Thyroid Hormone–Induced Alterations in Phospholamban-Deficient Mouse Hearts
Abstract—Alterations in the expression levels of the sarcoplasmic reticulum (SR) Ca2+-ATPase and its regulator, phospholamban, have been implicated in the effects of thyroxine hormone on cardiac function. To determine the role of phospholamban in these effects, hypothyroidism and hyperthyroidism were induced in phospholamban-deficient mice and their isogenic wild types. Hypothyroidism resulted in significant decreases of left ventricular contractility, which could be moderately stimulated by increases in preload or afterload, in both phospholamban-deficient and wild-type mice. However, the basal contractile parameters in hypothyroid phospholamban-deficient hearts were at least as high as those exhibited by hyperthyroid wild-type hearts. In hyperthyroidism, there was no further enhancement of the hyperdynamic contractile parameters in phospholamban-deficient hearts, although the wild-type hearts exhibited significantly increased contractile function compared with their respective euthyroid groups. Furthermore, increases in preload or afterload did not enhance contractility in either phospholamban-deficient or wild-type hyperthyroid hearts. Examination of the relative tissue levels of cardiac SR Ca2+-ATPase revealed increases in hyperthyroidism and decreases in hypothyroidism compared with euthyroidism, and these changes were similar between phospholamban-deficient and wild-type hearts. An opposite trend was observed for phospholamban expression levels in the wild-type group, which were depressed in hyperthyroid hearts but increased in hypothyroid hearts. These findings indicate that (1) thyroid hormones induce similar changes in the cardiac SR Ca2+-ATPase levels in either the presence or absence of phospholamban, (2) the thyroxine-induced increases in SR Ca2+-ATPase levels are not associated with any further stimulation of the hyperdynamic cardiac function in phospholamban-deficient mice, and (3) the decreased contractile parameters in hypothyroid phospholamban-deficient hearts associated with decreases in SR Ca2+-ATPase levels and myosin heavy chain isoform switches are at least as high as those of the stimulated hyperthyroid wild-type hearts. Thus, alterations in the phospholamban level or its activity may be a critical determinant of the contractile responses to altered thyroid states in the mammalian heart.
The sarcoplasmic reticulum (SR) Ca2+-ATPase is the enzyme that transports Ca2+ from the cytosol into the lumen of the SR, mediating relaxation in the mammalian myocardium.1 The activity of this enzyme is regulated by phospholamban. The dephosphorylated form of phospholamban has been shown to inhibit the SR Ca2+-ATPase through a decrease in the affinity of the enzyme for Ca2+.1 2 Phosphorylation of this regulatory protein, phospholamban, by β-adrenergic agonists in beating hearts temporarily relieves its inhibitory effects.3 4 Recently, direct evidence of the role of phospholamban in the regulation of left ventricular relaxation and contractility has been obtained, using a phospholamban-deficient mouse model.5 It was shown that ablation of phospholamban was associated with (1) significant increases in the affinity of the SR Ca2+ pump for Ca2+, (2) marked enhancement of all basal contractile parameters, and (3) attenuation of β-adrenergic stimulation.5 These findings indicate that phospholamban acts as a critical inhibitor of basal cardiac function, and removal of this protein from the SR results in a hyperdynamic heart.
Increases in thyroid hormone levels have also been reported to enhance myocardial contractility, speed of relaxation, cardiac output, and heart rate.6 7 On the other hand, decreases in these parameters were noted in hypothyroidism. The mechanisms for these changes have been suggested to include direct transcriptional regulation of cardiac genes.8 9 Among the transcriptional alterations, the most important ones are thyroid hormone–mediated effects on α-myosin heavy chain (α-MHC) and cardiac SR Ca2+-ATPase (SERCA2) genes.10 11 12 In the promoter region of both genes, thyroid-responsive elements were identified, indicating a critical role for these contractile and SR proteins in the regulation of cardiac function in hypothyroidism and hyperthyroidism. A switch from the β-MHC (slow) to α-MHC (fast) has been demonstrated in small mammals (rats and rabbits) in a transition from hypothyroid to hyperthyroid conditions. Changes in MHC isoforms correlated with alterations in myosin ATPase activity and actomyosin crossbridge cycle rate.13 The transcriptional activity of the SERCA2 gene has also been reported to be under the control of thyroid hormones. Moreover, it has been shown that the thyroid hormone–mediated changes in SERCA2 protein levels were inversely related to the alterations in phospholamban protein levels.14 Thus, it was suggested that the changes in the relative phospholamban/SERCA2 ratio were responsible for the altered contractility and speed of relaxation in the hypothyroid and hyperthyroid hearts.14 15 In addition, a close correlation was noted between the relative phospholamban/SERCA2 ratio and the affinity of SR Ca2+ pump for Ca2+ in animals with different thyroid conditions.14 On the basis of these findings, it was proposed that the thyroid hormone–mediated changes in the relative phospholamban/Ca2+-ATPase protein ratio may directly regulate the Ca2+ uptake rates by SR and the relaxation properties of the myocardium.14 16
However, because thyroid hormones regulate the expression levels of phospholamban and the SR Ca2+-ATPase in an opposite manner, it was of special interest to determine whether any “cross talk” between these genes occurs, such that genetic modification of one would result in some degree of compensation by the other. Thus, the phospholamban-deficient, along with their isogenic wild-type control, mice were used, and experiments were designed to answer the following questions: (1) Do alterations in thyroid hormones induce changes in SR Ca2+-ATPase expression levels in the hyperdynamic phospholamban-deficient hearts, which exhibit altered Ca2+ homeostasis? (2) Are the alterations in SR Ca2+-ATPase expression levels in the hyperdynamic phospholamban-deficient hearts similar in the absence as in the presence of phospholamban under different thyroid conditions? (3) Do alterations in SR Ca2+-ATPase levels in the absence of phospholamban reflect any alterations in contractile parameters? (4) Are the alterations in contractile parameters similar in the phospholamban-deficient and wild-type hearts under different thyroid conditions?
Materials and Methods
The generation of the phospholamban-deficient mouse was previously described.5 Hypothyroidism was induced in adult mice (7 weeks old) by feeding the animals with a 0.15% 5-propyl-2-thiouracil (PTU)-containing diet (Teklad Premier) for 5 weeks. The animals in the hyperthyroid group (11 weeks old) received a daily intraperitoneal injection of l-thyroxine (3 μg/g body weight). The treatment was carried out for 8 consecutive days. No spontaneous deaths were observed during the experimental period. The hypothyroid and hyperthyroid states of both wild-type and phospholamban-deficient groups were confirmed by serum thyroxine analysis. Average values obtained per deciliter of serum were 3.8±0.6 μg in euthyroid phospholamban-deficient mice and 4.8±0.6 μg in euthyroid wild-type mice; <0.5 μg in hypothyroid phospholamban-deficient and wild-type mice; and 9.3±1.2 μg in hyperthyroid phospholamban-deficient mice and 10.7±1.1 μg in hyperthyroid wild-type mice.
The experimental conditions for the work-performing mouse heart preparations were described previously.17 Briefly, mice of either sex were anesthetized with 30 mg/kg body weight pentobarbital sodium intraperitoneally. After thoracotomy, the hearts were first retrogradely perfused with oxygenated Krebs-Henseleit solution containing (in mmol/L) NaCl 118, CaCl2 2.5, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, Na2EDTA 0.5, NaHCO3 25, and glucose 11 at 37.4°C. For pressure measurements, a polyethylene catheter was inserted into the left ventricle and connected to a Cobe pressure transducer. After Langendorff mode retrograde perfusion, the opening of the pulmonary vein was connected to the venous return cannula, and anterograde work-performing perfusion was initiated at a workload of 250 mm Hg×mL/min, which was achieved with a venous return of 5 mL/min and an aortic pressure of 50 mm Hg. Coronary and aortic flows were separately measured. All recordings were monitored on a 6-channel P7 Grass polygraph. The signals were also digitized and analyzed by computer software on an IBM-compatible computer. The first derivatives of the intraventricular pressure curve (dP/dt) and the duration of contraction and relaxation (time to peak pressure [TPP] and time to half-relaxation [RT1/2]) were calculated at different loading conditions. Pressure and volume loadings were carried out in all work-performing heart preparations. First, afterload (aortic resistance) was kept constant at 50 mm Hg, and venous return (preload) was increased until the contractility (+dP/dt) was no longer elevated. Then the venous return (preload) was kept constant at 5 mL/min, and the aortic pressure (afterload) was increased to the point where the +dP/dt was not further elevated. The cardiac work at different preload or afterload conditions was calculated and expressed as mm Hg×mL/min.
The phospholamban monoclonal antibody was obtained from Upstate Biotechnology Inc. The SR Ca2+-ATPase polyclonal antibody was generated in rabbits using the 192 to 205 amino acid sequence portion of the SR Ca2+-ATPase. Anti–α- and β-MHC monoclonal antibodies were a generous gift from Dr J.J. Leger (Pharmacie Inserm. U. 300 LPM2, Montpellier, France). The relative tissue levels of phospholamban, SR Ca2+-ATPase, and MHC in wild-type and phospholamban-deficient hearts with different thyroid states were determined by quantitative immunoblotting.18 Polyacrylamide gel electrophoresis under denaturing conditions was performed according to Laemmli.19 Cardiac homogenates were separated on 8% (MHC) or 10% to 20% (phospholamban and SR Ca2+-ATPase) SDS polyacrylamide gradient gels and transferred to nitrocellulose membranes. The membranes were incubated with phospholamban monoclonal antibody (1:1000 dilution), SR Ca2+-ATPase polyclonal antibody (1:500 dilution), α-MHC antibody (1:1000 dilution), or β-MHC antibody (1:2500 dilution) and visualized with either 35S-labeled or peroxidase-labeled secondary antibody (Amersham). The degree of labeling was determined with the Phosphorimager (Molecular Dynamics) and a computer program (ImageQuant). Values were expressed as pixels/mg protein.
The protein content was assayed by the Bio-Rad assay using bovine serum albumin for the standard curve. Serum concentrations of total thyroxine were measured by standard radioimmunoassay. Data are presented as mean±SEM. Statistical analysis was carried out using ANOVA and Student t test for unpaired observations. Values of P<0.05 were regarded as statistically significant. The initial linear part of the cardiac function curves was calculated by linear regression, and statistical evaluation of the slopes was obtained using the Student t test for unpaired observations.
Hyperthyroidism was associated with significant increases in the heart/body weight ratio when compared with euthyroidism in both phospholamban-deficient and wild-type mice. Hypothyroidism, on the other hand, resulted in significantly lower ratios of heart weight to body weight in both groups. It is interesting to note that the alterations in heart/body weight ratios were similar between the phospholamban-deficient and wild-type mice. These ratios were (in mg/g) 6.36±0.16 (n=6) and 6.94±0.28 (n=5) for hypothyroid; 7.33±0.39 (n=6) and 7.63±0.13 (n=6) for euthyroid; and 8.55±0.36 (n=4) and 8.69±0.22 (n=9) for hyperthyroid wild-type and phospholamban-deficient mice, respectively. Hypothyroidism was also associated with downregulation of the α-MHC isoform and induction of the β-MHC isoform at the protein level (data not shown), consistent with previous observations at the mRNA level.17 The α-MHC protein levels normalized per calsequestrin levels were (in pixel values, n=3) 1.25±0.19 and 1.52±0.22 for euthyroid versus 0.39±0.03 and 0.46±0.05 for hypothyroid wild-type and phospholamban-deficient hearts, respectively. There was no β-MHC protein detected in euthyroid hearts whereas the levels of this isoform, normalized per calsequestrin levels, were (in pixel values, n=3) 1.71±0.08 and 2.11±0.13 in hypothyroid wild-type and phospholamban-deficient hearts, respectively. Thus, the alterations in the myosin isoforms were similar between wild-type and phospholamban-deficient hearts. Furthermore, the ratio of α-MHC/β-MHC was similar in hypothyroid wild-type (0.23±0.01) and phospholamban-deficient (0.22±0.03) hearts.
To examine the effects of altered thyroid status on basal cardiac function, isolated hearts from all groups were perfused with oxygenated Krebs solution at 37.4°C, and the contractile parameters were assessed under identical loading conditions. Hypothyroid mice exhibited largely depressed heart rates (≈250 bpm) compared with euthyroid animals (≈360 bpm), whereas the hyperthyroid groups had significantly elevated rates (≈450 bpm). Because of their decreases in heart rate, the hypothyroid wild-type and phospholamban-deficient hearts were paced at a rate of 350 bpm. These hearts could not tolerate pacing at higher rates, although the exhibited differences in their contractility were very small at heart rates between 350 and 400 bpm. Basal contractile parameters of hypothyroid, euthyroid, and hyperthyroid mouse hearts were assessed at an afterload (mean aortic pressure [MAP]) of 50 mm Hg and a preload (venous return) of 5 mL/min. Representative tracings of the data obtained in the 6 experimental groups are shown in Figure 1⇓. The euthyroid phospholamban-deficient hearts exhibited increases in the development of intraventricular pressure and the rates of contraction and relaxation compared with euthyroid wild-type hearts, in agreement with previous reports.5 Hypothyroidism was associated with depression of contractile function whereas hyperthyroidism stimulated contractility in wild-type hearts. Table 1⇓ summarizes the data obtained in several hypothyroid, euthyroid, and hyperthyroid phospholamban-deficient and wild-type hearts. Compared with the respective euthyroid groups, hypothyroidism resulted in significantly depressed left ventricular rates of contraction (+dP/dt and TPP) and speed of relaxation (−dP/dt and RT1/2) in both phospholamban-deficient and wild-type mice, assayed at a paced heart rate of 350 bpm. In the hyperthyroid condition, the phospholamban-deficient group showed no further increases in the already hyperdynamic cardiac function, whereas the wild-type group exhibited significant increases in contractile function when compared with its respective euthyroid group. The values of the contractile parameters in the phospholamban-deficient hearts significantly exceeded those in the wild-type hearts, assessed under either hyperthyroid or hypothyroid conditions. Interestingly, the depressed contractile parameters of the hypothyroid phospholamban-deficient hearts were significantly higher than the values in the euthyroid wild-type hearts and appeared to be similar to the contractile parameters exhibited by the hyperthyroid wild-type group, indicating that ablation of phospholamban keeps the myocardium at a stimulated state under any thyroid condition.
To evaluate the ability of hypothyroid, euthyroid, and hyperthyroid hearts to tolerate increased work, left ventricular Frank-Starling function curves were obtained in phospholamban-deficient and wild-type hearts. The hearts were loaded with increasing afterload (MAP) and/or volume (cardiac output [CO]) load resulting in varied cardiac minute work (MAP×CO, expressed as mm Hg×mL/min). The plots of cardiac work versus the rates of pressure development (+dP/dt or −dP/dt in mm Hg/s) in euthyroid and hypothyroid hearts are shown in Figure 2⇓. The hyperthyroid hearts of both phospholamban-deficient and wild-type mice were sensitive to increases in either preload or afterload, and only minor elevations in +dP/dt and −dP/dt were achieved on loading, indicating the lack of a Frank-Starling response due to the enhanced basal contractile state of these hearts. To examine the effects of different loading conditions in euthyroid and hypothyroid animals, the slope of the initial part of the Frank-Starling left ventricular function curve (range, 0 to 300 expressed as mm Hg×mL/min cardiac work) was calculated by linear regression analysis (Figure 2⇓). In euthyroidism, similar parallel slopes for +dP/dt and −dP/dt were obtained in both wild-type and phospholamban-deficient groups. However, all values for +dP/dt and −dP/dt in the phospholamban-deficient hearts were higher than the values in wild-type hearts, indicating that the myocardium of the phospholamban-deficient animals worked at a higher intrinsic contractile state. In the hypothyroid phospholamban-deficient and wild-type groups, the responses in contractility (+dP/dt) and speed of relaxation (−dP/dt) on increased afterload or preload were overall lower compared with their euthyroid counterparts (Figure 2⇓). However, all values for the phospholamban-deficient hearts were higher than those in wild-type hearts, similar to findings in euthyroid animals.
To determine whether the observed changes in cardiac functional parameters were associated with altered protein expression of the SR Ca2+-ATPase in phospholamban-deficient hearts and the SR Ca2+-ATPase and phospholamban in wild-type hearts, the relative levels of these proteins were determined at all thyroid conditions, using quantitative immunoblotting (Figure 3⇓). Administration of thyroid hormone to phospholamban-deficient and wild-type mice induced a significant increase in the expression levels of the cardiac SR Ca2+-ATPase protein compared with euthyroid wild-type hearts (Table 2⇓). On the other hand, the expression of the SR Ca2+-ATPase was depressed in both phospholamban-deficient and wild-type hypothyroid hearts. Interestingly, the alterations in the SR Ca2+-ATPase expression levels in hyperthyroidism and hypothyroidism were similar in phospholamban-deficient and wild-type hearts. Examination of the phospholamban protein levels in the wild-type hearts revealed an opposite trend in the expression pattern of this protein compared with the SR Ca2+-ATPase. There was a significant decrease in the phospholamban protein levels in the hyperthyroid hearts and an increase in the hypothyroid hearts compared with euthyroid ones (Table 2⇓). The relative ratio of phospholamban/SR Ca2+-ATPase was calculated in the wild-type group, and it was significantly increased in hypothyroidism and decreased in hyperthyroidism (Table 2⇓). When the relative changes in SR Ca2+-ATPase protein levels in hypothyroid, euthyroid, and hyperthyroid conditions were plotted against the changes in RT1/2 of the left ventricle, there was an apparent correlation observed for both the phospholamban-deficient and wild-type hearts (Figure 4⇓). It is interesting to note that the relaxant effect of thyroid hormones was markedly higher in the wild-type than in the phospholamban-deficient hearts, consistent with the presence and level of phospholamban expressed in these hearts (Figure 4⇓).
The hemodynamic alterations that occur in various thyroid states are well recognized. Increases in oxygen consumption, heart rate, cardiac output, left ventricular contractility, and velocity of relaxation in hyperthyroidism and decreases in these parameters in hypothyroidism have been documented.6 7 The mechanisms for these changes have been shown to include altered expression of proteins in the contractile apparatus, the SR, and the outer cell membrane. With respect to SR, hyperthyroidism was associated with increases in SR Ca2+-ATPase and decreases in phospholamban steady-state mRNA and tissue protein levels in rat hearts.14 16 On the other hand, opposite changes were noted for the expression of these 2 proteins in hypothyroidism.14 Furthermore, changes in the relative ratio of phospholamban/Ca2+-ATPase appeared to correlate with changes in the affinity of the SR Ca2+ uptake for Ca2+ and with the stimulatory effects of isoproterenol on the relaxation rate in beating rat hearts.14 Recently, similar observations were reported in baboons, in which case the administration of thyroxine was associated with increases in cardiac function and significant decreases in the phospholamban/Ca2+-ATPase ratio.20 On the basis of these findings, it has been suggested that thyroid hormones directly regulate SR protein levels and thus SR and cardiac function in hypothyroid and hyperthyroid conditions.14 15 16 In the present study, with the use of hypothyroid and hyperthyroid wild-type mouse hearts, we demonstrated similar changes in both left ventricular functional parameters and phospholamban or the SR Ca2+-ATPase protein expression levels as previously reported for the rat.14 Interestingly, the degree of change in the cardiac phospholamban/Ca2+-ATPase relative ratios in the hypothyroid and hyperthyroid conditions of the wild-type mouse was comparable to those observed in the rat.14
Furthermore, the availability of the phospholamban-deficient mouse enabled us to address the role of phospholamban in the responses of the heart to altered thyroid conditions. Specifically, it was of interest to examine whether the hyperdynamic phospholamban-deficient hearts5 could be stimulated further by increases in their thyroxine levels or whether they could be inhibited by decreases in thyroxine levels. Moreover, the effect of phospholamban ablation on the thyroid hormone–related alterations in the expression of the SR Ca2+-ATPase could be also examined. To the best of our knowledge, this is the first study that used a genetically altered animal model with modified cardiac function to elucidate the role of SR proteins in the regulation of myocardial contractility under pathological conditions. We observed that administration of thyroid hormones in the phospholamban-deficient mouse did not result in any significant stimulation of the basal cardiac contractile (TPP and +dP/dt) or relaxation (RT1/2 and −dP/dt) parameters, although the expression of the SR Ca2+-ATPase was stimulated to the same extent as in wild-type hearts. We further investigated the contractile reserve of these hearts by analysis of their Frank-Starling function. Increased loading conditions (preload or afterload) were associated with minimal left ventricular contractile response. The lack of response to increased workload by the hyperthyroid phospholamban-deficient hearts suggests that under baseline load conditions (250 mm Hg×mL/min), these hearts were functioning near their maximal capacity. On the other hand, the hypothyroid hearts of both phospholamban-deficient and wild-type mice exhibited only moderate responses to increased preload or afterload, which was probably due to depressed SR Ca2+-ATPase protein expression and intrinsic contractility in these hearts. Interestingly, the hypothyroid phospholamban-deficient hearts showed a greater response in +dP/dt and −dP/dt values on loading, suggesting that in the absence of phospholamban, decreases in the SR Ca2+-ATPase levels and switches in the α-MHC to β-MHC isoforms may not represent the major rate-limiting factors for contractility in hypothyroidism. Thus, phospholamban appears to be an important inhibitor of the intrinsic contractility, and reduction or ablation of this protein is associated with (1) preservation of the normal myocardial inotropic response (+dP/dt) to increases in preload or afterload and (2) shifts in the Frank-Starling curves to higher cardiac functional values in hypothyroidism. In fact, the basal contractile parameters in the hypothyroid phospholamban-deficient hearts were similar to those observed in the highly stimulated hyperthyroid wild-type hearts. These differences in contractile function between phospholamban-deficient and wild-type hearts were not due to different degrees of hypothyroidism, because the treatment and maintenance of the 2 mouse groups were the same and the serum total thyroxine levels were not significantly different.
In summary, our findings indicate that (1) thyroid hormones induce similar changes in the SR Ca2+-ATPase tissue levels in either the absence or presence of phospholamban, (2) the hyperdynamic cardiac function of the phospholamban-deficient mice cannot be further stimulated by thyroxine, although hypothyroidism depresses left ventricular function, (3) the relaxant effects of thyroid hormones are dependent on the presence and the levels of phospholamban, and (4) phospholamban is a critical determinant of the myocardial responses to altered thyroid states. Thus, it is interesting to propose that therapeutic approaches designed to either decrease the levels of phospholamban or disrupt the phospholamban/SR Ca2+-ATPase interaction may be more important than those designed to increase the levels of the SR Ca2+-ATPase in the treatment of depressed contractile function associated with cardiac diseases.
This study was supported by National Institutes of Health grants HL-26057, HL-22619, HL-52318, HL-07382, and P40 RR12358; Fogarty International Research Collaboration Award 1 R03 TW00861-01; Hungarian Ministry of Social Welfare grants ETT 582/1996 and 358/1996; and Hungarian Academy of Sciences grant OTKA T 020691.
- Received February 3, 1998.
- Accepted June 22, 1998.
- © 1998 American Heart Association, Inc.
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