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(Circulation Research. 2003;92:769.)
© 2003 American Heart Association, Inc.

Cellular Biology

Chronic SR Ca²⁺-ATPase Inhibition Causes Adaptive Changes in Cellular Ca²⁺ Transport

Angela G. Brittsan^*, Kenneth S. Ginsburg^*, Guoxiang Chu, Atsuko Yatani, Beata M. Wolska, Albrecht G. Schmidt, Michio Asahi, David H. MacLennan, Donald M. Bers, Evangelia G. Kranias

From the Department of Pharmacology and Cell Biophysics (A.G.B., G.C., A.G.S., E.G.K.), College of Medicine, University of Cincinnati, Cincinnati, Ohio; Department of Physiology (K.S.G., D.M.B.), Loyola University Chicago, Stritch School of Medicine, Maywood, Ill; Department of Cell Biology/Molecular Medicine (A.Y.), UMDNJ RWJ, Newark, NJ; Department of Medicine (B.M.W.), University of Illinois at Chicago, Chicago, Ill; and Banting and Best Department of Medical Research (M.A., D.H.M.), University of Toronto, Toronto, Ontario, Canada.

Correspondence to Evangelia G. Kranias, PhD, Department of Pharmacology and Cell Biophysics University of Cincinnati College of Medicine, 231 Albert Sabin Way, PO Box 670575, Cincinnati, OH 45267-0575. E-mail Litsa.Kranias{at}uc.edu

	Abstract

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Phospholamban, the critical regulator of the cardiac SERCA2a Ca²⁺ affinity, is phosphorylated at Ser¹⁶ and Thr¹⁷ during ß-adrenergic stimulation (eg, isoproterenol). To assess the impact of nonphosphorylatable phospholamban, a S16A, T17A double-mutant (DM) was introduced into phospholamban knockout mouse hearts. Transgenic lines expressing DM phospholamban at levels similar to wild types (WT) were identified. In vitro phosphorylation confirmed that DM phospholamban could not be phosphorylated, but produced the same shift in EC₅₀ of SERCA2a for Ca²⁺ as unphosphorylated WT phospholamban. Rates of basal twitch [Ca²⁺]_i decline were not different in DM versus WT cardiomyocytes. Isoproterenol increased the rates of twitch [Ca²⁺]_i decline in WT, but not DM myocytes, confirming the prominent role of phospholamban phosphorylation in this response. Increased L-type Ca²⁺ current (I_Ca) density, with unaltered characteristics, was the major compensation in DM myocytes. Consequently, the normal ß-adrenergic–induced increase in I_Ca caused larger dynamic changes in absolute I_Ca density. Isoproterenol increased Ca²⁺ transients to a comparable amplitude in DM and WT. There were no changes in myofilament Ca²⁺ sensitivity, or the expression levels and Ca²⁺ removal activities of other Ca²⁺-handling proteins. Nor was there evidence of cardiac remodeling up to 10 months of age. Thus, chronic inhibition of SERCA2a by ablation of phospholamban phosphorylation (abolishing its adrenergic regulation) results in a unique cellular adaptation involving greater dynamic I_Ca modulation. This I_Ca modulation may partly compensate for the loss in SERCA2a responsiveness and thereby partially normalize ß-adrenergic inotropy in DM phospholamban mice.

Key Words: sarcoplasmic reticulum • phospholamban • SR Ca²⁺-ATPase • cardiomyocytes • transgenic mice

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Contraction and relaxation in cardiac muscle are highly regulated by the sympathetic nervous system. Catecholamine-dependent activation of myocardial ß-adrenoreceptors (ß-AR) increases cAMP, which activates PKA and phosphorylation of key regulatory proteins. The main regulatory phosphoproteins include the following: (1) the L-type Ca²⁺ channel in the sarcolemmal membrane¹; (2) phospholamban (PLB)² and the ryanodine receptor (RyR)³ in the sarcoplasmic reticulum (SR); and (3) troponin I (TnI)² and C-protein in the myofibrils.⁴ Phosphorylation of L-type Ca²⁺ channels increases Ca²⁺ current (I_Ca) enhancing the Ca²⁺ trigger for SR Ca²⁺ release, as well as increasing cellular and SR Ca²⁺ content. PLB phosphorylation, in response to ß-AR stimulation, relieves its tonic inhibition on the Ca²⁺ affinity of the cardiac SR Ca²⁺-ATPase (SERCA2a), resulting in increases in SR Ca²⁺-uptake rates, enhanced relaxation, and increased SR Ca²⁺ content. PKA-dependent phosphorylation of the RyR has also been reported to increase the opening probability of this channel in lipid bilayers.R5-127150 ^5,6 On the other hand, TnI phosphorylation by PKA enhances the off-rate for Ca²⁺ from troponin C and may enhance relaxation rate.⁷

Ex vivo and in vivo studies have suggested that PLB, I_Ca, and TnI are the most critical functional substrates of the ß-AR pathway in the heart.R4-127150 R8-127150 R9-127150 ^4,8–10 Analysis of relative roles of these PKA targets has been aided by mouse models that are PLB-deficient,⁸ expressing phosphorylation site-specific PLB mutants,R11-127150 ^11,12 as well as expressing cardiac TnI phosphorylation site mutants¹³ or slow skeletal TnI in the PLB-KO.¹⁴ These studies indicated that PLB phosphorylation is a dominant factor in both the inotropic and lusitropic effect of ß-AR, and that TnI phosphorylation plays a minor but significant role in the lusitropic effect.R9-127150 R13-127150 ^9,13,14 Although PLB cannot be phosphorylated in the PLB-KO, interpretations can be complicated by an unphysiologically activated SR Ca²⁺-ATPase activity. Comparisons could be better made using endogenous TnI and the normal level but completely nonphosphorylatable PLB.

PLB phosphorylation is reduced in heart failure (human and animal),R15-127150 ^15,16 and this may be a contributing factor to the deteriorated function. However, long-term effects of dephosphorylated PLB on SR Ca²⁺ cycling and cardiac function are unknown. Thus, we developed a novel mouse model with a nonphosphorylatable double mutant form of PLB (PLB-DM, where both Ser¹⁶ and Thr¹⁷ are replaced by Ala) into the PLB-KO background (ie, creating tonic SERCA2a inhibition). This PLB-DM should chronically inhibit SERCA2a and limit the heart’s normal ability to increase SR Ca²⁺-ATPase activity in response to sympathetic activation.

The specific questions addressed were as follows: (1) What are the physiological and pathological consequences of chronic SERCA2a inhibition by PLB-DM? (2) How are maximal isoproterenol effects on myocyte Ca²⁺ transients and contractility changed in PLB-DM mice? (3) What is the relative contribution of PLB, versus other phosphoproteins, in cardiac myocytes during ß-AR stimulation? (4) What compensations in myocyte Ca²⁺ regulation occur when phosphorylation of PLB is unavailable? Our data indicate that there were no cardiac histological alterations up to 10 months of age, although the ß-AR–induced acceleration of myocyte relaxation and [Ca²⁺]_i decline were completely absent in the PLB-DM myocytes. On the other hand, I_Ca density was enhanced, which may provide an alternative (or surrogate) pathway for ß-AR to exert dynamic control over cardiac function.

	Materials and Methods

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In Vitro Coexpression Studies
Rabbit PLB wild-type (WT) or PLB-DM and SERCA2a cDNAs were cotransfected into human embryonic kidney cell line 293 (HEK-293), and microsomal Ca²⁺-transport activity was assayed.

Generation of Transgenic Mice
PLB-KO 129SvJ/CF-1 mice⁸ were mated with transgenic FVB/N mice expressing the S16A, T17A mutant murine PLB cDNA, specifically in cardiac muscle.¹⁷ F₁ heterozygous PLB offspring carrying the PLB mutant transgene were bred with PLB-KO mice to obtain F₂ pups. The PLB-KO offspring carrying the PLB mutant transgene were selected to backcross with PLB-KO mice to F₆ generation (see expanded Materials and Methods section in the online data supplement available at http://www.circresaha.org). Handling and maintenance of animals was approved by the ethics committee of the University of Cincinnati. Eight- to 13-week-old mice of either sex were used for the studies.

Quantitative Western Blot Analysis
Quantitative immunoblotting of cardiac homogenates and SR-enriched microsomes¹⁷ was used to assess the levels of PLB, SERCA2a, ryanodine receptor, - and ß-myosin heavy chain isoforms, calsequestrin, calreticulin, and TnI in WT and PLB-DM mouse hearts (see online data supplement).

SR Ca²⁺ Uptake
Mouse hearts were frozen in liquid nitrogen and later assayed for initial rates of oxalate-supported Ca²⁺ uptake⁸ (see on-line supplement).

In Vitro Phosphorylation
PKA or CaMK phosphorylation was performedR11-127150 ^11,12 using 45 µg of protein via 15% SDS-PAGE and autoradiography. Labeling with ³²P- was assessed using a PhosphorImager and ImageQuant software from Molecular Dynamics.

Left Ventricular Myocyte Measurements
Cell contraction was measured by video edge detection and intracellular-free [Ca²⁺] ([Ca²⁺]_i) was measured using fluo-3AM (see online data supplement). Cells were perfused with normal Tyrode’s (NT) solution (in mmol/L): NaCl 140, KCl 4, MgCl₂ 1, CaCl₂ 1, and HEPES 10, with pH 7.4, at 23°C. Twitches (steady state at 0.5 Hz) were field stimulated. To assess cytosolic Ca²⁺ removal by non-SR pathways and SR Ca²⁺ content, 10 mmol/L caffeine was applied for 10 seconds in NT. Isoproterenol (ISO; 100 nmol/L) was used to activate ß-AR. Under basal conditions, PLB phosphorylation was less than 5% at either P-Ser16 or P-Thr17, compared with the levels obtained on ISO stimulation (100%), assessed by the PLB phosphorylation site specific antibodies.

Twitch and caffeine-induced Ca²⁺ transient decline were fit with single exponential declines, described by time constants () or rate constants (=1/). SR Ca²⁺ content was calculated from caffeine-induced Ca²⁺ transient amplitude (after converting [Ca²⁺]_i values to total cytosolic [Ca²⁺] using buffering constants¹⁸).

³²P-Labeling of Isolated Ventricular Myocyte Proteins
Isolated myocytes were labeled with ³²P and stimulated with ISO.⁹ After boiling and electrophoresis, the degree of ³²P-incorporation was assessed (see online data supplement).

Electrophysiology of Isolated Left Ventricular Myocytes
Ventricular myocytes were studied using whole-cell patch clamp.¹⁹ I_Ca was recorded using Na⁺ and K⁺-free external solution (to prevent contaminating Na and K channel currents) containing (in mmol/L) CaCl₂ 2, MgCl₂ 1, TEA-Cl 135, 4-aminopyridine 5, glucose 10, and HEPES 10 (pH 7.3). The pipette solution contained (in mmol/L) Cs-aspartate 100, CsCl 20, MgCl₂ 1, MgATP 2, GTP 0.5, EGTA 5, and HEPES 5 (pH 7.3). I_Ca was activated by depolarization pulses (0.1 Hz) from a holding potential of -50 mV.¹⁹ To measure ISO-induced I_Ca response, 10 mmol/L BAPTA replaced pipette EGTA to minimize Ca²⁺-dependent inactivation. Outward Na⁺-Ca²⁺ exchanger current was activated (with membrane potential held at -40 mV) by rapidly switching the external solution to one in where equimolar LiCl was substituted for NaCl.²⁰

Force Measurements of Skinned Fiber Bundles
Skinned fiber bundles were isolated and the [Ca²⁺]-force relations (at sarcomere length of 2.0 µm) were measured²¹ and fit to the Hill equation to derive the -log [Ca²⁺] at half-maximal activation (pCa₅₀) and Hill coefficient.

Echocardiographic Measurements
Echocardiography studies were performed for noninvasive assessment of left ventricular function and dimensions, as described previously.²²

Statistics
Data are presented as mean±SEM. Statistical analysis was performed using linear regression and ANOVA. Mean data were compared using t tests, Newman-Keuls rank test, or Mann-Whitney test, as appropriate, and differences were considered significant if P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In Vitro Studies on PLB Double Mutant
Previous work in expression systems showed that single site-mutagenesis of Ser¹⁶ or Thr¹⁷ to Ala in PLB did not alter the interaction between PLB and SERCA2a. In the present study, we measured the in vitro effect of mutating both sites to Ala (PLB-DM).

Figure 1 shows the [Ca²⁺]-dependence of Ca²⁺ transport in microsomes isolated from HEK-293 cells transfected with SERCA2a alone or in combination with wild-type PLB (PLB-WT) or PLB-DM. Western blot analysis indicated similar SERCA2a expression in each case, and the two PLB forms were expressed equally. Therefore, the PLB/SERCA2a ratio was the same in cells expressing WT or mutant PLB. The apparent Ca²⁺ affinity of SERCA2a was reduced significantly and similarly when SERCA2a was coexpressed with either WT PLB or mutant PLB (SERCA2a alone, K_m=0.26±0.02 µmol/L; +WT-PLB, 0.69±0.04 µmol/L; +PLB-DM, 0.76±0.04 µmol/L). Thus, mutating both Ser¹⁶ and Thr¹⁷ to Ala do not alter SERCA2a inhibition by PLB in vitro.

View larger version (19K): [in this window] [in a new window]   Figure 1. Ca2+ uptake in HEK-293 cells. Microsomal Ca2+-uptake rates in HEK-293 cells transfected with (1) SERCA2a, (2) SERCA2a+PLB-WT, and (3) SERCA2a+PLB-DM. Data are mean±SEM from 3 separate transfections, fit by Hill curve.

Transgenic Mice With Cardiac Expression of PLB Double Mutant
To assess the functional significance of dual site PLB phosphorylation in vivo, nonphosphorylatable PLB was introduced into PLB-KO mouse hearts. Genomic tail DNA from transgenic mice (F₆ generation) was extracted, and PLB mutant cDNA was amplified by PCR and sequenced. Sequence analysis confirmed the presence of the expected mutant PLB transgene. WT mice, with the same genetic background as PLB-KO mice, were used as controls in the following studies.

Western blots of cardiac homogenates processed in parallel were used to analyze PLB expression levels. Figure 2A shows that PLB expression in lines 72 and 78 was 132% and 139% of that in WT. To assess SR localization of the mutant PLB, SR-enriched microsomal fractions (isolated by differential centrifugation) were analyzed by Western blot. Levels of PLB were similar between cardiac homogenates and microsomal preparations in both WT and transgenics (Figure 2B), indicating that mutant PLB targeted correctly to the SR.

View larger version (64K): [in this window] [in a new window]   Figure 2. Western blots of PLB and SERCA2a in WT and PLB-DM hearts. Representative immunoblots and pooled data from cardiac homogenates (A and C, 5 hearts each) and SR-enriched microsomal preparations (B and D, 3 hearts each) as mean±SEM. PLBL and PLBH indicate low and high molecular weight forms of PLB, respectively.

We also found that the levels of SERCA2a protein expression in both cardiac homogenates (Figure 2C) and SR-enriched microsomes (Figure 2D) were similar between transgenic and WT controls.

In Vitro Phosphorylation of PLB
To determine whether the expressed mutant PLB could be phosphorylated in vitro, cardiac homogenates were incubated with [-³²P] ATP and PKA catalytic subunit, or Ca²⁺ plus calmodulin. Autoradiographs (Figure 3) show that ³²P-incorporation into PLB was only detected in WT hearts. Thus, mutant PLB could not be phosphorylated in vitro by either PKA or CaMK.

View larger version (43K): [in this window] [in a new window]   Figure 3. In vitro phosphorylation of PLB. Autoradiograms of 32P-labeled cardiac homogenates from PLB-deficient, WT and PLB-DM mice exposed to either (A) the catalytic subunit of cAMP-dependent protein kinase A or (B) Ca2++Calmodulin to activate endogenous CaMK. PLBL indicates PLB monomer (on boiling); MLC, myosin light chain; cTnI, cardiac TnI; KO, PLB-KO; and DM, PLB-DM (with expression levels relative to WT).

Sarcoplasmic Reticulum Ca²⁺ Uptake
Initial rates of SR Ca²⁺ uptake were measured in cardiac homogenates at different [Ca²⁺]. The EC₅₀ values in PLB-DM hearts were slightly higher (0.34±0.01 µmol/L in line 72; 0.32±0.02 µmol/L in line 78) than those in WT control hearts (0.25±0.01 µmol/L), consistent with the higher levels of PLB in PLB-DM hearts. There were no differences in the maximal Ca²⁺-uptake velocity between WT (58.8±9.6 nmol/mg per min) and PLB-DM hearts (49.1±2.9 and 51.1±3.7 nmol/mg per min in lines 72 and 78, respectively). Thus, mutant PLB functionally interacts with SERCA2a in vivo. Because both transgenic lines expressed similar levels of PLB-DM protein and exhibited no significant differences in the EC₅₀ values for Ca²⁺ uptake, line 78 was used for further functional studies.

Cardiomyocyte Ca²⁺ Transients and Contractions
Figures 4A and 4B show that 100 nmol/L ISO increased Ca²⁺ transient amplitude in both WT and PLB-DM myocytes under steady state conditions (0.5 Hz). Normalized traces (Figures 4C and 4D) emphasize the prominent acceleration of [Ca²⁺]_i decline in WT, but not PLB-DM. In pooled data, ISO reduced by 50% in WT, but did not change in PLB-DM (Figure 4E). These results show that PLB is essential in the enhanced rate of [Ca²⁺]_i decline and SR Ca²⁺ uptake with ISO. However, Ca²⁺ transient amplitudes were increased even in PLB-DM myocytes. Thus, tonic SERCA2a inhibition by nonphosphorylatable PLB does not prevent the inotropic effect of ISO.

View larger version (32K): [in this window] [in a new window]   Figure 4. Ca2+ transients in WT and PLB-DM myocytes. Ca2+ transients obtained from WT (A) and PLB-DM (B) myocytes in the presence and absence of 100 nmol/L ISO. Traces were also normalized (C and D) and pooled data for twitch Ca2+ transient decline () and amplitude ([Ca2+]i) are shown (E and F). CTL indicates control conditions (n=21, 14, 19, and 19 for the 4 bars; *P<0.05 for subset of paired observations).

Myocyte contraction results agree with the [Ca²⁺]_i results. Twitch contraction amplitude of WT and PLB-DM myocytes was similar under control conditions and both increased comparably with ISO (Figure 5A). The maximum rate of control twitch contraction was slightly higher in PLB-DM than WT (not shown), consistent with the tendency toward higher [Ca²⁺]_i in PLB-DM (Figure 4F). With ISO, relaxation t_1/2 decreased (Figure 5B) and maximal relaxation rate (not shown) increased only in WT myocytes, while no change was observed in PLB-DM myocytes. These data indicate that PLB-DM myocytes have adapted mechanisms to increase contractility on ISO stimulation, presumably to compensate for the inability of PLB to be phosphorylated during sympathetic stimulation.

View larger version (28K): [in this window] [in a new window]   Figure 5. Myocyte contraction and Ca2+ transient analysis. Twitch contractions at 0.5 Hz steady state were assessed for amplitude (A) and relaxation half-time, t1/2 (B; n=33, 13, 29, and 17 for the 4 bars). Rate constants for [Ca2+]i decline during twitches (Tw) caffeine application (Caff) and the inferred fractional contribution of SR to twitch [Ca2+]i decline (SR/Tw) are shown (C-E; n= 18, 9, 14, and 15 for the 4 bars; *P<0.05).

SR Ca²⁺ content was also assessed by caffeine-induced Ca²⁺ transients on termination of steady state stimulation at 0.5 Hz. Without ISO, mean SR Ca²⁺ content was higher in PLB-DM versus WT myocytes (100.1±15.2 versus 64.3±13.0 µmol/l cytosol; n=12, 18), but the difference was not significant (P=0.08), presumably due to high cell-to-cell variation in this unpaired comparison. ISO increased SR Ca²⁺ content significantly in both PLB-DM and WT myocytes (by 22% and 24%, respectively; n=12, 9). Thus, ISO can still increase SR Ca²⁺ load, even when PLB cannot be phosphorylated.

Altered SR Ca²⁺ leak could also alter SR Ca²⁺ load. Resting Ca²⁺ sparks are indicative of this leak rate. We measured Ca²⁺ spark frequency and characteristics by confocal microscopy in both PLB-DM and WT myocytes.²³ There was no major difference in Ca²⁺ spark frequency or duration, although there was a slight increase in Ca²⁺ spark amplitude and a counterbalancing reduced spatial spread. Thus, there was little alteration in SR Ca²⁺ leak rate between PLB-DM and WT myocytes.

Ca²⁺ Removal Systems
The rate constant of twitch [Ca²⁺]_i decline (_Tw) was faster after ISO in WT, but unchanged in PLB-DM (Figure 5C). The rate constant of [Ca²⁺]_i decline during caffeine exposure (_caff) reflects primarily Ca²⁺ removal via Na⁺-Ca²⁺ exchange, and this was 10 times slower than _Tw but comparable between WT and PLB-DM (Figure 5D). We conclude that Na⁺-Ca²⁺ exchange function is unaltered in PLB-DM myocytes and unaffected by ISO. The rate constant of [Ca²⁺]_i decline attributed to SERCA2a (_SR) is inferred as _Tw-_Caff. In WT, _SR was significantly increased by ISO, but no difference was seen in PLB-DM. Thus, SERCA2a function could not be increased without PLB phosphorylation. Finally, the fraction of Ca²⁺ removal due to the SR (_SR/_Tw) was 88% in both wild-type and PLB-DM myocytes without ISO (Figure 5E). With ISO, this fraction increased (to 94%) only in WT.

Phosphorylation of PLB in Cardiac Myocytes
Possible compensatory phosphorylation of the mutant PLB on Ser¹⁰, the protein kinase C site, was examined in myocytes labeled with [³²P]-orthophosphate. Autoradiographs from WT myocytes exhibited low levels of basal PLB phosphorylation, which increased 2.3-fold upon ISO exposure (Figure 6A). However, the PLB-DM myocytes exhibited no PLB phosphorylation before or after ISO. TnI phosphorylation increased similarly in WT and PLB-DM.

View larger version (45K): [in this window] [in a new window]   Figure 6. Phosphorylation in intact myocytes and myofilament Ca2+ sensitivity. A, Representative autoradiogram showing 32P incorporation into myocytes (±ISO) in the presence of 1 mmol/L Ca2+ and 0.15 mCi [32P]-orthophosphate. Samples were boiled, run on SDS-PAGE (4% to 20% gradient gels), and autoradiographed. PLBL indicates PLB monomer (on boiling); MLC, myosin light chain; cTnI, cardiac TnI. B, Myofilament Ca2+sensitivity (in the absence of cAMP/PKA) at sarcomere length=2 µm. Force was normalized to value at 3 µmol/L Ca2+ (mean±SEM, n=9 each, from 6 and 5 hearts).

Compensatory Mechanisms
Quantitative immunoblots were used to assess possible compensatory changes in Ca²⁺ handling proteins in PLB-DM mice. The protein expression levels in PLB-DM (versus WT) were unaltered for SERCA2a (109±5%), calsequestrin (101±8%), calreticulin (101±6%), and ryanodine receptor (106±5%). Also, no alterations were seen in TnI or -myosin heavy chain in PLB-DM hearts versus WT. Expression of ß-myosin heavy chain was barely detectable in either WT or PLB-DM hearts.

Myofilament Ca²⁺ sensitivity was assessed in skinned fiber preparations. Figure 6B shows that [Ca²⁺]-force curves for PLB-DM and WT preparations were identical (pCa₅₀ was 5.70±0.01 for mutant and 5.69±0.01 for WT preparations). Thus, myofilament Ca²⁺ sensitivity was unaltered in PLB-DM.

Whole-cell I_Ca was measured using patch-clamp. Membrane capacitance was not different in PLB-DM (110±3 pF, n=65) versus WT myocytes (115±4 pF, n=35). The E_m-dependence of I_Ca was comparable in WT and PLB-DM (Figures 7A through 7C), but peak I_Ca density was 25% higher in PLB-DM versus WT (PLB-DM: 9.5±0.3 A/F, n=56; WT: 7.6±0.4 A/F, n=35; P<0.05). Figures 7B, inset, and 7F showed that I_Ca decline during the pulse was significantly faster (at +10 mV) in PLB-DM (15.5±0.8 ms, n=60) versus WT (20.9±0.9 ms, n=18) myocytes. Despite the faster I_Ca inactivation in PLB-DM, integrated I_Ca was still significantly higher in PLB-DM versus WT (Figure 7E). The more rapid inactivation could have been secondary to higher I_Ca in PLB-DM (and consequent SR Ca²⁺ loading and release). Thus, we measured I_Ca inactivation in the presence of 10 µmol/L ryanodine. Ryanodine significantly slowed I_Ca inactivation in both PLB-DM (33.1±2.5 ms, n=7) and WT (35.7±1.9 ms, n=5) cells, such that these were no longer different. ISO produced almost identical percent increases in I_Ca in both cell types (Figure 7D). Thus, there is no intrinsic difference in I_Ca characteristics, there is simply more I_Ca in PLB-DM. The expression level of L-type Ca²⁺ channel _1C subunit was also increased by 20±3% (n=4; data not shown), consistent with the increased I_Ca being due to more Ca²⁺ channels (rather than altered regulation). The enhanced I_Ca could be an important compensatory mechanism, in the PLB-DM, allowing a greater absolute increase in I_Ca density with ß-AR activation. This could cause the increased SR Ca²⁺ content and enhanced SR Ca²⁺ release during twitches with ISO (even without phosphorylatable PLB).

View larger version (29K): [in this window] [in a new window]   Figure 7. ICa in ventricular myocytes. Whole-cell ICa recorded in WT (A) and PLB-DM (B) myocytes. Holding potential was -50 mV and pulses were to the indicated test potentials at 0.1 Hz (Inset, normalized superimposed traces at +10 mV). C, Peak ICa-V relationships (mean±SEM, n=8 WT, 29 PLB-DM cells). D, Concentration-dependent ICa enhancement by ISO (normalized to basal ICa). Curves were fit to simple Michaelis relationship (EC50=36 and 27 nmol/L for WT and PLB-DM; mean±SE, n=9 WT and 14 PLB-DM myocytes). E, Average ICa integrals for the durations indicated for WT and DM myocytes (n=4 each). F, Mean half-times of ICa decline ±ryanodine. *P<0.05 vs WT controls.

Outward Na⁺-Ca²⁺ exchange current was induced by replacing external Na⁺ with Li⁺. When the peak outward current shift was normalized to cell capacitance, Na⁺-Ca²⁺ exchange current density was comparable for PLB-DM and WT (1.03±0.05 versus 0.93±0.07 A/F, respectively, n=40, 20). Thus, Na⁺-Ca²⁺ exchanger function was not altered in PLB-DM myocytes, consistent with unaltered [Ca²⁺]_i decline during caffeine exposure (Figure 5D).

In Vivo Assessment of Cardiac Function
Echocardiographic studies revealed no significant differences in fractional shortening or left ventricular dimensions between PLB-DM and WT mice at 10 to 12 weeks of age. Furthermore, there were no apparent differences during aging of PLB-DM mice to 10 months (Table).

View this table: [in this window] [in a new window]   Table 1. M-Mode Echocardiography Measurements of Basal Left Ventricular Systolic Function

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
PLB phosphorylation is a major determinant of ß-AR contractile response in the heart,R8-127150 R9-127150 R11-127150 ^8,9,11,12 and basal phosphorylation can be compensatory during depressed cardiac function. Thus, we expected that the absence of phosphorylatable PLB in the novel PLB-DM mouse would cause a pathological cardiac phenotype. Surprisingly, this tonic SERCA2a inhibition, by comparable levels of PLB-DM (versus WT), caused no pathophysiological phenotype up to 10 months of age. Rather, the inability to regulate PLB by phosphorylation seemed to be partially compensated by greater I_Ca density (which can be modulated by ß-AR). The enhanced I_Ca may allow animals to maintain a dynamic ß-AR–mediated inotropic response, even though the lusitropic effect is greatly diminished. Notably, the adaptation to nonphosphorylatable PLB does not involve altered expression or function of SERCA2a or Na⁺-Ca²⁺ exchange. This unique animal model is also a valuable tool for studying regulation of other Ca²⁺ transport systems without the complication of the prominent PLB effects (see following sections).

ß-Adrenergic Lusitropic Effect
Ventricular twitch relaxation and [Ca²⁺]_i decline are greatly accelerated by ß-AR stimulation (lusitropic effect; Figures 4E and 5B). The mechanism involves acceleration of SR Ca²⁺ uptake and reduced Ca²⁺ affinity of the myofilaments (due to phosphorylation of PLB and TnI, respectively).R10-127150 R13-127150 ^10,13,14 Studies in PLB-KO mice have demonstrated that PLB phosphorylation is responsible for the vast majority of the ß-AR lusitropic effect.⁹ Our results here with PLB-DM confirm this. Indeed, twitch relaxation and [Ca²⁺]_i decline rates are unaffected by ISO in either PLB-KO or PLB-DM myocytes. On the other hand, the role of TnI is more apparent when force (or pressure) is developed,R9-127150 ^9,14 and a somewhat larger fractional contribution by TnI phosphorylation was inferred using mice with mutant, nonphosphorylatable cardiac TnI.¹³ A unique advantage of the PLB-DM is that the SR Ca²⁺-ATPase is tonically inhibited by PLB, as in the normal basal WT state (in contrast to the tonic maximally active state in the PLB-KO). In balance, PLB phosphorylation is critical for the ß-AR–induced lusitropic effect, with TnI playing a complementary role.

Compensatory Changes in PLB-DM Mice
The PLB-DM heart cannot enhance the Ca²⁺ affinity of the SR Ca²⁺-ATPase during normal sympathetic tone and modulation. We explored compensations that may allow dynamic modulation of cardiac myocyte contractility (and perhaps explain the lack of pathophysiology). Interestingly, there was a small increase in the rates of contraction and a trend toward higher basal twitch Ca²⁺ transient amplitude and SR Ca²⁺ load in the PLB-DM myocytes. However, there were no alterations in the protein expression levels of SERCA2a, ryanodine receptor, calsequestrin, or calreticulin. Moreover, there was no increase in V_max of SR Ca²⁺ uptake in the PLB-DM, ruling out any contribution of CaMK-dependent phosphorylation of SERCA2a. Myofilament Ca²⁺ sensitivity was also unaltered in the PLB-DM (although it can be modulated by TnI phosphorylation).R4-127150 R7-127150 ^4,7,24 Thus, with slightly reduced SR Ca²⁺-ATPase function in the PLB-DM (see previous section), some other compensatory change in Ca²⁺ transport may have indirectly prevented a decline in SR Ca²⁺.

The Na⁺-Ca²⁺ exchanger normally competes with the SR Ca²⁺-ATPase for Ca²⁺ during relaxation, and reduced Na⁺-Ca²⁺ exchange function could increase SR Ca²⁺load (all other things being equal). The converse is observed in heart failure, where upregulated Na⁺-Ca²⁺ exchange can lower SR Ca²⁺ load and cause contractile dysfunction.²⁵ However, in the PLB-DM there was no change in either Na⁺-Ca²⁺ exchange current or its ability to cause relaxation and [Ca²⁺]_i decline during caffeine exposure.

On the other hand, I_Ca density was significantly increased in the PLB-DM versus WT, similar to previous observations in mice overexpressing a superinhibitory PLB-mutant (V49G).²⁶ Such an increase would enhance both the trigger for SR Ca²⁺ release as well as the SR Ca²⁺ load (by increasing Ca²⁺ influx per beat). I_Ca is normally the other major functional target for PKA (other than PLB), with respect to enhancing Ca²⁺ transients in the ß-AR inotropic response. Upregulated I_Ca may be a unique compensatory mechanism, in that it partially replaces the ß-AR–induced modulation of PLB with an alternative PKA target (enhanced I_Ca). Thus, the enhanced I_Ca can produce the higher SR Ca²⁺ content and release in PLB-DM compared with WT myocytes. Moreover, the greater absolute increase in I_Ca with ISO may directly offset (in part) the loss of PLB phosphorylation in the dynamic ß-AR inotropic response. Indeed, there was still a very substantial inotropic effect of ß-AR stimulation (Figures 4F and 5A), although the lusitropic effect was abolished (Figures 4E and 5B) in the PLB-DM. Notably, the relative ISO stimulation of Ca²⁺ transients in PLB-DM (versus WT) is much better maintained than we have seen for the PLB-KO mouse where I_Ca was not increased.R9-127150 ^9,19 Although the increase in I_Ca seems clearly to be a compensatory factor in the PLB-DM, we cannot yet rule out other prospects (eg, ryanodine receptor or sorcin properties).

The larger I_Ca and SR Ca²⁺load in the PLB-DM may work synergistically to enhance the fraction of SR Ca²⁺ release, which may also accelerate I_Ca inactivation.²⁷ Consistent with this notion, when ryanodine inhibited SR Ca²⁺ release, the difference in I_Ca inactivation (PLB-DM versus WT) was abolished. Thus, increased I_Ca may be the crucial compensatory mechanism that keeps twitch and Ca²⁺ transient amplitudes from declining in the face of chronically inhibited SR Ca²⁺-ATPase.

Reduced levels of PLB phosphorylation (and higher levels of type-1 phosphatase expression) have been implicated in contractile dysfunction in failing human hearts.R15-127150 ^15,28 In this context, we expected that PLB-DM mice might exhibit depressed Ca²⁺ transients and cardiac function. However, in vivo cardiac function, assessed by echocardiography, revealed no significant differences between PLB-DM and age-matched WT controls even up to 10 months of age. Furthermore, no signs of cardiac hypertrophy were detected at the organ and cellular levels. We conclude that the detrimental effect of chronic SERCA2a inhibition by nonphosphorylatable PLB is ameliorated by compensatory mechanisms (eg, enhanced I_Ca) during development. These results in our PLB-DM model are different from recent findings in another transgenic model with cardiac overexpression of the PLB-Arg9Cys human mutant, which traps protein kinase A and prevents PLB phosphorylation.²⁹ Chronic inhibition of SERCA2a in the PLB-R9C hearts was associated with dilated cardiomyopathy and terminal heart failure at 4 to 8 months of age.²⁹ The reason for these apparent different phenotypes between our PLB-DM and the PLB-R9C models may include differences in the following: (1) compensatory mechanisms; (2) the degree of PLB expression levels (overexpression of PLB-R9C versus normal levels of PLB-DM); and (3) the mouse genetic background. Alternatively, lack of PLB phosphorylation may not be the only mechanism underlying the phenotype of the PLB-R9C mutant, which sequesters PKA and may prevent phosphorylation of other key phosphoproteins in the microenvironment of SERCA2a, thereby inhibiting cardiac function and triggered remodeling.

PLB-DM as a New Investigative Tool
Because PLB phosphorylation is so important in cardiac ß-AR effects, preventing phosphorylation, while retaining the normal inhibitory role of PLB on SR Ca²⁺-ATPase, has distinct advantages. For example, we have already used these PLB-DM myocytes to more clearly evaluate the effects of ryanodine receptor versus PLB phosphorylation in the cellular environment.²³ These studies suggest that the main effects of PKA on SR were mediated by PLB and not ryanodine receptor phosphorylation.

In summary, this is the first study to replace native cardiac PLB with a nonphosphorylatable form in vivo. This dual site PLB phosphorylation mutant abolished the lusitropic effect of ß-AR stimulation, without greatly altering basal contraction, relaxation, or Ca²⁺ transients. A key compensatory mechanism may be enhanced I_Ca density. This could both offset the reduced SR Ca²⁺-uptake rate in the PLB-DM (retaining higher SR Ca²⁺ load) and increase the fraction of SR Ca²⁺ released. Since I_Ca is strongly enhanced by ß-AR activation, this compensation may also help the heart retain the dynamic ß-AR modulation necessary during sympathetic activation.

	Acknowledgments

 
This work was supported by NIH grants HL-26057, HL-64018, HL-52318, and P40RR12358 (to E.G.K.), 5 T32 HL-07382 (to A.G.B.), HL-30077 and HL-64098 (to D.M.B.), HL58591 and HL64209 (to B.M.W.), and the Heart and Stroke Foundation of Ontario (to D.H.M.). We wish to thank B.A. Mitton and Q.Y. Yuan for assistance with some of the experiments.

	Footnotes

 
*Both authors contributed equally to this study.

Received November 7, 2002; revision received March 4, 2003; accepted March 5, 2003.

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