Impaired Ca2+ handling and contraction in cardiomyocytes from mice with a dominant negative thyroid hormone receptor α1

Journal of Molecular and Cellular Cardiology 38 (2005) 655–663 www.elsevier.com/locate/yjmcc

Original article

Impaired Ca2+ handling and contraction in cardiomyocytes from mice with a dominant negative thyroid hormone receptor a1 Pasi Tavi a,b, Maria Sjögren c, Per Kristian Lunde d, Shi-Jin Zhang a, Fabio Abbate a, Björn Vennström c, Håkan Westerblad a,* a

Department of Physiology and Pharmacology, Karolinska Institute, SE-171 77 Stockholm, Sweden b Department of Physiology and Biocenter Oulu, University of Oulu, Oulu, Finland c Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden d Institute for Experimental Medical Research, Ullevaal University Hospital, Oslo, Norway Received 10 December 2004; received in revised form 18 January 2005; accepted 4 February 2005 Available online 17 March 2005

Abstract The profound effects of thyroid hormone (TH) on heart development and function are mediated by the thyroid hormone receptors (TR) a1 and b1. While numerous patients with TRb1 mutations have been identified, patients with similar mutations in TRa1 are yet to be discovered. Recently generated heterozygous mice with a dominant negative mutation in TRa1 (TRa1+/m mice) have normal TH levels, which may have hampered the discovery of patients with such mutations. We now measure intracellular Ca2+ and contraction in cardiomyocytes isolated from TRa1+/m mice and wildtype littermates (WT). TRa1+/m cardiomyocytes showed a phenotype similar to that in hypothyroidism with significant slowing of voltage-activated Ca2+ transients and contractions. Increased stimulation frequency (from 0.5 to 3 Hz) or b-adrenergic stimulation reduced the differences between TRa1+/m and WT cardiomyocytes. However, in TRa1+/m cells stimulation at 3 Hz gave a marked increase in diastolic Ca2+ and b-adrenergic stimulation triggered spontaneous Ca2+ release events during relaxation. Both TRa1+/m and WT cardiomyocytes responded to TH treatment by displaying a “hyperthyroid” phenotype with faster and larger Ca2+ transients and contractions. Excised TRa1+/m hearts showed an increased expression of phospholamban (PLB). In conclusion, isolated TRa1+/m cardiomyocytes display major dysfunctions with marked slowing of the Ca2+ transients and contractions. © 2005 Elsevier Ltd. All rights reserved. Keywords: Excitation-contraction coupling; Phospholamban; Ca2+ transients; Arrhythmia; Thyroid hormone

1. Introduction Thyroid hormone (TH) has profound effects on heart development and function. Hypothyroidism, with decreased 3,5,3′triiodothyronine (T3) levels, is characterized by systolic and diastolic dysfunction with reduced force and slowed relaxation, whereas hyperthyroidism induces the opposite. These functional alterations result from quantitative changes in pro-

Abbreviations: CaMKII, calmodulin kinase II; MyHC, myosin heavy chain; PKA, protein kinase A; PLB, phospholamban; RCL, resting cell length; RyR, ryanodine receptor; SERCA, sarcoplasmic reticulum Ca2+-ATPase; SR, sarcoplasmic reticulum; T3, 3,5,3′-triiodothyronine; TH, thyroid hormone; TR, thyroid hormone receptor; WT, wildtype. * Corresponding author. Tel.: +46 8 524 87253; fax: +46 8 32 7026. E-mail address: [email protected] (H. Westerblad). 0022-2828/$ - see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2005.02.008

tein expression, where TH induces either increased or decreased expression. Proteins that are up-regulated by TH include the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA), Na+/K+ ATPase, and the fast cardiac isoform of myosin heavy chain (a-MyHC), whereas down-regulated proteins include the SERCA inhibitory protein phospholamban (PLB) and the slow b-myosin heavy chain (b-MyHC) [1,2]. TH exerts direct effects on cardiac gene expression by acting as a ligand for specific nuclear receptors [3]. These thyroid hormone receptors (TRs) are encoded by two genes, TRa and TRb. The mammalian heart expresses one TH binding isoform of TRa (a1) and one of TRb (b1) [3]. TRs exert dual actions on the expressions of target genes in that both liganded and un-liganded receptor can modulate target gene expression. Based on cardiac function in mice lacking either TRa or TRb, TRa seems to have the largest impact on TH-

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dependent cardiac remodeling [4]. For instance, proper TRa1 function is essential for promoting normal myocyte growth [5] and suppression of b-MyHC expression [6]. The profound role for TRa1 in cardiac function suggested by the use TR knock out mice has been supported by recent studies on mice carrying a dominant negative mutation in TRa1 [7,8]. When cotransfected in human 293 cells, a TRa1 mutation with an arginine to cysteine change (R384C) exhibits an about 10-fold lower affinity for T3 [7]. Homozygous TRa1 mutant mice die before 3 weeks of age. On the other hand, heterozygous mice survive, are responsive to TH, and exhibit a phenotype akin to that seen in hypothyroid animals, with the distinction that the disorder is caused by the mutant TRa1 and not by low TH levels [7]. This is in contrast to patients with a dominant negative TRb, who usually show elevated TH levels and exhibit tachycardia apparently caused by TH-induced activation of TRa1 [9]. To gain further insight in the mechanisms by which TRa1 affects cardiac function, we have studied intracellular Ca2+ handling and contraction in isolated cardiomyocytes from mice heterozygous for a dominant negative mutation in TRa1 (TRa1+/m mice) [7]. We show a marked slowing of voltageactivated Ca2+ transients and contractions in TRa1+/m cardiomyocytes, which are defects similar to those observed in hypothyroidism.

2. Materials and methods 2.1. Mice Heterozygous (TRa1+/m) mice with an arginine to cysteine mutation at position 384 in TRa1 were generated as described previously [7] and compared to wildtype littermates (WT). Adult mice were killed by rapid neck disarticulation and the heart excised. Hormone treatment was done by addition of T3 and bovine serum albumin to the drinking water (0.05 µg/ml and 0.01%, respectively) for about 14 days. The water was changed daily. The free serum T3 concentration was measured as described elsewhere [7]. All animal experiments were approved by the local animal ethics committee. 2.2. Isolation of cardiac myocytes and confocal microscopy Ca2+ imaging Cardiomyocytes were isolated according to the protocols developed by the Alliance for Cellular Signaling (AfCS Procedure Protocol ID PP00000 125) [10]. Isolated cardiomyocytes were incubated in medium containing 20 µmol/l fluo3 AM for 60 min at room temperature followed by 10 min in medium without fluo-3. After being loaded, cardiomyocytes were placed in a perfusion chamber on glass coverslips precoated with laminin (Sigma, 1 µg/1 µl in phosphate-buffered saline (PBS) for 2 h and washed with PBS). They were superfused with standard Tyrode solution (mmol/l): NaCl, 121; KCl, 5.0; CaCl2, 2.2; MgCl2, 0.5; NaH2PO4, 0.4; NaHCO3,

24.0; EDTA, 0.1; glucose, 5.5. To study the effect of b-adrenergic stimulation, isoproterenol (100 nmol/l) was added to the solution in some experiments. The solution was bubbled with 5% CO2/95% O2, which gives a bath pH of 7.4. A three-way solenoid valve system allowed for rapid exchange of solutions. Experiments were performed at room temperature (~24 °C). Cells were stimulated with two platinum electrodes, one on each side of the perfusion chamber. Measurements were performed on cardiomyocytes of similar size and a resting cell length (RCL) of ~100 µm. To measure Ca2+ signals, we used a BioRad MRC 1024 confocal unit equipped with a krypton/argon laser run at 15 mW (BioRad Microscopy Division, Hertfordshire, UK). The confocal unit was attached to a Nikon Diaphot 200 inverted microscope and a Nikon Plan Apo 40× oil immersion objective (N.A. 1.3) was used. Fluo-3 was excited at 488 nm and the emitted light was collected through a 522 nm narrow band filter. The laser power used (3–6% of the maximum) did not have any noticeable deleterious effect on the fluorescent signal or cell function over the time-course of an experiment. Fluo-3 intensity was measured by line scanning at 6 ms intervals along the long axis and with focus in the middle of the cell. Stored images were analyzed with ImageJ (NIH, USA; http://www.rsb.info.nih.gov/ij/). Changes in the fluo-3 fluorescent signal at each time point (F) are expressed relative to that measured in the rested state (F0 = diastolic F at 0.5 Hz stimulation). This procedure allowed comparison of the fluo-3 signal in different cardiomyocytes. Cell shortening was measured from the line-scan images and related to RCL. 2.3. Protein immunoblot analysis (Western blot) Total homogenate were prepared from hearts as described elsewhere [11]. Protein concentration was determined by the bicinchoninic acid assay (Pierce 23235, Pierce Biotechnology, Rockford, IL, USA) using bovine serum albumin as standard. Proteins were separated by 15% (PLB) or 7% (SERCA2A and Na,K-ATPase a1 and a2 subunits) SDSpolyacrylamid gelelectrophoresis, respectively, and transferred to polivinyldifluoride membranes by 100 V for 2 h at 4 °C. The primary antibodies used were anti-SERCA2A (MA3-919, Affinity Bioreagents), anti-PLB (MA3-922, Affinity Bioreagents), anti-Na,K-ATPase a1 subunit (05–369, Upstate) and anti-Na,K-ATPase a2 subunit (kind gift from professor Alicia A. McDonough, University of Southern California). The immunoreactive bands on the membranes were visualized by enhanced chemiluminiscence method (RPN2106, Amersham), and the signal intensities detected by LAS-1000 (Fujifilm) and quantified by Imagegauge software (Fujifilm). Measurements of signal intensities were normalized to the mean value in WT, which was set to 100%. 2.4. Statistics Values are presented as mean ± S.E.M. Student’s paired t-tests were used to establish significant differences between

P. Tavi et al. / Journal of Molecular and Cellular Cardiology 38 (2005) 655–663

measurements obtained pre- and post-application of isoproterenol. Unpaired t-tests were used when comparing two groups. For comparison between multiple groups, we used one-way ANOVA followed by the Bonferroni test. P < 0.05 was considered statistically significant.

3. Results 3.1. Heart and body weight and their response to T3 treatment TRa1+/m mice were smaller and had thinner left ventricular walls than WT mice, whereas heart weight (HW) and heart to body weight (BW) ratios were not significantly different between the groups (Table 1). Addition of T3 to the drinking water for 14 days increased the free T3 concentration in serum from 10.3 ± 0.4 pM (n = 16) to 68.2 ± 3.3 (n = 6) in TRa1+/m mice and from 9.1 ± 0.8 pM (n = 10) to 61.6 ± 7.9 (n = 5) in WT mice. The response to T 3 treatment was larger in TRa1+/m than in WT mice with an increase in BW of 26% vs. 9% and an increase in the HW of 122% vs. 76%. Thus, the only significant difference between TRa1+/m and WT mice that remained after T3 treatment was a 15% lower BW in TRa1+/m mice. 3.2. Ca2+ transients, shortening parameters, and protein expression Table 2 shows that compared to WT, TRa1+/m cells displayed significantly impaired contractions with 28% decrease in total shortening, 47% decrease in shortening speed (V; measured as the extent of shortening divided by the time to peak shortening), and 125% increase in relaxation time (RT90%; measured as the time from peak shortening until the cell had

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relaxed by 90% of the shortening amplitude). Fig. 1A shows representative Ca2+ transients in WT and TRa1+/m cardiomyocytes stimulated at 0.5 Hz and mean data are shown in Table 2. When directly comparing TRa1+/m than in WT cells, the amplitude of the Ca2+ transient was smaller in the former (P < 0.01 in unpaired t-test). Furthermore, TRa1+/m showed a significantly slower Ca2+ decline than WT cells, as judged from a 86% longer time constant (sCa; obtained by fitting the decline phase to a single exponential function) and a 56% longer half width (1/2DCa; measured as the duration at 50% of the amplitude). T3 treatment of the animals resulted in a “hyperthyroid” phenotype in both WT and TRa1+/m cardiomyocytes (Fig. 1B and Table 2). Thus, the shortening amplitude, the maximum speed of shortening, the rate of relaxation, the amplitude of the Ca2+ transient (DF/F0), the rate of Ca2+ decline, and the half width of the Ca2+ transient were all markedly increased. The only significant difference between WT and TRa1+/m that remained after T3 treatment was a 33% longer relaxation time in TRa1+/m. To study the T3 effect on intracellular Ca2+ handling proteins and to link this to the observed changes in SR Ca2+ reuptake, we used Western blot to measure the expression of SERCA2A and PLB protein, the genes of which are known targets of TRs [1,12]. While the expression of SERCA2A was not different between groups (mean values ranging between 86% and 109% of that in WT), TRa1+/m hearts expressed ~20% more PLB than WT hearts and T3 treatment resulted in decreased PLB expression in both TRa1+/m and WT hearts (Fig. 1C). To assess the functional importance of these changes in PLB expression, we plotted differences in relative PLB expression against sca (all values normalized to untreated WT where the mean was set to 1). Fig. 1D shows that the differences in the rate of Ca2+ reuptake between TRa1+/m

Table 1 BW, HW, body to heart weight ratio (HW/BW), and left ventricular wall thickness in WT and TRa1+/m mice ± T3 treatment

BW (g) HW (mg) HW/BW (mg/g) Wall thickness (mm)

WT (n = 6) 34.0 ± 1.4 169.0 ± 10.7 4.9 ± 0.2 1.79 ± 0.05

TRa1+/m (n = 6) 25.2 ± 1.1* 119.3 ± 5.5 4.7 ± 0.15 1.47 ± 0.08*

WT +T3 (n = 6) 37.1 ± 0.4 297.8 ± 21.8* 8.0 ± 0.5* 1.74 ± 0.06

TRa1+/m +T3 (n = 6) 31.7 ± 0.8# 264.5 ± 17.5* 8.3 ± 0.6* 1.64 ± 0.04

* Indicates statistical difference (P < 0.05) compared to WT. # Shows difference (P < 0.05) between T3 treated groups. Table 2 Ca2+ transient and shortening parameters of isolated cardiomyocytes from WT and TRa1+/m mice ± T3 treatment

Shortening (%RCL) V (%RCL/s) RT90% (ms) DF/F0 sCa (ms) 1/2DCa (ms)

WT (n = 12) 11.0 ± 0.7 129 ± 12 138 ± 11 4.1 ± 0.1 177 ± 11 194 ± 9

TRa1+/m (n = 16) 7.9 ± 0.6* 69 ± 6* 311 ± 17* 3.4 ± 0.2 330 ± 14* 302 ± 10*

WT + T3 (n = 10) 15.7 ± 0.4* 228 ± 14* 78 ± 9* 5.6 ± 0.4* 109 ± 5* 136 ± 6*

TRa1+/m + T3 (n = 14) 16.4 ± 0.5* 212 ± 13* 104 ± 6 # 5.5 ± 0.4* 115 ± 7* 145 ± 5*

The extent of shortening (%RCL); the speed of shortening (V); the 90% relaxation time (RT90%); Ca2+ transient amplitude (F/F0); the time constant of Ca2+ decay (sCa); the duration at 50% of the amplitude half (1/2DCa). * and # indicate statistical difference (P < 0.05) compared to WT and between T3 treated groups, respectively.

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calmodulin kinase II (CaMKII) and phosphorylation of PLB (at Thr-17) or SERCA2A (at Ser-38), leading to a faster SR Ca2+ uptake [13–17]. Fig. 2A, B show representative Ca2+ records at 0.5 and 3 Hz stimulation of WT and TRa1+/m cardiomyocytes, respectively. When WT cardiomyocytes were stimulated at increasing frequencies from 0.5 to 3 Hz, they responded with slight increases in Ca2+ amplitude, diastolic Ca2+ (measured immediately before the next Ca2+ transient; Fig. 2C), rate of Ca2+ reuptake (measured as changes in 1/2DCa; Fig. 2D), and rate of relaxation (Fig. 2E). When comparing 3–0.5 Hz, TRa1+/m cardiomyocytes showed a more than twofold increase in diastolic Ca2+ and a corresponding increase in systolic Ca2+, resulting in a minor (~10%) decrease in the amplitude (Fig. 2C). Furthermore, TRa1+/m cardiomyocytes showed a significantly larger increase in the rate of Ca2+ reuptake and relaxation with increasing stimulation frequency than WT cells (Fig. 2D, E) but even at 3 Hz, 1/2DCa and RT90% were significantly longer in TRa1+/m than in WT cells. As described above, T3 treatment resulted in larger and faster Ca2+ transients and contractions in both TRa1+/m and WT cardiomyocytes. On the other hand, cells of T3 treated mice showed little frequency-dependence (Fig. 2A, B) and none of the parameters measured showed any significant difference between 0.5 and 3 Hz stimulation in either TRa1+/m or WT cells. 3.4. Effects of b-adrenergic stimulation

Fig. 1. Cardiomyocytes from TRa1+/m mice display slower Ca2+ transients. A. Representative Ca2+ transients obtained at 0.5 Hz stimulation from a WT (dotted line) and a TRa1+/m (full line) cardiomyocyte. B. Ca2+ transients obtained under the same conditions from cardiomyocytes isolated from T3 treated animals. C. Representative immunoblots of PLB and SERCA2. D. Comparison between relative time constants of the Ca2+ transient decay (sCa; left axis, black bars) and PLB expression (right axis, white bars). Data from T3 treated and non-treated TRa1+/m and WT mice as indicated; x-axis labeling refers also to C. sCa and PLB expression are presented relative to the mean value in WT, which was set to 1.0. sCa data from 10 to 16 cells of three to four animals in each group; PLB data from six hearts in each group.

and WT cells and the effect of T3 treatment correlate well with the changes in PLB expression. Since the protein expression of Na+/K+ ATPase is known to be affected by TH [2], we performed Western blot analyses of the Na+/K+ ATPase a1 and a2 subunits. The expression of the a1 subunit showed no difference between the groups (mean values ranging between 88% and 106% of that in WT), whereas there was a decrease in the a2 subunit expression in TRa1+/m hearts (mean expression ~25% lower than in WT and T3 treated animals). 3.3. Responses to changes in stimulation frequency An increased stimulation frequency shortens the relaxation phase in cardiomyocytes, presumably via activation of

b-adrenergic stimulation activates cAMP-dependent protein kinase (PKA), which phosphorylates several target proteins including the SR Ca2+ release channels (ryanodine receptors, RyR) and PLB leading to an increased SR Ca2+ release and faster SR Ca2+ uptake [15,18]. Cardiomyocytes were exposed to a high concentration of the b-receptor agonist isoproterenol (100 nM). The sensitivity to b-adrenergic stimulation followed the same pattern as the frequency sensitivity; that is, TRa1+/m cardiomyocytes were more affected than WT cells and there was no significant response in the two T3 treated groups (Fig. 3A). The maximal increase in Ca2+ amplitude induced by isoproterenol was ~30% in WT cells and ~61% in TRa1+/m cells (Fig. 3B). Moreover, b-adrenergic stimulation had a larger effect on sca, the extent of shortening, the shortening speed, and RT90% in TRa1+/m compared to WT cells (Fig. 3C). However, even with b-adrenergic stimulation, the rate of Ca2+ decline and relaxation were somewhat lower in TRa1+/m than in WT cells. At the cellular level, impaired SR Ca2+ release and removal result in a slower Ca2+ transient and relaxation. At the subcellular level, this defect can be manifested as spatial and temporal inhomogeneity of SR Ca2+ release [19]. Inhomogeneous Ca2+ release was a characteristic feature in TRa1+/m cardiomyocytes, where excitation generally failed to fully trigger Ca2+ release along the whole length of the cell (Fig. 4A, upper part). In agreement with previous results [19], Ca2+ release became more synchronized by b-adrenergic stimula-

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Fig. 2. TRa1+/m cardiomyocytes are more sensitive to altered stimulation frequency than WT cells. Representative Ca2+ transients at 0.5 and 3 Hz from WT (A) and TRa1+/m (B) cardiomyocytes; lower panels obtained from T3 treated mice. C. Relative changes in the Ca2+ transient amplitude (upper part) and diastolic Ca2+ (lower part) induced by increasing the stimulation frequency from 0.5 to 3 Hz. The Ca2+ transient half width (1/2DCa; D) and relaxation time (RT90%; E) as a function of the stimulation frequency (left) and the relative change 3–0.5 Hz (right). Values in C–E are expressed as mean ± S.E.M. (n = 6 cells from three animals in each group). Filled and open symbols/bars represent WT and TRa1+/m, respectively. Stars indicate statistical significance; * P < 0.05, ** P < 0.01, *** P < 0.001.

tion (Fig. 4A, lower part). To quantify these changes, we measured the standard deviation (S.D.) of the fluorescence signal along the cell at the time of peak fluorescent intensity (Fig. 4B). b-Adrenergic stimulation had no effect on this parameter in WT cardiomyocytes, whereas TRa1+/m cells became significantly more synchronized (decreased S.D.) with b-agonist exposure (P < 0.01). While the actual excitation-induced Ca2+ release was improved by b-adrenergic stimulation in TRa1+/m cardiomyocytes, frequent spontaneous Ca2+ release events (“Ca2+ sparks”) were observed during the Ca2+ decay phase and the diastolic period (see Fig. 4A). The frequency of these Ca2+ sparks was quantified by identifying local peak elevations where the fluorescent intensity was 1.5 times the surrounding background levels [20]. Few such Ca2+ sparks were detected in WT cells even in the presence of b-agonist (Fig. 4C). On the other hand, b-adrenergic stimulation increased the frequency of Ca2+ sparks about 10-fold in TRa1+/m cardiomyocytes (P < 0.001).

4. Discussion The results of this study show that cardiomyocytes of mice heterozygous for a point mutation in TRa1 display marked functional changes with a major slowing of Ca2+ transients

and contractions. The difference between TRa1+/m and WT cardiomyocytes generally became smaller with increased stimulation frequency (Fig. 2) or with b-adrenergic stimulation (Fig. 3). However, these modes of stimulation could not fully restore the WT phenotype in TRa1+/m cardiomyocytes and hence this mutation would cause defect cardiac function in vivo [7]. PLB, a 52-kDa phosphoprotein, is associated with SERCA2 in cardiomyocytes. PLB inhibits SR Ca2+ pumping in its dephosphoryated form and phosphorylation of serine16 and/or threonine-17 may relieve this inhibition [16,17,21]. Thus, the relation between PLB and SERCA2 expression defines the basic efficiency of cardiomyocyte Ca2+ uptake and therefore has a fundamental role in regulating cardiac Ca2+ signaling and contraction [22–24]. The expression of both these proteins is regulated by TH, which induces an upregulation of SERCA2 whereas PLB is down-regulated [1,2]. In the present study we observed an increased PLB protein expression in TRa1+/m cardiomyocytes, whereas the expression of SERCA2 was not changed. These results are similar to those previously obtained in rat cardiomyocytes, where T3 treatment resulted in a marked decrease in the protein level of PLB whereas SERCA2 remained unchanged [25]. Thus, at the protein level, PLB seems to be more sensitive than SERCA2 to changes in TR activity. The consequence of the

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Fig. 3. TRa1+/m cardiomyocytes are more sensitive to b-adrenergic stimulation than WT cells. A. Representative calcium transient at 1 Hz before (Control, dotted line) and after applying isoproterenol (ISO 100 nM, full line) of WT (left) and TRa1 +/m (right) cardiomyocytes. Lower panels show records from T3 treated mice. B. Relative changes (isoproterenol/control) in Ca2+ transient amplitude in WT (filled bar) and TRa1+/m (open bar) cardiomyocyte. Stars indicate significant difference between groups (** P < 0.01). C. The effect of isoproterenol on the decay of Ca2+ (sCa), the extent of shortening, the shortening speed (V), and the relaxation time (RT90%). Open bars before and filled bars after isoproterenol application. Stars indicate the statistical significance between before and after isoproterenol exposure; * P < 0.05, ** P < 0.01, *** P < 0.001. # Indicates statistical significance between isoproterenol treated groups; ## P < 0.01. Values in B and C are expressed as mean ± S.E.M.; n = 6 cells from three animals in each group.

change in PLB expression was studied in the basal state (low stimulation frequency, no b-agonist present), where PLB would be largely dephosphorylated [16,21] and hence exert its full inhibition of SR Ca2+ uptake. We then noticed a clear correlation between the rate of Ca2+ decline and PLB expressions, with TRa1+/m cardiomyocytes showing the slowest Ca2+ decay and the highest expression of PLB (Fig. 1C). Thus, insufficient TH binding to the mutant TRa1 resulted in increased PLB expression and slowed SR Ca2+ uptake under basal conditions. However, the mutant TRa1 can respond to T3 since treatment with this hormone led to decreased PLB expression and faster Ca2+ uptake. Healthy ventricular myocardium responds to an increased heart rate with an increased contractile strength and faster relaxation. Experiments on isolated cardiomyocytes indicate that CaMKII-induced threonine-17 phosphorylation of PLB has a key role in this positive force–frequency relationship. For instance, a recent study by Zhao et al. [16] showed a severely decreased contractile response to increased stimulation frequency in isolated cardiomyocytes from transgenic mice with a point mutation in threonine-17 as compared to WT cells and cells with a mutation on serine-16. They also showed a frequency dependent increase in threonine-17 phosphorylation in WT cells that was blocked by the CaMKII inhibitor KN-93. However, some positive response to in-

creased stimulation frequency remained in the threonine17 mutants, which indicates that other mechanisms are operating in parallel, e.g. CaMKII phosphorylation of SERCA2 and RyR [14]. When the stimulation frequency was increased in the present study, TRa1+/m cardiomyocytes showed a significantly larger increase in the rate of SR Ca2+ uptake and relaxation as compared to WT cells (Fig. 2). This might be explained by the higher PLB concentration in TRa1+/m cardiomyocytes and hence an increased PLBinduced inhibition of SR Ca2+ pumping in the basal state (0.5 Hz stimulation). However, even at the highest stimulation frequency studied (3 Hz), the rate of SR Ca2+ uptake and relaxation was significantly lower in TRa1+/m compared to WT cells, and TRa1+/m cells showed a marked increase in diastolic Ca2+. Thus, based on these results it can be predicted that hearts of TRa1+/m mice would display impaired diastolic relaxation, which is a prominent feature of hypothyroidism [1,2,26]. During b-agonist stimulation, PLB is phosphorylated both at Ser-16 by PKA and at Thr-17 by CaMKII, but phosphorylation of Ser-16 alone might be sufficient to get the maximal contractile response [21]. PLB is a principal regulator of the b-agonist-induced positive inotropy and lusitropy; in fact, hearts from PLB deficient mice show no or a markedly reduced response to b-agonists [22,27,28], whereas

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Fig. 4. b-Adrenergic stimulation synchronizes the calcium release of TRa1+/m myocytes. A. Representative line-scan images (1 Hz stimulation) from TRa1+/m myocyte before (top) and after (bottom) application of isoproterenol (100 nmol/l). Right part shows the time-course of Ca2+ changes at the indicated locations. Note the marked difference in amplitude before isoproterenol exposure and the frequent appearance of spontaneous Ca2+ events (“sparks”) during the decay phase after isoproterenol application. B. To quantify the spatial difference in amplitude, we measured the S.D. of the fluorescence signal along the cell at the time of peak fluorescent intensity. Data for each transient are normalized to the maximal amplitude of the transient. C. The frequency of Ca2+ sparks was quantified by identifying local peak elevations where the fluorescent intensity was 1.5 times the surrounding background levels [20]. Data in B and C are expressed as mean ± S.E.M.; n = 6 cells from three animals in each group. Stars indicate differences between before and after isoproterenol exposure; ** P < 0.01, *** P < 0.001.

b-adrenergic stimulation restores the normal function in PLB overexpressing mouse hearts [23]. Similar to the results with increased stimulation frequency, b-adrenergic stimulation had larger effects in TRa1+/m than WT cardiomyocytes (Fig. 3). However, even in the presence of b-agonist, the rate of SR Ca2+ uptake and relaxation were lower in TRa1+/m cells. These results indicate that an increased basal b-adrenergic drive would improve cardiac function in TRa1+/m mice in vivo. Interestingly, TRa1+/m mice showed a deceased cardiac response to injection of the b-agonist isoprenalin in vivo [7], which is opposite to the increased response we observe in isolated TRa1+/m cardiomyocytes and might indicate an increased b-adrenergic stimulation already in the basal state in vivo. Increased b-adrenergic-induced phosphorylations as a compensatory mechanism can have potentially harmful side effects in the heart. For example, rapid restoring of the SR Ca2+ stores with b-agonist stimulation can result in spontaneous Ca2+ release events during the diastolic decay of the Ca2+ transient that may predispose the myocytes to arrhyth-

mias [29]. In line with this, we observed frequent Ca2+ sparks in TRa1+/m cells but not in WT cells in the presence of b-agonist (Fig. 4C). This indicates that larger animals expressing this type of TRa1 mutation would be more susceptible to arrhythmias when stressed. Furthermore, chronic increase of b-adrenergic stimulation may induce cardiac dysfunction due to hyperphosphorylation of the RyR and dissociation of the RyR-associated regulatory protein FKBP12.6 [30], eventually leading to dilated cardiomyopathy and sudden cardiac death [18,31]. Several functional properties of TRa1+/m ventricular myocytes are qualitatively identical to those of myocytes from failing or pathologically hypertrophied hearts. Both are characterized by decreased Ca2+ transient amplitude, slowed Ca2+ transient and relaxation, and decreased cell shortening [32]. On the other hand, TRa1+/m myocytes showed enhanced response to both increased stimulation frequency and b-agonist stimulation, while isolated cardiac preparations from failing hearts show blunted responses to both these interventions [30,33], indicating differences in the underlying

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mechanisms. The augmented response of the TRa1+/m cardiomyocytes to b-adrenergic stimulation indicates intact components in the subsequent b-adrenergic signaling cascade. In the failing heart, on the other hand, the decreased response to b-adrenergic stimulation is due to uncoupling of downstream signaling components [24]. Patients harboring a dominant negative for of TRa1 have not yet been discovered, despite the identification of more than 300 patient families with similar mutations in TRb [34]. A major purpose behind developing the TRa1+/m mice was to aid the discovery of corresponding patients. The previously characterized TRa1+/m mouse phenotype includes a retarded postnatal development with, e.g. delayed eye opening, bone growth and ossification, and maturation of the immune system [7]. It was recently found that these mice also exhibit severe behavioral and neuromuscular problems that can be ameliorated by appropriately timed T3 treatment (Björn Vennström, unpublished observation). In this context, the present data are important for several reasons. They provide clear-cut data on deleterious effects of the mutant receptor on cardiomyocyte function. These defects may be masked in vivo, for instance, by an increased b-adrenergic drive that, together with normal TH levels [7,8], may preclude the discovery of patients with TRa1 mutations. Thus, patients with a dominant negative TRa1 may not be detected until they suffer from serious abnormalities in cardiac function that could have been avoided by early TH treatment.

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Acknowledgements Funds were received from the Swedish Research Council (proj. no. 10842 and 14453) and the Swedish Heart Lung Foundation (H.W.), the Swedish Cancer Society and Karobio AB (B.V.), Finnish Heart Research Foundation, Emil Aaltonen Foundation, Sigrid Juselius Foundation, Helsingin Sanomat, Orion-Farmos Scientific Fund, Biovitrum Partner Fund, and funds at the Karolinska Institute. We thank Kristina Nordström for help with animal breeding and hormone assays.

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