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Am J Physiol Heart Circ Physiol 291: H797-H803, 2006. First published March 24, 2006; doi:10.1152/ajpheart.01334.2005
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STAT-3 activation is necessary for ischemic preconditioning in hypertrophied myocardium

Karyn L. Butler,1 Lynn C. Huffman,1 Sheryl E. Koch,1 Harvey S. Hahn,2 and Judith K. Gwathmey3,4

Departments of 1Surgery and 2Medicine, University of Cincinnati, Cincinnati, Ohio; and 3Harvard Medical School, Department of Medicine, and 4Gwathmey, Incorporated, Boston, Massachusetts

Submitted 19 December 2005 ; accepted in final form 9 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The JAK-STAT pathway is activated in the early and late phases of ischemic preconditioning (IPC) in normal myocardium. The role of this pathway and the efficacy of IPC in hypertrophied hearts remain largely unknown. We hypothesized that phosphorylated STAT-3 (pSTAT-3) is necessary for effective IPC in pressure-overload hypertrophy. Male Sprague-Dawley rats 8 wk after thoracic aortic constriction (TAC) or sham operation underwent echocardiography and Langendorff perfusion. Randomized hearts were subjected to 30 min of global ischemia and 120 min of reperfusion with or without IPC in the presence or absence of the JAK-2 inhibitor AG-490 (AG). Functional recovery and STAT activation were assessed. TAC rats had a 31% increase in left ventricular mass (1,347 ± 58 vs. 1,028 ± 43 mg, TAC vs. sham, P < 0.001), increased anterior and posterior wall thickness but no difference in ejection fraction compared with sham-operated rats. In TAC, IPC improved end-reperfusion maximum first derivative of developed pressure (+dP/dtmax; 4,648 ± 309 vs. 2,737 ± 343 mmHg/s, IPC vs. non-IPC, P < 0.05) and minimum –dP/dt (–dP/dtmin; –2,239 ± 205 vs. –1,215 ± 149 mmHg/s, IPC vs. non-IPC, P < 0.05). IPC increased nuclear pSTAT-1 and pSTAT-3 in sham-operated rats but only pSTAT-3 in TAC. AG in TAC significantly attenuated +dP/dtmax (4,648 ± 309 vs. 3,241 ± 420 mmHg/s, IPC vs. IPC + AG, P < 0.05) and –dP/dtmin (–2,239 ± 205 vs. –1,323 ± 85 mmHg/s, IPC vs. IPC + AG, P < 0.05) and decreased only nuclear pSTAT-3. In myocardial hypertrophy, JAK-STAT signaling is important in IPC and exhibits a pattern of STAT activation distinct from nonhypertrophied myocardium. Limiting STAT-3 activation attenuates the efficacy of IPC in hypertrophy.

ischemia-reperfusion; Janus-activated kinase; signal transducer and activator of transcription; cardiac preconditioning; myocardial hypertrophy

CARDIAC HYPERTROPHY represents an adaptive response to increased workload on the heart. Clinically, the increased load is commonly due to systemic hypertension; however, cardiac responses to neural and humoral factors can also incite hypertrophic changes independent of increases in afterload or vascular resistance (15). Initially, pathological myocardial hypertrophy is adaptive, allowing the heart to maintain normal cardiac output despite mechanical loading. This compensatory response eventually develops into a maladaptive form of hypertrophy and, if untreated, is a precursor to heart failure. Recent findings are beginning to illuminate the signaling pathways involved in this putative adaptive response as well as those that may trigger the transition to decompensation and heart failure.

Cardioprotection or preconditioning is a powerful endogenous form of cellular adaptation that has been reproduced in numerous species and demonstrated in noncardiac tissue, such as liver, intestine, lung, kidney, and endothelium (16, 23). Despite the numerous experimental investigations over the last several decades on ischemic protection in animals, a paucity of studies has focused on understanding the phenomenon of preconditioning in animal models of clinically relevant cardiac disease. Furthermore, few strategies of cardioprotection identified in the laboratory have been translated into clinically useful tools (2). The imbalance between cardiac mass and coronary blood flow may render hypertrophied hearts more susceptible to ischemia, underscoring the need to investigate preconditioning in animal models with clinically relevant cardiac dysfunction (15).

Previous studies have demonstrated that the JAK-STAT pathway is activated in the early and late phases of ischemic preconditioning (IPC) (7, 28) in normal myocardium. There is also increasing evidence that JAK-STAT signaling plays an important role in cardiac myocyte adaptation to stress stimuli, such as ischemia or pressure overload (13, 25). Furthermore, the pathway has been implicated in cardiac hypertrophy, apoptosis, angiotensin signaling, and ischemia-reperfusion injury (4, 12, 21). Activation of JAK-2 and STAT-3 appears to be protective in ex vivo as well as in vivo models of ischemia-reperfusion injury (1, 11). Despite the elegant studies in normal myocardium, the effects of JAK-STAT signaling and IPC in hypertrophied myocardium have not been described.

In this study we demonstrate that preconditioning is an effective cardioprotective strategy in the hypertrophied heart, a clinically relevant pathological model. Furthermore, we find that activation of the JAK-STAT pathway is necessary for this cardioprotection.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal protocols conformed to 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 were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati. Animals were provided a standard rat chow diet and water ad libitum.

Animals. Male Sprague-Dawley (Taconic, Germantown, NY) rats at 6 wk of age underwent thoracic aortic constriction (TAC) or sham operation via a left thorocotomy. The aorta was partially constricted with a silk suture to the diameter of an 18-gauge needle. Age-matched animals underwent sham operation and served as controls.

In vivo echocardiography. Before isolated heart experimentation, animals were anesthetized with isoflurane, and echocardiographic images of the left ventricle were obtained by using a 7.5-MHz transducer at the level of the papillary muscles to assess left ventricular geometry and performance. Anterior and posterior wall thickness was measured during diastole utilizing the leading-edge method as recommended by the American College of Cardiology (24). Fractional shortening was calculated after measuring left ventricular end-diastolic and -systolic dimension (LVEDD and LVESD, respectively) using the formula: LVEDD – LVESD/LVEDD x 100.

Ex vivo cardiac perfusion. The stability of the isolated heart preparation on the Langendorff apparatus was observed over 3.5 h of continuous perfusion to confirm that the perfusion setup would support the isolated heart for the duration of the longest experimental protocol (n = 3 animals). For ex vivo cardiac perfusion studies, randomly selected animals 8 wk after surgery were anesthetized (ketamine 90 mg/kg and xylazine 10 mg/kg) and heparinized (500 U) via intraperitioneal injection. The hearts were rapidly excised and arrested in iced, oxygenated (95% O2-5% CO2) buffer, mounted on a Langendorff apparatus, and perfused with Krebs-Henseleit buffer containing (in mmol/l) 10 glucose, 118 NaCl, 2.5 CaCl2, 4.7 KCl, and 25.0 NaHCO3 (pH 7.4) in a nonrecirculating mode at a constant pressure of 90 mmHg as previously described (5). All hearts were mounted and perfused within 40 s of removal from the chest. The buffer was maintained at 37°C and oxygenated throughout the duration of the experiments. A water-filled latex balloon connected to a pressure transducer (AD Instruments) was inserted into the left ventricle through an incision in the left atrium. The pressure transducer was coupled to a Powerlab 4SP (AD Instruments) data recording system. Left ventricular end-diastolic pressure was set at 6–8 mmHg, and the volume of the balloon was left unchanged during the experiment. Hearts were equilibrated for 10 min before any intervention. Hearts that could not achieve a minimal developed pressure of 85 mmHg at the end of the equilibration period were discarded. Hearts from aortic-banded rats and sham-operated animals were randomized into three treatment groups (Fig. 1). Hearts in group I (TAC and sham, n = 6), the control group, were subjected to 30 min of global, normothermic ischemia, and 120 min of reperfusion. Group II hearts (TAC, n = 6; and sham, n = 8) were subjected to IPC (5 min of ischemia, followed by 5 min of perfusion) before 30 min of global ischemia and 120 min of reperfusion. Group III hearts (TAC and sham, n = 6) were treated with the JAK-2 inhibitor AG-490 (AG; 10 µM by infusion for 20 min after equilibration), IPC, and then 30 min of global ischemia and 120 min of reperfusion. Cardiac function was assessed at the end of equilibration and again at the end of reperfusion by determining the maximum and minimum first derivative of developed pressure (+dP/dtmax and –dP/dtmin, respectively, in mmHg/s). Coronary flow was determined by timed collection of cardiac effluent throughout the reperfusion phase.


Figure 1
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  		Fig. 1. Time course of experimental protocols. Hearts from animals undergoing thoracic aortic constriction (TAC) or sham operation were randomized to three experimental protocols. IPC, ischemic preconditioning; n, number of hearts.

 
Protein isolation and Western blot analysis of STAT proteins. At the completion of the perfusion experiments, the left ventricles were frozen in liquid nitrogen, and nuclear fractions were isolated to identify activated and translocated STAT protein. Preparation of nuclear extracts were performed by using NXTRACT (Sigma-Aldrich Chemical, St. Louis, MO), according to the manufacturer's recommendations with the addition of phosphatase inhibitors containing (in mM) 200 imidazole, 100 NaFl, 115 sodium molybdate, 100 sodium orthovandate, and 400 sodium tartrate dehydrate, comparable to Phosphatase Inhibitor Cocktail Set II (Calbiochem, LaJolla, CA). Total protein concentrations were determined by using the BCA Protein Assay Kit (Pierce, Rockford, IL).

Aliquots of nuclear fractions, corresponding to 75 µg of protein, were separated on a 10% SDS-PAGE gel (Gene Mate Express gels, ISC Bioexpress, Salt Lake City, UT), transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA), blocked with 5% bovine serum albumin-TBS-Tween, and analyzed with a primary polyclonal antibody against phosphorylated STAT-1 (1:600, Chemicon, Temecula, CA) and primary monoclonal antibodies against STAT-3 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA) and STAT-5 (1:400, Chemicon, Temecula, CA). The same immunoblots were stripped and reprobed with primary polyclonal antibody against total STAT-1 (1:200, Santa Cruz Biotechnology), STAT-3 (1:1,000, Chemicon), and STAT-5 (1:200, Santa Cruz Biotechnology). To normalize protein loading, the membranes were cut, and the lower molecular weight portion was analyzed with the primary antibody for GAPDH (1:2,500, Santa Cruz Biotechnology). Immunoreactive signals were visualized with chemiluminescence luminal reagents (ECL; Amersham Pharmacia Biotech, Piscataway, New Jersey).

Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed by two-tailed t-test or one-way ANOVA with Bonferonni post hoc analysis for multiple comparisons (SigmaStat 2.03). A P ≤ 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Myocardial response to pressure overload. After 8 wk of pressure overload, transthoracic echocardiography demonstrated a 31% increase in left ventricular mass in TAC hearts compared with sham-operated hearts (1,347 ± 58 vs. 1,028 ± 43 mg, TAC vs. sham, P < 0.001), as well as an increase in anterior (1.89 ± 0.08 vs. 1.55 ± 0.06 mm, TAC vs. sham, P < 0.005) and posterior wall thickness (1.87 ± 0.06 vs. 1.55 ± 0.04 mm, TAC vs. sham, P < 0.001). There were no significant differences in ejection fraction (64 ± 2% vs. 63 ± 2%, TAC vs. sham, P > 0.05) or fractional shortening (40 ± 2% vs. 39 ± 1%, TAC vs. sham, P > 0.05). The time of ventricular ejection (E-time) was significantly prolonged in aortic banded rats (78.23 ± 1.7 vs. 69.11 ± 1.0 ms, TAC vs. sham, P < 0.001). Preperfusion heart weight-to-body weight ratios were significantly increased in banded animals compared with sham-operated rats (5.0 ± 0.1 vs. 3.8 ± 0.07 g/kg, TAC vs. sham, P < 0.001).

Baseline cardiac function. Isolated heart perfusion for 3.5 h resulted in a modest 14% reduction in +dP/dtmax and a 16% reduction in –dP/dtmin from baseline, confirming that the perfusion system supports experimental protocols over a length of 3.5 h. Baseline cardiodynamics of all hearts in the three experimental groups were not statistically different (Table 1).


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  		Table 1. Baseline cardiodynamics in nonhypertrophied and hypertrophied hearts

 
Effect of ischemia-reperfusion on nonhypertrophied hearts. Without IPC, 30 min of global ischemia and 120 min of reperfusion resulted in considerable impairment in contractile function. At end reperfusion, nonhypertrophied hearts recovered only 34% of their baseline developed pressure (mean baseline developed pressure, 140 ± 7 mmHg; and mean end-reperfusion developed pressure, 48 ± 6 mmHg). When compared with baseline function, there was a 28% reduction in mean +dP/dtmax (1,748 ± 183 from 6,126 ± 329 mmHg/s, end reperfusion from baseline, P < 0.001) and a 31% reduction in mean –dP/dtmin (–813 ± 120 from –2,664 ± 109 mmHg/s, end reperfusion from baseline, P < 0.001). IPC significantly improved end-reperfusion +dP/dtmax and –dP/dtmin when compared with nonpreconditioned hearts (Fig. 2).


Figure 2
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  		Fig. 2. Effect of ischemia-reperfusion and preconditioning on end-reperfusion myocardial performance as reflected by maximum and minimum first derivative of pressure (+dP/dtmax and –dP/dtmin, respectively) in hearts from sham-operated [individual rats ({circ}) and means ± SE ({blacksquare})]; n, number of hearts. *P < 0.05 vs. sham + IPC + dP/dtmax; **P < 0.05 vs. sham + IPC –dP/dtmin.

 
STAT activation and ischemia-reperfusion in nonhypertrophied myocardium. Immunoblotting with phospho-specific antibodies against nuclear STAT-1, STAT-3, and STAT-5 demonstrated increased phosphorylation of STAT-1 and STAT-3 but not STAT-5 in preconditioned hearts compared with control (Fig. 3A). Treatment with AG, a JAK-2 inhibitor, before preconditioning significantly reduced the phosphorylation of STAT-3, but there was no significant effect on STAT-1 or STAT-5 phosphorylation (Fig. 3B). Furthermore, pretreatment with AG significantly attenuated protection of +dP/dtmax (3,381 ± 368 vs. 2,044 ± 362 mmHg/s, IPC vs. IPC + AG, P < 0.05) and –dP/dtmin (–1,612 ± 156 vs. –959 ± 95 mmHg/s, IPC vs. IPC + AG, P < 0.01).


Figure 3
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  		Fig. 3. A: effect of ischemia-reperfusion alone (control) and after preconditioning (IPC) on STAT-1, STAT-3, and STAT-5 protein levels. Hearts from sham-operated animals were frozen in liquid nitrogen, and left ventricles were separated into cytosolic and nuclear fractions. Nuclear fractions were separated on a 10% SDS-PAGE gel, and membranes were probed with antibodies against total (t) and phosphorylated (p) STAT-1, STAT-3, and STAT-5 protein. There was a significant increase in pSTAT-1 and pSTAT-3 in preconditioned myocardium. No significant effect on pSTAT-5 was seen. Representative gel of pSTAT-3 is shown. *P < 0.01 vs. pSTAT sham-operated control. B: effect of AG-490 (AG) treatment after preconditioning on STAT-1, STAT-3, and STAT-5 protein levels in hearts from sham-operated animals. AG significantly attenuated the expression of pSTAT-3. There was no significant effect on pSTAT-1 or pSTAT-5. A representative gel of pSTAT-3 is shown. **P < 0.01 vs. pSTAT-3 sham + IPC.

 
Effect of ischemia-reperfusion on hypertrophied hearts. In the absence of preconditioning, hypertrophied hearts subjected to the same 30 min of ischemia and 120 min of reperfusion challenge as nonhypertrophied hearts recovered 50% of baseline +dP/dtmax compared with only 29% in nonhypertrophied hearts (2,737 ± 343 vs. 1,748 ± 183 mmHg/s, hypertrophied vs. nonhypertrophied, P < 0.05). Furthermore, IPC in hypertrophied hearts significantly improved end-reperfusion +dP/dtmax (4,648 ± 309 vs. 2,737 ± 343 mmHg/s, IPC vs. non-IPC, P < 0.005) and –dP/dtmin (–2,239 ± 205 vs. –1,215 ± 149 mmHg/s, IPC vs. non-IPC, P < 0.005) compared with the nonpreconditioned state.

STAT activation and ischemia-reperfusion in hypertrophied myocardium. Hypertrophied hearts subjected to the ischemia-reperfusion challenge without preconditioning exhibited increased expression of pSTAT-1 and pSTAT-3 but not pSTAT-5 compared with sham-operated hearts (Fig. 4A). IPC further increased nuclear pSTAT-3 expression in hypertrophied myocardium compared with nonpreconditioned hypertrophied myocardium. IPC did not significantly increase nuclear pSTAT-1 or pSTAT-5 (Fig. 4B) over that exhibited in response to ischemia-reperfusion (control) alone. Expression of activated STAT-3 in hypertrophied hearts treated with AG before the preconditioning stimulus was significantly attenuated; however, there was no significant effect on the phosphorylation of STAT-1 or STAT-5 proteins. Correspondingly, treatment with AG significantly reduced myocardial performance, +dP/dtmax (4,648 ± 309 vs. 3,241 ± 420 mmHg/s, TAC + IPC vs. TAC + IPC + AG, P < 0.05), and –dP/dtmin (–2,239 ± 205 vs. 1,323 ± 85 mmHg/s, TAC + IPC vs. TAC + IPC + AG, P < 0.05) in hypertrophied preconditioned hearts compared with preconditioned hypertrophied hearts without the inhibitor (Fig. 5).


Figure 4
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  		Fig. 4. A: effect of ischemia-reperfusion without preconditioning (control) on STAT-1, STAT-3, and STAT-5 protein levels in nonhypertrophied compared with hypertrophied hearts. Ischemia-reperfusion resulted in a significant increase in pSTAT-1 and pSTAT-3 in TAC hearts compared with sham-operated hearts. No significant effect on the pSTAT-5 was seen. *P < 0.05 vs. pSTAT sham-operated control; {dagger}P < 0.05 vs. tSTAT sham-operated control. B: effect of ischemia-reperfusion alone (control) and after preconditioning (IPC) on STAT-1, STAT-3, and STAT-5 protein levels in hearts from animals subjected to TAC. Unlike the pattern found in hearts from sham-operated animals, these hypertrophied hearts exhibited a further increase in only pSTAT-3 in response to IPC. A representative gel of pSTAT-3 is shown. *P < 0.05 vs. pSTAT-3 TAC control.

 

Figure 5
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  		Fig. 5. Effect of AG on pSTAT-3 expression and myocardial performance in hypertrophied hearts. Hypertrophied hearts were subjected to 30 min of ischemia, followed by 120 min of reperfusion without IPC (TAC control), 30 min of ischemia and 120 min of reperfusion with IPC (TAC + IPC), or 30 min of ischemia and 120 min of reperfusion with IPC, and the JAK-2 inhibitor AG (TAC + IPC + AG). Left: Western blot analysis with antibody against nuclear pSTAT-3 revealed significantly increased expression in the IPC group and reduced expression, similar to control, in the presence of AG. A representative gel of pSTAT-3 is shown. Right: recovery of dP/dt in the IPC + AG group compared with IPC alone was attenuated [individual rats ({circ}) and means ± SE ({blacksquare})]. Data are means ± SE. {dagger}P < 0.05 vs. TAC control, TAC + IPC + AG; *P < 0.05 vs. TAC + IPC +dP/dtmax; **P < 0.05 vs. TAC + IPC –dP/dtmin.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results presented here provide important new information on ischemic tolerance in myocardial hypertrophy. We find that IPC is effective in protecting myocardial function from ischemia-reperfusion injury in compensated pressure-overload hypertrophy and that this protection is associated with activation of the JAK-STAT pathway. Moreover, the efficacy of IPC was attenuated when STAT-3 activation was limited by treatment with AG, the JAK-2 inhibitor. Unexpectedly, when compared with nonhypertrophied hearts, hypertrophied hearts exhibited improved ischemic tolerance even in the absence of preconditioning.

JAK-STAT signaling and myocardial protection. The JAK-STAT pathway was originally described as a mechanism for interferon signaling in immune cells. This ostensibly simple pathway is, in fact, a complex dual-function pathway that integrates membrane-to-nuclear signal transduction and gene transcription. Each of the STAT proteins can exert a different effect depending on the stimulus involved in its activation, and the opposing effects between the different subtypes may also occur (29). For example, in cardiac myocytes, STAT-1 has been reported to possess proapoptotic effects (26) in response to ischemia, whereas STAT-3 expression is cardioprotective, in part, through its antiapoptotic effect (7). In response to stimuli, such as ischemia, STAT-3 activation limited the occurrence of apoptosis in a model of permanent coronary occlusion in rats (18). This cytoprotection, as evidenced by suppression of caspase-3 activity and Bax expression, was attenuated after pretreatment with AG, the JAK-2 inhibitor, before coronary ligation.

Other reports utilizing genetically engineered animal models have similarly demonstrated that JAK-STAT signaling, in nonhypertrophied hearts, plays an important role in cardioprotection. Transgenic mice with cardiac-specific deletion of STAT-3 develop heart failure with age and exhibit greater susceptibility to doxorubicin-induced cardiac injury (10). Conversely, mice expressing cardiac-specific, constitutively active STAT-3 exhibited a 60% reduction in infarct size and diminished reactive oxygen species generation in response to ischemia-reperfusion stress compared with nontransgenic mice (20). After exposure to simulated ischemia/reoxygenation, myocytes with cardiac depletion of STAT-3 exhibited decreased steady-state STAT-3 protein levels and reduced myocyte viability compared with wild-type myocytes. Interestingly, IPC and other agents, previously reported to pharmacologically precondition, were ineffective at improving viability in the STAT-3 knockout myocytes (25). These results provide evidence that STAT-3 activation is an important mediator in regulating cell survival in response to ischemia-reperfusion stress.

JAK-STAT signaling and myocardial hypertrophy. Hypertrophic stimuli (pressure overload and mechanical stretch), ischemia and ischemia/reoxygenation can activate cardiac JAK-STAT signaling (21, 26). In fact, angiotensin II production via the cardiac renin-angiotensin system may serve as an endogenous initiator of the JAK-STAT pathway via G protein-coupled receptor signaling (4). Although functional protection or postischemic myocyte viability was not investigated, Omura et al. (19) demonstrated STAT-3 phosphorylation in rats induced by coronary occlusion and prevented by pretreatment with angiotensin II receptor blockade.

Transgenic mice with cardiac-specific overexpression of STAT-3 develop myocardial hypertrophy by 12 wk of age and are protected from doxorubicin-induced cardiomyopathy (12). These data and others (14, 27) suggest that constitutive JAK-STAT activation in pressure-overloaded animals may exert a cardioprotective signal that induces ischemic tolerance. Our results showed that after ischemia-reperfusion challenge (in the absence of IPC), nonhypertrophied and hypertrophied hearts demonstrated increased phosphorylation of STAT-3 with no effect on activation of STAT-5. In the hypertrophied group, ischemia-reperfusion in the absence of preconditioning increased activation of STAT-1 over that seen in nonhypertrophied hearts. However, IPC in hypertrophied hearts did not significantly increase the phosphorylation of STAT-1 over that already present in hypertrophied, nonpreconditioned hearts. This suggests that STAT-1 may be maximally upregulated, perhaps in response to hypertrophy and/or ischemia-reperfusion, and further activation of STAT-1 by IPC may not be detectable. This profile of STAT activation is different from that identified in nonhypertrophied hearts and may account for the improved ischemic tolerance exhibited in our model of pressure-overload hypertrophy.

Possible mechanisms. It is unclear how the JAK-STAT pathway affects cardioprotection after early-phase (classic) preconditioning. After late-phase preconditioning, JAK-STAT signaling may promote cell survival by diminishing myocyte apoptosis (1). However, the response to early-phase preconditioning develops quickly and is characterized by posttranslational protein modification, not de novo protein synthesis (6). Other possible mechanisms include activation of supplementary intracellular kinases, e.g., MAPK (7), modification of antioxidant defenses through an upregulation of superoxide dismutase (17), or upregulation of inducible nitric oxide synthase (3).

The ability of STAT molecules to form heterodimers with other STAT subtypes, in particular STAT-1, may also alter the cellular response to stress and, in fact, may be ligand dependent (9). The specificity of STAT signaling depends on the interaction between the STAT SH2 domain and receptor phosphotyrosine motifs (8). The effect of STAT activation may trigger different cellular responses depending on many factors, and it is this variability that may account for different patterns of protein expression reported from studies using diverse experimental protocols and models. Obviously, further in-depth work is necessary to identify the posttranslational role for STAT-3 in early-phase preconditioning and to clarify its influence in hypertrophied myocardium.

In conclusion, our data suggest a compelling role for activated STAT-3 in the cardioadaptive response to ischemia-reperfusion injury in a clinically relevant model of myocardial hypertrophy. The present results emphasize that a hypertrophied heart is capable of responding counterintuitively to an ischemia-reperfusion stress in a favorable manner. Furthermore, we have demonstrated that this stress response is facilitated by a pattern of JAK-STAT activation different from that identified in nonhypertrophied myocardium. The finding of decreased STAT-3 levels in left ventricle samples from patients with dilated cardiomyopathy compared with nonfailing controls (22) suggests that the JAK-STAT pathway is an important link in the transition from compensated hypertrophy to heart failure. Further characterization of this pathway may potentially minimize the deleterious effects of cardiac remodeling in response to stress.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a National Heart, Lung, and Blood Institute Grant K-08-HL-68867 (to K. L. Butler).


   ACKNOWLEDGMENTS
 
The authors thank Dr. Geraldine Fuller-Bicer for assistance in preparation of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. L. Butler, Univ. of Cincinnati, Dept. of Surgery, Division of Trauma/Critical Care, Inst. of Molecular Pharmacology & Biophysics, 231 Albert B. Sabin Way, Cincinnati, OH 45267-0828 (e-mail: karyn.butler{at}uc.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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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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