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
|
---|
The cardiac-specific tetracycline-regulated gene expression system (tet-system) is a powerful tool using double-transgenic mice. The cardiac
-myosin heavy chain promoter (
MHC) drives lifetime expression of a tetracycline-inhibited transcription activator (tTA). Crossing
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
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
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
MHC-tTA mice. Compared with WT littermates,
MHC-tTA mice had a greater heart-to-body weight ratio (
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
MHC-tTA with WT myocardium. These findings demonstrate that introduction of the
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
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
MHC-tTA myocardium.
calcium; trabeculae; tet-system; cardiomyopathy; mouse
 |
INTRODUCTION
|
---|
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, 3437, 42, 43). Cardiac-specific tetracycline-regulated gene expression uses two lines of transgenic mice. In one line, the cardiac
-myosin heavy chain promoter (
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).
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
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
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
MHC-tTA construct modifies the background and function of single-transgenic
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
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,
MHC-tTA mice had increased heart weight, ventricular dilation, and decreased ejection fraction, suggesting early-stage cardiomyopathy. Furthermore,
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
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
|
---|
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
-myosin heavy chain gene (
MHC-tTA) were backcrossed in an FVB/N background for over 10 generations (99.9% congenic) (34, 43). tTA expression in
MHC-tTA mice is able to induce cardiac-restricted expression of tTA-dependent target genes in all chambers of the heart (34, 43). Typically,
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 24 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 2530 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
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. MA3919; 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;
MHC-tTA, n = 8). A second cohort of animals was placed back on the Dox diet for 4 wk (WT, n = 4;
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
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
|
---|
Except as noted below, all experiments were performed in the absence of doxycycline.
In vivo
MHC-tTA heart function.
Table 1 shows that, for
MHC-tTA mice, blood pressure, heart rate, and cardiac output did not differ from WT. However,
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.
MHC-tTA hearts have increased in vitro function.
Consistent with the in vivo assessment, Table 2 indicates that
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.
In contrast to decreased indexes of contraction in vivo,
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
MHC-tTA hearts were greater than for WT (even after normalizing these measures to the developed pressure). Faster contraction and relaxation kinetics for
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
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
MHC-tTA hearts did not involve increased maximum pressure. Instead, the data indicate that, compared with WT,
MHC-tTA hearts were relatively more activated during pacing.
MHC-tTA trabeculae have increased myofilament Ca2+ sensitivity.
To investigate the basis for increased pressure development in vitro for
MHC-tTA hearts, we monitored force and Ca2+ transients using ventricular trabeculae (Fig. 1). Increased submaximal contraction for
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
MHC-tTA trabeculae was appreciably greater than for WT myocardium (40 ± 1 vs. 11 ± 3 mN/mm2; P < 0.001, n = 6/group). For
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
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).
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
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
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
MHC-tTA myocardium was
30% higher than that for WT (P < 0.05, ANOVA).
Figure 3 shows the relationship between peak systolic [Ca2+]c and force for the experiments described in Fig. 2. The data for
MHC-tTA myocardium were shifted to the left of the data for WT, suggesting that
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
MHC-tTA were shifted to the left of the data for WT, indicating that
MHC-tTA had increased myofilament Ca2+ sensitivity.
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
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
MHC-tTA hearts had less phospho-TnI compared with WT (Fig. 5).
MHC-tTA hearts have faster Ca2+ removal from the cytosol.
As noted above, the decline phase of the Ca2+ transient was appreciably faster for
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
MHC-tTA myocardium compared with WT (P < 0.01). Thus, at higher levels of activation, clearance of Ca2+ from the cytosol was faster for
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).
For mouse myocardium, Ca2+ removal from the cytosol is mediated predominantly by SERCA and NCX. To investigate the mechanism of improved Ca2+ removal in
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
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
MHC-tTA myocardium is mediated by SERCA function. Furthermore, with SERCA inhibited, Ca2+ removal by the remaining NCX function was not different between
MHC-tTA and WT myocardium.
MHC-tTA hearts have altered gene expression.
We used DNA microarrays to assess gene expression profiles in WT and
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
MHC-tTA construct caused modification of the genetic background.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 7. 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
MHC-tTA vs. WT myocardium, the most highly regulated genes are summarized in Table 3. Genes for the myofilament proteins ß-tropomyosin and skeletal
-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
-actin was upregulated with hypertrophy (25).
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
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.
View this table:
[in this window]
[in a new window]
|
Table 4. Analysis of GO terms representing transcripts differentially regulated by genotype ( MHC-tTA vs. WT myocardium)
|
|
Influence of doxycycline on the phenotype of
MHC-tTA hearts.
The transcription activator tTA is inhibited by doxycycline. To investigate whether the phenotype of
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
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
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
MHC-tTA genotype rather than to doxycycline. Together, these findings suggest that, compared with WT, alterations in the physiology and gene expression of
MHC-tTA hearts were independent of doxycycline.
 |
DISCUSSION
|
---|
The major finding of this study was that the presence of the
MHC-tTA construct caused significant effects on myocardial gene expression and function. Compared with WT, hearts from
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,
MHC-tTA myocardium had increased submaximal contractions and enhancement of both myofilament Ca2+ sensitivity and Ca2+ handling. Finally, gene expression analysis revealed that
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
MHC-tTA mouse hearts differ markedly from true WTs. Thus, using the tet-system, WT animals may not be suitable controls. Instead, single-transgenic
MHC-tTA mice are preferred controls for double-transgenic mice containing the
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
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
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
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
MHC-tTA mice that otherwise appear phenotypically normal. Failure to appreciate these abnormalities of
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
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,
MHC-tTA myocardium had increased submaximal contractions but maximal contractions were unchanged. It is not clear why
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
MHC-tTA hearts differed substantially from WT hearts in terms of function (in vivo or in vitro) and gene expression.
Compared with WT,
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
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
MHC-tTA myocardium did not involve increased intracellular pH. However,
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
MHC-tTA myocytes compared with WT. Because contracting myocardium will likely have some basal Gs tone, decreased TnI phosphorylation for
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
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
MHC-tTA myocardium (L. Turnbull and A. J. Baker, unpublished observations).
Some changes in gene expression observed for
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
MHC-tTA myocardium, we found mild hypertrophy and upregulation of skeletal
-actin gene expression. Upregulation of skeletal
-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
-actin) (11) were downregulated rather than upregulated. For
MHC-tTA myocardium, we found cardiomyopathy and increased Ca2+ sensitivity. As noted above, the ß-tropomyosin gene was upregulated in
MHC-tTA myocardium, and increased levels of this myofilament protein have been associated with cardiomyopathy and increased Ca2+ sensitivity.
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,
MHC-tTA myocardium also had a faster Ca2+ transient decline, which suggested improved SERCA function.
Compared with WT,
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
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
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
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
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
|
---|
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. 
 |
REFERENCES
|
---|
- Backx PH, Gao WD, Azan-Backx MD, and Marban E. The relationship between contractile force and intracellular [Ca2+] in intact rat cardiac trabeculae. J Gen Physiol 105: 119, 1995.[Abstract]
- Baker AJ, Redfern CH, Harwood MD, Simpson PC, and Conklin BR. Abnormal contraction caused by expression of Gi-coupled receptor in transgenic model of dilated cardiomyopathy. Am J Physiol Heart Circ Physiol 280: H1653H1659, 2001.[Abstract/Free Full Text]
- Beggah AT, Escoubet B, Puttini S, Cailmail S, Delage V, Ouvrard-Pascaud A, Bocchi B, Peuchmaur M, Delcayre C, Farman N, and Jaisser F. Reversible cardiac fibrosis and heart failure induced by conditional expression of an antisense mRNA of the mineralocorticoid receptor in cardiomyocytes. Proc Natl Acad Sci USA 99: 71607165, 2002.[Abstract/Free Full Text]
- Bendall JK, Heymes C, Wright TJ, Wheatcroft S, Grieve DJ, Shah AM, and Cave AC. Strain-dependent variation in vascular responses to nitric oxide in the isolated murine heart. J Mol Cell Cardiol 34: 13251333, 2002.[CrossRef][ISI][Medline]
- Bowman JC, Steinberg SF, Jiang T, Geenen DL, Fishman GI, and Buttrick PM. Expression of protein kinase C beta in the heart causes hypertrophy in adult mice and sudden death in neonates. J Clin Invest 100: 21892195, 1997.[Abstract/Free Full Text]
- Brooks WW and Apstein CS. Effect of treppe on isovolumic function in the isolated blood-perfused mouse heart. J Mol Cell Cardiol 28: 18171822, 1996.[CrossRef][ISI][Medline]
- Chen J, Kelz MB, Zeng G, Sakai N, Steffen C, Shockett PE, Picciotto MR, Duman RS, and Nestler EJ. Transgenic animals with inducible, targeted gene expression in brain. Mol Pharmacol 54: 495503, 1998.[Abstract/Free Full Text]
- de Tombe PP. Altered contractile function in heart failure. Cardiovasc Res 37: 367380, 1998.[CrossRef][ISI][Medline]
- Doniger SW, Salomonis N, Dahlquist KD, Vranizan K, Lawlor SC, and Conklin BR. MAPPFinder: using Gene Ontology and GenMAPP to create a global gene-expression profile from microarray data. Genome Biol 4: R7, 2003.[CrossRef][Medline]
- Dor Y, Djonov V, Abramovitch R, Itin A, Fishman GI, Carmeliet P, Goelman G, and Keshet E. Conditional switching of VEGF provides new insights into adult neovascularization and pro-angiogenic therapy. EMBO J 21: 19391947, 2002.[Abstract/Free Full Text]
- Friddle CJ, Koga T, Rubin EM, and Bristow J. Expression profiling reveals distinct sets of genes altered during induction and regression of cardiac hypertrophy. Proc Natl Acad Sci USA 97: 67456750, 2000.[Abstract/Free Full Text]
- Furth PA, St Onge L, Boger H, Gruss P, Gossen M, Kistner A, Bujard H, and Hennighausen L. Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc Natl Acad Sci USA 91: 93029306, 1994.[Abstract/Free Full Text]
- Gao MH, Bayat H, Roth DM, Yao Zhou J, Drumm J, Burhan J, and Kirk Hammond H. Controlled expression of cardiac-directed adenylylcyclase type VI provides increased contractile function. Cardiovasc Res 56: 197204, 2002.[CrossRef][ISI][Medline]
- Gao WD, Perez NG, and Marban E. Calcium cycling and contractile activation in intact mouse cardiac muscle. J Physiol 507: 175184, 1998.[Abstract/Free Full Text]
- Ghersa P, Gobert RP, Sattonnet-Roche P, Richards CA, Merlo Pich E, and Hooft van Huijsduijnen R. Highly controlled gene expression using combinations of a tissue-specific promoter, recombinant adenovirus and a tetracycline-regulatable transcription factor. Gene Ther 5: 12131220, 1998.[CrossRef][ISI][Medline]
- Go LO, Moschella MC, Watras J, Handa KK, Fyfe BS, and Marks AR. Differential regulation of two types of intracellular calcium release channels during end-stage heart failure. J Clin Invest 95: 888894, 1995.[ISI][Medline]
- Hess J, Nielsen PJ, Fischer KD, Bujard H, and Wirth T. The B lymphocyte-specific coactivator BOB.1/OBF1 is required at multiple stages of B-cell development. Mol Cell Biol 21: 15311539, 2001.[Abstract/Free Full Text]
- Huang WY, Aramburu J, Douglas PS, and Izumo S. Transgenic expression of green fluorescence protein can cause dilated cardiomyopathy. Nat Med 6: 482483, 2000.[CrossRef][ISI][Medline]
- Hwang DY, Chae KR, Shin DH, Hwang JH, Lim CH, Kim YJ, Kim BJ, Goo JS, Shin YY, Jang IS, Cho JS, and Kim YK. Xenobiotic response in humanized double transgenic mice expressing tetracycline-controlled transactivator and human CYP1B1. Arch Biochem Biophys 395: 3240, 2001.[CrossRef][ISI][Medline]
- Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, and Speed TP. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res 31: e15, 2003.[Abstract/Free Full Text]
- Ishizaka S, Sievers RE, Zhu BQ, Rodrigo MC, Joho S, Foster E, Simpson PC, and Grossman W. New technique for measurement of left ventricular pressure in conscious mice. Am J Physiol Heart Circ Physiol 286: H1208H1215, 2004.[Abstract/Free Full Text]
- Ju H, Gros R, You X, Tsang S, Husain M, and Rabinovitch M. Conditional and targeted overexpression of vascular chymase causes hypertension in transgenic mice. Proc Natl Acad Sci USA 98: 74697474, 2001.[Abstract/Free Full Text]
- Kostin S, Dammer S, Hein S, Klovekorn WP, Bauer EP, and Schaper J. Connexin 43 expression and distribution in compensated and decompensated cardiac hypertrophy in patients with aortic stenosis. Cardiovasc Res 62: 426436, 2004.[CrossRef][ISI][Medline]
- Lerman I, Harrison BC, Freeman K, Hewett TE, Allen DL, Robbins J, and Leinwand LA. Genetic variability in forced and voluntary endurance exercise performance in seven inbred mouse strains. J Appl Physiol 92: 22452255, 2002.[Abstract/Free Full Text]
- Long CS, Ordahl CP, and Simpson PC. Alpha 1-adrenergic receptor stimulation of sarcomeric actin isogene transcription in hypertrophy of cultured rat heart muscle cells. J Clin Invest 83: 10781082, 1989.[ISI][Medline]
- MacGowan GA and Koretsky AP. Inotropic and energetic effects of altering the force-calcium relationship: mechanisms, experimental results, and potential molecular targets. J Card Fail 6: 144156, 2000.[ISI][Medline]
- Malleret G, Haditsch U, Genoux D, Jones MW, Bliss TV, Vanhoose AM, Weitlauf C, Kandel ER, Winder DG, and Mansuy IM. Inducible and reversible enhancement of learning, memory, and long-term potentiation by genetic inhibition of calcineurin. Cell 104: 675686, 2001.[ISI][Medline]
- Mayford M, Bach ME, Huang YY, Wang L, Hawkins RD, and Kandel ER. Control of memory formation through regulated expression of a CaMKII transgene. Science 274: 16781683, 1996.[Abstract/Free Full Text]
- McCloskey D, Rokosh D, O'Connell T, Keung E, Simpson P, and Baker A.
1-Adrenoceptor subtypes mediate negative inotropy in myocardium from
1A/C-knockout and wild type mice. J Mol Cell Cardiol 34: 10071017, 2002.[CrossRef][ISI][Medline]
- McCloskey DT, Turnbull L, Swigart P, O'Connell TD, Simpson PC, and Baker AJ. Abnormal myocardial contraction in alpha(1A)- and alpha(1B)-adrenoceptor double-knockout mice. J Mol Cell Cardiol 35: 12071216, 2003.[CrossRef][ISI][Medline]
- Mungrue IN, Gros R, You X, Pirani A, Azad A, Csont T, Schulz R, Butany J, Stewart DJ, and Husain M. Cardiomyocyte overexpression of iNOS in mice results in peroxynitrite generation, heart block, and sudden death. J Clin Invest 109: 735743, 2002.[Abstract/Free Full Text]
- O'Connell TD, Ishizaka S, Nakamura A, Swigart PM, Rodrigo MC, Simpson GL, Cotecchia S, Rokosh DG, Grossman W, Foster E, and Simpson PC. The alpha(1A/C)- and alpha(1B)-adrenergic receptors are required for physiological cardiac hypertrophy in the double-knockout mouse. J Clin Invest 111: 17831791, 2003.[Abstract/Free Full Text]
- Pieske B, Sutterlin M, Schmidt-Schweda S, Minami K, Meyer M, Olschewski M, Holubarsch C, Just H, and Hasenfuss G. Diminished post-rest potentiation of contractile force in human dilated cardiomyopathy. Functional evidence for alterations in intracellular Ca2+ handling. J Clin Invest 98: 764776, 1996.[Abstract/Free Full Text]
- Redfern CH, Coward P, Degtyarev MY, Lee EK, Kwa AT, Hennighausen L, Bujard H, Fishman GI, and Conklin BR. Conditional expression and signaling of a specifically designed Gi-coupled receptor in transgenic mice. Nat Biotechnol 17: 165169, 1999.[CrossRef][ISI][Medline]
- Redfern CH, Degtyarev MY, Kwa AT, Salomonis N, Cotte N, Nanevicz T, Fidelman N, Desai K, Vranizan K, Lee EK, Coward P, Shah N, Warrington JA, Fishman GI, Bernstein D, Baker AJ, and Conklin BR. Conditional expression of a Gi-coupled receptor causes ventricular conduction delay and a lethal cardiomyopathy. Proc Natl Acad Sci USA 97: 48264831, 2000.[Abstract/Free Full Text]
- Sanbe A, Gulick J, Hanks MC, Liang Q, Osinska H, and Robbins J. Reengineering inducible cardiac-specific transgenesis with an attenuated myosin heavy chain promoter. Circ Res 92: 609616, 2003.[Abstract/Free Full Text]
- Suzuki J, Shen WJ, Nelson BD, Patel S, Veerkamp JH, Selwood SP, Murphy GM Jr, Reaven E, and Kraemer FB. Absence of cardiac lipid accumulation in transgenic mice with heart-specific HSL overexpression. Am J Physiol Endocrinol Metab 281: E857E866, 2001.[Abstract/Free Full Text]
- Terzic A, Puceat M, Clement O, Scamps F, and Vassort G. Alpha 1-adrenergic effects on intracellular pH and calcium and on myofilaments in single rat cardiac cells. J Physiol 447: 275292, 1992.[Abstract]
- Turnbull L, McCloskey DT, O'Connell TD, Simpson PC, and Baker AJ.
1-Adrenergic receptor responses in
1AB-AR knockout mouse hearts suggest the presence of
1D-AR. Am J Physiol Heart Circ Physiol 284: H1104H1109, 2003.[Abstract/Free Full Text]
- van der Velden J, Papp Z, Zaremba R, Boontje NM, de Jong JW, Owen VJ, Burton PB, Goldmann P, Jaquet K, and Stienen GJ. Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins. Cardiovasc Res 57: 3747, 2003.[CrossRef][ISI][Medline]
- Wolska BM and Wieczorek DM. The role of tropomyosin in the regulation of myocardial contraction and relaxation. Pflügers Arch 446: 18, 2003.[CrossRef][ISI][Medline]
- Yang LL, Gros R, Kabir MG, Sadi A, Gotlieb AI, Husain M, and Stewart DJ. Conditional cardiac overexpression of endothelin-1 induces inflammation and dilated cardiomyopathy in mice. Circulation 109: 255261, 2004.[Abstract/Free Full Text]
- Yu Z, Redfern CS, and Fishman GI. Conditional transgene expression in the heart. Circ Res 79: 691697, 1996.[Abstract/Free Full Text]