121. Compensatory STAT Subtype Activation does not Limit Ischemia-Reperfusion Injury in Mice with Cardiac Restricted STAT-3 Deletion

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS and protection from LPS-mediated apoptosis (ctrl: 11⫾2%, LPS: 27⫾5%, LPS⫹heat: 18⫾2%, p⬍0.05), while inhibition of Hsp70 using the specific inhibitor quercetin (10uM, 2h) or Hsp70 siRNA (100nM) significantly increased TLR4 signaling as measured by expression of iNOS relative to that of Actin (control: 1, LPS 6h: 7.09, LPS⫹ Hsp70 siRNA 6h: 11.57, p⬍0.05 ANOVA) and fold increase of IL-6 release relative to untreated controls (Quercetin: 1, Quercetin ⫹ LPS 6h: 5.5, p⬍0.05 ). In support of the potential physiological relevance of Hsp70 induction as an inhibitor of TLR4 signaling, LPS-mediated enterocyte apoptosis was significantly increased after the inhibition of Hsp70 (% apoptosis: control 11⫾2%, LPS 27⫾5%, LPS⫹Hsp70 siRNA 24⫾5%, p⬍0.05 ANOVA), while the expression of Hsp70 was reduced in the intestinal mucosa of mice and humans with NEC in which enterocyte apoptosis was increased. Strikingly, LPS injection caused a significant increase in enterocyte apoptosis in Hsp70 knockout mice compared to wild-type counterparts, confirming the in vivo relevance of these findings. Conclusions: These data reveal a novel mechanism by which the host regulates TLR4 signaling in enterocytes through the induction of Hsp70. These findings also shed light on the pathways that lead to exaggerated TLR4-mediated intestinal inflammation in which Hsp70 expression is impaired, such as NEC. PL8. INSULIN REGULATES THE EFFICACY OF NITRIC OXIDE IN TYPE I DIABETES. V. Varu, S. S. Ahanchi, H. A. Bhikhapurwala, A. C. Chen, J. Martinez, M. R. Kibbe; Northwestern University, Chicago, IL Introduction: Diabetes mellitus (DM) is responsible for a myriad of negative effects in the vasculature, leading to greater restenosis from neointimal hyperplasia following vascular interventions. Recently, we found that local application of nitric oxide (NO) decreased neointimal hyperplasia more in type II DM animals versus controls. However, the effect of NO in type I DM is unknown. Therefore, the aim of this study is to determine the efficacy of NO at inhibiting neointimal hyperplasia following arterial injury in vivo and its effects on vascular smooth muscle cell (VSMC) proliferation in vitro in a rodent model of type I DM with and without exogenous insulin administration. We hypothesize that NO will inhibit neointimal hyperplasia more effectively in a type I DM model. Methods: For in vivo experiments, type I DM was induced in male 11-week-old Lean Zucker (LZ) rodents with a single injection of streptozotocin (STZ; 60 mg/kg). Animals with glucose concentrations ⬎300 mg/dL were considered diabetic and included in the study. Non-STZ LZ animals served as controls. Neointimal hyperplasia was assessed using the rat carotid artery injury model. Treatment groups for LZ and LZ⫹STZ animals included: injury, injury⫹insulin, injury⫹the NO donor PROLI/NO, and injury⫹insulin⫹PROLI/NO. Vessels were harvested at 14 days for morphometric analysis. For in vitro experiments, aortic VSMC from LZ rodents were exposed to the NO donor DETA/NO (0.5-1 mM) ⫾ glucose (5-25 mM), ⫾ insulin (100-200 nm). Proliferation was assessed by [3H]-thymidine incorporation. Results: Cholesterol, triglyceride, insulin, and glucose levels were measured confirming the type I DM disease state. Following arterial injury, neointimal hyperplasia was significantly lower in the type I DM rodents versus LZ controls as measured by the intima/media area ratio (I/M; 0.603 vs 0.866, p⬍0.05). Surprisingly, the type I DM animals experienced no significant reduction in neointimal hyperplasia following treatment with PROLI/NO (19% increase, p⫽0.133), while PROLI/NO significantly inhibited neointimal hyperplasia in LZ controls (51%, p⬍0.05). In type I DM animals that received daily injections of insulin, neointimal hyperplasia was significantly higher than LZ control rodents (I/M 1.59 vs 0.866, p⬍0.05). Treatment with PROLI/NO in animals receiving insulin resulted in an unexpected and significant reduction in neointimal hyperplasia (62% reduction, p⬍0.05.) In vitro experiments supported these data. DETA/NO inhibited proliferation in a concentration-dependent manner to a greater extent in euglycemic (5mM) VSMC (56%, p⬍0.05) compared to hyperglycemic (25mM) VSMC (type I DM model) (46%, p⬍0.05).

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With the addition of exogenous insulin, DETA/NO inhibited proliferation more effectively in both euglycemic and hyperglycemic environments (65% and 64%, resp, p⬍0.05). Conclusion: NO was completely ineffective at inhibiting neointimal hyperplasia in an uncontrolled model of type I DM; however, following administration of insulin, the efficacy of NO was restored. These data demonstrate the markedly different effects of NO in type I versus type II DM, and reveal that insulin may regulate the downstream beneficial effects of NO. Further research is required to elucidate the mechanism by which NO exerts its effects in these different disease states, as many patients with vascular disease have type I and type II DM and have an even greater need of therapy to reduce restenosis rates after interventions.

ORAL SESSION: THURSDAY 2/5 7:30 AM CARDIOTHORACIC 2: ISCHEMIA/ REPERFUSION 121. COMPENSATORY STAT SUBTYPE ACTIVATION DOES NOT LIMIT ISCHEMIA-REPERFUSION INJURY IN MICE WITH CARDIAC RESTRICTED STAT-3 DELETION. M. D. Goodman1, S. E. Koch1, M. R. Afzal1, K. L. Butler2; 1University of Cincinnati, Cincinnati, OH; 2Hartford Hospital, Hartford, CT Objective: We have previously demonstrated that activation of STAT-3 is essential for ischemic preconditioning. The role of other STAT subtypes in conferring ischemic tolerance however, is unclear. We hypothesized that compensatory upregulation of other STAT subtypes in the presence of myocardial STAT-3 deletion would protect myocardial contractile function against ischemia-reperfusion injury. Methods: Wild-type (WT), male C57BL/6 mice or mice with cardiomyocyte-specific STAT-3 deletion (KO) were evaluated with transthoracic 2D echocardiography to determine baseline in vivo myocardial performance, defined by fractional shortening (%FS) and ejection fraction (%EF). Animals were then randomized to receive ischemic preconditioning (IPC) or no-IPC (control) prior to undergoing ischemia/ reperfusion (I/R) challenge. Following 10 minutes of equilibration, Langendorff perfused hearts were subjected to 30 minutes global ischemia/40 minutes reperfusion ⫾ IPC (5 minutes ischemia/5 minutes reperfusion, n⫽6 per group). Cardiac performance was assessed throughout each perfusion protocol and expressed as the maximum first derivative of the developed pressure (dp/dtmax, mmHg/s). Following ex vivo perfusion, hearts were analyzed for STAT-5 and ⫺6 phosphorylation (p) by Western immunoblotting of nuclear fractions. Data are expressed as mean ⫾SEM. Results: Echocardiography revealed no differences in cardiac function (29⫾4% vs. 24⫾3% FS, WT vs. KO, P⫽0.35; 62⫾7% vs. 55⫾5% EF, WT vs. KO, P⫽0.4) between WT and KO hearts. Similarly, there were no differences in ex vivo performance between WT and KO hearts at baseline (3042⫾97 mmHg/s vs. 2753⫾161 mmHg/s, WT vs. KO, P⫽0.13). The expression of pSTAT-5 and pSTAT-6 was similar in WT and KO hearts prior to perfusion. Myocardial contractile function in WT and KO hearts was significantly impaired following I/R challenge in the absence of IPC (929⫾180 mmHg/s vs. 732⫾211mmHg/s, WT/no-IPC vs. KO/no-IPC, P⫽0.45). In WT hearts IPC significantly improved recovery of dp/dt max compared to hearts subjected to I/R alone (2760⫾188 mmHg/s vs. 929⫾180 mmHg/s, WT/IPC vs. WT/no-IPC, P⬍0.001). By contrast, IPC was not effective in protecting contractile function against I/R injury in KO hearts (1319⫾229 mmHg/s vs. 732⫾211 mmHg/s, KO/IPC vs. KO/no-IPC, P⫽0.07). IPC more effectively improved end-reperfusion dp/dt max in WT hearts compared with KO hearts (2760⫾188 mmHg/s vs. 1319⫾229 mmHg/s, WT/IPC vs. KO/IPC, P⬍0.001). Following ischemia/ reperfusion, preconditioned and non-preconditioned KO hearts exhibited increased pSTAT-5 and ⫺6 expression compared to WT. The increased activation of these STAT subtypes did not improve the efficacy of IPC in KO hearts. Conclusions: Baseline cardiac performance is preserved in hearts with cardiac-restricted STAT-3 deletion. STAT-3

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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS

deletion attenuates preconditioning and is not associated with compensatory upregulation of STAT-5 and-6 subtypes. Moreover, activation of STAT-5 and ⫺6 in KO hearts following ischemic challenge does not provide functional compensation for the loss of STAT-3. JAK-STAT signaling via STAT-3 is essential for effective ischemic preconditioning and may represent an important target for pharmacologic modulation of cardiac recovery from ischemia/reperfusion injury. 122. BOTH ENDOGENOUS AND EXOGENOUS TESTOSTERONE DECREASE MYOCARDIAL STAT3 ACTIVATION AND SOCS3 EXPRESSION FOLLOWING ACUTE ISCHEMIA AND REPERFUSION. M. Wang, Y. Wang, J. Tan, J. Herrmann, A. Abarbanell, B. Weil, D. R. Meldrum; Indiana University School of Medicine, Indianapolis, IN Background: Signal transducer and activator of transduction 3 (STAT3) pathway has been shown to be cardioprotective. We also observed decreased STAT3/suppressor of cytokine signaling 3 (SOCS3) in male hearts, which was associated with worse post-ischemic myocardial function compared to females. However, it is unknown whether this down-regulation of myocardial STAT3/SOCS3 is due to testosterone in males. We hypothesized that following I/R: 1) endogenous testosterone decreases myocardial STAT3 activation and SOCS3 expression in male hearts; 2) administration of exogenous testosterone reduces myocardial STAT3/SOCS3 in female and castrated male hearts. Methods: To study this, Langendorff perfused rat hearts subjected to I/R injury were homogenized and assessed for phosphorylated-STAT3 (p-STAT3), totalSTAT3 (T-STAT3), SOCS3 and GAPDH by western blot. Groups: agematched adult males, females, castrated males (castr), males with flutamide (Flut) implantation (3-week release pellet), females and Castr males supplement with chronic (3-week) exogenous testosterone via 5alpha-dihydrotestosterone (DHT) release pellet implantation (n⫽ 5-8/group). Data were analyzed with student’s t-test, p⬍0.05⫽ statistically significant. Results: Castration or androgen receptor blockade (Flut) significantly increased SOCS3 expression (% of SOCS3/ GAPDH: Castr 98.8⫾16.1%, Flut 46.7⫾12.1% vs. 15.4⫾2.5%) in male hearts after I/R. However, only castration increased myocardial STAT3 activation (% of p-STAT3/T-STAT3: 92.2⫾24.3% vs. male 37.2⫾7.0%), but not flutamide treatment (39.9⫾11.3%). Notably, DHT replacement markedly decreased myocardial STAT3 activation and SOCS3 expression in castrated males (STAT3: DHT 42.1⫾10.1% vs. 79.9⫾13.8%; SOCS3: DHT 46.7⫾12.1% vs. 98.8⫾16.1%) and females (STAT3: DHT 39.3⫾5.5% vs. 65.9⫾7.7%; SOCS3: DHT 22.5⫾3.9% vs. 29.6⫾2.5%)) subjected to I/R. Conclusion: These results suggest that endogenous and exogenous testosterone decrease myocardial STAT3 activation and SOCS3 expression following I/R. This represents the initial demonstration of testosterone-downregulated STAT3/SOCS3 signaling in myocardium. 123. ACUTE POST-ISCHEMIC TREATMENT WITH ESTROGEN RECEPTOR-BETA AGONIST 2,3-BIS(4-HYDROXYPHENYL)-PROPIONITRILE IMPROVES MYOCARDIAL RECOVERY. N. Vornehm, M. Wang, A. Abarbanell, J. Herrmann, B. Weil, Y. Wang, D. Meldrum; Indiana University, Indianapolis, IN Background: Gender differences exist in cardiovascular disease. Female proestrus rats subjected to ischemia-reperfusion (I/R) injury developed less myocardial dysfunction than their male counterparts. Knocking out either estrogen receptor alpha or beta abolishes this effect; however, it remains unknown whether: 1) Post-ischemic treatment with the ER-␤ agonist, 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) induces cardioprotection following I/R injury, 2) Post-ischemic infusion of the ER-␣ agonist 4,4’,4”-(4-propyl-[H]-pyrazole-1,3,5triyl)trisphenol (PPT) does not induce protection following myocardial I/R injury. Methods: Isolated, perfused rat hearts (Langendorff) from adult males (n⫽4-6/group) were subjected to 25 minutes of ischemia followed by 40 minutes of reperfusion, during which time myocardial

contractile function was continuously monitored. During the entire 40 minutes of reperfusion, experimental hearts where infused with either ER-␣ agonist or ER-␤ agonist (1, 10, and 100 nM, respectively). Control hearts (n⫽7) where infused with a perfusate vehicle. Data were analyzed with two-way ANOVA, p⬍0.05⫽statistically significant. Results: Post-ischemic treatment with 10 and 100 nM of DPN significantly increased myocardial functional recovery following I-R exhibited as LVDP (60⫾9%-10nM, 52%⫾4-100nM vs. control 39%⫾5), ⫹dP/dt (67⫾10%-10nM, 56⫾2%-100nM vs. control 45⫾5%) and ⫺dP/dt (⫺65⫾9%-10nM, ⫺53⫾4%-100nM vs. control ⫺45⫾6%), with the maximum protection at 10nM. Groups treated with 10 and 100 nM DPN significantly improved myocardial function earlier during reperfusion (30 minute: 58⫾9%-10nM, 50⫾6%-100nM vs. control-27⫾3%). However, there was no significant functional recovery observed in PPT treated hearts following I-R. Conclusions: These results demonstrate that administration of ER-␤ agonist, DPN, decreases recovery time and attenuates myocardial dysfunction following I/R injury. Further clarification of these mechanisms may lead to therapeutic manipulation of estrogen receptors for clinical application in the treatment of myocardial I/R. 124. MITOCHONDRIAL RESPIRATORY COMPLEX I AND II ACTIVITIES ARE PRESERVED BY ISCHEMIC PRECONDITIONING. D. Lee, G. Steinbaugh, J. Zweier, J. A. Crestanello; The Ohio State University, Columbus, OH Mitochondrial respiratory complexes are damaged by ischemia reperfusion injury. While IPC preserves overall mitochondrial respiratory function after ischemia reperfusion, it is unclear which is the effect of IPC on individual mitochondria respiratory complexes. The purpose of this experiment was to determine the effect of IPC on mitochondrial respiratory complexes. Isolated rat hearts (n⫽6/ group) were subjected to either A) 40 minutes (min) of equilibration (EQ), 30 min of ischemia (I), and 30 min of reperfusion (RP) (CONTROL) or B) 10 min of EQ, three 5 min episodes of IPC, 30 min I, and 30 min of RP (IPC group). Interfibrillar (IF) and subsarcolemmal (SS) mitochondria were isolated at end reperfusion. Mitochondria complex I, II, and IV activities were assessed by polarography using specific substrates and inhibitors for each complex. Substrates for Complex I, II, and IV were glutamate and malate (4.7 mM), succinate (7 mM), tetramethyl-p-phenylenediamine (0.4 mM) respectively. Inhibitors for complex I, II, and III used were rotenone (188 nM), thenoyltrifluoroacetone (0.4 mM), and antimycin A (18.8 nM). Data is expressed as mean⫾SEM. Ischemia reperfusion impairs mitochondrial complex I, II and IV activity. IPC preserves both complex I and II activity in subsarcolemmal and interfibrillar mitochondria at end reperfusion. Complex IV activity is not affected by IPC. Selective protection of mitochondrial complex I and II during reperfusion is the mechanism responsible for preservation of respiration by IPC. Complex I

CONTROL IPC

Complex II

Complex IV

SS

IF

SS

IF

SS

IF

84 ⫾ 5 130 ⫾ 7*

55 ⫾ 4 99 ⫾ 12*

52 ⫾ 2 71 ⫾ 3*

24 ⫾ 2 35 ⫾ 3*

58 ⫾ 2 60 ⫾ 4

46 ⫾ 4 66 ⫾ 6

Data expressed in ng atoms O/mg protein. * p⬍0.05 vs CONTROL. 125. EUROSCORE FOR THE PATIENT UNDERGOING OFFPUMP CORONARY ARTERY BYPASS PREDICTS POSTOPERATIVE MORTALITY, CERTAIN MORBIDITIES, AND LENGTH OF STAY. H. Hirose, K. Tambara, H. Inaba, T. Yamamoto, M. Yamasaki, K. Kikuchi, A. Amano; Department of Cardiovascular Surgery, Juntendo University Hospital, Tokyo, Japan