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Physiol. Genomics 22: 118-126, 2005. First published March 29, 2005; doi:10.1152/physiolgenomics.00016.2005 Free Article
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Received 19 January 2005; accepted in final form 24 March 2005.
Physiological Genomics 22:118-126 (2005)
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Cardiac transgenesis with the tetracycline transactivator changes myocardial function and gene expression

Diana T. McCloskey1,5,*, Lynne Turnbull1,5,*, Philip M. Swigart2,5, Alexander C. Zambon4, Sally Turcato1,5, Shuji Joho2, William Grossman2,3, Bruce R. Conklin2,4, Paul C. Simpson2,3,5 and Anthony J. Baker1,5

1 Department of Radiology, University of California, San Francisco
2 Department of Medicine, University of California, San Francisco
3 Department of Cardiovascular Research Institute, University of California, San Francisco
4 Department of Gladstone Institute of Cardiovascular Disease, University of California, San Francisco
5 Department of Veterans Affairs Medical Center, San Francisco, California


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The cardiac-specific tetracycline-regulated gene expression system (tet-system) is a powerful tool using double-transgenic mice. The cardiac {alpha}-myosin heavy chain promoter ({alpha}MHC) drives lifetime expression of a tetracycline-inhibited transcription activator (tTA). Crossing {alpha}MHC-tTA mice with mice containing a tTA-responsive promoter linked to a target gene yields double-transgenic mice having tetracycline-repressed expression of the target gene in the heart. Using the tet-system, some studies use nontransgenic mice for the control group, whereas others use single-transgenic {alpha}MHC-tTA mice. However, previous studies found that high-level expression of a modified activator protein caused cardiomyopathy. Therefore, we tested whether cardiac expression of tTA was associated with altered function of {alpha}MHC-tTA mice compared with wild-type (WT) littermates. We monitored in vivo and in vitro function and gene expression profiles for myocardium from WT and {alpha}MHC-tTA mice. Compared with WT littermates, {alpha}MHC-tTA mice had a greater heart-to-body weight ratio ({approx}10%), ventricular dilation, and decreased ejection fraction, suggesting mild cardiomyopathy. In vitro, submaximal contractions were greater compared with WT and were associated with greater myofilament Ca2+ sensitivity. Gene expression profiling revealed that the expression of 153 genes was significantly changed by >20% when comparing {alpha}MHC-tTA with WT myocardium. These findings demonstrate that introduction of the {alpha}MHC-tTA construct causes significant effects on myocardial gene expression and major functional abnormalities in vivo and in vitro. For studies using the tet-system, these results suggest caution in the use of controls, since {alpha}MHC-tTA myocardium differs appreciably from WT. Furthermore, the results raise the possibility that the phenotype conferred by a target gene may be influenced by the modified genetic background of {alpha}MHC-tTA myocardium.

calcium; trabeculae; tet-system; cardiomyopathy; mouse


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
CARDIAC-TARGETED conditional gene expression is a powerful tool to control the expression levels of particular genes in the heart. The technique allows transgenes to be turned on or off at specific developmental stages, thus allowing study of genes in young or adult animals while avoiding potentially adverse effects due to transgene expression during development. Furthermore, after the emergence of a particular phenotype, for example cardiomyopathy (35), target gene expression can be terminated to study the process of recovery.

Conditional expression using a tetracycline-regulated system (tet-system) was described by Furth et al. (12) and has now been widely used. The tet-system was implemented in the mouse heart to study the roles of numerous genes (5, 10, 19, 34–37, 42, 43). Cardiac-specific tetracycline-regulated gene expression uses two lines of transgenic mice. In one line, the cardiac {alpha}-myosin heavy chain promoter ({alpha}MHC) derived from rat (43) or mouse (36) drives lifetime expression of a transcription activator (tTA). Transactivation by tTA is inhibited by tetracycline, which prevents tTA binding to DNA (12). {alpha}MHC-tTA mice are crossed with a second line of mice containing a tTA-responsive promoter linked to a target gene. This cross yields double-transgenic mice having tetracycline-repressed expression of the target gene in the heart. Thus removal of tetracycline from the diet initiates expression of the target gene.

For studies using the tet-system, several different control animals have been used to assess the effect of a target gene expressed in double-transgenic mice, including wild-type (WT) mice (13, 15, 17, 27, 28), single-transgenic tTA mice (2, 10, 27, 34, 35, 37), and single-transgenic mice containing the target gene (5, 7, 22, 27, 31, 42). However, since the first use of the tet-system by Bujard and colleagues, it has been hypothesized that expression of tTA alone could alter gene expression. Indeed, high-level expression of a modified activator protein in the heart caused a lethal cardiomyopathy within 2 mo (36). Thus, despite {alpha}MHC-tTA mice appearing overtly normal, there may be myocardial effects that would necessitate use of appropriate controls. Furthermore, because genetic background can affect physiological function (4, 24), significant modification of the background and function of single-transgenic {alpha}MHC-tTA mice could potentially also modify the phenotype conferred by a target gene in double-transgenic animals.

Thus, despite the importance and utility of the tet-system, it is not clear whether the introduction of the {alpha}MHC-tTA construct modifies the background and function of single-transgenic {alpha}MHC-tTA mice compared with WT littermates. Therefore, the goal of the present study was to determine whether myocardium from tTA-expressing mice was different from that of WT littermates. We compared contractions (both in vivo and in vitro) and gene expression profiles of myocardium from WT and {alpha}MHC-tTA mice developed by Fishman and coworkers (43) and now used extensively by others (2, 3, 10, 13, 34, 35, 37, 42).

We found that, compared with WT littermates, {alpha}MHC-tTA mice had increased heart weight, ventricular dilation, and decreased ejection fraction, suggesting early-stage cardiomyopathy. Furthermore, {alpha}MHC-tTA myocardium had increased in vitro contractions, increased myofilament Ca2+ sensitivity, and major alterations of gene expression. Thus, for studies using the tet-system, these complex effects of the {alpha}MHC-tTA construct on the myocardium may need to be considered for selection of controls and for evaluating the phenotype caused by a target gene.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal methods.
This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 85-23, revised 1996) and was approved by the Institutional Animal Care and Use Committee of the Veterans Affairs Medical Center, San Francisco. WT littermates and mice expressing a tetracycline-controlled transactivator (tTA) under the regulatory control of 2.9 kb of 5'-flanking sequence from the rat {alpha}-myosin heavy chain gene ({alpha}MHC-tTA) were backcrossed in an FVB/N background for over 10 generations (99.9% congenic) (34, 43). tTA expression in {alpha}MHC-tTA mice is able to induce cardiac-restricted expression of tTA-dependent target genes in all chambers of the heart (34, 43). Typically, {alpha}MHC-tTA mice are used for experiments involving tetracycline-regulated gene expression, where gene expression is initiated by removal of tetracycline (or the analog doxycycline) from the diet. For this study, all animals were bred and maintained on a diet containing doxycycline (200 mg/kg, Dox diet no. S3888; Bio-Serve, Frenchtown, NJ). Except as noted below, 8-wk-old animals were removed from the Dox diet and experiments performed 2–4 wk later.

In vivo measurements.
Systolic blood pressure and heart rate were measured in conscious mice, as previously described (32), using a noninvasive computerized tail cuff system.

Echocardiography in conscious mice was performed as previously described (21). Two-dimensional long-axis images of the left ventricle (LV) were obtained in parasternal long- and short-axis views with guided M-mode recordings at the midventricular level in both views.

Langendorff-perfused hearts.
Male or female adult mice (weight 25–30 g) were anesthetized with pentobarbital sodium (1 mg/g; Abbott Laboratories, Chicago, IL) and heparinized (2 U/g; Elkins-Sinn, Cherry Hill, NJ), and hearts were rapidly removed and placed in cold arrest solution. Hearts were mounted on a Langendorff perfusion apparatus and retrogradely perfused with Krebs-Henseleit solution at 37°C as previously described (39). Ventricular pressure was monitored with the use of a fluid-filled balloon placed in the LV. Balloon volume was adjusted to set diastolic pressure to 10 mmHg. Hearts were electrically paced at 6 Hz, since rates closer to physiological (10 Hz) may cause ischemia, using crystalloid perfusate (6).

Right ventricle trabeculae.
Thin, unbranched trabeculae were removed from the right ventricle (RV), mounted on a force transducer, and superfused with Krebs-Henseleit solution as previously described (30). Muscles were stretched to a diastolic sarcomere length of 2.1 µm (monitored with laser diffraction), and measures were made of force, cytosolic Ca2+ concentration ([Ca2+]c) (using fura-2 loaded to the cytosol by iontophoretic injection), and intracellular pH [using the fluorescent pH indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)]. To minimize rundown of the preparation and washout of fura-2, in vitro experiments were performed at low temperature (22°C) and therefore low pacing rate (0.5 Hz). Steady-state contractions were induced by tetanic stimulation for several seconds (1). Thus a limitation is that these in vitro conditions are far from physiological temperature and frequency; therefore, caution is required in extrapolating to more physiological conditions.

The width and thickness of trabeculae from WT mice (172 ± 16 and 72 ± 8 µm, respectively; n = 18) were not statistically different (P > 0.05) from those from {alpha}MHC-tTA mice (162 ± 17 and 85 ± 12 µm, respectively; n = 15).

Western blots.
As previously described, we monitored levels of total and phospho-troponin I using Western blots (30). To monitor levels of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) and Na/Ca exchanger (NCX), isolated hearts were rinsed in cold saline and homogenized in RIPA buffer containing protease inhibitors (no. 1836153, Roche) and phosphatase inhibitors (P-2850 and P-5726, Sigma). After low-speed centrifugation of homogenate, samples of supernatant were processed for Western blotting as previously described (30) using 40 µg of protein per lane run on a 7.5% SDS-PAGE gel (Bio-Rad) and antibodies for SERCA2 ATPase (Cat. MA3–919; Affinity BioReagents, Golden, CO) and NCX (Cat. NCX11-S; Alpha Diagnostic, San Antonio, TX).

Gene expression analysis.
Animals were raised on doxycycline-containing chow for 6 wk, and then doxycycline was removed for 8 wk (WT, n = 3; {alpha}MHC-tTA, n = 8). A second cohort of animals was placed back on the Dox diet for 4 wk (WT, n = 4; {alpha}MHC-tTA, n = 5). Animals were anesthetized with 0.02 ml of 2.5% Avertin/g body wt. Chest cavities were opened, and hearts were removed and quickly rinsed in 1x PBS two times and placed in 1 M KCl. Atria and apex were removed, and hearts were flash frozen in liquid nitrogen (35). From ~75 mg of frozen tissue, total RNA was extracted, and 15 µg of RNA were reverse transcribed and converted to double-stranded cDNA; then biotinylated cRNA was generated by in vitro transcription. For each heart, 15 µg of fragmented cRNA were hybridized to a separate Affymetrix murine MG-U74Av2 array (n = 20). Arrays were hybridized and scanned with a GeneArray Scanner (Hewlett-Packard/Affymetrix). For each array, the ".cel" files were generated with Affymetrix Microarray Suite 5.0 and analyzed with robust microarray analysis (RMA) (20). Array results were similar among animals within each experimental group: for the {alpha}MHC-tTA group (n = 8), the standard error per gene was 2.4% of the mean expression signal per gene. RMA signal values for each array have been uploaded to the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) and can be retrieved using the series accession number GSE986.

Statistical analysis.
Results are presented as means ± SE. For physiology studies, comparisons between groups were made with Student's t-test and two-way ANOVA, where values of P < 0.05 were considered statistically significant. For gene expression studies, two-way ANOVA was used to distinguish between the effects of genotype and doxycycline on gene expression. Genes were significantly changed with P < 0.05, fold change >20%, and interaction P > 0.05 (interaction P > 0.05 indicates that the effects of genotype on gene expression were independent of doxycycline status). Significantly changed genes were annotated with the MAPPFinder program (9).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Except as noted below, all experiments were performed in the absence of doxycycline.

In vivo {alpha}MHC-tTA heart function.
Table 1 shows that, for {alpha}MHC-tTA mice, blood pressure, heart rate, and cardiac output did not differ from WT. However, {alpha}MHC-tTA hearts had increased LV mass (114% of WT), were dilated (end-diastolic volume 127% of WT), and had reduced fractional shortening and ejection fraction (89 and 94% of WT, respectively). These abnormalities suggest mild cardiomyopathy.


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  		Table 1. Echocardiography in conscious WT and {alpha}MHC-tTA mice

 
{alpha}MHC-tTA hearts have increased in vitro function.
Consistent with the in vivo assessment, Table 2 indicates that {alpha}MHC-tTA hearts had mild hypertrophy (heart-to-body wt ratio was 10% greater than for WT). Other organs weights (kidney or liver) were not significantly changed.


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  		Table 2. Body and organ weights for WT and {alpha}MHC-tTA mice and in vitro Langendorff heart function

 
In contrast to decreased indexes of contraction in vivo, {alpha}MHC-tTA hearts in vitro had increased (128% of WT) LV developed pressure (systolic minus diastolic). Furthermore, the maximum rates of tension rise and fall for {alpha}MHC-tTA hearts were greater than for WT (even after normalizing these measures to the developed pressure). Faster contraction and relaxation kinetics for {alpha}MHC-tTA hearts were also reflected in a shorter time to peak pressure and time from peak to half relaxation (Table 2).

In some experiments, we interrupted pacing stimulation with a brief rest and used the postrest contraction as an index of maximum pressure development (33). For {alpha}MHC-tTA hearts compared with WT, there was not a significant increase in postrest systolic pressure (291 ± 5 mmHg, n = 16, vs. 276 mmHg ± 7, n = 14; P > 0.05). Thus increased submaximal pressure for {alpha}MHC-tTA hearts did not involve increased maximum pressure. Instead, the data indicate that, compared with WT, {alpha}MHC-tTA hearts were relatively more activated during pacing.

{alpha}MHC-tTA trabeculae have increased myofilament Ca2+ sensitivity.
To investigate the basis for increased pressure development in vitro for {alpha}MHC-tTA hearts, we monitored force and Ca2+ transients using ventricular trabeculae (Fig. 1). Increased submaximal contraction for {alpha}MHC-tTA myocardium was also evident in trabeculae. Figure 1 and the pooled data show that at 1.5 mM bath [Ca2+], force development for {alpha}MHC-tTA trabeculae was appreciably greater than for WT myocardium (40 ± 1 vs. 11 ± 3 mN/mm2; P < 0.001, n = 6/group). For {alpha}MHC-tTA trabeculae, there was a trend toward an increase in systolic cytosolic [Ca2+] ([Ca2+]c) vs. WT (829 ± 67 vs. 617 ± 113 nM; P > 0.05, n = 6 per group), which became significant over a wider range of bath [Ca2+] (see below). The time to peak [Ca2+]c for {alpha}MHC-tTA trabeculae was not different from WT (65 ± 10 vs. 67 ± 9 ms; P > 0.05, n = 6/group). However, the time from peak to 50% decline was considerably faster than for WT (130 ± 21 vs. 218 ± 26 ms; P < 0.05, n = 6/group) (see below).



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  		Fig. 1. {alpha}MHC-tTA trabeculae have increased submaximal force and Ca2+ transients. Representative recordings of force (A) and [Ca2+]c (B) during contractions of WT (thin trace) and {alpha}MHC-tTA (bold trace) trabeculae at 1.5 mM bath Ca2+. MHC, myosin heavy chain promoter; tTA, tetracycline-inhibited transcription activator; [Ca2+]c, cytosolic Ca2+ concentration; WT, wild type.

 
We monitored force and Ca2+ transients over the full range of activation by varying extracellular [Ca2+] ([Ca2+]e) (Fig. 2). Figure 2A shows the effect of increasing [Ca2+]e on peak twitch force. Similar to previous studies, WT mouse myocardium required high levels of extracellular Ca2+ to fully activate (14, 29, 30). In contrast, similar to our previous study (2), Fig. 2A shows that {alpha}MHC-tTA myocardium was close to fully activated at 2 mM [Ca2+]e. Like the perfused heart, for trabeculae at full activation, maximum force developed was similar for {alpha}MHC-tTA and WT myocardium. Figure 2B shows corresponding [Ca2+]c at peak systole. For contractions between 0.75 and 2 mM [Ca2+]e, systolic [Ca2+]c for {alpha}MHC-tTA myocardium was {approx}30% higher than that for WT (P < 0.05, ANOVA).



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  		Fig. 2. {alpha}MHC-tTA trabeculae have increased responsiveness to [Ca2+]e. Pooled data for force (A) and systolic [Ca2+]c (B) during contractions at varied [Ca2+]e for WT (n = 6, {circ}) and {alpha}MHC-tTA (n = 6, •). Force levels were normalized to the maximum for each experiment and scaled to the maximum for all experiments (shown at right of break in x-axis). To avoid injury, [Ca2+]e did not exceed that producing maximum force. Data are presented as means ± SE. [Ca2+]e, extracellular [Ca2+].

 
Figure 3 shows the relationship between peak systolic [Ca2+]c and force for the experiments described in Fig. 2. The data for {alpha}MHC-tTA myocardium were shifted to the left of the data for WT, suggesting that {alpha}MHC-tTA myocardium had increased myofilament Ca2+ sensitivity. To confirm this suggestion, we monitored the steady-state relationship between [Ca2+]c and force using tetanization (during which [Ca2+]c and force reach steady-state levels) (1). Figure 4 shows the relationship between steady-state tetanic force and [Ca2+]c for contractions performed at various [Ca2+]e. Again, the data for {alpha}MHC-tTA were shifted to the left of the data for WT, indicating that {alpha}MHC-tTA had increased myofilament Ca2+ sensitivity.



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  		Fig. 3. {alpha}MHC-tTA trabeculae have increased myofilament Ca2+ sensitivity. Relationship between peak force vs. peak systolic [Ca2+]c during contraction of WT and {alpha}MHC-tTA trabeculae (data from Fig. 2).

 


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  		Fig. 4. {alpha}MHC-tTA trabeculae have increased steady-state myofilament Ca2+ sensitivity during tetanization. Each data point represents a single tetanic contraction during which force and systolic [Ca2+]c achieved a steady state for several seconds for {alpha}MHC-tTA (n = 6, •) and WT trabeculae (n = 6, {circ}).

 
To investigate the basis for increased myofilament Ca2+ sensitivity, we monitored intracellular pH using the fluorescent probe BCECF. Intracellular alkalinization is one factor that can increase myofilament Ca2+ sensitivity (38). However, intracellular pH for {alpha}MHC-tTA was not different from that for WT (7.33 ± 0.05, n = 5, vs. 7.32 ± 0.06, n = 7; P > 0.05). Therefore, we also monitored the phosphorylation status of troponin I (TnI) using phospho-specific antibodies and Western blots (30). Decreased phospho-TnI has been associated with increased myofilament Ca2+ sensitivity (for review see Ref. 26). For quiescent myocytes, there was no detectable level of phospho-TnI (data not shown). However, after stimulation with isoproterenol or forskolin, myocytes from {alpha}MHC-tTA hearts had less phospho-TnI compared with WT (Fig. 5).



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  		Fig. 5. {alpha}MHC-tTA myocytes have decreased phospho-TnI. Phospho-TnI levels in {alpha}MHC-tTA and WT myocytes after stimulation with isoproterenol or forskolin. Phospho-TnI levels were normalized to total TnI levels and scaled relative to the WT response. TnI, troponin I. **P < 0.01 (n = 3 per group).

 
{alpha}MHC-tTA hearts have faster Ca2+ removal from the cytosol.
As noted above, the decline phase of the Ca2+ transient was appreciably faster for {alpha}MHC-tTA myocardium compared with WT. Figure 6 shows the relationship between the time to half decline of the Ca2+ transient and systolic [Ca2+]c, where systolic [Ca2+]c was varied using different levels of [Ca2+]e. The slope of this relationship was steeper for {alpha}MHC-tTA myocardium compared with WT (P < 0.01). Thus, at higher levels of activation, clearance of Ca2+ from the cytosol was faster for {alpha}MHC-tTA myocardium than for WT myocardium. Faster Ca2+ transient decline was not associated with changes in the abundance of SERCA, phospho-phospholamban, or NCX by Western blot analysis (data not shown).



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  		Fig. 6. {alpha}MHC-tTA hearts have faster Ca2+ transient decline. Summary of the relationship between the time to half decline of the Ca2+ transient and systolic [Ca2+]c for {alpha}MHC-tTA (n = 6, •) and WT trabeculae (n = 6, {circ}). Data are presented as means ± SE.

 
For mouse myocardium, Ca2+ removal from the cytosol is mediated predominantly by SERCA and NCX. To investigate the mechanism of improved Ca2+ removal in {alpha}MHC-tTA myocardium, we inhibited SERCA using ryanodine (1 µM) and cyclopiazonic acid (100 nM) and monitored [Ca2+]c decline after brief tetanization (2 mM bath [Ca2+]) (1). With SERCA inhibited, there was no difference in the initial rate of [Ca2+]c decline between {alpha}MHC-tTA vs. WT myocardium (92 ± 14 nM/s, n = 6, vs. 107 ± 19 nM/s, n = 5; P > 0.05). This suggests that the more rapid [Ca2+]c decline in {alpha}MHC-tTA myocardium is mediated by SERCA function. Furthermore, with SERCA inhibited, Ca2+ removal by the remaining NCX function was not different between {alpha}MHC-tTA and WT myocardium.

{alpha}MHC-tTA hearts have altered gene expression.
We used DNA microarrays to assess gene expression profiles in WT and {alpha}MHC-tTA myocardium. Figure 7 shows results of an ANOVA among the treatment groups to identify significant differences in gene expression attributable to the different genotypes and doxycycline treatment (with fold changes >20%, P < 0.05, and interaction P > 0.05). The greatest difference in gene expression was associated with the different genotypes (75 genes upregulated and 78 genes downregulated), with fewer differences in gene expression attributable to doxycycline (18 genes upregulated and 4 genes downregulated). These findings suggest that the presence of the {alpha}MHC-tTA construct caused modification of the genetic background.



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  		Fig. 7. {alpha}MHC-tTA hearts have altered gene expression. No. of genes with statistically significant differences in expression attributable to genotype and doxycycline (determined using 2-way ANOVA, where differences in expression had fold change >20%, P < 0.05, and interaction P > 0.05).

 
For {alpha}MHC-tTA vs. WT myocardium, the most highly regulated genes are summarized in Table 3. Genes for the myofilament proteins ß-tropomyosin and skeletal {alpha}-actin were upregulated. Both genes were significantly upregulated with or without doxycycline; however, expression levels were also affected by doxycycline (hence significant interaction P values). Previously, ß-tropomyosin was associated with increased myofilament Ca2+ sensitivity (41), and skeletal {alpha}-actin was upregulated with hypertrophy (25).


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  		Table 3. Most highly regulated genes in {alpha}MHC-tTA myocardium vs. WT myocardium

 
To relate all 153 significantly regulated genes to biologically relevant groupings, we used the Gene Ontology (GO) (16) Consortium (9) terms that describe biological processes, cellular components, and molecular functions of genes. We also used MAPPFinder, a software program that links gene expression data to the GO hierarchy (9). The 153 most affected genes were distributed among a number of different biological processes (Table 4). Compared with WT, for {alpha}MHC-tTA myocardium there was an upregulation of genes involved in intracellular transport, intracellular signaling cascade, and kinase, transferase, and heat shock protein activities; furthermore, there was a downregulation of genes involved in lipid, carbohydrate, and protein metabolism and mitochondria and transporter activity.


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  		Table 4. Analysis of GO terms representing transcripts differentially regulated by genotype ({alpha}MHC-tTA vs. WT myocardium)

 
Influence of doxycycline on the phenotype of {alpha}MHC-tTA hearts.
The transcription activator tTA is inhibited by doxycycline. To investigate whether the phenotype of {alpha}MHC-tTA hearts was influenced by doxycycline, in a separate set of experiments, mice were not removed from the doxycycline diet. In the presence of doxycycline, there was a greater heart weight-to-body weight ratio for {alpha}MHC-tTA vs. WT hearts (4.64 ± 0.19, n = 7, vs. 3.98 ± 0.23, n = 7; P < 0.05), similar to the data without doxycycline (Table 2). Furthermore, with doxycycline there was greater LV systolic pressure for {alpha}MHC-tTA vs. WT hearts (142 ± 6 mmHg, n = 7, vs. 122 ± 3 mmHg, n = 11; P < 0.01), similar to the data without doxycycline (Table 2). Finally, as noted above, significant effects on gene expression were attributable to the {alpha}MHC-tTA genotype rather than to doxycycline. Together, these findings suggest that, compared with WT, alterations in the physiology and gene expression of {alpha}MHC-tTA hearts were independent of doxycycline.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major finding of this study was that the presence of the {alpha}MHC-tTA construct caused significant effects on myocardial gene expression and function. Compared with WT, hearts from {alpha}MHC-tTA mice had mild hypertrophy and were dilated, and in vivo measures showed decreased ejection fraction but with normal cardiac output. In vitro measures showed that, compared with WT, {alpha}MHC-tTA myocardium had increased submaximal contractions and enhancement of both myofilament Ca2+ sensitivity and Ca2+ handling. Finally, gene expression analysis revealed that {alpha}MHC-tTA myocardium had wide-ranging changes in gene expression compared with WT.

The significance of these findings is, first, that they suggest that studies using the tet-system may require careful selection of appropriate control animals, since single-transgenic {alpha}MHC-tTA mouse hearts differ markedly from true WTs. Thus, using the tet-system, WT animals may not be suitable controls. Instead, single-transgenic {alpha}MHC-tTA mice are preferred controls for double-transgenic mice containing the {alpha}MHC-tTA and target gene constructs. Second, genetic background can profoundly influence the phenotype conferred by genetic manipulation (4, 24). Thus, using the tet-system, the phenotype conferred by a target gene may be influenced by the modified background caused by the {alpha}MHC-tTA construct. Finally, the findings underscore the concept that genetic manipulation can cause unintended side effects on gene expression and function (18).

The findings of this study are limited to the particular {alpha}MHC-tTA line studied (43). However, this line has now been widely used by others. Other lines used for conditional gene expression would have to be separately evaluated.

Abnormalities of {alpha}MHC-tTA myocardium.
Previously, Sanbe et. al. (36) found that high-level expression of a modified activator protein in the heart caused a lethal cardiomyopathy within 2 mo. Furthermore, a low level of expression of tTA protein was sufficient for inducible gene expression but was not associated with disease (36). Here we extend their findings to show that significant myocardial abnormalities exist in {alpha}MHC-tTA mice that otherwise appear phenotypically normal. Failure to appreciate these abnormalities of {alpha}MHC-tTA myocardium could lead to erroneous conclusions in studies using the tet-system to control the expression of a target gene in the heart.

For {alpha}MHC-tTA hearts, the phenotype of modest hypertrophy, dilation, and decreased ejection fraction suggests a mild cardiomyopathy. In vitro function, assessed using Langendorff hearts and RV trabeculae, showed that compared with WT, {alpha}MHC-tTA myocardium had increased submaximal contractions but maximal contractions were unchanged. It is not clear why {alpha}MHC-tTA hearts had decreased indexes of contraction in vivo but increased contraction in vitro. Possibly the in vitro measures are more reflective of intrinsic myocardial properties without regulatory influences exerted by the whole animal. Nevertheless, for this study, a central finding was that {alpha}MHC-tTA hearts differed substantially from WT hearts in terms of function (in vivo or in vitro) and gene expression.

Compared with WT, {alpha}MHC-tTA myocardium had increased myofilament Ca2+ sensitivity. Interestingly, there is growing evidence for increased Ca2+ sensitivity with heart failure (2, 30, 40). Thus increased Ca2+ sensitivity for {alpha}MHC-tTA myocardium may reflect the cardiomyopathic state suggested by our in vivo studies. On the other hand, as noted above, in vitro function was not impaired; moreover, the issue of Ca2+ sensitivity in heart failure remains unclear, because other studies report that, with heart failure, Ca2+ sensitivity was unchanged or even decreased (reviewed in Ref. 8).

The mechanism for increased myofilament Ca2+ sensitivity in {alpha}MHC-tTA myocardium did not involve increased intracellular pH. However, {alpha}MHC-tTA had increased expression of ß-tropomyosin, which previous studies found to play a role in increased myofilament Ca2+ sensitivity and cardiomyopathy (41). In addition, we found that stimulation of Gs signaling in quiescent myocytes was associated with decreased phosphorylation of TnI for {alpha}MHC-tTA myocytes compared with WT. Because contracting myocardium will likely have some basal Gs tone, decreased TnI phosphorylation for {alpha}MHC-tTA myocardium could also contribute to increased myofilament Ca2+ sensitivity compared with WT. Previous studies found decreased TnI phosphorylation and increased Ca2+ sensitivity in human heart failure (2, 30, 40).

There were 153 genes that were significantly changed >20% in {alpha}MHC-tTA hearts compared with WT hearts. These genes were linked to multiple GO terms. Interestingly, upregulation of heat shock genes and downregulation of genes involved in metabolism may relate to the better protection against ischemic injury that we have also observed in {alpha}MHC-tTA myocardium (L. Turnbull and A. J. Baker, unpublished observations).

Some changes in gene expression observed for {alpha}MHC-tTA myocardium are consistent with the observed phenotype and with previous studies of cardiomyopathy. Other changes in gene expression do not fit such a pattern. For {alpha}MHC-tTA myocardium, we found mild hypertrophy and upregulation of skeletal {alpha}-actin gene expression. Upregulation of skeletal {alpha}-actin gene expression was previously associated with cardiac myocyte hypertrophy (25). In contrast, two other hypertrophy genes (natriuretic peptide precursor type B and vascular smooth muscle {alpha}-actin) (11) were downregulated rather than upregulated. For {alpha}MHC-tTA myocardium, we found cardiomyopathy and increased Ca2+ sensitivity. As noted above, the ß-tropomyosin gene was upregulated in {alpha}MHC-tTA myocardium, and increased levels of this myofilament protein have been associated with cardiomyopathy and increased Ca2+ sensitivity. {alpha}MHC-tTA myocardium had downregulation of genes for connexin-43 and the ryanodine receptor. Similar changes were associated with some forms of cardiomyopathy (16, 23) and could lead to impaired excitation-contraction coupling. However, {alpha}MHC-tTA myocardium also had a faster Ca2+ transient decline, which suggested improved SERCA function.

Compared with WT, {alpha}MHC-tTA hearts manifested alterations in gene expression and physiology (mild hypertrophy, higher systolic pressure in vitro) that were independent of the presence of doxycycline. Because doxycycline inhibits the interaction of tTA with DNA, our findings suggest that altered gene expression for {alpha}MHC-tTA myocardium does not depend on the conformational change in the DNA-binding domain that occurs when tTA binds doxycycline. Possibly altered gene expression in {alpha}MHC-tTA myocardium could arise because tTA contains a VP16 domain that is a powerful transcription activator that interacts with a large number of other transcription factors. Alternatively, effects on DNA caused by integration of the {alpha}MHC-tTA construct at a particular site(s) could play a role.

In conclusion, controlling gene expression in the mouse heart using the tet-system can be complicated by unintended gene expression and physiological effects caused by introduction of the {alpha}MHC-tTA construct. This suggests that caution is required in the selection of control animals and in interpreting the effects of expression of a target gene.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants P01-HL-68738 (project 3; A. J. Baker) and HL-31113 (P. C. Simpson) and Postdoctoral Fellowship HL-10422 (D. T. McCloskley), and an Established Investigator Award from the American Heart Association (A. J. Baker).


   ACKNOWLEDGMENTS
 
We thank Tiffany Yap, Gregory Simpson, and Eli Shalenberg for expert technical assistance and Karen Vranizan for statistical advice.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: A. Baker, Univ. of California-San Francisco, VA Medical Center (111C), 4150 Clement St, San Francisco, CA 94121 (e-mail: ajbaker{at}itsa.ucsf.edu).

* D. T. McCloskey and L. Turnbull contributed equally to this study. Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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