphorylation of PLN Thr17 in response to {beta}-adrenergic regulation of cardiac function is not clear. Several studies have demonstrated that PLN Ser16 is the direct and dominant site for PKA-mediated phosphorylation underlying the {beta}-adrenergic relaxant effect (5, 6, 18). The LSR-AIP4 mouse model demonstrated that under the condition of the ablation Thr17 phosphorylation in response to Iso (Fig. 6A), Iso still produced a dose-dependent response of contractile parameters (±dP/dt) in isolated work-performing hearts and in cardiomyocytes (±dL/dt). These data further support the possibility that Ser16 in PLN is the major phosphorylation site for {beta}-adrenergic stimulation. However, there is increasing evidence showing that phosphorylation of PLN Thr17 participates in the relaxant action of Iso (30–300 nM) in isolated rat perfused hearts (28) or in isolated cardiomyocytes subjected to sustained (24 h) {beta}1-adrenergic stimulation (30). In addition, a more recent study demonstrates that CaMKII is a crucial downstream signal for cardiomyopathic responses to chronic {beta}-adrenergic stimulation (33). Thus Thr17, as a target of CaMKII, may play an important role in the pathophysiological response to chronic {beta}-adrenergic stimulation.

In summary, our study demonstrates that targeted inhibition of LSR CaMKII activity results in alterations in cardiac contractility and Ca2+ handling. These effects are mediated, at least in part, by inhibiting SR Ca2+ transport function via inhibition of PLN Thr17 phosphorylation. Imbalances of intracellular Ca2+ homeostasis play a central role in the development of heart failure (2, 25). This LSR-AIP4 TG mouse model provides a unique opportunity to study the role of SR CaMKII in the regulation of excitation-contraction coupling as well as in the development of heart failure.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-46433 and an American Heart Association (AHA) Grant-in-Aid (to J. R. Dedman), National Heart, Lung, and Blood Institute (NHLBI) Grant HL-30077 (to D. M. Bers), NHLBI Grants HL-26057 and HL-64018 (to E. G. Kranias), AHA Beginning Grant-in-Aid 0465391B (to Y. Ji), and an AHA Postdoctoral Fellowship (to E. Picht).


   ACKNOWLEDGMENTS
 
We thank Dr. Ingrid L. Grupp for excellent advice on the work-performing heart experiments and Gilbert Newman for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. R. Dedman, Dept. of Genome Science, Univ. of Cincinnati, 2180 E. Galbraith Road, Cincinnati, OH 45237-0505 (e-mail: john.dedman{at}uc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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