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Am J Physiol Heart Circ Physiol 283: H958-H965, 2002; doi:10.1152/ajpheart.00078.2002
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Vol. 283, Issue 3, H958-H965, September 2002

Altered dose response to beta -agonists in SERCA1a-expressing hearts ex vivo and in vivo

Sabine Huke1, Vikram Prasad1, Michelle L. Nieman2, Kalpana J. Nattamai1, Ingrid L. Grupp3, John N. Lorenz2, and Muthu Periasamy1

1 Department of Physiology and Cell Biology, Ohio State University College of Medicine and Public Health, Columbus 43210; and Departments of 2 Molecular and Cellular Physiology and 3 Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

H965, 2002; 10.1152/ajpheart.00078.2002. ---In this study we evaluated the contractile characteristics of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)1a-expressing hearts ex vivo and in vivo and in particular their response to beta -adrenergic stimulation. Analysis of isolated, work-performing hearts revealed that transgenic (TG) hearts develop much higher maximal rates of contraction and relaxation than wild-type (WT) hearts. Addition of isoproterenol only moderately increased the maximal rate of relaxation (+20%) but did not increase contractility or decrease relaxation time in TG hearts. Perfusion with varied buffer Ca2+ concentrations indicated an altered dose response to Ca2+. In vivo TG hearts displayed fairly higher maximal rates of contraction (+ 25%) but unchanged relaxation parameters and a blunted but significant response to dobutamine. Our study also shows that the phospholamban (PLB) level was decreased (-40%) and its phosphorylation status modified in TG hearts. This study clearly demonstrates that increases in SERCA protein level alter the beta -adrenergic response and affect the phosphorylation of PLB. Interestingly, the overall cardiac function in the live animal is only slightly enhanced, suggesting that (neuro)hormonal regulations may play an important role in controlling in vivo heart function.

transgenic mice; contractility; sarco(endo)plasmic reticulum calcium adenosinetriphosphatase; phospholamban


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SEVERAL STUDIES HAVE SHOWN that sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity is decreased in animal models of cardiac hypertrophy and failure as well as in human heart failure (11). This decrease in sarcoplasmic reticulum (SR) Ca2+ transport function was thought to contribute to abnormalities in Ca2+ handling and contractile dysfunction in failing hearts (14). Adenoviral gene transfer of SERCA is currently being explored as a new therapeutic approach for the treatment of heart failure (7, 26-29).

Recent studies have shown that the fast-twitch skeletal muscle isoform SERCA1 has a higher Ca2+ uptake activity than the cardiac isoform SERCA2a because of a higher Ca2+ turnover rate when expressed in cardiac myocytes (34). To test whether SERCA1a can functionally substitute for SERCA2a and increase cardiac contractility, we have developed a transgenic (TG) mouse model expressing SERCA1a in the heart (16, 19, 23). In this model, SERCA1a expression resulted in a 2.5-fold increase in total SERCA pump protein level, but the expression of the endogenous SERCA2a isoform was reduced to ~50% in TG hearts. As a consequence 20% of the total number of SERCA pumps in TG hearts were type 2a whereas the majority of 80% were type 1a pumps. The contractility of isolated TG hearts was clearly increased despite the reduced expression of SERCA2a (23). SERCA1a pump expression was well tolerated without any signs of hypertrophy or other pathophysiological changes in the heart (19). Together these data demonstrate that SERCA1a can functionally substitute for SERCA2a in the heart.

However, not much is known about how SERCA1a expression affects the cardiac response to neurohormonal stimulation (beta -agonists) and the phosphorylation status of phospholamban (PLB). The phosphorylation of PLB and subsequent activation of SERCA pump activity are considered to be key events in the signal transduction of beta -agonists, leading to increased contractility and faster relaxation of the heart (4, 20, 32). Therefore, the major goal of this study was to understand how SERCA1a expression modifies the ability of the heart to respond to beta -adrenergic stimulation ex vivo and in vivo. In particular, we wanted to clarify three important issues: 1) How do isolated, perfused SERCA1a (TG) hearts respond to beta -adrenergic stimulation? 2) How does cardiac function in the isolated heart differ from function in vivo? 3) What is the role of PLB in this model?

This study reports a number of important observations on SERCA1a TG hearts. 1) In isolated heart preparations SERCA1a hearts showed a drastic increase in cardiac contractility. 2) Importantly, the beta -adrenergic response is minimal in TG hearts, whereas contractility remains at a high level. 3) Functional analysis in vivo shows that TG hearts exhibit a modest increase in the maximal rates of contraction (+dP/dt), with no significant change in the maximal rates of relaxation (-dP/dt). 4) The beta -adrenergic response is blunted in TG hearts in vivo. 5) Our studies additionally reveal decreased expression and phosphorylation of PLB. Together these studies suggest that cardiac function in vivo may be subjected to modulation by additional parameters including the (neuro)hormonal system and have the ability to partially mask the effects of TG perturbations.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TG mice. The generation of SERCA1a TG mice was described previously (23). All experiments were performed with line no. 38. Eleven- to fifteen-week-old mice of either sex were used for all experiments. High expression of SERCA1a in the heart of TG mice was confirmed with SERCA1a-specific antibodies (data not shown). All animal procedures followed were in accordance with institutional guidelines.

Isolated heart perfusions ("work-performing hearts"). The experimental conditions for the work-performing mouse heart preparations were described previously (36). 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 (PE)-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). Hearts were allowed to equilibrate for 30 min before isoproterenol (Iso) was infused or extracellular calcium concentration ([Ca2+]o) was changed. Myocardial oxygen consumption was calculated by the formula MVO2 = [PO2 (arterial perfusate) - PO2 (coronary sinus effluent)] × [coronary flow (ml/min) × (0.0239/760) × 1,000]/heart wet weight (g).

A cumulative Iso dose-response curve was obtained with Isoprel (0.2 mg/ml; Abbott, Chicago, IL) infusion from 0.8 up to 40 nmol/l; each dose was applied for 5 min. To achieve exact and stable changes in [Ca2+]o, a second set of containers was used providing oxygenized KHS with a different calcium content. The exact free Ca2+ concentration was calculated with WEBMAXC v2.10 (http://www.stanford.edu/~cpatton/maxc.html), as described previously (2) and is indicated in parentheses. One set of hearts worked initially for 30 min at 1.5 (1.47) mmol/l free Ca2+ and was switched to 0.8 (0.80) mmol/l; another set started with 2 (1.96) mmol/l extracellular free Ca2+, and Ca2+ was increased to 2.5 (2.45) mmol/l after 30 min. Baseline loading conditions were maintained during all experiments.

In vivo catheterization. The experiments were done as described previously (22). Mice were anesthetized (ketamine-thiobutabarbital) and placed on a thermally controlled surgical table. A tracheotomy (PE-90) was performed to protect the airway. The right femoral artery was cannulated with custom-fashioned PE tubing and connected to a pressure transducer to measure blood pressure. The right femoral vein was cannulated and connected to a microinjection pump for the infusion of the beta -agonist dobutamine (Dob). To assess myocardial performance, a 1.4-Fr Millar Mikro-Tip transducer was carefully introduced into the left ventricle via the right carotid artery. After the animals were allowed to stabilize for 20-30 min, increasing doses of Dob were administered as a constant infusion over a range of 1-32 ng · min-1 · g body wt-1. Each dose was administered for 3 min, and the animals were allowed to recover between doses. After completion of the dose-response protocol and restabilization of heart function, a bolus dose of propranolol (Prop; 100 ng/g body wt) was administered to evaluate baseline cardiac function in the absence of endogenous beta -adrenergic activity.

Quantitation of beta -receptors. The total receptor density was determined essentially as previously described (8). Briefly, an enriched membrane fraction derived from whole heart homogenates via two centrifugation steps was used for radioligand binding with 125I-labeled cyanopindolol. Membranes were incubated with a saturating concentration of this ligand in the absence (total binding) or presence (nonspecific binding) of 1 µM alprenolol. Binding reactions were terminated by dilution and rapid filtration.

Quantitative immunoblotting analysis. To study PLB phosphorylation, wild-type (WT; n = 8) and TG (n = 8) hearts were perfused with the isolated, work-performing heart setup. One set of hearts (4 WT and 4 TG) was freeze-clamped after 30 min without Iso, and the other set was treated with Iso (40 nmol/l) for 5 min after 25 min.

Hearts were homogenized in buffer containing (in mmol/l) 10 Tris, 1 dithiothreitol, and 50 NaF with 0.25% NP-40 and protease inhibitors. Proteins were separated by SDS polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and probed with a monoclonal anti-PLB antibody (Affinity Bioreagents, Golden, CO) or polyclonal anti-phosphorylated PLB-Ser16 or Thr17 antibody (Cyclacel, Dundee, UK). Binding of the primary antibody was detected by peroxidase-conjugated secondary antibodies (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and visualized with the SuperSignal substrate kit (Pierce, Rockford, IL).

Statistical analysis. Results are expressed as means ± SE. Significance was estimated by Student's t-test for paired and unpaired observations and one-way repeated-measures ANOVA followed by a Bonferroni t-test for comparison of a pair of points as appropriate (SPSS for Windows 11.0). P <=  0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Loss of response to Iso in isolated SERCA1a TG hearts. beta -Adrenergic regulation is a major signaling mechanism in the control of cardiac function. beta -Adrenergic stimulation is associated with an increase in contractility and a decrease in relaxation time. An important step in the signal transduction is the activation of the SR-Ca2+ uptake function because of PLB phosphorylation and consequent activation of SERCA pump activity. Therefore, changes in SERCA protein expression level and PLB-to-SERCA ratio could modify the beta -adrenergic response.

Isolated work-performing WT and TG hearts were perfused with cumulative doses of the beta -agonist Iso (0.08-40 nmol/l in the presence of 2 mmol/l [Ca2+]o). Stimulation with Iso increased the heart rate similarly in both groups (Fig. 1). In WT hearts (n = 5) the maximal rates of contraction (+dP/dt; 4,305 ± 111 to 6,702 ± 220 mmHg/s) and relaxation (-dP/dt; 3,695 ± 119 to 6,491 ± 247 mmHg/s) increased and the time to peak pressure (TPP; 37.1 ± 0.9 to 28.8 ± 0.8 ms) and the time to 50% relaxation (RT1/2; 28.0 ± 0.4 to 18.8 ± 0.2 ms) decreased in response to Iso, as expected, whereas in isolated TG hearts (n = 5) Iso did not increase +dP/dt (7,083 ± 205 to 6,538 ± 172 mmHg/s) or decrease TPP (30.3 ± 0.5 to 29.7 ± 0.8 ms) or RT1/2 (20.6 ± 1.0 to 18.1 ± 0.8 ms). Only a moderate increase in -dP/dt (+20%; -5,584 ± 273 to -6,705 ± 99) and a decrease in the diastolic pressure (-11.2 ± 1.0 to -22.3 ± 0.7) were observed. Remarkably, the baseline unstimulated contractile parameters of isolated TG hearts were comparable to those of WT hearts that were maximally stimulated with Iso. The high baseline contractility of TG hearts was reflected in MVO2 under baseline conditions. WT hearts extracted 145 ± 10 µl O2 · min-1 · g-1 (n = 10), whereas TG hearts used 205 ± 11 µl O2 · min-1 · g-1 (n = 10).


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  		Fig. 1.   Cumulative dose-response to isoproterenol (Iso; 0.08-40 nmol/l) in isolated work-performing hearts. Heart rate (A), time to 50% relaxation (RT1/2; B), and rates of contraction (+dP/dt; C) and relaxation (-dP/dt; D) are shown. Data are derived from 5 wild-type (WT) and 5 transgenic (TG) hearts.

Altered dose response to [Ca2+]o in isolated SERCA1a TG hearts. To determine how SERCA1a TG hearts respond to changes in [Ca2+]o, [Ca2+]o was increased from 2 to 2.5 mmol/l (Table 1). The contractile parameters of TG hearts remained unchanged (+dP/dt, P = 0.098; RT1/2, P = 0.809), whereas WT hearts showed an increase in contractility (+dP/dt; <=  0.001) and a decrease in relaxation time (RT1/2; P <=  0.001). To gain a more complete picture of the dose response to [Ca2+]o, cardiac function was also analyzed at 1.5 and 0.8 mmol/l [Ca2+]o. At 0.8 mmol/l [Ca2+]o, only the TG hearts were able to function, whereas the WT hearts failed to perform the basal workload. +dP/dt and -dP/dt were clearly increased in TG hearts at all [Ca2+]o tested (0.8, 1.5, 2.0, 2.5 mmol/l; see also Fig. 2). Together these data strongly suggest that in TG hearts the dose response to [Ca2+]o is shifted leftward with a maximum at 2 mmol/l [Ca2+]o at physiological heart rates.

                              
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  		Table 1.   Functional parameters of isolated work-performing hearts at varied [Ca2+]o



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  		Fig. 2.   Effect of extracellular Ca2+ concentration ([Ca2+]o) on maximal +dP/dt (A) and -dP/dt (B) in isolated work-performing hearts. Data derived from 5 WT and 4 TG hearts are plotted at varied [Ca2+]o. Note that the values for 0.8, 1.5, and 2.5 mmol/l were statistically compared with the baseline values at 2 mmol/l [Ca2+]o. Statistical differences between the groups are not indicated (see Table 1). n.s., Not significant.

Changes in heart function in SERCA1a TG mice in vivo. To determine how contractile function in isolated, work-performing hearts compares with that in in vivo hearts, we assessed the beta -adrenergic response additionally in the living animal with Dob (Fig. 3). Basally, TG hearts showed higher maximal +dP/dt [7,960 ± 557 in WT (n = 7) vs. 10,503 ± 587 mmHg/s in TG (n = 8)] but unchanged -dP/dt (8,127 ± 475 in WT vs. 8,624 ± 422 mmHg/s in TG) compared with WT hearts. Heart rate (397 ± 20 beats/min in WT vs. 335 ± 31 beats/min in TG) and mean arterial pressure under control conditions (73.3 ± 2.9 mmHg in WT vs. 70.5 ± 5.2 mmHg in TG) were unaltered between groups. In the living animals both WT and TG hearts responded significantly to Dob infusion with increases in +dP/dt (at 32 ng · min-1 · g body wt-1 to 19,481 ± 1,356 mmHg/s in WT and 17,805 ± 1,253 mmHg/s in TG) and -dP/dt (maximal effect at 8 ng · min-1 · g body wt-1 to 12,576 ± 670 mmHg/s in WT and 10,667 ± 527 mmHg/s in TG). The highest maximal +dP/dt values reached during stimulation with Dob were similar in WT and TG hearts, but the WT hearts developed higher maximal -dP/dt than the TG hearts. The highest maximal +dP/dt and -dP/dt in response to Dob, when expressed as a percentage of basal values, were reduced in TG hearts (maximal effect: +dP/dt, +144% in WT and +70% in TG; -dP/dt, +55% in WT and +24% in TG).


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  		Fig. 3.   Dose response to dobutamine (1-32 ng · min-1 · g body wt-1) in anesthetized mice. Graphs show heart rate (A), +dP/dt (C), and -dP/dt (D) determined with a catheter advanced into the left ventricle and mean arterial pressure (B) measured in the right femoral artery. Values for each animal were determined from at least 50 consecutive beats during the final 30 s of each 3-min dose. Data are derived from 7 WT and 8 TG mice.

To exclude the possibility of considerable endogenous beta -adrenergic activity, the beta -blocker Prop was given as a bolus dose after completion of the Dob dose-response protocol. Prop did not affect +dP/dt or -dP/dt in either WT or TG [+dP/dt: (recontrol) 8,080 ± 572 mmHg/s vs. (+Prop) 8,124 ± 540 mmHg/s in WT and 10,955 ± 1,075 mmHg/s vs. 10,733 ± 832 mmHg/s in TG; -dP/dt: 8,978 ± 522 mmHg/s vs. 8,769 ± 429 mmHg/s in WT and 9,110 ± 478 mmHg/s vs. 9,182 ± 548 mmHg/s in TG], demonstrating the absence of a tonic sympathetic drive in these mice. The effectiveness of the beta -blockade was verified by a second perfusion with Dob (32 ng · min-1 · g body wt-1). During this subsequent perfusion Dob did not increase +dP/dt or -dP/dt above recontrol values in WT and TG.

Moreover, dP/dt40 (rate of contraction at intraventricular pressure of 40 mmHg), an index that attempts to correct for the preload dependence of +dP/dt (31), and the relaxation time constant tau , unlike -dP/dt not dependent on afterload, were calculated (Fig. 4). In comparison, the results with loading-dependent parameters (Fig. 3) versus loading-independent parameters (Fig. 4) are very similar.


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  		Fig. 4.   Dose response to dobutamine (1-32 ng · min-1 · g body wt-1) in anesthetized mice. Graphs show additional parameters (see Fig. 4) of contraction [rate of contraction at intraventricular pressure of 40 mmHg (dP/dt40); A] and relaxation (relaxation constant tau ; B). Values for each animal were determined from at least 50 consecutive beats during the final 30 s of each 3-min dose. Data are derived from 7 WT and 8 TG mice.

Taken together, in vivo, maximal +dP/dt values were increased in TG hearts (+25%) and the response to Dob was blunted. However, compared with our observations in isolated hearts the baseline contractile parameters in TG hearts were only moderately increased compared with those in WT hearts. This was not due to a tonic sympathetic drive in WT and/or TG hearts or major differences in pre- or afterload in the living animal.

No change in beta -receptor density. To determine whether the blunted response to beta -adrenergic stimulation is, at least in part, due to a decrease in beta -receptor expression we performed 125I-cyanopindolol binding studies with membrane-enriched fractions. There was no change in beta -receptor expression in TG compared with WT mice [27.6 ± 2.6 fmol/mg protein in WT vs. 29.2 ± 4.3 fmol/mg protein in TG (both n = 4)].

SERCA1a hearts show a decrease in PLB protein and lower PLB phosphorylation at Thr17. SERCA1a is not a natural partner of PLB. However, it was shown that SERCA1a interacts with PLB in vitro (35) and in vivo when PLB is ectopically expressed in fast-twitch skeletal muscle, where SERCA1a is naturally found (33).

PLB exists as a monomer and a pentamer with the pentameric state as the predominant state in cardiac tissue (10, 18). The pentamer can be converted into monomer when protein samples are boiled. Total PLB expression level was analyzed with boiled (data not shown) and nonboiled samples from perfused hearts. Our studies revealed a decrease of ~40% in TG hearts (Fig. 5, A and D). In nonboiled samples the amount of monomer was below our detection limit in both groups.


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  		Fig. 5.   Phospholamban (PLB) expression (A and D) and phosphorylation at Ser16 (PLB-S-16; B and E) and Thr17 (PLB-T-17; C and F) in isolated, work-performing hearts before (-Iso) and after (+Iso) Iso perfusion. A, B, and C are representative blots. Data in D are derived from 8 WT and 8 TG mice (independent of Iso treatment), whereas E is derived from 4 hearts and F from 3 hearts in each group. Note that in E and F the phosphorylation signals are normalized to the expression level of PLB in each individual heart.

We further analyzed the basal phosphorylation status of Ser16 and Thr17 and that in response to Iso stimulation in WT and TG hearts. PLB Ser16 was completely dephosphorylated after 30 min of equilibration, whereas basal phosphorylation at PLB Thr17 was detectable (in WT ~15% of what became phosphorylated because of Iso stimulation). In response to Iso, PLB became phosphorylated at Ser16 in WT as well as TG hearts (Fig. 5B), whereas the extent of phosphorylation in the TG hearts was slightly less than in the WT hearts (-14%; Fig. 5E). Stimulation with Iso (40 nM, 5 min) did not increase PLB Thr17 phosphorylation in TG hearts, whereas it increased Thr17 phosphorylation in WT hearts by more than fivefold. PLB Thr17 phosphorylation in TG hearts was lower than in WT hearts basally (by ~56%) and after stimulation with Iso (by >90%) (Fig. 5, C and F). These data indicate that cardiac SERCA1a expression results, in addition to the decrease in expression of the endogenous SERCA2a, in decreased PLB expression and reduced phosphorylation at the Thr17 site.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SERCA1a TG hearts show blunted response to beta -adrenergic stimulation. An important finding of this study is that the beta -adrenergic response is drastically reduced in isolated TG hearts. As expected, beta -agonists had positive chronotropic, inotropic, and lusitropic effects in WT hearts whereas in TG hearts the chronotropic effect was fully maintained but most other contractile parameters remained unchanged. The only exception was that Iso moderately increased maximal -dP/dt (+20%).

The minimal response to beta -agonists in isolated hearts could be due to a change in the PLB-to-SERCA ratio (-40% PLB/+150% SERCA), i.e., most of the SERCA pumps are not regulated by PLB and further deinhibition of the PLB-regulated SERCA pumps has no more impact on cardiac function. On the other hand, the TG hearts may already have reached maximum contractility under baseline conditions, which cannot be further increased by the effects of Iso stimulation on SR Ca2+ uptake and release. The second idea is supported by two observations. First, the contractility of WT hearts never surpassed the contractility of TG hearts even when stimulated with high doses of Iso. Second, stimulation with >2 mmol/l [Ca2+]o ([Ca2+]o at which the Iso response was performed) fails to increase cardiac contractility in TG hearts. A third possibility is that the beta -adrenoceptor expression is decreased in TG mice and accounts for the lack of response, but 125I-cyanopindolol binding studies revealed an unchanged level of receptors. A loss of beta -adrenergic response was also seen in PLB-null mice (25), in which the response of beta -agonists was partly restored when the hearts were unloaded from Ca2+ at very low [Ca2+]o (30).

Response to [Ca2+]o is altered at physiological heart rates. The data derived from the perfusion of the isolated hearts with varied [Ca2+]o suggest that the dose response to [Ca2+]o is shifted leftward in TG hearts, which in turn means a lower EC50 of [Ca2+]o at physiological heart rates (~5 Hz). Studies in isolated trabeculae from SERCA2a TG mice (1.5-fold SERCA expression) found no difference in the dose response to [Ca2+]o when stimulated at 0.5 Hz (12). Ca2+ transients and twitch force increased almost simultaneously as [Ca2+]o increased. Thus the functional effects of increased SERCA expression appear to manifest at higher stimulation rates.

Our studies revealed that when [Ca2+]o was decreased to 0.8 mmol/l, only the TG hearts were able to function and perform the basal workload whereas the WT hearts failed. Interestingly, TG hearts at 0.8 mmol/l [Ca2+]o develop maximal +dP/dt and -dP/dt comparable to those of WT hearts at 2 mmol/l [Ca2+]o. This concurs with our earlier finding (19) that the L-type Ca2+ channel current is decreased in TG hearts by ~50%. On the basis of this reduction it can be interpreted that considerably less amount of "trigger" Ca2+ is needed in TG hearts to develop the same contractility as in WT hearts. This could be a direct consequence of the increased SR Ca2+ load. It has been shown that intraluminal Ca2+ regulatory mechanisms cause a potentiation of SR Ca2+ release in cardiac cells in response to increases in SR Ca2+ load (1, 5, 24). It was reported that this relation is very steep at high SR Ca2+ loads (31). The increased SR Ca2+ load in TG hearts might enable them to maintain sufficient function at low [Ca2+]o, whereas the WT hearts cannot. Moreover, our data show that TG hearts tolerate higher than physiological [Ca2+]o (2.5 mmol/l) without any signs of Ca2+ overload and failure.

SERCA1a hearts in vivo show moderately increased cardiac contractility and blunted response to Dob. In vivo baseline contractility in TG hearts was moderately increased, whereas relaxation parameters, in contrast to the observations in the isolated hearts, were unchanged. Overall, the contractile parameters were enhanced to a lesser degree than in the ex vivo experiments. This indicates the presence of in vivo compensatory mechanisms that "normalize" cardiac function in TG mice. The data obtained from isolated hearts show that the contractile parameters of WT and TG converge in the presence of beta -adrenergic stimulation. Thus a tonic sympathetic drive could partly account for smaller differences between the groups. Because beta -blockade does not affect the basal contractile parameters in both groups, this possibility can be excluded.

On the other hand, the loading of the heart cannot be controlled in vivo and could account, at least in part, for the discordance between ex vivo and in vivo function. However, the finding that less loading-dependent indexes of contractility and relaxation are showing largely the same magnitude of differences as loading-dependent indexes does not support this idea.

A discordance between ex vivo and in vivo function is less apparent in other transgenic mouse models, in which the isolated muscle or heart function is largely reflected in vivo. In mice with moderately increased levels of the endogenous cardiac isoform SERCA2a (+20% in SERCA protein level), in vivo hemodynamic measurements revealed significantly increased maximal +dP/dt and -dP/dt, similar to what was observed in isolated myocytes and isolated papillary muscles (13). Similarly, PLB-null hearts were hypercontractile and had a severely blunted response to stimulation with beta -agonists ex vivo as well as in vivo (21, 25).

Role of PLB in SERCA1a TG mice. Our current studies with quantitative immunoblotting revealed that the PLB expression level was actually decreased in TG hearts by ~40%. Interestingly, this decrease is comparable to the decrease in SERCA2a expression in this model, the natural in vivo partner of PLB (23). Thus we could argue that the SERCA2a pump level can affect the expression level of PLB to maintain a constant PLB-to-SERCA2a ratio. This idea is supported by our recent findings (15) in the SERCA2 (+/-) ablated mice, in which the SERCA2a protein level is decreased by ~35%. As a secondary effect, the PLB protein level in this model is reduced by about the same amount. Thus the PLB-to-SERCA ratio remains unchanged (15). Although this idea is not supported by findings in failing hearts (3, 11), this mechanism might be functional solely in nonfailing hearts.

In addition to the decrease in the PLB expression level in TG hearts, PLB phosphorylation at Thr17 by calmodulin (CaM) kinase was significantly decreased. Phosphorylation at both sites, Ser16 and Thr17, relieves the inhibitory effect of PLB on SERCA and enhances SR Ca2+ uptake, although the exact contribution of each phosphorylation site and its specific effect on heart function is not clear (3, 6, 9). Interestingly, in SERCA2 (+/-) mice we found an increase in the phosphorylation status of PLB. The phosphorylation at PLB Thr17 was enhanced, and in accordance with this finding the activity of SR associated CaM kinase was increased (15, 17). The opposite changes in PLB Thr17 phosphorylation in genetically manipulated mice with increased SERCA expression (phosphorylation decrease) and mice with decreased SERCA expression (phosphorylation increase) allow us to speculate that changes in SR Ca2+ transport and SR Ca2+ load affect the activity or translocation of SR-associated CaM kinase. This could be a general mechanism regulating SR Ca2+ uptake independent of beta -adrenergic stimulation. These data indicate a specific role for the phosphorylation at PLB Thr17 that is not yet understood and requires additional studies.


    ACKNOWLEDGEMENTS

The authors thank Gilbert Newman and Kari M. Brown for expert technical assistance. We are grateful to Dr. Stephen B. Liggett for allowing us to perform the beta -adrenoceptor expression studies in his laboratory.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-64140-02 and American Heart Association (AHA) Grant 9950570N. S. Huke was supported by postdoctoral fellowships from the Deutsche Forschungsgemeinschaft (HU 898/1-1) and the AHA (0120149B).

Address for reprint requests and other correspondence: M. Periasamy, Dept. of Physiology and Cell Biology, Ohio State Univ. College of Medicine and Public Health, 302 Hamilton Hall, 1645 Neil Ave, Columbus, OH 43210 (E-mail: periasamy.1{at}osu.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.

10.1152/ajpheart.00078.2002

Received 30 January 2002; accepted in final form 6 May 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 283(3):H958-H965
0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society


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