Published online before print February 16, 2004, doi: 10.1161/01.CIR.0000117254.68497.39
(Circulation. 2004;109:1154-1160.)
© 2004 American Heart Association, Inc.
Basic Science Reports |
Altered Calcium Handling Is Critically Involved in the Cardiotoxic Effects of Chronic ß-Adrenergic Stimulation
From the Institute of Pharmacology and Toxicology (S.E., L.H., M.J.L.), University of Wuerzburg, Germany; Institute of Physiology (V.D., G.I.), Martin-Luther-University Halle, Germany; and Department of Pharmacology and Cell Biophysics (E.G.K.), University of Cincinnati College of Medicine, Cincinnati, Ohio.
Correspondence to Stefan Engelhardt, Rudolf-Virchow-Center for Experimental Biomedicine, DFG Forschungszentrum der Universitaet Wuerzburg, Versbacher Straße 9, 97078 Wuerzburg, Germany. E-mail stefan.engelhardt{at}virchow.uni-wuerzburg.de
Received July 18, 2003; revision received October 24, 2003; accepted October 28, 2003.
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Abstract |
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Background— Chronic adrenergic stimulation leads to cardiac hypertrophy and heart failure in experimental models and contributes to the progression of heart failure in humans. The pathways mediating the detrimental effects of chronic ß-adrenergic stimulation are only partly understood. We investigated whether genetic modification of calcium handling through deletion of phospholamban in mice would affect the development of heart failure in mice with transgenic overexpression of the ß1-adrenergic receptor.
Methods and Results— We crossed ß1-adrenergic receptor transgenic (ß1TG) mice with mice homozygous for a targeted deletion of the phospholamban gene (PLB-/-). Phospholamban ablation dramatically enhanced survival of ß1TG mice. The decrease of left ventricular contractility typically observed in ß1TG mice was reverted back to normal by phospholamban ablation. Cardiac hypertrophy and fibrosis were significantly inhibited in ß1TG/PLB-/- mice compared with ß1TG mice, and the heart failure–specific gene expression pattern was normalized. Analysis of intracellular calcium transients revealed increased diastolic calcium levels and decreased rate constants of diastolic calcium decline in ß1TG mice. In ß1TG/PLB-/- mice, diastolic calcium concentration was normal and rate constants of diastolic calcium decline were greater than in wild-type mice.
Conclusions— We conclude that modification of abnormal calcium handling in ß1TG mice through ablation of phospholamban resulted in a rescue of functional, morphological, and molecular characteristics of heart failure in ß1-adrenergic receptor–transgenic mice. These results imply altered calcium handling as critical for the detrimental effects of ß1-adrenergic signaling.
Key Words: calcium • heart failure • sarcoplasmic reticulum • hypertrophy • heart failure
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Introduction |
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The incidence of heart failure has been constantly increasing during the past decades, and heart failure is among the most frequent causes of morbidity and mortality in the Western world. Most patients with heart failure show chronic activation of the sympathetic nervous system, and the resulting elevation of plasma norepinephrine concentration correlates with the severity of the disease.1 Mimicking chronic adrenergic stimulation in transgenic animal models results in progressive hypertrophy and heart failure.2 The importance of this dysregulation of the sympathetic nervous system has been additionally underlined by several large clinical trials demonstrating beneficial effects of ß-adrenergic receptor antagonists.3 Thus, whereas chronic adrenergic stimulation initially serves to compensate for the functional impairment of cardiac function, it ultimately results in deterioration of cardiac structure and function. The mechanisms by which ß-adrenergic receptor stimulation contributes to the progression of heart failure are still not clear. Several signaling pathways have been shown to be involved in adrenergically induced hypertrophy and heart failure. Many of these pathways are linked to cardiac sarcoplasmic reticulum (SR) Ca2+ cycling directly or indirectly.
In the present study, we tested whether alterations in SR Ca2+ handling are a critical step involved in the detrimental effects of chronic ß-adrenergic stimulation. To investigate this hypothesis, we used a genetic strategy to enhance SR Ca2+ uptake and deleted the principal inhibitor of SR Ca2+ uptake, phospholamban, in ß1-adrenergic receptor (ß1AR) transgenic (ß1TG) mice.
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Methods |
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Transgenic Animals
The generation of transgenic mice overexpressing human ß1AR under the control of the
-myosin heavy chain promoter has been described previously.4 For this study, we crossed heterozygous ß1TG-mice (line TG4) with mice carrying a homozygous deletion of the phospholamban gene (both maintained on the same genetic background, FVB/N). Heterozygous intercrosses for the phospholamban deletion and the ß1AR transgene were then mated to heterozygous phospholamban knockout mice. From the resultant F2 generation, we followed a total of 17 ß1TG+/-/PLB+/+ (termed ß1TG), 28 ß1TG+/-/PLB-/- (termed ß1TG/PLB-/-), 8 ß1TG-/-/PLB-/- (termed PLB-/-), and 20 ß1TG-/-/PLB+/+ mice (wild-type controls [WT]) for survival analysis. Four to 7 mice per genotype (aged 12 months) were used for experiments determining cardiac function, gene expression, and morphology. All animals were housed under specified pathogen-free conditions. All animal experiments were approved by the responsible authorities (protocol No. 621-2531.01-10/98 and 28/01).
Morphological Analysis
Sections (4 µm) were stained with H&E and picric acid/Sirius red, essentially as described.5 For the determination of myocyte cross-sectional area, 50 individual cells per genotype from at least 3 different animals were analyzed morphometrically. Only nucleated cardiac myocytes from areas of transversely cut muscle fibers were included in the analysis. Quantification of left ventricular fibrosis was achieved by Sirius red staining followed by semiautomated image analysis, as described.6
Left Ventricular Catheterization
Left ventricular catheterization was carried out via the right carotid artery, as previously described.4 Data analysis was carried out with Chart software (Chart 4.2, AD Instruments).
Cells and Solutions
After the aorta was cannulated in situ, the heart was retrogradely perfused and cells were enzymatically dissociated as described previously.7 Isolated ventricular myocytes were loaded with 7 µmol/L Indo-1 AM (Molecular Probes) at 37°C for 30 minutes. During the experiment, cells were continuously superfused by a physiological salt solution containing (in mmol/L) NaCl 150, KCl 5.4, CaCl2 1.8, MgCl2 1.2, glucose 10, and HEPES/NaOH 10 (pH 7.4, 37°C).
[Ca2+]c Measurements
Cells were washed in Indo-1 free physiological salt solution for at least 20 minutes. Fluorescence (F) of Indo-1 was excited at 350 nm. Emitted F was collected through 20-nm bandpass filters at 400 and 490 nm with a pair of photomultipliers.8 Time-dependent F400 and F490 were filtered at 100 Hz, sampled at 3 kHz (CED1401 interface), and stored in a personal computer (customer written software). Cells were voltage clamped with 40-ms pulses from -45 to 0 mV at a rate of 1 Hz. After a 4-minute equilibration period, measurements started. Offline, 10 traces of stimulation were averaged, and [Ca2+]c was calculated from the ratio of F400 to F490.9 Diastolic and peak systolic [Ca2+]c were identified by computer algorithms. The rate constant of the diastolic decay of [Ca2+]c was obtained by nonlinear fit of [Ca2+]c after the end of the ms pulse. The number of animals was 
4 for each group, and the number of cells was 10 for each animal.
RNase Protection Analysis of Atrial Natriuretic Factor and SERCA
RNase protection analysis was carried out as described.5 The 18S band intensities of total RNA isolated from WT and transgenic animals determined after denaturing agarose gel electrophoresis were essentially identical and were used to normalize the specific RNA levels. The size of the unprotected fragments was 60 nucleotides longer than that of the protected fragments, thus excluding the contribution of undigested probe to the signal.
Statistical Analysis
Average data are presented as mean±SEM. Statistical analysis was carried out using the Prism software package (GraphPad). ANOVA followed by Bonferroni’s test was used for comparisons unless indicated otherwise. Differences were considered significant when P<0.05.
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Results |
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PLB Deletion Enhances Survival of ß1AR Transgenic Mice
To determine the role of phospholamban in the development of heart failure in mice with cardiac-specific overexpression of the ß1AR, we generated 4 different transgenic lines by crossing of ß1TG with mice homozygous for deletion of the phospholamban gene (PLB-/-). We followed a total of 73 mice for 12 months and monitored survival by Kaplan-Meier analysis (Figure 1). No deaths occurred in the WT group (n=20) and in PLB-/- mice (n=8). Survival of ß1TG mice was significantly reduced, with 10 deaths during the study period. In sharp contrast, concomitant ablation of the phospholamban gene in ß1TG mice (ß1TG/PLB-/-, n=28) led to a complete rescue from premature death (Figure 1). All animals from this group also appeared clinically healthy, as opposed to the 7 surviving ß1TG animals that showed clinical signs of heart failure, including tachypnea and reduced physical activity. The surviving mice of all genotypes were then used for the subsequent investigations.
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Inhibition of Cardiac Hypertrophy, Fibrosis, and Pulmonary Congestion
Morphological analysis indicated marked hypertrophy of left ventricular myocardium in ß1TG mice compared with WT mice (Figure 2A). This increase in ventricular weight was completely abolished in ß1TG/PLB-/- mice. Likewise, the ratio of heart weight to body weight of PLB-/- mice showed no signs of cardiac hypertrophy. During pathological examination, severe pulmonary congestion paired with pleural effusions became apparent in ß1TG mice (Figure 2B, inset). In accordance with these findings, the lung weights of ß1TG mice were more than doubled compared with WT mice (Figure 2B). However, on PLB ablation, the morphology and lung weights of ß1TG/PLB-/- mice were in the normal range and did not differ from WT animals.
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The histological analysis of left ventricular myocardium revealed severely hypertrophic cardiac myocytes throughout the whole left ventricular myocardium of ß1TG mice (Figure 2C). In addition, the cellular arrangement of cardiac myocytes was disturbed with large areas of cellular disarray. Nuclear morphology was highly abnormal, with numerous pleomorphic cardiomyocyte nuclei. Quantitative morphometrical analysis revealed marked cardiomyocyte hypertrophy in ß1TG mice compared with WT mice (Figure 2E). In sharp contrast, cardiomyocyte hypertrophy was absent when phospholamban was deleted in ß1TG/PLB-/- mice.
A hallmark of heart failure in ß1TG mice is the development of profound interstitial fibrosis (Figure 2D). We stained left ventricular sections with picric acid/Sirius red and quantified the percentage of left ventricular fibrosis by semiautomated image analysis. ß1TG mice showed an increase of left ventricular fibrosis to 19±2% of the myocardium compared with 3±0.5% in WT animals (Figure 2F). ß1TG/PLB-/- mice showed significantly decreased left ventricular fibrosis (5±1%) compared with ß1TG mice. Phospholamban ablation alone had no detectable impact on the formation of left ventricular fibrosis.
Rescue of Left Ventricular Function in ß1TG Mice
Left ventricular function was determined in vivo by ventricular catheterization of anesthetized animals. Deletion of phospholamban in ß1TG mice prevented the decrease of left ventricular systolic pressure observed in the surviving ß1TG mice (Figure 3A). Phospholamban deletion also markedly improved left ventricular congestion, as evidenced by a normalization of left ventricular end-diastolic pressure (LVEDP) (Figure 3B). Furthermore, the decrease of left ventricular contractility observed in ß1TG mice (Figure 3C) was completely inhibited by phospholamban ablation. This beneficial effect of phospholamban deletion was also observed for diastolic dysfunction in these mice (Figure 3D). Again, ß1TG/PLB-/- mice did not show impairment of left ventricular relaxation compared with wild-type animals.
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Reversal of Heart Failure–Specific Changes in Cardiac Gene Expression
We then assessed the influence of phospholamban ablation on cardiac gene expression by RNase protection analysis of mRNAs for atrial natriuretic factor (ANF) and the SR Ca2+-ATPase (SERCA). We found expression of ANF to be significantly enhanced in ß1TG mice (Figures 4A and 4B). The principal effector molecule of phospholamban, SERCA2A, was downregulated in ß1TG mice (Figures 4A and 4C), a finding observed in several models of heart failure and also in human heart failure. Deletion of phospholamban reversed this pathological gene expression pattern back to normal (Figure 4C).
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Rescue of Calcium Handling Defects
To determine the potential mechanisms underlying the detrimental effects of chronic ß-adrenergic stimulation on cardiac myocytes, we assessed intracellular calcium signaling. We isolated cardiac myocytes from mice at a very young age (8 weeks) to detect crucial defects occurring early in the development of the disease. At this age, ß1TG mice do not show any signs of cardiac impairment. Cells were loaded with the fluorescent Ca2+-indicator Indo-1, and we detected 2 major alterations of the intracellular calcium transient (Figure 5). First, ß1TG mice displayed significantly prolonged calcium transients compared with WT mice (Figure 5A). Analysis of the rate constant of diastolic calcium decline demonstrated a significant impairment of diastolic calcium decline (Figure 5B). Second, ß1TG mice displayed enhanced diastolic calcium levels, whereas peak systolic calcium was unaltered (Figures 5A, 5C, and 5D). Concomitant deletion of phospholamban resulted in a complete reversal of both abnormalities (Figures 5A through 5C), resulting in intracellular calcium transients decaying even faster than calcium transients from wild-type controls (Figures 5A and 5B). Diastolic calcium levels from ß1TG/PLB-/- mice were significantly decreased both versus ß1TG mice and versus WT mice (Figure 5C). As a result, the Ca2+ load, ie, the time average of systolic and diastolic [Ca2+]c, was higher in ß1TG than in WT mice. Again, ablation of phospholamban in ß1TG/PLB-/- mice reduced the calcium load significantly (Figure 5E). Analysis of voltage-clamp data indicated no significant differences in the amplitude, rate of inactivation, and time of the L-type calcium currents (not shown), suggesting that the influx of extracellular Ca2+ was comparable between the 3 types of cells.
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Discussion |
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The main result of the present study is that abnormal intracellular calcium handling underlies the detrimental effects of chronic ß1-adrenergic signaling in the heart. Modification of the abnormal calcium transients through ablation of phospholamban protects from the detrimental functional, morphological, and molecular consequences of chronically enhanced ß1AR signaling in the heart.
Enhancement of Cardiac Contractility Through Inhibition of Phospholamban Is Beneficial Rather Than Detrimental
Acute enhancement of myocardial performance is regarded as the main effect of the cardiac ß-adrenergic signaling pathway. However, chronic activation of the ß adrenergic receptor–Gs–protein kinase A (PKA) axis in the heart leads to progressive myocyte hypertrophy, myocyte damage, and fibrosis and eventually heart failure.2 Inhibition of phospholamban via phosphorylation and thereby enhancement of SERCA function is one of the key effectors of this signaling pathway,10,11 and it might therefore be expected to be an integral part of the detrimental signaling cascade. We have previously shown phospholamban phosphorylation to be significantly enhanced in this mouse model.5 Accordingly, complete phospholamban inhibition through ablation of the gene might be expected to aggravate the phenotype of ß1AR transgenic mice. Surprisingly, histological analysis revealed that hypertrophy and interstitial fibrosis, indicators of tissue damage, were significantly decreased in ß1TG/PLB-/- mice compared with ß1TG animals. Isoproterenol-stimulated adenylyl cyclase activity (data not shown) was preserved in ß1TG/PLB-/- mice compared with ß1TG mice, indicating that phospholamban ablation did not interfere with the ß1AR-mediated cAMP signal. Thus, neither the enhancement of cardiac contractility nor phosphorylation of phospholamban can be regarded as part of the toxic signal transduction cascade originating from the cardiac ß1AR. It appears more likely that additional signaling pathways are involved, possibly via enhanced Ca2+ entry into cardiac myocytes or enhanced release from internal stores, eg, through the ryanodine Ca2+ release channel. In accordance with the latter hypothesis, leakiness of the ryanodine Ca2+ release channel has been described in heart failure through PKA-mediated hyperphosphorylation.12
Calcium Signaling in Heart Failure
Similar alterations in cardiomyocyte SR Ca2+ handling with slow rate constants and enhanced diastolic calcium concentrations have been identified in a variety of animal models as well as in human heart failure.13,14 Specifically, in the human disease, prolonged intracellular Ca2+ transients have been described and have been attributed to a decreased reuptake of diastolic calcium into the SR.15,16 This impairment of Ca2+ reuptake has been attributed to alterations in the function of several SR proteins, including a decreased ratio of the expression of the SR calcium ATPase (SERCA) to phospholamban.13,17 Studies with overexpression of SERCA2a and inhibition of phospholamban18 have demonstrated beneficial effects in a variety of heart failure models as well as in isolated myocytes from failing human hearts. The enhancement of SERCA function through gene knockout of phospholamban has led to less clear results. Whereas phospholamban ablation markedly improved the phenotype of some heart failure models,19–21 it did not attenuate the phenotype of tropomodulin transgenic mice22 and mice with transgenic overexpression of Gq and a mutant myosin-binding protein C.23 Thus, some forms of cardiomyopathy seem to be resistant to the therapeutic inhibition of phospholamban function, whereas others benefit from phospholamban inhibition. Additional complexity is suggested by 2 recent reports studying phospholamban mutations in humans.24,25 Although a premature stop-codon is described to be associated with cardiomyopathy in the homozygous state,24 Schmitt et al25 report that a heterozygous R9C mutation in phospholamban leads to local trapping of PKA, thereby minimizing PKA-mediated inhibition of the remaining phospholamban allele. The disinhibited WT allele becomes functionally dominant, and thereby SERCA function is decreased. Thus, the 2 studies found either complete absence or hyperactivity of phospholamban to be associated with the development of heart failure. At present it is not clear what might explain these differences and the divergent effects of phospholamban inhibition in different transgenic mouse models of heart failure. We observed downregulation of SERCA in ß1TG mice and a significant upregulation of SERCA in ß1TG/PLB-/- mice, which may contribute to the rescue of the detrimental effects of ß-adrenergic stimulation. However, our results cannot prove a causal role for SERCA expression changes in this model, because phospholamban ablation may also improve cardiac function independent of changes in expression and also reverse remodeling indirectly through other pathways that may be altered in PLB-deficient hearts. Thus, in most heart failure models tested to date and in cardiac myocytes from failing human hearts, enhancement of SERCA function and inhibition of phospholamban exerted markedly beneficial effects. A direct comparison of these alternative concepts performed in a model of chronic heart failure is needed to decide which represents the most promising therapeutic principle to follow.
Thus, a paradigm evolves, with signaling pathways enhancing diastolic calcium being detrimental and strategies preventing calcium entry into the cell either directly (L-type Ca2+ channel blockade26,27) or indirectly (Na+/H+ exchange inhibition6) being beneficial for the heart. Importantly, it is now clear that this occurs independently of hemodynamic unloading or negative inotropy. What might be the downstream mechanisms exerting the detrimental action of cytosolic calcium? During the past few years, a multitude of experimental results has refined our picture of how enhanced calcium levels might chronically harm cardiac myocytes.28 Specifically, activation of calcineurin29 and the calmodulin/calmodulin kinase pathway seem to be essentially involved in Ca2+-mediated cardiomyocyte hypertrophy. Recently, the signaling cascade downstream of calmodulin kinase has been additionally explored in cardiac myocytes and has been shown to involve regulation of histon deacetylases,30 potent inhibitors of cardiac gene transcription.
Phospholamban Inhibition as Therapeutic Strategy to Remove the Cardiotoxic Effects of ß1AR Stimulation
Heart failure is a complex clinical syndrome originating from a wide variety of different causes, including longstanding pressure overload, ischemic damage, and less common familial forms of cardiomyopathies. Because of this diversity of disease origins, effective therapeutic strategies focus on common pathophysiological mechanisms crucial for the progression of the disease. Among these mechanisms, chronic stimulation of the ß-adrenergic receptor system is common in most patients with heart failure,1 and blockade of this detrimental mechanism represents the most effective therapeutic principle to date.3 However, treatment of hemodynamically compromised patients with heart failure is clinically challenging, because the negative inotropic effect of this therapy acutely impairs cardiac function and as a direct result excludes a significant number of patients from the application of this therapy. Our data suggest an alternative therapeutic strategy by inhibition of phospholamban, which rescues the detrimental effects of ß1AR signaling without the negative inotropic effects of ß-blockade.
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Acknowledgments |
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These studies were supported by grants from the Deutsche Forschungsgemeinschaft (SFB 355, SFB 598, and Leibniz award), the Fonds der Chemischen Industrie, and NIH HL26057, HL64018, and HL52318 (to Dr Kranias). The authors would like to thank Lydia Vlaskin for help with cross-breeding and genotyping of the animals. The excellent technical assistance of Ursula Keller is gratefully acknowledged.
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