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Endocrinology Vol. 148, No. 6 2870-2877
Copyright © 2007 by The Endocrine Society

Adeno-Associated Virus-Mediated Expression of Thyroid Hormone Receptor Isoforms-{alpha}1 and -ß1 Improves Contractile Function in Pressure Overload-Induced Cardiac Hypertrophy

Darrell D. Belke, Bernd Gloss, Eric A. Swanson and Wolfgang H. Dillmann

Department of Medicine, University of California, San Diego, La Jolla, California 92093-0618

Address all correspondence and requests for reprints to: Wolfgang H. Dillmann, Department of Medicine, 5063 Basic Sciences Building, University of California, San Diego, La Jolla, California 92093-0618. E-mail: wdillmann{at}ucsd.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pressure overload-induced cardiac hypertrophy leads to decreased contractile performance, frequently progressing to heart failure. Cardiac hypertrophy and heart failure can be accompanied by the so-called sick thyroid syndrome, resulting in decreased serum T3 levels along with decreased expression of thyroid hormone receptors (TR{alpha}1 and TRß1) and sarco(endo)plasmic reticulum Ca-ATPase (SERCA). Because the binding of T3 occupied receptors to the thyroid response elements in the SERCA promotor can increase gene expression, we wanted to determine whether increasing TR expression in the hypertrophied heart could also improve SERCA expression and cardiac function. Mice subjected to aortic constriction to generate pressure overload-induced hypertrophy were also subjected to gene therapy using adeno-associated virus (AAV) expressing either TR{alpha}1 or TRß1, with LacZ expressing AAV serving as control. After 8 wk of aortic constriction, a similar degree of hypertrophy was observed in all three groups; however, mice treated with TR{alpha}1 or TRß1 showed improved contractile function. Administration of a physiological dose of T3 increased serum T3 levels only into the lower range of normal. This T3 dose, with or without AAV TR treatment, did not result in any significant increase in contractile performance. Calcium transients measured in isolated myocytes also exhibited an enhanced rate of decay associated with TR{alpha}1 or TRß1 treatment. Western blot analysis showed increased SERCA expression in the TR{alpha}1- or TRß1-treated groups relative to the LacZ-treated control group. These results demonstrate that increasing TR expression in the hypertrophied heart is associated with an improvement in contractile function and increased SERCA expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PATHOLOGICAL CARDIAC HYPERTROPHY is associated with decreased contractile performance of the heart, limiting cardiac output and decreasing cardiac efficiency. If left untreated, hypertrophy can progress to overt heart failure with its associated increase in mortality. Hypertrophy is associated with remodeling of the myocardium on a number of levels, from changes in ventricular chamber dimensions and increased fibrosis to increased myocyte size and alterations in protein expression (1). Specifically, alterations in myosin heavy chain (MHC) isoform expression (from MHC{alpha} to MHCß) and a sharp reduction in sarco(endo) plasmic reticulum Ca-ATPase (SERCA) expression contribute to the decreased contractile phenotype and altered intracellular calcium transients (2, 3, 4). Interestingly, these changes represent a reversal of the protein expression observed during maturation of the heart from its fetal to adult phenotype shortly after birth (5). These observations, along with other phenotypic changes such as an increased reliance on glucose for energy metabolism (6), suggest that the hypertrophied heart reverts to a more fetal-like state. Normally, maturation of the heart with respect to MHC and SERCA expression coincides with an increase in plasma levels of thyroid hormone shortly after birth (7, 8).

Recent studies have shown hypertrophy and heart failure to be associated with a decrease in circulating thyroid hormone or the development of sick thyroid syndrome (3, 9). In addition, heart failure itself has been shown to be associated with a reduction in the actual number of thyroid hormone receptors in the cardiac myocyte (10, 11, 12, 13), suggesting that even though the level of thyroid hormone may not change, the heart itself may be less responsive to signaling by thyroid hormone. Whereas some studies have examined the use of thyroid hormone treatment as a means for improving contractile performance in the hypertrophied heart (13, 14), the development of adverse characteristics associated with increased circulating levels of thyroid hormone such as increased heart rate may limit the effectiveness of the treatment and could eventually exacerbate the heart failure phenotype (13). In contrast, increasing thyroid hormone receptor expression in the heart could help revert the phenotype without the need for large doses of thyroid hormone. In this study we used adeno-associated virus (AAV) expressing thyroid hormone receptor (TR{alpha}1 or TRß1) in a mouse model of pressure overload-induced hypertrophy to examine the efficacy of such a treatment on contractile phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of the AAV expressing TR-{alpha} and TR-ß
The adeno-associated shuttle vector (AAV shuttle) used in viral construction was derived by taking an XbaI fragment of the pSub201 plasmid (15) and ligating it to a human cytomegalovirus enhancer/promoter. The sequences coding for the rat TR-{alpha} and TR-ß (a kind gift of Dr. Howard Towle, Michigan State University, Minneapolis, MN) were cloned into AAV shuttle and verified by DNA sequencing. Large amounts of the plasmid along with the plasmid pDG as described by Grimm et al. (16) were purified using a CsCl protocol.

For production of virus particles (AAV type 2), both the AAV shuttle plasmid and pDG plasmid were transfected into 293T cells. After 3 d incubation in DMEM containing 10% fetal bovine serum, the cells were harvested and lysed using three freeze-thaw cycles. After this, 100 µg of DNase I and RNase A were added to the suspension and incubated at 37 C for 30 min. After centrifugation at 3000 x g, 0.5% deoxycholate was added to the supernatant and incubated for 30 min at 37 C. The supernatant was then filtered sequentially through a 5- and 0.8-µm syringe filter and mixed gently at room temperature for 1 h with 3 ml of a suspension of heparin-agarose. The suspension was then loaded onto a glass column, and the heparin-agarose resin was washed with 25 ml PBS containing 0.254 M NaCl. Virus particles were eluted with PBS containing 0.554 M NaCl, and fractions with the highest titer were pooled and dialyzed against PBS at 4 C. The LacZ expressing AAV used as the control viral vector in this study, and green fluorescent protein (GFP) expressing AAV used to identify transfected myocytes was produced by Genethon (Nantes, France) using similar methods.

Titration of AAV particles
For AAV titering, 10 µl of virus was diluted with DMEM to a final volume of 200 µl, and 5 U of DNase I was added. Samples were incubated at 37 C for 1 h and then digested with Proteinase K (100 µg per sample) for 2 h at 37 C. The samples were extracted with phenol/chloroform (50:50) and the DNA in the aqueous phase was precipitated by the addition of sodium acetate (300 mM), 40 µg of glycogen and 2 volumes of ethanol. The DNA samples were centrifuged in the cold for 15 min, washed with 80% ethanol, and air dried. The DNA was denatured in 300 µl of 400 mM NaOH/10 mM EDTA, and various amounts were transferred to a Gene Screen Membrane using a Slot Blot apparatus (Bio-Rad Laboratories, Hercules, CA). The reference plasmid was also denatured in 300 mM NaOH/10 mM EDTA and a range from 5 ng to 1.6 pg was transferred to the Slot Blot membrane. The membrane was then air dried, UV cross-linked, and hybridized to a radiolabeled fragment from either TR-{alpha} or TR-ß. After a stringent wash, the membrane was exposed to x-ray film and the signals were quantified by densitometry. From the values of the double-stranded plasmid DNA that were used to plot a standard curve, the amount of single-stranded recombinant AAV DNA in each fraction could be calculated. Assuming that each virus particle contains one single-stranded genome, the number of virus particles in each column fraction could be calculated. Usually the titers were higher than 1011 particles/ml and were adjusted to exactly 1011 particles/ml for gene therapy experiments by dilution of the fractions with PBS.

Animals and surgical procedures
This investigation conforms with the Guide for the Care and Use of Laboratory Animals as published by the National Institutes of Health (NIH). The mice used in this study were NIH Swiss male mice, approximately 8 wk of age (25 g body weight). Mice were anesthetized with a ketamine (100 mg/kg) and xylazine (8 mg/kg) mixture, intubated, and ventilated with room air. Access to the thoracic cavity was obtained at the level of the second intercostal. Aortic constriction was performed by placing a 6-O Ethalon ligature around the ascending aorta and tightening the ligature around a 26-gauge needle (template) that was subsequently removed. Sham operated mice underwent the same procedure; only the 6-O Ethalon ligature was passed behind the aorta and removed. After banding, the chest cavity was closed and the mouse was taken off the ventilator and allowed to recover. For those mice also undergoing AAV-mediated gene therapy, both procedures were performed during the same surgery with the gene therapy being performed before aortic banding. The gene therapy was performed as outlined previously (17). In mice destined for experiments involving the measurement of calcium transients in individual myocytes, AAV expressing LacZ or TR was coinjected with AAV expressing GFP (lower titer) to aid in the identification of transfected myocytes as described previously (17). All mice were allowed to recover for 8 wk; however, after 5 wk of recovery some of those receiving gene therapy (TR{alpha}, TRß, or LacZ) were started on a series of single daily injections of a physiological dose of T3 (3.5 ng/g body weight), which continued for the remaining 3 wk. In addition, some of the mice undergoing AAV-mediated gene therapy (TR{alpha}, TRß, or LacZ) were omitted from the daily T3 treatments to gauge the impact of this treatment.

Isolated perfused heart
After 8 wk of recovery from surgery, ex vivo left ventricular function [+dP/dt (change in pressure/change in time), –dP/dt, and developed pressure] was measured in isolated Langendorff perfused hearts as describe previously (18) using a small balloon made from polyethylene (19). Hearts were paced at 400 bpm and perfused with Kreb’s Henseleit buffer at a pressure of 60 mm Hg. At the end of the experiment, hearts were trimmed of atria and great vessels, blotted, and weighed for determination of heart weight (HW) to body weight (BW) ratios. The right ventricle was then dissected away and the left ventricle frozen in liquid nitrogen and stored at –80 C for further analysis.

Myocyte isolation and calcium transient
Individual myocytes were isolated from the excised hearts by collagenase digestion according to the method outlined in Belke et al. (20). At the end of the collagenase digestion, the right ventricle was trimmed from the heart and discarded so that only left ventricular myocytes were used for the measurement of calcium transients. Calcium transients were measured in myocytes loaded with the fluorescent calcium indicator indo 1 (AM ester) according to the methods outlined in Suarez et al. (21). Transfected cells were located by scanning the slide for GFP expressing myocytes.

Northern, Western, and RNase protection assay
Total RNA for RNase protection assay was prepared using the RNA easy Kit from QIAGEN (Valencia, CA). Typically, 2 µg of total RNA were used in an RNase protection experiment using probes for the mouse TRß. Hybridization conditions for RNase protection and the generation of radiolabeled antisense RNA transcripts was done as previously described (22). TRß and a control transcript (calsequestrin) were analyzed in one hybridization reaction.

Isolation of tissue RNA for Northern blot was performed as described by Chomczynski and Sacchi (23). Hybridization was performed using random primed cDNA probes for rat SERCA2 and the full-length TR{alpha}1 (rat). An oligonucleotide probe to 28S ribosomal RNA was labeled with [32P]dCTP using terminal deoxynucleotide transferase and hybridized as previously described (24).

For Western blot analysis, protein separation was achieved using a 4–12% Bis-Tris polyacrylamide gel (Invitrogen, Carlsbad, CA) and transferring to a nitrocellulose membrane. The nitrocellulose membrane was probed using primary antibodies directed against SERCA (rabbit polyclonal, made in laboratory), TR{alpha}1 (rabbit polyclonal; Affinity BioReagents Inc., Golden, CO), TRß1 (mouse monoclonal; Affinity Bioreagents) and secondary antibodies conjugated to horseradish peroxidase for chemiluminescence. Western and Northern blots were scanned into high-resolution image files and analyzed using NIH Image J.

Statistical analysis
Values shown represent mean ± SEM. Comparisons between two groups were performed using an unpaired Student’s t test. All multigroup comparisons were performed using ANOVA followed by a Student-Newman-Keuls post hoc analysis. Differences of P < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Our initial study compared functional characteristics of hypertrophied [aortic constriction (AC)] and sham-treated (control) mice. After 8 wk of ascending AC, the average HW to BW ratio was increased by over 70% (Table 1Go). When cardiac function was subsequently measured in Langendorff perfused hearts using an intraventricular balloon, the rates of contraction and relaxation as well as developed pressure were all significantly lower in the AC group. In addition, analysis of serum T3 levels of blood collected at the time the animals were killed were also significantly lower in the AC group.


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  		TABLE 1. Cardiac function after 8 wk of aortic constriction

 
Northern blot analysis of SERCA mRNA expression showed a large (74%) decrease in the AC group, which was mirrored by a decrease in the level of SERCA protein (40%) as observed by Western blot analysis (Fig. 1Go A and B). With respect to thyroid hormone receptor mRNA expression in the heart, we observed a 35% decrease in band intensity of TR{alpha}1 in the AC group (Fig. 1BGo) and a 73% decrease in TRß1 expression as determined by an RNase protection assay (Fig. 1CGo). We also observed a small (19%) decrease in TR{alpha}2 expression on the Northern blot (Fig 1BGo); however, this decrease was not sufficient to be statistically different from control values.


Figure 1
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  		FIG. 1. Changes in protein and mRNA expression after AC. A, An example of SERCA protein expression in control and hypertrophied hearts after 8 wk of AC. B, Northern blot showing SERCA, TR{alpha}1 and -{alpha}2 along with 28sRNA loading standard in control and AC hearts. C, An RNase protection assay showing TRß and calsequestrin (CS) loading standard in control and AC hearts.

 
Reasoning that the decreased TR expression may partially mediate the decreased contractile phenotype observed in the AC group, we proceeded to examine the effect of increased TR expression in hearts with pressure overload-induced hypertrophy. To achieve increased TR expression in the hypertrophied state, the mice underwent gene therapy using AAV vectors at the same time as the AC surgery. AAV-mediated expression of TR{alpha}1 and TRß1 could be observed in Western and Northern blots of hearts after 8 wk of AC (Figs. 2AGo and B, and 3Go). When examining the effects of AAV-mediated TR expression in hypertrophied hearts, we observed an improvement in contractile function including a significant increase in the rates of contraction and relaxation as well as a greater level of developed pressure (Fig. 4Go). Although hearts were paced at 400 bpm for data collection involving the measurement of contractile function, endogenous heart rates measured in the isolated perfused hearts before pacing were similar for all AAV-treated groups (AAV-LacZ 333 ± 14 bpm, AAV-TR{alpha}1 330 ± 22 bpm, AAV-TRß1 334 ± 10 bpm), indicating that increased expression of TR itself did not lead to an increase in heart rate. Plasma T3 levels measured in a separate cohort of banded mice 24 h after the final injection of T3 were significantly (P < 0.05) increased relative to untreated mice (69 ± 3 ng/dl vs. 54 ± 3 ng/dl). Treatment raised the plasma T3 levels to within the lower range of the control group (see Table 1Go). Whereas treatment with T3 did lead to a slight, although not statistically significant, improvement in the contractile performance of LacZ-treated hearts, the addition of T3 treatment also had no further effect on the AAV-TR-treated hearts, indicating that increased TR expression itself was principally responsible for the improvement in contractile function. Significantly, this improvement in contractile performance occurred in the absence of any change in HW to BW ratio, which was similar for all groups (Table 2Go). The various groups did not differ with respect to either body mass or heart mass by 8 wk of aortic banding, and the HW to BW ratio was approximately 2-fold greater than values observed in nonbanded control mice (see Table 1Go). This indicates that TR expression did not affect the development of hypertrophy (i.e. alter the HW to BW ratio) in these mice and cannot account for the improved contractile phenotype through moderation of the extent to which the hearts hypertrophied. Due to the nature of the banding, the greatest increase in heart mass occurs primarily in the left ventricle (left ventricle: 220 ± 10 mg vs. 132 ± 12 mg; right ventricle: 39 ± 2 mg vs. 33 ± 2 mg; banded vs. nonbanded, respectively).


Figure 2
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  		FIG. 2. Western blots showing TR expression in AAV-treated mouse hearts. A, TR{alpha}1 expression (arrow) in hearts infected with AAV-TR{alpha}1. B, TRß1 expression (arrow) in hearts treated with AAV-TRß1.

 

Figure 3
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  		FIG. 3. Northern blots showing SERCA and TR expression in AAV-treated mouse hearts. Increased SERCA mRNA expression is observed in hearts showing increased TR{alpha}1 or TRß1 expression. Glyceraldehyde phosphate dehydrogenase (GAPDH) expression is shown as control.

 

Figure 4
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  		FIG. 4. Left ventricular function in AC hearts after AAV-TR gene therapy. Rates of contraction and relaxation and developed pressure measured in isolated mouse hearts after gene therapy and 8 wk of AC. Values represent mean ± SEM for n = 9 LacZ AAV, n = 9 TR{alpha} AAV, and n = 10 TRß AAV mice for the T3-treated group, and n = 8 LacZ AAV, n = 6 TR{alpha} AAV, and n = 2 TRß AAV mice for the non-T3-treated group. *, Values significantly different (P < 0.05) from their respective LacZ AAV-treated group.

 

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  		TABLE 2. Body weight and heart weight in AAV-treated hearts

 
Because SERCA expression is generally decreased in hypertrophy and SERCA expression is regulated, in part, through the actions of the TRs, we examined SERCA expression in the AAV-treated hearts. In the mice receiving gene therapy and supplementary injections of T3, SERCA expression measured by Western blot analysis and normalized to porin as a loading standard was significantly increased by 44% in hearts treated with AAV TRß1 and increased by 62% in AAV TR{alpha}1-treated hearts when compared with the AAV LacZ-treated hearts (Fig. 5Go, A and B). A similar effect was observed in those mice receiving gene therapy without additional T3 injections (Fig. 5Go, C and D). In those mice SERCA expression was increased by 56% for TRß1-treated mice and 76% for TR{alpha}1-treated mice. The difference in SERCA expression between TR{alpha}1- or TRß1-treated hearts was not statistically significant. Similarly, we observed increased SERCA expression on Northern blots from TR-treated hearts relative to LacZ-treated hearts (Fig. 3Go).


Figure 5
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  		FIG. 5. Western blots showing SERCA expression in AAV-treated mouse hearts. For the T3- and no T3-treated group: A and C, SERCA expression measured in AAV TRß, TR{alpha} and LacZ-treated hearts along with porin loading standard. B and D, The ratio of SERCA expression to porin loading standard. *, Values significantly different (P < 0.05) from the LacZ AAV-treated group.

 
To determine whether increased expression of TR{alpha}1 or TRß1 had any effect on the calcium transient, myocytes were isolated from hypertrophied hearts 8 wk after aortic banding and AAV injection. Calcium transients measured using indo-1 fluorescence revealed identical transients for TR{alpha}1- or TRß1-treated hearts and a longer transient for LacZ-treated hearts (Fig. 6Go, A and B). The significantly decreased (P < 0.05) transient decay time measured in the TR{alpha}1- or TRß1-treated hearts matches the increased level of SERCA expression observed in those groups.


Figure 6
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  		FIG. 6. Normalized calcium transients recorded from myocytes isolated from AAV-treated hearts. A, Normalized indo-1 measured calcium transients from AAV TRß, TR{alpha}, and LacZ-treated hearts. B, Transient decay times measured from n = 68 LacZ-treated myocytes, n = 65 TR{alpha}1-treated myocytes, and n = 51 TRß1-treated myocytes. *, Values significantly different (P < 0.05) from the LacZ AAV-treated group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Pathological hypertrophy is associated with a decreased expression of SERCA along with remodeling of the heart and other changes in protein expression that contribute to the contractile dysfunction (2, 3, 4). Recent studies have shown that hypertrophy leading to heart failure is associated with a decrease in circulating thyroid hormone levels or subclinical evidence of abnormal regulation of thyroid hormone levels (3, 9). The condition termed nonthyroidal illness or sick thyroid syndrome can also be initiated by cardiovascular disease such as myocardial infarction (25), and recent findings have shown that low T3 levels are a predictor of mortality in patients with heart failure (26). In addition, recent studies have also shown a decrease in TR expression in the hypertrophied heart, which could help to manifest the appearance of a more fetal/hypothyroid state that characterizes the failing heart (10, 11, 12, 13).

Our mouse model of pressure overload-induced heart failure was associated with both a decrease in circulating T3 levels and a significant decrease in the expression of TR{alpha}1 and TRß1, the two T3 binding TRs in the heart (22). In addition, we observed a decrease in contractile performance associated with a decrease in SERCA expression. In an attempt to mitigate the depressed contractile phenotype, we used AAV-expressing TR{alpha}1, TRß1, or LacZ (control) to obtain long-term expression (27) so that gene therapy and aortic banding could be accomplished in the same surgery. Treatment with AAV-TR did not affect cardiac remodeling with respect to the development of hypertrophy because both the LacZ- and TR-treated groups showed a similar increase in heart mass and HW to BW ratio that was increased 2-fold in comparison to nonbanded mice (compare Tables 1Go and 2Go). We do note, however, that this model used to develop hypertrophy (aortic band) is associated with a rapid (10–14 d) increase in heart size, whereas AAV-mediated expression is generally slower (27), which precludes us from deriving any conclusions as to the ability of increased TR expression in moderating the development of hypertrophy. Nevertheless, we were able to observe a significant improvement in contractile performance of TR{alpha} and TRß-treated hearts in the present study, and whereas the addition of a low-dose T3 treatment in the later stages of the study leads to a slight, but not statistically significant, improvement in contractile function in the LacZ control group, there was no further increase in function in the TR-treated group. It should be noted, however, that our treatment of mice by daily T3 injection resulted only in an increase in plasma T3 to a level that was comparable with the lower range of plasma T3 values reported for euthyroid control mice. Consequently, we cannot rule out the possibility that a larger increase in plasma T3 (to the average value reported for control mice) may have some further beneficial effect in the AAV-TR-treated hearts. Furthermore, recent studies examining the mode of T3 delivery (injection vs. osmotic minipump) indicate that continuous delivery via constant infusion has a greater effect on cardiac gene expression than the discontinuous delivery via injection (28). As a result, some caution should be used in assessing the effects of the low-dose T3 treatment in the current study.

Several studies have recently examined the actions of thyroid hormone in different models of cardiac hypertrophy. Using a rat model of pressure overload hypertrophy, Chang et al. (14) noted increased SERCA expression and an improvement in calcium handling when animals were treated with large doses of T4. A recent study using a spontaneously hypertensive rat model of hypertrophy treated with various doses of thyroid hormone led to an increase in left ventricular pressure development but was also associated with some adverse effects including an increase in heart mass and the development of tachycardia (13). Other studies have used cardiomyopathic BIO T-O2 hamsters exhibiting heart failure and subclinical signs of thyroid dysfunction (9). Heart failure in this model occurs as a result of a mutation in {delta}-sarcoglycan. In this study, treatment with thyroid hormone also resulted in adverse effects such as increased heart rate and heart mass. Interestingly, prolonged treatment with thyroid hormone decreased ejection fraction and contractility in control hamsters but resulted in neither an improvement or exacerbation of heart failure in myopathic hamsters. SERCA expression was increased only by thyroid hormone treatment in the myopathic hamsters but not in the control hamsters. We have recently shown in another line of cardiomyopathic hamsters (BIO 14.6) that increasing SERCA activity through removal of the actions of phospholamban, which inhibits SERCA activity in its unphosphorylated state, is associated with improved calcium handling and contractile characteristics without any change in remodeling of the heart (29). Together, these studies indicate that treatment of heart failure with thyroid hormone can result in some beneficial effects with respect to improving contractile performance; however, this effect is variable within the different models and may be associated with some adverse side effects, depending on the dose of thyroid hormone given.

In this study we used aortic banding to induce pressure overload and hypertrophy to avoid the use of models of heart failure arising from genetic mutations. We used banding to create a chronic increase in pressure within the left ventricle (50 mm Hg higher when measured under anesthetized conditions; results not shown) and focus on increasing expression of the diminished thyroid hormone receptor through gene therapy rather than using large doses of thyroid hormone. As observed in larger species, we report that our mouse model of pressure overload-induced hypertrophy also leads to a decreased expression in both TR{alpha}1 and TRß1 along with a decrease in SERCA expression and contractile performance. The main consequence of aortic banding in mice is primarily a large increase in left ventricular mass (with the right ventricle showing only a small increase in mass). Accordingly, we used a gene therapy involving AAV-mediated expression of TR{alpha}1 and TRß1, along with LacZ expression as control, delivered to the left ventricular free wall of the heart (17). AAV-mediated expression of either TR{alpha}1 or TRß1 led to an improvement in contractile performance in comparison with LacZ-treated hearts. Whereas treatment with T3 did lead to a slight (not significant) improvement in the LacZ-treated control group, the improvement in contractile performance was smaller than that obtained with the treatment of TR ({alpha}1 or ß1) alone. Interestingly, although T3 treatment did lead to a significant increase in plasma T3 levels, we observed no further increase in contractile performance of the AAV-TR-treated hearts. Western and Northern blot analysis indicated an increased SERCA expression in the AAV TR-treated hearts, suggesting an improvement in calcium handling in these hearts. Subsequent analysis of calcium transients in myocytes from AAV-treated hearts indicated a faster rate of decay of the calcium transient in TR-treated hearts, suggesting a faster rate of calcium removal from the cytosol. This observation could be explained by an increase in SERCA activity. We have recently shown, using doxycyline-inducible SERCA expressing transgenic mice, that enhancing SERCA expression leads to an improvement in contractile function in mice subjected to aortic banding (30).

The influence of virally expressed TR on the function of adult cardiac myocytes under in vivo conditions has not been previously studied. The effect of adenovirus-mediated expression of TR{alpha}1 and TRß1 has been studied only in neonatal rat cardiac myocytes in cell culture (10, 31). These studies did show a differential effect between TR{alpha}1 and TRß1 expression of the development of neonatal cardiac myocyte hypertrophy, with TR{alpha}1 being able to induce a hypertrophic growth phenotype whereas TRß1 does not. Increased TR{alpha}1 expression induces a fetal gene expression program with increased MHCß expression and decreased SERCA expression, whereas increased TRß1 expression leads to the opposite effect. In contrast, the present study was conducted using adult mice subjected to aortic banding for 8 wk to induce hypertrophy, with TR{alpha}1 and TRß1 expression being mediated in vivo and showing a similar ability to improve contractile function. This improvement in contractile function is accompanied by an increase in SERCA expression. Specifically, calcium transients measured in our study were made within 6 h using freshly isolated myocytes to avoid changes in calcium handling by the myocytes that can result from cell culture (32, 33). We observed an increased rate of calcium removal in myocytes isolated from TR-treated mice consistent with the improved contractile function measured in whole hearts and the increased SERCA expression. Similarly, our previous studies have shown decreased SERCA expression in TR{alpha} knockout mice (in which TRß is the dominant T3 binding isoform) but no decrease in SERCA expression in TRß knockout mice (made euthyroid to avoid the hyperthyroid phenotype resulting from knocking out TRß in the pituitary) in which TR{alpha}1 is the dominant T3 binding isoform in the heart (22). These observations suggest that results obtained in cell culture using neonatal myocytes in which adenoviral expression of one TR makes it the dominant receptor does not necessarily lead to the same effect being observed in adult myocytes.

Although some studies suggest that thyroid hormone supplementation causes remodeling of the myocardium with respect to both the interior chamber dimensions (decreased wall stress) and myocyte size (9, 13, 34), we did not examine either of those parameters in the current study. Although we did not follow up the progression of hypertrophy over time, we note that no difference in HW to BW ratio could be observed between the groups (neither HW nor BW was independently different between the groups). Furthermore, we have previously observed a rapid increase in myocyte size over the first 8–12 d after banding, indicating that the development of hypertrophy in this model is rapid (Belke, D., unpublished observations). We should note, however, that although the development of hypertrophy is rapid in the mouse model, the diminished contractile performance associated with decompensated heart failure takes longer to develop (2). Because AAV mediated protein expression takes some time to occur, it is likely that the onset of significant TR expression occurred after the development of hypertrophy. This would make any effect of TR expression on the physical development of hypertrophy unlikely. Still we cannot rule out the possibility that TR expression led to a favorable remodeling of the heart to improve contractile performance. Due to the angiogenic effect of T3, some studies have suggested an increased coronary flow as being beneficial to remodeling of the heart and improving contractile function (34).

The contractile performance of the pressure overload hypertrophied hearts and corresponding abnormalities in calcium handing were significantly improved by AAV-mediated increases in TR{alpha}1 and TRß1 levels, even without additional T3 supplementation. T3 ligand-occupied thyroid receptors are the essential component in mediating transcription-based thyroid hormone effects (35), and TR interaction with T3 is governed by the law of mass actions (36). Obtaining a significant improvement in cardiac function by TR supplementation itself implies that the pressure overload-induced decline in TR expression levels is the crucial limiting component for normal TR action in pressure overloaded hearts.

In summary, we have observed a decrease in TR expression with severe cardiac hypertrophy, which can be corrected by AAV-mediated gene therapy, leading to increased expression of either TR{alpha}1 or TRß1. Expression of either TR isoform is sufficient to increase SERCA expression and improve contractile function in hypertrophied hearts. Currently thyroid hormone signaling system-based interventions are not recommended for patients with chronic heart failure. The finding reported here may be an impetus for further research in this area.


   Acknowledgments
 
We thank the Vector Core of the University Hospital of Nantes supported by the Association Française Contre les Myopathies for providing the LacZ-AAV vector.


   Footnotes
 
This work was supported by National Institutes of Health Grant HL25022 (to W.H.D.).

Present address for B.G.: Duke University, Department of Neurobiology, Durham, North Carolina 27710.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 22, 2007

Abbreviations: AAV, Adeno-associated virus; AC, aortic constriction; BW, body weight; dP/dt, change in pressure/change in time; GFP, green fluorescent protein; HW, heart weight; MHC, myosin heavy chain; SERCA, sarco(endo)plasmic reticulum Ca-ATPase; TR, thyroid hormone receptor.

Received January 4, 2007.

Accepted for publication February 14, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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