Sex differences in the myocardial inflammatory response to ischemia-reperfusion injury

The myocardium generates inflammatory mediators during ischemia-reperfusion (I/R), and these mediators contribute to cardiac functional depression and apoptosis. The great majority of these data have been derived from male animals and humans. Sex has a profound effect over many inflammatory responses; however, it is unknown whether sex affects the cardiac inflammatory response to acute myocardial I/R. We hypothesized the existence of inherent sex differences in myocardial function, expression of inflammatory cytokines, and activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway after I/R. Isolated rat hearts from age-matched adult males and females were perfused (Langendorff), and myocardial contractile function was continuously recorded. After I/R, myocardium was assessed for expression of TNF-alpha, IL-1beta, and IL-6 (RT-PCR, ELISA); IL-1alpha and IL-10 mRNA (RT-PCR); and activation of p38 MAPK (Western blot). All indexes of postischemic myocardial function [left ventricular developed pressure, left ventricular end-diastolic pressure, and maximal positive (+dP/dt) and negative (-dP/dt) values of the first derivative of pressure] were significantly improved in females compared with males. Compared with males, females had decreased myocardial TNF-alpha, IL-1beta, and IL-6 (mRNA, protein) and decreased activation of p38 MAPK pathway. These data demonstrate that hearts from age-matched adult females are relatively protected against I/R injury, possibly due to a diminished inflammatory response.     

Deficiency of TNFR1 protects myocardium through SOCS3 and IL-6 but not p38 MAPK or IL-1beta

Tumor necrosis factor-alpha (TNF-alpha) plays an important role in the development of heart failure. There is a direct correlation between myocardial function and myocardial TNF levels in humans. TNF may induce local inflammation to exert tissue injury. On the other hand, suppressors of cytokine signaling (SOCS) proteins have been shown to inhibit proinflammatory signaling. However, it is unknown whether TNF mediates myocardial inflammation via STAT3/SOCS3 signaling in the heart and, if so, whether this effect is through the type 1 55-kDa TNF receptor (TNFR1). We hypothesized that TNFR1 deficiency protects myocardial function and decreases myocardial IL-6 production via the STAT3/SOCS3 pathway in response to TNF. Isolated male mouse hearts (n = 4/group) from wild-type (WT) and TNFR1 knockout (TNFR1KO) were subjected to direct TNF infusion (500 pg.ml(-1).min(-1) x 30 min) while left ventricular developed pressure and maximal positive and negative values of the first derivative of pressure were continuously recorded. Heart tissue was analyzed for active forms of STAT3, p38, SOCS3 and SOCS1 (Western blot analysis), as well as IL-1beta and IL-6 (ELISA). Coronary effluent was analyzed for lactate dehydrogenase (LDH) activity. As a result, TNFR1KO had significantly better myocardial function, less myocardial LDH release, and greater expression of SOCS3 (percentage of SOCS3/GAPDH: 45 +/- 4.5% vs. WT 22 +/- 6.5%) after TNF infusion. TNFR1 deficiency decreased STAT3 activation (percentage of phospho-STAT3/STAT3: 29 +/- 6.4% vs. WT 45 +/- 8.8%). IL-6 was decreased in TNFR1KO (150.2 +/- 3.65 pg/mg protein) versus WT (211.4 +/- 26.08) mice. TNFR1 deficiency did not change expression of p38 and IL-1beta following TNF infusion. These results suggest that deficiency of TNFR1 protects myocardium through SOCS3 and IL-6 but not p38 MAPK or IL-1beta.     

STAT-3 activation is necessary for ischemic preconditioning in hypertrophied myocardium

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/dt(max); 4,648 +/- 309 vs. 2,737 +/- 343 mmHg/s, IPC vs. non-IPC, P < 0.05) and minimum -dP/dt (-dP/dt(min); -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/dt(max) (4,648 +/- 309 vs. 3,241 +/- 420 mmHg/s, IPC vs. IPC + AG, P < 0.05) and -dP/dt(min) (-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.     

Inhibition of p38 reduces myocardial infarction injury in the mouse but not pig after ischemia-reperfusion

The MAPK family member p38 is activated in the heart after ischemia-reperfusion (I/R) injury. However, the cardioprotective vs. proapoptotic effects associated with p38 activation in the heart after I/R injury remain unresolved. Another issue to consider is that the majority of past studies have employed the rodent as a model for assessing p38's role in cardiac injury vs. protection, while the potential regulatory role in a large animal model is even more uncertain. Here we performed a parallel study in the mouse and pig to directly compare the extent of cardiac injury after I/R at baseline or with the selective p38 inhibitor SB-239063. Infusion of SB-239063 5 min before ischemia in the mouse prevented ischemia-induced p38 activation, resulting in a 25% reduction of infarct size compared with vehicle-treated animals (27.9 +/- 2.9% vs. 37.5 +/- 2.7%). In the pig, SB-239063 similarly inhibited myocardial p38 activation, but there was no corresponding effect on the degree of infarction injury (43.6 +/- 4.0% vs. 41.4 +/- 4.3%). These data suggest a difference in myocardial responsiveness to I/R between the small animal mouse model and the large animal pig model, such that p38 activation in the mouse contributes to acute cellular injury and death, while the same activation in pig has no causative effect on these parameters.     

SDF-1/CXCR4 mediates acute protection of cardiac function through myocardial STAT3 signaling following global ischemia/reperfusion injury

Stromal cell-derived factor-1α (SDF-1) has been reported to mediate cardioprotection through the mobilization of stem cells into injured tissue and an increase in local angiogenesis after myocardial infarction. However, little is known regarding whether SDF-1 induces acute protection following global myocardial ischemia/reperfusion (I/R) injury and if so, by what molecular mechanism. SDF-1 binding to its cognate receptor CXCR4 has been shown to activate STAT3 in a variety of cells. STAT3 is a cardioprotective factor and may mediate SDF-1/CXCR4-induced acute protection. We hypothesized that SDF-1 would improve myocardial function through CXCR4-increased STAT3 activation following acute I/R. Isolated mouse hearts were subjected to 25-min global ischemia/40-min reperfusion and divided into groups of 1) vehicle; 2) SDF-1; 3) AMD3100, a CXCR4 inhibitor; 4) SDF-1 + AMD3100; 5) Stattic, a STAT3 inhibitor; 6) SDF-1 + Stattic; 7) cardiomyocyte-restricted ablation of STAT3 (STAT3KO); 8) STAT3KO + SDF-1; 9) Ly294002, an inhibitor of the Akt pathway; and 10) SDF-1 + Ly294002. Reagents were infused into hearts within 5 min before ischemia. SDF-1 administration significantly improved postischemic myocardial functional recovery in a dose-dependent manner. Additionally, pretreatment with SDF-1 reduced cardiac apoptotic signaling and increased myocardial STAT3 activation following acute I/R. Inhibition of the SDF-1 receptor CXCR4 neutralized these protective effects by SDF-1 in hearts subjected to I/R. Notably, inhibition of the STAT3 pathway or use of STAT3KO hearts abolished SDF-1-induced acute protection following myocardial I/R. Our results represent the first evidence that the SDF-1/CXCR4 axis upregualtes myocardial STAT3 activation and, thereby, mediates acute cardioprotection in response to global I/R.     

STAT subtype specificity and ischemic preconditioning in mice: is STAT-3 enough?

The role of other STAT subtypes in conferring ischemic tolerance is unclear. We hypothesized that in STAT-3 deletion alternative STAT subtypes would protect myocardial function against ischemia-reperfusion injury. Wild-type (WT) male C57BL/6 mice or mice with cardiomyocyte STAT-3 knockout (KO) underwent baseline echocardiography. Langendorff-perfused hearts underwent ischemic preconditioning (IPC) or no IPC before ischemia-reperfusion. Following ex vivo perfusion, hearts were analyzed for STAT-5 and -6 phosphorylation by Western blot analysis of nuclear fractions. Echocardiography and postequilibration cardiac performance revealed no differences in cardiac function between WT and KO hearts. Phosphorylated STAT-5 and -6 expression was similar in WT and KO hearts before perfusion. Contractile function in WT and KO hearts was significantly impaired following ischemia-reperfusion in the absence of IPC. In WT hearts, IPC significantly improved the recovery of the maximum first derivative of developed pressure (+dP/dtmax) compared with that in hearts without IPC. IPC more effectively improved end-reperfusion dP/dtmax in WT hearts compared with KO hearts. Preconditioned and nonpreconditioned KO hearts exhibited increased phosphorylated STAT-5 and -6 expression compared with WT hearts. The increased subtype activation did not improve the efficacy of IPC in KO hearts. In conclusion, baseline cardiac performance is preserved in hearts with cardiac-restricted STAT-3 deletion. STAT-3 deletion attenuates preconditioning and is not associated with a compensatory upregulation of STAT-5 and -6 subtypes. The 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 IPC.     

Inhibition of p38 MAPK and AMPK restores adenosine-induced cardioprotection in hearts stressed by antecedent ischemia by altering glucose utilization

p38 mitogen-activated protein kinase (MAPK) and 5'-AMP-activated protein kinase (AMPK) are activated by metabolic stresses and are implicated in the regulation of glucose utilization and ischemia-reperfusion (IR) injury. This study tested the hypothesis that inhibition of p38 MAPK restores the cardioprotective effects of adenosine in stressed hearts by preventing activation of AMPK and the uncoupling of glycolysis from glucose oxidation. Working rat hearts were perfused with Krebs solution (1.2 mM palmitate, 11 mM [(3)H/(14)C]glucose, and 100 mU/l insulin). Hearts were stressed by transient antecedent IR (2 x 10 min I/5 min R) before severe IR (30 min I/30 min R). Hearts were treated with vehicle, p38 MAPK inhibitor (SB-202190, 10 microM), adenosine (500 microM), or their combination before severe IR. After severe IR, the phosphorylation (arbitrary density units) of p38 MAPK and AMPK, rates of glucose metabolism (micromol x g dry wt(-1) x min(-1)), and recovery of left ventricular (LV) work (Joules) were similar in vehicle-, SB-202190- and adenosine-treated hearts. Treatment with SB-202190 + adenosine versus adenosine alone decreased p38 MAPK (0.03 +/- 0.01, n = 3 vs. 0.48 +/- 0.10, n = 3, P < 0.05) and AMPK (0.00 +/- 0.00, n = 3 vs. 0.26 +/- 0.08, n = 3 P < 0.05) phosphorylation. This was accompanied by attenuated rates of glycolysis (1.51 +/- 0.40, n = 7 vs. 3.95 +/- 0.65, n = 7, P < 0.05) and H(+) production (2.12 +/- 0.76, n = 7 vs. 6.96 +/- 1.48, n = 7, P < 0.05), and increased glycogen synthesis (1.91 +/- 0.25, n = 6 vs. 0.27 +/- 0.28, n = 6, P < 0.05) and improved recovery of LV work (0.81 +/- 0.08, n = 7 vs. 0.30 +/- 0.15, n = 8, P < 0.05). These data indicate that inhibition of p38 MAPK abolishes subsequent phosphorylation of AMPK and improves the coupling of glucose metabolism, thereby restoring adenosine-induced cardioprotection.     

Simvastatin reverses cardiac hypertrophy caused by disruption of the bradykinin 2 receptor

Bradykinin 2 receptor (B2R) deficiency predisposes to cardiac hypertrophy and hypertension. The pathways mediating these effects are not known. Two-month-old B2R knockout (KO) and wild-type (WT) mice were assigned to 4 treatment groups (n = 12-14/group): control (vehicle); nitro-l-arginine methyl ester (l-NAME) an NO synthase inhibitor; simvastatin (SIM), an NO synthase activator; and SIM+l-NAME. Serial echocardiography was performed and blood pressure (BP) at 6 weeks was recorded using a micromanometer. Myocardial eNOS and mitogen-activated protein kinase (MAPK, including ERK, p38, and JNK) protein expression were measured. Results showed that (i) B2RKO mice had significantly lower ejection fraction than did WT mice (61% +/- 1% vs. 73% +/- 1%), lower myocardial eNOS and phospho-eNOS, normal systolic BP, and higher LV mass, phospho-p38, and JNK; (ii) l-NAME increased systolic BP in KO mice (117 +/- 19 mm Hg) but not in WT mice and exacerbated LV hypertrophy and dysfunction; and (iii) in KO mice, SIM decreased hypertrophy, p38, and JNK, improved function, increased capillary eNOS and phospho-eNOS, and prevented l-NAME-induced LV hypertrophy without lowering BP. We conclude that disruption of the B2R causes maladaptive cardiac hypertrophy with myocardial eNOS downregulation and MAPK upregulation. SIM reverses these abnormalities and prevents the development of primary cardiac hypertrophy as well as hypertrophy secondary to l-NAME-induced hypertension.     

Regulating RISK: a role for JAK-STAT signaling in postconditioning?

Postconditioning (POC), a novel strategy of cardioprotection against ischemia-reperfusion injury, is clinically attractive because of its therapeutic application at the predictable onset of reperfusion. POC activates several intracellular kinase signaling pathways, including phosphatidylinositol 3-kinase (PI3K)-Akt (RISK). The regulation of POC-induced survival kinase signaling, however, has not been fully characterized. JAK-STAT activation is integral to cardiac ischemic tolerance and may provide upstream regulation of RISK. We hypothesized that POC requires the activation of both JAK-STAT and RISK signaling. Langendorff-perfused mouse hearts were subjected to 30 min of global ischemia and 40 min of reperfusion, with or without POC immediately after ischemia. A separate group of POC hearts was treated with AG 490, a JAK2 inhibitor, Stattic, a specific STAT3 inhibitor, or LY-294002, a PI3K inhibitor, at the onset of reperfusion. Cardiomyocyte-specific STAT3 knockout (KO) hearts were also subjected to non-POC or POC protocols. Myocardial performance (+dP/dt(max), mmHg/s) was assessed throughout each perfusion protocol. Phosphorylated (p-) STAT3 and Akt expression was analyzed by Western immunoblotting. POC enhanced myocardial functional recovery and increased expression of p-STAT3 and p-Akt. JAK-STAT inhibition abrogated POC-induced functional protection. STAT3 inhibition decreased expression of both p-STAT3 and p-Akt. PI3K inhibition also attenuated POC-induced cardioprotection and reduced p-Akt expression but had no effect on STAT3 phosphorylation. Interestingly, STAT3 KO hearts undergoing POC exhibited improved ischemic tolerance compared with KO non-POC hearts. POC induces myocardial functional protection by activating the RISK pathway. JAK-STAT signaling, however, is insufficient for effective POC without PI3K-Akt activation.     

Coronary effluent from a preconditioned heart activates the JAK-STAT pathway and induces cardioprotection in a donor heart

Preconditioning (PC) protects against ischemia-reperfusion (I/R) injury via the activation of the JAK-STAT pathway. We hypothesized that the mediators responsible for PC can be transferred to naive myocardium through the coronary effluent. Langendorff-perfused hearts from male Sprague-Dawley rats were randomized to paired donor/acceptor protocols with or without PC in the presence or absence of the JAK-2 inhibitor AG-490 (n = 6 for each group). Warmed, oxygenated coronary effluent collected during the reperfusion phases of PC (3 cycles of 5 min ischemia and 5 min reperfusion) was administered to acceptor hearts. The hearts were then subjected to 30 min ischemia and 40 min reperfusion. The left ventricles were analyzed for phosphorylated (p)STAT-1, pSTAT-3, Bax, Bcl, Bcl-X(L)/Bcl-2-associated protein (BAD), and caspase-3 expression by Western blot. A separate group of hearts (n = 6) was analyzed for STAT activation immediately after the transfer of the PC effluent (no I-R). Baseline cardiodynamics were not different among the groups. End-reperfusion maximal change in pressure over time (+dP/dt(max)) was significantly (P < 0.05) improved in acceptor PC (3,637 +/- 199 mmHg/s) and donor PC (4,304 +/- 347 mmHg/s) hearts over non-PC donor (2,020 +/- 363 mmHg/s) and acceptor (2,624 +/- 345 mmHg/s) hearts. Similar differences were seen for minimal change in pressure over time (-dP/dt(min)). STAT-3 activation was significantly increased in donor and acceptor PC hearts compared with non-PC hearts. Conversely, pSTAT-1 and Bax expression was decreased in donor and acceptor PC hearts compared with non-PC hearts. No differences in Bcl, BAD, or caspase-3 expression were observed. Treatment with AG-490 attenuated the recovery of +/-dP/dt in acceptor PC hearts and significantly reduced pSTAT-3 expression. The PC coronary effluent activates JAK-STAT signaling, limits apoptosis, and protects myocardial performance from I/R injury.     

The intrinsic resistance of female hearts to an ischemic insult is abrogated in primary cardiac hypertrophy

Important sex differences in cardiovascular disease outcomes exist, including conditions of hypertrophic cardiomyopathy and cardiac ischemia. Studies of sex differences in the extent to which load-independent (primary) hypertrophy modulates the response to ischemia-reperfusion (I/R) damage have not been characterized. We have previously described a model of primary genetic cardiac hypertrophy, the hypertrophic heart rat (HHR). In this study the sex differences in HHR cardiac function and responses to I/R [compared to control normal heart rat (NHR)] were investigated ex vivo. The ventricular weight index was markedly increased in HHR female (7.82 +/- 0.49 vs. 4.80 +/- 0.10 mg/g; P < 0.05) and male (5.76 +/- 0.22 vs. 4.62 +/- 0.07 mg/g; P < 0.05) hearts. Female hearts of both strains exhibited a reduced basal contractility compared with strain-matched males [maximum first derivative of pressure (dP/dt(max)): NHR, 4,036 +/- 171 vs. 4,258 +/- 152 mmHg/s; and HHR, 3,974 +/- 160 vs. 4,540 +/- 259 mmHg/s; P < 0.05]. HHR hearts were more susceptible to I/R (I = 25 min, and R = 30 min) injury than NHR hearts (decreased functional recovery, and increased lactate dehydrogenase efflux). Female NHR hearts exhibited a significantly greater recovery (dP/dt(max)) post-I/R relative to male NHR (95.0 +/- 12.2% vs. 60.5 +/- 9.4%), a resistance to postischemic dysfunction not evident in female HHR (29.0 +/- 5.6% vs. 25.9 +/- 6.3%). Ventricular fibrillation was suppressed, and expression levels of Akt and ERK1/2 were selectively elevated in female NHR hearts. Thus the occurrence of load-independent primary cardiac hypertrophy undermines the intrinsic resistance of female hearts to I/R insult, with the observed abrogation of endogenous cardioprotective signaling pathways consistent with a potential mechanistic role in this loss of protection.     

Hydrogen sulfide contributes to cardioprotection during ischemia-reperfusion injury by opening K ATP channels

The present study was undertaken to investigate the protective effect of H2S against myocardial ischemia-reperfusion (I/R) injury and its possible mechanism by using isolated heart perfusion and patch clamp recordings. Rat isolated hearts were Langendorff-perfused and subjected to a 30-minute ischemia insult followed by a 30-minute reperfusion. The heart function was assessed by measuring the LVDP, +/-dP/dt max, and the arrhythmia score. The results showed that the treatment of hearts with a H2S donor (40 micromol/L NaHS) during reperfusion resulted in significant improvement in heart function compared with the I/R group (LVDP recovered to 85.0% +/- 6.4% vs. 35.0% +/- 6.1%, +dP/dt max recovered to 80.9% +/- 4.2% vs. 43.0% +/- 6.4%, and -dP/dt max recovered to 87.4% +/- 7.3% vs. 53.8% +/- 4.9%; p < 0.01). The arrhythmia scores also improved in the NaHS group compared with the I/R group (1.5 +/- 0.2 vs. 4.0 +/- 0.4, respectively; p < 0.001). The cardioprotective effect of NaHS during reperfusion could be blocked by an ATP-sensitive potassium channel (K ATP) blocker (10 micromol/L glibenclamide). In single cardiac myocytes, NaHS increased the open probability of K ATP channels from 0.07 +/- 0.03 to 0.15 +/- 0.08 after application of 40 mumol/L NaHS and from 0.07 +/- 0.03 to 0.36 +/- 0.15 after application of 100 mumol/L NaHS. These findings provide the first evidence that H2S increases the open probability of K ATP in cardiac myocytes, which may be responsible for cardioprotection against I/R injury during reperfusion.     

LOX-1 inhibition in myocardial ischemia-reperfusion injury: modulation of MMP-1 and inflammation

A recently identified lectin-like oxidized low-density lipoprotein receptor (LOX-1) mediates endothelial cell injury and facilitates inflammatory cell adhesion. We studied the role of LOX-1 in myocardial ischemia-reperfusion (I/R) injury. Anesthetized Sprague-Dawley rats were subjected to 60 min of left coronary artery (LCA) ligation, followed by 60 min of reperfusion. Rats were treated with saline, LOX-1 blocking antibody JXT21 (10 mg/kg), or nonspecific anti-goat IgG (10 mg/kg) before I/R. Ten other rats underwent surgery without LCA ligation and served as a sham control group. LOX-1 expression was markedly increased during I/R (P < 0.01 vs. sham control group). Simultaneously, the expression of matrix metalloproteinase-1 (MMP-1) and adhesion molecules (P-selectin, VCAM-1, and ICAM-1) was also increased in the I/R area (P < 0.01 vs. sham control group). There was intense leukocyte accumulation in the I/R area in the saline-treated group. Treatment of rats with the LOX-1 antibody prevented I/R-induced upregulation of LOX-1 and reduced MMP-1 and adhesion molecule expression as well as leukocyte recruitment. LOX-1 antibody, but not nonspecific IgG, also reduced myocardial infarct size (P < 0.01 vs. saline-treated I/R group). To explore the link between LOX-1 and adhesion molecule expression, we measured expression of oxidative stress-sensitive p38 mitogen-activated protein kinase (p38 MAPK). The activity of p38 MAPK was increased during I/R (P < 0.01 vs. sham control), and use of LOX-1 antibody inhibited p38 MAPK activation (P < 0.01). These findings indicate that myocardial I/R upregulates LOX-1 expression, which through p38 MAPK activation increases the expression of MMP-1 and adhesion molecules. Inhibition of LOX-1 exerts an important protective effect against myocardial I/R injury.     

p38 MAPK-dependent small HSP27 and αB-crystallin phosphorylation in regulation of myocardial function following cardioplegic arrest

We previously demonstrated that myocardial p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27) are phosphorylated following cardioplegic arrest in patients undergoing cardiac surgery and correlate with reduced cardiac function. The following studies were performed to determine whether inhibition of p38 MAPK and/or overexpression of nonphosphorylatable HSP27 improves cardiac function following cardioplegic arrest. Langendorff-perfused isolated rat hearts were subjected to 2 h of intermittent cold cardioplegia followed by 30 min of reperfusion. Hearts were treated with (CP+SB) or without (CP) the p38 MAPK inhibitor SB-203580 (5 μM) supplied in the cardioplegia. Sham-treated hearts served as controls. In separate experiments, isolated rat ventricular myocytes infected with either green fluorescent protein (GFP) or a nonphosphorylatable HSP27 mutant (3A-HSP27) were subjected to 3 h of cold hypoxic cardioplegia and simulated reperfusion (CP) followed by video microscopy and length change measurements. Baseline parameters of cardiac function were similar between groups [left ventricular developed pressure (LVDP), 119 ± 4.9 mmHg; positive and negative first derivatives of LV pressure (± dP/dt), 3,139 ± 245 and 2, 314 ± 110 mmHg/s]. CP resulted in reduced cardiac function (LVDP, 72.2 ± 5.8 mmHg; ± dP/dt, 2,076 ± 231 and -1,317 ± 156 mmHg/s) compared with baseline. Treatment with 5 μM SB-203580 significantly improved CP-induced cardiac function (LVDP, 101.9 ± 0 mmHg; ± dP/dt, 2,836 ± 163 and -2,108 ± 120 mmHg/s; P = 0.03, 0.01, and 0.04, CP+SB vs. CP). Inhibition of p38 MAPK significantly lowered CP-induced p38 MAPK, HSP27, and αB-crystallin (cryAB) phosphorylation. In vitro CP decreased myocyte length changes from 10.3 ± 1.5% (GFP) to 5.7 ± 0.8% (GFP+CP). Infection with 3A-HSP27 completely rescued CP-induced decreased myocyte contraction (11.1 ± 1.0%). However, infection with 3A-HSP27 did not block the endogenous HSP27 response. We conclude that inhibition of p38 MAPK and subsequent HSP27 and cryAB phosphorylation and/or overexpression of nonphosphorylatable HSP27 significantly improves cardiac performance following cardioplegic arrest. Modulation of HSP27 phosphorylation may improve myocardial stunning following cardiac surgery.     

Role of endogenous testosterone in myocardial proinflammatory and proapoptotic signaling after acute ischemia-reperfusion

Myocardial ischemia is the leading cause of death in both men and women; however, very little information exists regarding the effect of testosterone on the response of myocardium to acute ischemic injury. We hypothesized that testosterone may exert deleterious effects on myocardial inflammatory cytokine production, p38 MAPK activation, apoptotic signaling, and myocardial functional recovery after acute ischemia-reperfusion (I/R). To study this, isolated, perfused rat hearts (Langendorff) from adult males, castrated males, and males treated with a testosterone receptor blocker (flutamide) were subjected to 25 min of ischemia followed by 40 min of reperfusion. Myocardial contractile function (left ventricular developed pressure, left ventricular end-diastolic pressure, positive and negative first derivative of pressure) was continuously recorded. After reperfusion, hearts were analyzed for expression of tissue TNF-alpha, IL-1beta, and IL-6 (ELISA) and activation of p38 MAPK, caspase-1, caspase-3, caspase-11, and Bcl-2 (Western blot). All indices of postischemic myocardial functional recovery were significantly higher in castrated males or flutamide-treated males compared with untreated males. After I/R, castrated male and flutamide-treated male hearts had decreased TNF-alpha, IL-1beta, and IL-6; decreased activated p38 MAPK; decreased caspase-1, caspase-3, and caspase-11; and increased Bcl-2 expression compared with untreated males. These results show that blocking the testosterone receptor (flutamide) or depleting testosterone (castration) in normal males improves myocardial function after I/R. These effects may be attributed to the proinflammatory and/or the proapoptotic properties of endogenous testosterone. Further understanding may allow therapeutic manipulation of sex hormone signaling mechanisms in the treatment of acute I/R.     

cGMP-dependent protein kinase type I inhibits TAB1-p38 mitogen-activated protein kinase apoptosis signaling in cardiac myocytes

Cardiac myocyte apoptosis during ischemia and reperfusion (I/R) is tightly controlled by a complex network of stress-responsive signaling pathways. One pro-apoptotic pathway involves the interaction of the scaffold protein TAB1 with p38 mitogen-activated protein kinase (p38 MAPK) leading to the autophosphorylation and activation of p38 MAPK. Conversely, NO and its second messenger cGMP protect cardiac myocytes from apoptosis during I/R. We provide evidence that the cGMP target cGMP-dependent protein kinase type I (PKG I) interferes with TAB1-p38 MAPK signaling to protect cardiac myocytes from I/R injury. In isolated neonatal cardiac myocytes, activation of PKG I inhibited the interaction of TAB1 with p38 MAPK, p38 MAPK phosphorylation, and apoptosis induced by simulated I/R. During I/R in vivo, mice with a cardiac myocyte-restricted deletion of PKG I displayed a more pronounced interaction of TAB1 with p38 MAPK and a stronger phosphorylation of p38 MAPK in the myocardial area at risk during reperfusion and more apoptotic cardiac myocytes in the infarct border zone as compared with wild-type littermates. Notably, adenoviral expression of a constitutively active PKG I mutant truncated at the N terminus(PKGI-DeltaN1-92) did not inhibit p38 MAPK phosphorylation and apoptosis induced by simulated I/R in vitro, indicating that the N terminus of PKG I is required. As shown by co-immunoprecipitation experiments in HEK293 cells, cGMP-activated PKG I, but not constitutively active PKG I-DeltaN1-92 or PKG I mutants carrying point mutations in the N-terminal leucine-isoleucine zipper, interacted with p38 MAPK, and prevented the binding of TAB1 to p38 MAPK. Together, our data identify a novel interaction between the cGMP target PKG I and the TAB1-p38 MAPK signaling pathway that serves as a defense mechanism against myocardial I/R injury.     

Molecular and pharmacological approaches to inhibiting nitric oxide after burn trauma

Whereas controversial, several studies have suggested that nitric oxide (NO) alters cardiac contractility via cGMP, peroxynitrite, or poly(ADP ribose) synthetase (PARS) activation. This study determined whether burn-related upregulation of myocardial inducible NO synthase (iNOS) and NO generation contributes to burn-mediated cardiac contractile dysfunction. Mice homozygous null for the iNOS gene (iNOS knockouts) were obtained from Jackson Laboratory. iNOS knockouts (KO) as well as wild-type mice were given a cutaneous burn over 40% of the total body surface area by the application of brass probes (1 x 2 x 0.3 cm) heated to 100 degrees C to the animals' sides and back for 5 s (iNOS/KO burn and wild-type burn). Additional groups of iNOS KO and wild-type mice served as appropriate sham burn groups (iNOS/KO sham and wild-type sham). Cardiac function was assessed 24 h postburn by perfusing hearts (n = 7-10 mice/group). Burn trauma in wild-type mice impaired cardiac function as indicated by the lower left ventricular pressure (LVP, 67 +/- 2 mmHg) compared with that measured in wild-type shams (94 +/- 2 mmHg, P < 0.001), a lower rate of LVP rise (+dP/dtmax, 1,620 +/- 94 vs. 2,240 +/- 58 mmHg/s, P < 0.001), and a lower rate of LVP fall (-dP/dtmax, 1,200 +/- 84 vs. 1,800 +/- 42 mmHg/s, P < 0.001). Ventricular function curves confirmed significant contractile dysfunction after burn trauma in wild-type mice. Burn trauma in iNOS KO mice produced fewer cardiac derangements compared with those observed in wild-type burns (LVP: 78 +/- 5 mmHg; +dP/dt: 1,889 +/- 160 mmHg/s; -dP/dt: 1,480 +/- 154 mmHg/s). The use of a pharmacological approach to inhibit iNOS (aminoguanidine, given ip) in additional wild-type shams and burns confirmed the iNOS KO data. Whereas the absence of iNOS attenuated burn-mediated cardiac contractile dysfunction, these experiments did not determine the contribution of cardiac-derived NO versus NO generated by immune cells. However, our data indicate a role for NO in cardiac dysfunction after major trauma.     

VEGFR1 (Flt-1+/-) gene knockout leads to the disruption of VEGF-mediated signaling through the nitric oxide/heme oxygenase pathway in ischemic preconditioned myocardium

This report demonstrates that mice deficient in Flt-1 failed to establish ischemic preconditioning (PC)-mediated cardioprotection in isolated working buffer-perfused ischemic/reperfused (I/R) hearts compared to wild type (WT) subjected to the same PC protocol. WT and Flt-1+/- mice were divided into four groups: (1) WT I/R, (2) WT + PC, (3) Flt-1+/- I/R, and (4) Flt-1+/- + PC. Group 1 and 3 mice were subjected to 30 min of ischemia followed by 2 h of reperfusion and group 2 and 4 mice were subjected to four episodes of 4-min global ischemia followed by 6 min of reperfusion before ischemia/reperfusion. For both wild-type and Flt-1+/- mice, the postischemic functional recovery for the hearts was lower than the baseline, but the recovery for the knockout mice was less compared to the WT mice even in preconditioning. The myocardial infarction and apoptosis were higher in Flt-1+/- compared to wild-type I/R. Flt-1+/- KO mice demonstrated pronounced inhibition of the expression of iNOS, p-AKT & p-eNOS. Significant inhibition of STAT3 & CREB were also observed along with the inhibition of HO-1 mRNA. Results demonstrate that Flt-1+/- mouse hearts are more susceptible to ischemia/reperfusion injury and also document that preconditioning is not as effective as found in WT and therefore suggest the importance of VEGF/Flt-1 signaling in ischemic/reperfused myocardium.