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Am J Physiol Heart Circ Physiol 290: H1103-H1109, 2006. First published October 21, 2005; doi:10.1152/ajpheart.00732.2005
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Sustained preconditioning induced by cardiac transgenesis with the tetracycline transactivator

Lynne Turnbull,1,5 Hui-Zhong Zhou,2,5 Philip M. Swigart,2,5 Sally Turcato,1,5 Joel S. Karliner,2,3,5 Bruce R. Conklin,2,4 Paul C. Simpson,2,3,5 and Anthony J. Baker1,5

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

Submitted 11 July 2005 ; accepted in final form 13 October 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preconditioning protocols that protect the heart from ischemic injury may aid in the development of new therapies. However, the temporal window of cardioprotection is limited to a few days after the preconditioning stimulus. Here we report a sustained cardioprotected phenotype in mice expressing a tetracycline transactivator (tTA) transcription factor under the control of the {alpha}-myosin heavy chain ({alpha}MHC) promoter. {alpha}MHC-tTA mice were originally designed for tetracycline-regulated gene expression in the heart (Tet system). However, we found that after 45 min of global ischemia at 37°C, left ventricular developed pressure (LVDP) of Langendorff-perfused {alpha}MHC-tTA mouse hearts rapidly recovered in 5 min to 60% of initial levels, whereas LVDP of wild-type (WT) littermates recovered to only 10% of the initial level. Improved postischemic recovery of function for {alpha}MHC-tTA hearts was associated with a 50% decrease of infarct size and a significantly smaller release of lactate dehydrogenase to the coronary effluent. Improved postischemic recovery was not attributable to differences in coronary flow that was similar for WT- and {alpha}MHC-tTA hearts during recovery. Moreover, improved postischemic recovery of {alpha}MHC-tTA hearts was not abolished by inhibitors of classical cardioprotective effectors (mitochondrial ATP-sensitive K+ channels, PKC, or adenosine receptors), suggesting a novel mechanism. Finally, the tetracycline analog doxycycline, which inhibits binding of tTA to DNA, did not abolish improved recovery for {alpha}MHC-tTA hearts. The sustained cardioprotected phenotype of {alpha}MHC-tTA hearts may have implications for developing new therapies to minimize cardiac ischemic injury. Furthermore, investigations of cardioprotection using the Tet system may be aberrantly influenced by sustained preconditioning induced by cardiac transgenesis with tTA.

tetracycline-regulated gene expression system; Langendorff; mouse; contraction; cardioprotection; {alpha}-myosin heavy chain

MYOCARDIAL ISCHEMIA caused by acute coronary artery occlusion is a leading cause of death and morbidity worldwide (7). Ischemia rapidly produces profound metabolic and functional derangements followed by myocyte injury and death when ischemia is prolonged. The search for cardioprotective strategies to attenuate ischemia-reperfusion (I/R) injury is a keen area of research interest with important clinical implications (e.g., for patients with ischemic heart disease who are at risk for acute myocardial infarction or during interventions involving myocardial ischemia, such as stent deployment, bypass surgery, or heart transplantation) [see review (31)].

The classical cardioprotective strategy of ischemic preconditioning (IPC) was first described by Murry and colleagues (22) in dog heart. IPC involves one or more brief periods of coronary artery occlusion that results in increased resistance to injury from subsequent prolonged ischemia. Multiple preconditioning agents have now been described. These include {alpha}1-adrenergic receptor agonists, B2 bradykinin receptor agonists, A1 adenosine receptor agonists, opioid receptor agonists, inhibitors of the Na+/H+ exchanger, cytokines, PKC activators, and activators of the mitochondrial ATP-sensitive K+ (mitoKATP) channel. Understanding the mechanisms responsible for preconditioning may lead to new therapeutic approaches to protect the heart (10).

Unfortunately, cardioprotection conferred by IPC or most preconditioning protocols has a temporal window that is limited to a few days. Thus a cardioprotected phenotype that was sustained indefinitely would be a major advantage. Sustained cardioprotection has been shown to result from ethanol consumption and, similar to IPC, was abolished by inhibitors of PKC, mitoKATP channels, or adenosine receptors (20, 21, 33). Here we report a novel sustained cardioprotected phenotype in transgenic mice that express the tetracycline transactivator (tTA) (11, 12) driven by an {alpha}-myosin heavy chain ({alpha}MHC) promoter (32). The single transgenic {alpha}MHC-tTA mouse was developed to allow tetracycline-regulated gene expression in the mouse heart [see review (9)] and has now been widely used (2, 4, 5, 8, 19, 24, 25). Unexpectedly, we find that Langendorff-perfused {alpha}MHC-tTA hearts display a remarkably rapid and potent recovery after prolonged periods of global ischemia. Improved functional recovery for {alpha}MHC-tTA myocardium was not abolished by conventional inhibitors of IPC, suggesting that it was not mediated by classical preconditioning mechanisms. Sustained preconditioning inherent to {alpha}MHC-tTA myocardium may suggest future therapies to minimize cardiac ischemic injury.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal methods. The Animal Studies Subcommittee of the San Francisco Veterans Affairs Medical Center approved all procedures. Male and female mice expressing a tTA under the regulatory control of 2.9 kb of 5' flanking sequence from the {alpha}MHC-tTA have been previously described (24, 32) and were maintained in an FVB/N background for over 10 generations (99.9% congenic). Wild-type (WT) littermates were used as controls. Typically, {alpha}MHC-tTA mice are used in studies involving tetracycline-regulated gene expression where expression is initiated by removal of tetracycline (or the analog doxycycline) from the diet. Animals were bred and maintained on a diet containing doxycycline (200 mg/kg, Dox diet, No. S3888, Bio-Serve, Frenchtown, NJ). Except as noted in RESULTS, 8- to 12-wk-old animals used in this study were removed from the Dox diet, and experiments were performed 2 wk later.

Male and female mice expressing tTA under the control of the human early cytomegalovirus promoter (CMV-tTA) (15) were also used. WT littermates were used as controls (CMV-WT). These mice were obtained from Jackson Laboratories [stock TgN(tTAhCMV)3Uh, Stock No. 003271, Bar Harbor, MA] and maintained in a NMRI strain.

Isolated heart preparation and I/R protocol. We used the Langendorff perfused mouse heart model that has the advantage of constant heart rate, avoidance of the effects of anesthesia, control of systolic and diastolic pressures, and the absence of systemic neurohormonal responses.

Hearts were excised, mounted, and perfused as described previously (29). To study I/R responses, after a 30-min equilibration period and 45 min of no flow, normothermic (37°C) ischemia was induced. Electrical stimulation was stopped after 1 min of ischemia. The heart chamber was sealed to maintain a humid environment, and warm perfusate was slowly dripped onto the heart surface to prevent desiccation. At the end of ischemia, electrical stimulation was restarted 1 min before reperfusion, and hearts were reperfused for 45 min. To study IPC, during the 30-min equilibration period, the hearts were subjected to a brief (3 min) ischemia (starting at minute 12).

After I/R, left ventricular (LV) infarct size was determined by the triphenyltetrazolium chloride (TTC)-staining technique as previously described (14). TTC stains viable tissue a dark red color, whereas necrotic myocardium appears pale.

Inhibitors. Stock solutions of the preconditioning inhibitors chelerythrine chloride (Chel), 5-hydroxydecanoate (5-HD) and 8-(p-sulfophenyl)theophylline (8-SPT) were freshly prepared in Krebs-Henseleit perfusate. Inhibitors were infused (at 1% of coronary flow) for 5–10 min just before the major ischemic insult. All chemicals were analytical grade and were purchased from Sigma-Aldrich (St. Louis, MO).

Lactate dehydrogenase assay. Cellular damage was assessed from measurements of lactate dehydrogenase (LDH) release from perfused hearts to the coronary effluent during reperfusion. Perfusate was collected for three 15-min periods during reperfusion, LDH content in each sample was measured by using a commercially available kit (CytoTox 96 Non-Radioactive Cytotoxicity Assay, Promega, WI), and data were expressed as absorbance units released per milliliter per minute per gram of heart tissue.

Western blot analysis. To monitor tTA in the myocardium, freshly isolated myocytes were processed for Western blot analysis as previously described (18). A 12.5% SDS-PAGE gel with molecular mass markers was used with equal loading of 40,000 myocytes/lane; tTA was detected with an antibody to the virion protein 16 (VP16) domain (Cat. No. 3844–1, BD Biosciences, Franklin Lakes, NJ), diluted 1:2,000.

Statistics. Values are means ± SE. To determine statistical differences between the means of two groups, a Student’s t-test was used. To determine statistical differences between the means of multiple groups, a one-way ANOVA was used with a Bonferroni test for post hoc analysis. For experiments involving repeated measures on the same subject over time, a two-way repeated-measures ANOVA was used for the group analysis and a Tukey’s test for the post hoc analysis. Values of P < 0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
{alpha}MHC-tTA hearts were protected against I/R injury. Baseline hemodynamics for {alpha}MHC-tTA hearts were recently described (19). Figure 1 shows representative examples of contractions from WT littermate (Fig. 1A) and {alpha}MHC-tTA (Fig. 1B) hearts immediately before ischemia (pre-I/R) and after 45 min of ischemia and 45 min of reperfusion (post-I/R). Figure 1 shows that pre-I/R {alpha}MHC-tTA hearts developed greater pressure than WT littermate hearts as recently described (19). Interestingly, post-I/R {alpha}MHC-tTA hearts recovered substantially greater developed pressure and had lower diastolic pressure than WT hearts.


Figure 1
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  		Fig. 1. {alpha}-Myosin heavy chain ({alpha}MHC)-tetracycline transactivator (tTA) hearts were protected against ischemia-reperfusion (I/R) injury. Representative fast time-based (1-kHz sampling) pressure recordings from wild-type (WT) (A) and {alpha}MHC-tTA (B) hearts show left ventricular (LV) pressure (LVP) before (solid line) and after (dashed line) 45 min of ischemia and 45 min of reperfusion.

 
Figure 2 shows pooled data for postischemic pressure recovery over the 45-min reperfusion period for WT and {alpha}MHC-tTA hearts. Figure 2A shows recovery of LV developed pressure (LVDP = systolic – diastolic pressure) as a percentage of the pre-I/R LVDP. For WT hearts, LVDP recovered slowly during reperfusion. At 5 min of reperfusion, WT hearts recovered only to {approx}10% of pre-I/R LVDP with a gradual increase to 34 ± 4% (n = 26 hearts) of pre-I/R LVDP at 45 min of reperfusion. In sharp contrast, {alpha}MHC-tTA hearts displayed a remarkably rapid recovery to {approx}60% of pre-I/R LVDP at 5 min of reperfusion with a small increase thereafter to 65 ± 3% (n = 20 hearts) of pre-I/R LVDP at 45 min of reperfusion. Consistent with a more rapid recovery of {alpha}MHC-tTA hearts, the time required for contractions to resume during reperfusion was considerably shorter for {alpha}MHC-tTA hearts than for WT hearts (27 ± 9 s, n = 20 vs. 363 ± 68 s, n = 26, respectively; P < 0.001).


Figure 2
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  		Fig. 2. {alpha}MHC-tTA hearts recovered rapidly and had lower diastolic pressure than WT hearts. Pooled data (means ± SE) show postischemic recovery of LV developed pressure (LVDP, normalized to pre-I/R values, A) and LV end-diastolic pressure (LVEDP, B). Data from {alpha}MHC-tTA hearts (solid line, n = 20) are statistically different from WT hearts (dashed line, n = 26); P < 0.001, repeated-measures ANOVA. Differences became statistically significant after 1 min of reperfusion (P < 0.01, Tukey’s post hoc test).

 
Figure 2B summarizes postischemic recovery of LV end-diastolic pressure (LVEDP) for {alpha}MHC-tTA and WT hearts. At the end of ischemia and before reperfusion, LVEDP was elevated but was lower for {alpha}MHC-tTA than for WT hearts (44 ± 3 mmHg, n = 20 vs. 52 ± 2 mmHg, n = 26; P < 0.05). On reperfusion, for WT hearts there was a substantial increase of LVEDP (to 76 ± 5 mmHg, n = 26, P < 0.001) at 5 min of reperfusion. However, for {alpha}MHC-tTA hearts, there was no appreciable increase of LVEDP during reperfusion. At 45 min of reperfusion, LVEDP for {alpha}MHC-tTA hearts recovered to a significantly lower level than for WT hearts (22 ± 2 mmHg, n = 20 vs. 39 ± 3 mmHg, n = 26, respectively; P < 0.001).

Coronary flow was measured during reperfusion of WT and {alpha}MHC-tTA hearts. After 10 min of reperfusion, there was no difference in coronary flow of WT versus {alpha}MHC-tTA hearts (96 ± 13 ml·min–1·g–1, n = 5 vs. 82 ± 11 ml·min–1·g–1, n = 4; P = not significant). In contrast, LVDP recovery differed greatly at this time (Fig. 2A). This result demonstrates that the improved pressure recovery for {alpha}MHC-tTA hearts was not due to improved recovery of coronary flow.

{alpha}MHC-tTA hearts had less injury. We used several measures to assess ischemic injury in WT and {alpha}MHC-tTA hearts: contracture development, infarct size, and the release of LDH in the coronary effluent. Figure 3A shows representative recordings of LV pressure during contracture development for WT and {alpha}MHC-tTA hearts during ischemia. Ischemic contracture pressure was lower and developed later for the {alpha}MHC-tTA heart compared with the WT heart. The pooled data show that the lower ischemic contracture pressure (Fig. 3B) and delayed contracture development (Fig. 3C) for {alpha}MHC-tTA hearts were statistically significant.


Figure 3
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  		Fig. 3. {alpha}MHC-tTA hearts were more resistant to ischemic contracture. A: single representative slow time-based LVP recordings from WT and {alpha}MHC-tTA hearts during 45 min of ischemia. Arrows indicate peak contracture. B and C: pooled data for maximum LV ischemic contracture pressure and time to reach maximum contracture pressure, respectively. Data are means ± SE for WT (open bars, n = 32) and {alpha}MHC-tTA (solid bars, n = 28) hearts. *P < 0.05 and **P < 0.01 for {alpha}MHC-tTA vs. WT (Student’s t-test).

 
We assessed whether the resistance to ischemic contracture and the improved postischemic recovery of {alpha}MHC-tTA hearts compared with WT hearts were associated with decreased cellular injury. Cellular injury was monitored from measurements of infarct size using TTC staining and also from the appearance of the cellular enzyme LDH in the coronary effluent. At the end of reperfusion, the infarct size for {alpha}MHC-tTA hearts was only half of that for WT hearts (Fig. 4A). Moreover, {alpha}MHC-tTA hearts had appreciably less LDH release to the coronary effluent compared with WT hearts (Fig. 4B). Together these findings indicate that in response to the ischemia and reperfusion protocol, there was considerably less cellular injury and necrosis in {alpha}MHC-tTA hearts compared with WT hearts.


Figure 4
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  		Fig. 4. {alpha}MHC-tTA hearts were less damaged during I/R. Ischemic injury assessed by infarct size (A) as percentage of whole heart for {alpha}MHC-tTA (n = 9) and WT (n = 10) hearts after 45 min of ischemia and 45 min of reperfusion, release of lactate dehydrogenase (LDH) (B; {alpha}MHC-tTA, solid squares, n = 5; and WT, open circles, n = 4) to coronary effluent during reperfusion. Coronary outflow was collected for three 15-min periods during reperfusion and sampled for LDH content. abs, absolute. Data points represent average LDH release per minute for the preceding 15-min period. All data are means ± SE. *P < 0.05 and **P < 0.01 for {alpha}MHC-tTA vs. WT.

 
Sustained preconditioning versus IPC. The sustained cardioprotected phenotype inherent to {alpha}MHC-tTA hearts was compared with cardioprotection conferred by IPC. Figure 5 summarizes postischemic LVDP recovery (% pre-I/R) during reperfusion. For WT hearts, IPC improved postischemic LVDP recovery as expected (58 ± 6%, n = 8, vs. 34 ± 4%, n = 26, P < 0.01). However, for {alpha}MHC-tTA hearts, IPC did not further improve postischemic LVDP recovery. Thus, for LVDP recovery, the beneficial effects of IPC and inherent preconditioning were not additive in {alpha}MHC-tTA hearts.


Figure 5
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  		Fig. 5. {alpha}MHC-tTA cardioprotection was not improved by ischemic preconditioning (IPC). Pooled data show recovery of LVDP from onset of reperfusion in WT and {alpha}MHC-tTA hearts with and without IPC (3 min of transient ischemia 18 min before sustained ischemia). Pressure recovery in {alpha}MHC-tTA hearts is shown as solid line without IPC (open squares, n = 20) and with IPC (solid squares, n = 7). Pressure recovery in WT hearts is shown as dashed line without IPC (open circles, n = 27) and with IPC (solid circles, n = 8). LVDP data are normalized to pre-I/R values and are means ± SE.

 
Figure 5 also shows that the time course of LVDP recovery for {alpha}MHC-tTA hearts was considerably faster than for WT hearts with IPC. For WT hearts, cardioprotection due to IPC mediated a gradual increase in LVDP during reperfusion. In contrast, the cardioprotection inherent to {alpha}MHC-tTA hearts mediated a rapidly acting beneficial effect on LVDP recovery early during reperfusion.

IPC can be abolished by inhibitors of mitoKATP, PKC, and adenosine receptors (17, 23). Therefore, we investigated whether inhibitors of these classical cardioprotective effectors (100 µmol/l 5-HD for mitoKATP, 3 µmol/l Chel for PKC, and 50 µmol/l 8-SPT for adenosine receptors) could also abolish the improved functional recovery manifested by {alpha}MHC-tTA hearts. Figure 6 summarizes the effects of these inhibitors on WT and {alpha}MHC-tTA hearts. For WT hearts, as expected, improved recovery of LVDP due to IPC was abolished by each of the three inhibitors. In contrast, Fig. 6 also shows that improved LVDP recovery for {alpha}MHC-tTA hearts compared with that for WT hearts was not abolished by the inhibitors. Furthermore, as noted above, when compared with WT hearts, {alpha}MHC-tTA hearts had lower ischemic contracture pressure, more rapid postischemic resumption of contractions, and lower postischemic LVEDP. Table 1 shows that the inhibitors did not abolish these additional indicators of an improved response to I/R injury in {alpha}MHC-tTA hearts compared with WT hearts. In the presence of the inhibitors, significant differences remained between values for WT versus {alpha}MHC-tTA hearts, including in the presence of Chel and 8-SPT, which resulted in higher LVEDP for both WT and {alpha}MHC-tTA hearts.


Figure 6
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  		Fig. 6. {alpha}MHC-tTA cardioprotection was not inhibited by preconditioning antagonists. Pooled data from WT (open bars) and {alpha}MHC-tTA (solid bars) hearts show LVDP as percentage of preischemic LVDP after I/R. Hearts underwent 45 min of I/R after no pretreatment (I/R45), IPC, or pretreatment (for 5–10 min before sustained ischemia) with 100 µmol/l 5-hydroxydecanoate (5-HD), 3 µmol/l chelerythrine chloride (Chel), or 50 µmol/l 8-(p-sulfophenyl)theophylline (8-SPT). All inhibitor-treated WT hearts underwent IPC before inhibitor incubation. Data are means ± SE with numbers per group in parentheses. **P < 0.01 vs. I/R45 (1-way ANOVA with Bonferroni post hoc test).

 

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  		Table 1. Preconditioning inhibitors did not abolish functional differences in ischemia-reperfusion responses between WT versus {alpha}MHC-tTA hearts

 
{alpha}MHC-tTA cardioprotection was not abolished by doxycycline. With the use of the Tet system, control of gene expression involves a doxycycline-induced conformational change in tTA that inhibits its interaction with DNA. We determined whether cardioprotection observed in {alpha}MHC-tTA hearts was dependent on the conformation of the DNA-binding domain of tTA. We compared our findings with the I/R responses of hearts from mice that were maintained on a diet containing doxycycline from birth. Figure 7 summarizes LVDP during reperfusion of hearts from WT and {alpha}MHC-tTA mice maintained on a doxycycline-containing diet. For comparison, data in the absence of doxycycline are also shown (from Fig. 2). Figure 7 shows that improved recovery of {alpha}MHC-tTA hearts compared with that of WT was not abolished by doxycycline. This indicates that cardioprotection in {alpha}MHC-tTA hearts was not related to an effect of doxycycline on the DNA-binding domain of tTA. However, doxycycline caused improved recovery of LVDP (%pre-I/R) at 45 min of reperfusion compared with untreated hearts for both {alpha}MHC-tTA hearts (79 ± 4%, n = 7 vs. 65 ± 3%, n = 20; P < 0.05) and WT hearts (51 ± 8%, n = 7 vs. 34 ± 4%, n = 26; P = 0.05). This finding is consistent with a previous report that doxycycline protects the heart via inhibition of matrix metalloproteinase-2, a contributor to myocardial I/R injury (6).


Figure 7
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  		Fig. 7. {alpha}MHC-tTA cardioprotection was not abolished by doxycycline (Dox). Pooled data show recovery of LVDP from onset of reperfusion in WT and {alpha}MHC-tTA hearts from animals maintained on Dox. Pressure recovery in {alpha}MHC-tTA hearts is shown as solid line without Dox (open squares, n = 20) and with Dox (solid squares, n = 7). Pressure recovery in WT hearts is shown as dashed line without Dox (open circles, n = 27) and with Dox (solid circles, n = 7). LVDP data are normalized to pre-I/R values and are means ± SE.

 
CMV-tTA hearts were not protected against I/R injury. We compared results from {alpha}MHC-tTA mice with those from mice where tTA was driven by the human CMV promoter (CMV-tTA). Figure 8A summarizes the time course of postischemic LV pressure recovery for hearts from CMV-tTA mice and WT littermates. Unlike {alpha}MHC-tTA hearts, CMV-tTA hearts did not have improved postischemic recovery compared with WT hearts.


Figure 8
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  		Fig. 8. No inherent cardioprotection in cytomegalovirus (CMV)-tTA hearts. A: pooled data showing recovery of LVDP from onset of reperfusion in CMV-WT hearts (open triangles, n = 6) and CMV-tTA hearts (solid triangles, n = 6). LVDP data are normalized to pre-I/R values and are means ± SE. B: Western blot analysis using antibody to the virion protein 16 (VP16) domain of tTA. Lanes are indicated for samples from hearts of WT, {alpha}MHC-tTA, and CMV-tTA mice (n = 3 animals/group).

 
Figure 8B shows results of Western blot analysis to detect tTA expression using an antibody to the VP16 domain and an equal number of cells (40,000) loaded per lane. As expected, VP16 was not detectable in WT hearts. Figure 8B shows that VP16 was detectable in {alpha}MHC-tTA hearts. According to molecular mass markers, the VP16 signal corresponded to a molecular mass of ~40 kDa, close to the value (37 kDa) reported for tTA (12).

Surprisingly, VP16 was not detected in CMV-tTA hearts (Fig. 8B), suggesting that tTA expression in CMV-tTA hearts allows doxycycline-dependent transactivation (15) but that the level of tTA in CMV-tTA hearts was below the detection threshold for our assay. These results suggest a dose effect where tTA expression in {alpha}MHC-tTA hearts was associated with cardioprotection, but lower level tTA expression in CMV-tTA hearts was not associated with cardioprotection.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major finding in this study was that hearts from mice expressing the tTA (11, 12), driven by the rat {alpha}MHC promoter (32), displayed a remarkably rapid and appreciable level of functional recovery after a prolonged period of global ischemia. Improved postischemic recovery for {alpha}MHC-tTA hearts was associated with reduced myocyte necrosis compared with that for WT hearts. Improved postischemic recovery for {alpha}MHC-tTA hearts could not be abolished by conventional preconditioning antagonists, suggesting that cardioprotection inherent to {alpha}MHC-tTA hearts involved a nonclassical mechanism. These findings demonstrate a potent and novel form of cardioprotection that may represent a new substrate for therapies to protect the ischemic heart. Moreover, because most preconditioning protocols have a limited temporal window of cardioprotection, the sustained cardioprotected phenotype is a notable feature of {alpha}MHC-tTA hearts.

The {alpha}MHC-tTA mouse line used in this study (32) forms one arm of a binary transgenic Tet system (11, 12) for tetracycline-regulated gene expression in the heart (5, 8, 13, 19, 24–26, 28, 30, 32). Ideally, the phenotype of {alpha}MHC-tTA mice would be identical to that of WT mice. However, Baker’s laboratory (19) previously found that {alpha}MHC-tTA mice have complex changes in physiology and gene expression, including increased contractions in vitro, increased myofilament Ca2+ sensitivity, increased Ca2+-transient amplitude with faster Ca2+-transient decline, and changes in the expression of more than 150 genes. The present study identifies an additional and interesting property of {alpha}MHC-tTA hearts: their inherent resistance to I/R injury.

We used a long (45 min) period of normothermic (37°C) ischemia that significantly injured WT hearts and resulted in a limited and slow recovery of LVDP during reperfusion. In contrast, {alpha}MHC-tTA hearts were much less injured by the I/R protocol as evidenced by an almost doubling of LVDP recovery compared with that in WT hearts. Interestingly, for {alpha}MHC-tTA hearts, recovery of LVDP was remarkably rapid, being virtually complete after only 5 min of reperfusion. In contrast, WT hearts had little postischemic recovery of LVDP at this time. Thus the disparity in functional recovery between WT and {alpha}MHC-tTA hearts was particularly prominent early in reperfusion. Therefore, the rapid rate of postischemic recovery, and not just its extent, is a key feature of the cardioprotection inherent in {alpha}MHC-tTA hearts.

For WT hearts, IPC significantly increased LVDP recovery close to that of {alpha}MHC-tTA hearts at 45 min of reperfusion. However, with IPC, the rate of LVDP recovery for WT hearts remained relatively slow. Consequently, early in reperfusion, a great disparity persisted in functional recovery between {alpha}MHC-tTA and WT hearts treated by IPC. Thus cardioprotection inherent to {alpha}MHC-tTA hearts appeared more beneficial compared with cardioprotection conferred by IPC on account of the more rapid postischemic recovery of {alpha}MHC-tTA hearts.

Improved postischemic recovery of function in {alpha}MHC-tTA hearts compared with WT hearts was associated with reduced myocyte injury as reflected by several measures. When compared with WT hearts, {alpha}MHC-tTA hearts had less necrosis during the I/R protocol as indicated by reduced infarct size and decreased release of LDH to the coronary effluent. Furthermore, {alpha}MHC-tTA hearts were slower to develop ischemic contracture, and the contracture pressure was lower, suggesting decreased cell injury (16, 27).

Like other forms of cardioprotection, the mechanism of cardioprotection for {alpha}MHC-tTA hearts is uncertain. However, we investigated and excluded several possibilities. Cardioprotection of {alpha}MHC-tTA hearts was not attributable to coronary flow that did not differ between WT and {alpha}MHC-tTA hearts. Improved postischemic functional recovery for {alpha}MHC-tTA hearts was not abolished by nonspecific inhibitors of PKC, adenosine receptors, or inhibition of mitoKATP channels. These findings suggest that these classical effectors, which play pivotal roles in other forms of cardioprotection, are not involved in the sustained cardioprotection in {alpha}MHC-tTA hearts. This is in contrast to the sustained cardioprotected phenotype observed with chronic ethanol treatment, where sustained cardioprotection was abolished by inhibitors of PKC, mitoKATP channels, or adenosine receptors (20, 21, 33).

Cardioprotection for {alpha}MHC-tTA hearts was not abolished by a conformational change in the DNA-binding domain of tTA induced by doxycycline. This suggests cardioprotection for {alpha}MHC-tTA hearts did not involve binding of tTA to DNA. Interestingly, we previously found that {alpha}MHC-tTA hearts exhibited differences in expression of numerous genes compared with WT hearts, including upregulation of heat shock genes and downregulation of metabolism genes for carbohydrates, proteins, and lipids (19). Such changes could protect the heart during ischemia by reducing metabolic demands. Furthermore, {alpha}MHC-tTA myocardium also had more rapid removal of Ca2+ from the cytosol during diastole that could limit postischemic injury by reducing Ca2+ overload (19).

A direct insertional effect of the transgene could not be ruled out, despite multiple attempts to isolate the insertion site via inverse PCR (data not shown). The failure to identify an insertion site could be due to the incomplete nature of the mouse genome sequence and the lack of a strain-specific sequence for the FVB/N mouse line.

A possible cause of phenotypic effects associated with tTA expression is the fact that this artificial transactivator has the potent VP16 domain that is known to bind multiple components of the core transcriptional machinery (1, 3). The overexpression of the VP16 domain alone has been predicted to have biological effects (1, 3). Moreover, VP16 effects should not be influenced by doxycycline, which affects the DNA-binding domain of tTA, consistent with our finding of sustained preconditioning in {alpha}MHC-tTA hearts in the presence of doxycycline. Our results also suggested a dose effect, where tTA expression in {alpha}MHC-tTA hearts resulted in sustained preconditioning but lower level tTA expression in CMV-tTA hearts did not. It will be interesting to identify VP16-binding partners in myocytes, because they might reveal potential novel targets for cardioprotection.

In conclusion, these studies show that {alpha}MHC-tTA hearts manifest a sustained cardioprotected phenotype that results in a striking and rapid functional recovery after a long period of normothermic global ischemia. Improved postischemic functional recovery for {alpha}MHC-tTA hearts was not abolished by inhibitors of classical cardioprotective effectors (mitoKATP channels, PKC, or adenosine receptors), suggesting a novel mechanism with implications for future therapies to minimize cardiac ischemic injury. Obviously, the cardioprotected phenotype of {alpha}MHC-tTA hearts would complicate studies of cardioprotection using the Tet system with this line of mice.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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This work was supported by National Heart, Lung, and Blood Institute Grants P01-HL-68738 (to A. J. Baker and J. S. Karliner), HL-60664 (to B. R. Conklin), and HL-31113 (to P. C. Simpson) and by an Established Investigator Award from the American Heart Association (to A. J. Baker).


   ACKNOWLEDGMENTS
 
We thank Manoj Rodrigo and Marietta Paningbatan for expert technical assistance.


   FOOTNOTES
 

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

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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