The use of transgenic mice to study cytoprotection by the stress proteins

Heat shock or stress proteins (HSPs) have been shown to be able to confer cytoprotection in a diversity of cell types and organisms. We were interested in assessing if HSPs, in particular HSP70, were protective against pathophysiological stresses such as myocardial ischemia. Our approach was to generate a transgenic mouse line that would constitutively express high levels of an inducible rat HSP70 isoform in the heart. The hearts of the transgenic mice were then used in an isolated perfused mouse heart model to assess whether increased expression of HSP70 alone was protective against ischemia-reperfusion injury. Our study showed that there was a significant improvement in contractile recovery, less cellular damage, and a reduction in infarct size in the hearts of transgenic mice as compared to non-transgenic mice following global ischemia in our isolated perfused mouse heart model. Additional studies have since shown that increased expression of HSP70 as well as other stress proteins in transgenic mice protects against different forms of pathological stresses. We present here the methods we used to generate HSP70 transgenic mice and assess their increased tolerance to ischemia-reperfusion injury.     

Role of heat shock protein 70 in hepatic ischemia-reperfusion injury in mice

It is well established that liver ischemia-reperfusion induces the expression of heat shock protein (HSP) 70. However, the biological function of HSP70 in this injury is unclear. In this study, we sought to determine the role of HSP70 in hepatic ischemia-reperfusion injury in mice. Male mice were subjected to 90 min of partial hepatic ischemia followed by up to 8 h of reperfusion. HSP70 was rapidly upregulated after reperfusion. To explore the function of HSP70, sodium arsenite (8 mg/kg iv) was injected before surgery. We found that this dose induced HSP70 expression within 6 h of treatment. Induction of HSP70 with arsenite resulted in a >50% reduction in liver injury as determined by serum transaminases and histology. In addition, arsenite similarly reduced liver neutrophil recruitment and liver nuclear factor-kappaB activation, and attenuated serum levels of tumor necrosis factor-alpha and macrophage inflammatory protein-2, but increased levels of interleukin (IL)-6. In HSP70 knockout mice, arsenite did not protect against liver injury but did reduce liver neutrophil accumulation. Arsenite-induced reductions in neutrophil accumulation in HSP70 knockout mice were found to be mediated by IL-6. To determine whether extracellular HSP70 contributed to the injury, recombinant HSP70 was injected before surgery. Intravenous injection of 10 microg of recombinant HSP70 had no effect on liver injury after ischemia-reperfusion. The data suggest that intracellular HSP70 is directly hepatoprotective during ischemia-reperfusion injury and that extracellular HSP70 is not a significant contributor to the injury response in this model. Targeted induction of HSP70 may represent a potential therapeutic option for postischemic liver injury.     

Transgenic overexpression of HSP56 does not result in cardiac hypertrophy nor protect from ischaemia/reperfusion injury

Heat shock proteins are known to be induced during and following different forms of cardiac stress. It has previously been shown that their expression is beneficial for the heart following trauma such as ischaemia-reperfusion (I/R) injury. Heat shock protein 56 (HSP56) belongs to the family of FK506-binding immunophilin proteins and is found in steroid receptor complexes, notably the glucocorticoid receptor. We have previously shown that HSP56 and other HSPs are induced in cardiac myocytes treated with cardiotrophin-1, a cytokine with potent hypertrophic and protective properties on cardiac cells. The hypertrophic action of cardiotrophin-1 on cardiac cells is dependent on HSP56 induction and overexpression of HSP56 is sufficient for inducing hypertrophy in cardiac cells. To investigate this phenomenon in vivo, we have generated transgenic mice overexpressing HSP56 and assessed them for the development cardiac hypertrophy and resistance of their hearts to I/R-injury by Langendorff perfusion. Mice generated demonstrated stable, yet varying expression levels of HSP56. Initial characterisation identified a sex-specific phenotype where male overexpressing mice exhibited a moderate, but significant, reduced body weight compared to wild-type controls. In ex vivo stress analyses we found, unexpectedly, that significant overexpression of HSP56 does not induce myocardial hypertrophy and nor does it protect the intact heart from I/R-injury. These observations now suggest a more intricate HSP56-Sp. Cardiophenotype that requires further studies to determine if HSP56 is necessary in mediating hypertrophy induced by other myocardial stimuli.     

Histidine button engineered into cardiac troponin I protects the ischemic and failing heart

The myofilament protein troponin I (TnI) has a key isoform-dependent role in the development of contractile failure during acidosis and ischemia. Here we show that cardiac performance in vitro and in vivo is enhanced when a single histidine residue present in the fetal cardiac TnI isoform is substituted into the adult cardiac TnI isoform at codon 164. The most marked effects are observed under the acute challenges of acidosis, hypoxia, ischemia and ischemia-reperfusion, in chronic heart failure in transgenic mice and in myocytes from failing human hearts. In the isolated heart, histidine-modified TnI improves systolic and diastolic function and mitigates reperfusion-associated ventricular arrhythmias. Cardiac performance is markedly enhanced in transgenic hearts during reperfusion despite a high-energy phosphate content similar to that in nontransgenic hearts, providing evidence for greater energetic economy. This pH-sensitive 'histidine button' engineered in TnI produces a titratable molecular switch that 'senses' changes in the intracellular milieu of the cardiac myocyte and responds by preferentially augmenting acute and long-term function under pathophysiological conditions. Myofilament-based inotropy may represent a therapeutic avenue to improve myocardial performance in the ischemic and failing heart.     

Inhibition of Mas G-protein signaling improves coronary blood flow, reduces myocardial infarct size, and provides long-term cardioprotection

The Mas receptor is a class I G-protein-coupled receptor that is expressed in brain, testis, heart, and kidney. The intracellular signaling pathways activated downstream of Mas are still largely unknown. In the present study, we examined the expression pattern and signaling of Mas in the heart and assessed the participation of Mas in cardiac ischemia-reperfusion injury. Mas mRNA and protein were present in all chambers of human hearts, with cardiomyocytes and coronary arteries being sites of enriched expression. Expression of Mas in either HEK293 cells or cardiac myocytes resulted in constitutive coupling to the G(q) protein, which in turn activated phospholipase C and caused inositol phosphate accumulation. To generate chemical tools for use in probing the function of Mas, we performed a library screen and chemistry optimization program to identify potent and selective nonpeptide agonists and inverse agonists. Mas agonists activated G(q) signaling in a dose-dependent manner and reduced coronary blood flow in isolated mouse and rat hearts. Conversely, treatment of isolated rat hearts with Mas inverse agonists improved coronary flow, reduced arrhythmias, and provided cardioprotection from ischemia-reperfusion injury, an effect that was due, at least in part, to decreased cardiomyocyte apoptosis. Participation of Mas in ischemia-reperfusion injury was confirmed in Mas knockout mice, which had reduced infarct size relative to mice with normal Mas expression. These results suggest that activation of Mas during myocardial infarction contributes to ischemia-reperfusion injury and further suggest that inhibition of Mas-G(q) signaling may provide a new therapeutic strategy directed at cardioprotection.     

Detrimental effects of thyroid hormone analog DITPA in the mouse heart: increased mortality with in vivo acute myocardial ischemia-reperfusion

There is emerging evidence that treatment with thyroid hormone (TH) can improve postischemic cardiac function. 3,5-Diiodothyropropionic acid (DITPA), a TH analog, has been proposed to be a safer therapeutic agent than TH because of its negligible effects on cardiac metabolism and heart rate. However, conflicting results have been reported for the cardiac effects of DITPA. Importantly, recent clinical trials demonstrated no symptomatic benefit in patients with DITPA despite some improved hemodynamic and metabolic parameters. To address these issues, dose-dependent effects of DITPA were investigated in mice for baseline cardiovascular effects and postischemic myocardial function and/or salvage. Mice were treated with subcutaneous DITPA at 0.937, 1.875, 3.75, or 7.5 mg·kg(-1)·day(-1) for 7 days, and the results were compared with untreated mice for ex vivo and/or in vivo myocardial ischemia-reperfusion (I/R). DITPA had no effects on baseline body temperature, body weight, or heart rate; however, it mildly increased blood pressure. In isolated hearts, baseline contractile function was significantly impaired in DITPA-pretreated mice; however, postischemic recovery was comparable between untreated and DITPA-treated groups. In vivo baseline cardiac parameters were significantly affected by DITPA, with increased ventricular dimensions and decreased contractile function. Importantly, DITPA-treated mice demonstrated high prevalence of fatal cardiac rhythm abnormalities during in vivo ischemia and/or reperfusion. There were no improvements in myocardial infarction and postischemic fractional shortening with DITPA. Myocardial sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), phospholamban (PLB), and heat shock protein (HSP) levels remained unchanged with DITPA treatment. Thus DITPA administration impairs baseline cardiac parameters in mice and can be fatal during in vivo acute myocardial I/R.     

HSP25 in isolated perfused rat hearts: Localization and response to hyperthermia

Recent investigations concentrate on the correlation between the myocardial expression of the inducible 70-kDa heat shock protein (HSP70i) by different stress conditions and its possible protective effects. Only few studies have focused on the involvement of small heat shock proteins in this process. We analyzed the location of the small heat shock protein HSP25 in isolated cardiomyocytes as well as its location and induction in isolated perfused hearts of rats. By immunofluorescence microscopy HSP25 was found to colocalize with actin in the I-band of myofibrils in cardiomyocytes of isolated perfused hearts as well as in isolated neonatal and adult cardiomyocytes. Hyperthermic perfusion of isolated hearts for 45 min resulted in modulation of different parameters of heart function and in induction of HSP25 and HSP70i. Temperatures higher than 43°C (44–46°C) were lethal with respect to the contractile function of the hearts. Compared to control hearts perfused at 37°C, significant increases during hyperthermic perfusion at 42°C and 43°C were obtained for heart rate, contraction velocity and relaxation velocity. In response to hyperthermia at 43°C and after subsequent normothermic perfusion for 135 min at 37°C, left ventricular pressure, contraction velocity and relaxation velocity remained significantly elevated. However, heart rate returned to control values immediately after the period of heat treatment. HSP25 is constitutively expressed even in normothermic perfused hearts as shown by Western blotting. Hyperthermia increased the content of HSP25 only in the left ventricular tissue. In contrast, HSP70i was strongly induced in all analyzed parts of the myocardium (left ventricle, right ventricle, septum). Our findings suggest a differential regulation of HSP25 and HSP70i expression in response to hyperthermia in isolated perfused hearts. The constitutively expressed HSP25 seems to be located adjacent to the myofibrils which implies a specific role of this protein even under unstressed conditions for the contractile function of the myocardium.     

Inhibition of cyclooxygenase-2 enhances myocardial damage in a mouse model of viral myocarditis

To determine critical role of cyclooxygenase-2 (COX-2) for development of viral myocarditis, a mouse model of encephalomyocarditis virus-induced myocarditis was used. The virus was intraperitoneally given to COX-2 gene-deficient heterozygote mice (COX-2+/-) and wild-type mice (WT). We examined differences in heart weights, cardiac histological scores, numbers of infiltrating or apoptotic cells in myocardium, cardiac expression levels of COX-2, tumor necrosis factor-alpha (TNF-alpha), and adiponectin mRNA, immunoreactivity of COX-2, TNF-alpha, and adiponectin in myocytes, cardiac concentrations of TNF-alpha and adiponectin, prostaglandin E2 (PGE2) levels in hearts, and viral titers in tissues between COX-2+/- and WT. We observed significantly decreased expression of COX-2 mRNA and reactivity in hearts from COX-2+/- on day 8 after viral inoculation as compared with that from WT, together with elevated cardiac weights and severe inflammatory myocardial damage in COX-2+/-. Cardiac expression of TNF-alpha mRNA, reactivity, and protein on day 8 was significantly higher in COX-2+/- than in WT, together with reciprocal expression of adiponectin mRNA, reactivity, and protein in hearts. Significantly reduced cardiac PGE2 levels on day 8 were found in COX-2+/- compared with those in WT. There was no difference in local viral titers between both groups on day 4. Infected WT treated with a selective COX-2 inhibitor, NS-398, also showed the augmented myocardial damage on day 8. These results suggest that inhibition of COX-2 may enhance myocardial damage through reciprocal cardiac expression of TNF-alpha and adiponectin in a mouse model of viral myocarditis.     

Gender differences in the expression of heat shock proteins: the effect of estrogen

The heat shock proteins (HSPs) are an important family of endogenous, protective proteins that are found in all tissues. In the heart, HSP72, the inducible form of HSP70, has been the most intensely studied. It is well established that HSP72 is induced with ischemia and is cardioprotective. Overexpression of other HSPs also is protective against cardiac injury. Recently, we observed that 17beta-estradiol increases levels of HSPs in male rat cardiac myocytes. We hypothesized that there were gender differences in HSP72 expression in the heart secondary to estrogen. To test this hypothesis, we examined cardiac levels of HSP72 by ELISA in male and female Sprague-Dawley rats. In addition, three other HSPs were assessed by Western blot (HSP27, HSP60, and HSP90). To determine whether estrogen status affected HSP72 expression in other muscles or tissues, two other muscle tissues, slow twitch muscle (soleus muscle) and fast twitch muscle (gastrocnemius muscle), were studied as well as two other organs, the kidney and liver. Because HSP72 is cardioprotective, and females are known to have less cardiovascular disease premenopause, the effects of ovariectomy were examined. We report that female Sprague-Dawley rat hearts have twice as much HSP72 as male hearts. Ovariectomy reduced the level of HSP72 in female hearts, and this could be prevented by estrogen replacement therapy. These data show that the expression of cardiac HSP72 is greater in female rats than in male rats, due to upregulation by estrogen.     

电转联合热休克蛋白佐剂疫苗引发抗HBV的免疫应答

针对HBV感染的治疗性DNA疫苗虽然具有很好的应用前景,但目前抗病毒效果并不高,表明在病毒长期感染过程中存在免疫抑制机制。以HBV的表面蛋白(HBsAg)和核心蛋白(HBcAg)为DNA疫苗抗原,采用gp96和HSP70作为佐剂联合电转以提高疫苗的活性。将gp96为佐剂的HBsAg/HBcAg DNA疫苗免疫HBV转基因鼠后引发抗原特异性的细胞免疫和体液免疫应答。使用gp96和HSP70佐剂引起Treg下调20%。与没有免疫的小鼠相比,以gp96和HSP70为佐剂的DNA疫苗显著降低血清中病毒S抗原水平和DNA拷贝数,大幅降低小鼠肝脏中HBc的表达。该研究为设计以gp96为佐剂的乙肝治疗性DNA疫苗提供了依据。     

Proteomic analysis reveals significant elevation of heat shock protein 70 in patients with chronic heart failure due to arrhythmogenic right ventricular cardiomyopathy

As proteins are the ultimate biological determinants of phenotype of disease, we screened altered proteins associated with heart failure due to arrhythmogenic right ventricular cardiomyopathy (ARVC) to identify biomarkers potential for rapid diagnosis of heart failure. By 2-dimensional gel electrophoresis and mass spectrometry, we identified five commonly altered proteins with more than 1.5 fold changes in eight ARVC failing hearts using eight non-failing hearts as reference. Noticeably, one of the altered proteins, heat shock protein 70 (HSP70), was increased by 1.64 fold in ARVC failing hearts compared with non-failing hearts. The increase of cardiac HSP70 was further validated by Western blot, immunochemistry, and enzyme-linked immunosorbent assay (ELISA) in failing hearts due to not only ARVC, but also dilated (DCM, n = 18) and ischemic cardiomyopathy (ICM, n = 8). Serum HSP70 was also observed to be significantly increased in heart failure patients derived from the three forms of cardiomyopathies. In addition, we observed hypoxia/serum depletion stimulation induced significantly elevation of intracellular and extracellular HSP70 in cultured neonatal rat cardiomyocytes. For the first time to our knowledge, we revealed and clearly demonstrated significant up-regulation of cardiac and serum HSP70 in ARVC heart failure patients. Our results indicate that elevated HSP70 is the common feature of heart failure due to ARVC, DCM, and ICM, which suggests that HSP70 may be used as a biomarker for the presence of heart failure due to cardiomyopathies of different etiologies and may hold diagnostic/prognostic potential in clinical practice.     

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.     

Cardiomyocyte-specific gene expression following recombinant adeno-associated viral vector transduction

Recombinant adeno-associated viral (rAAV) vectors hold promise for delivering genes for heart diseases, but cardiac-specific expression by the use of rAAV has not been demonstrated. To achieve this goal rAAV vectors were generated expressing marker or potentially therapeutic genes under the control of the cardiac muscle-specific alpha myosin heavy chain (MHC) gene promoter. The rAAV-MHC vectors expressed in primary cardiomyocytes with similar kinetics to rAAV-CMV; however, expression by the rAAV-MHC vectors was restricted to cardiomyocytes. rAAV vectors have low cytotoxicity, and it is demonstrated here that rAAV fails to induce apoptosis in cardiomyocytes compared with a recombinant adenoviral vector. rAAV-MHC or rAAV-CMV vectors were administered to mice to determine the specificity of expression in vivo. The rAAV-MHC vectors expressed specifically in cardiomyocytes, whereas the control rAAV-CMV vector expressed in heart, skeletal muscle, and brain. rAAV-MHC transduction resulted in long term (16 weeks) expression of human growth hormone following intracardiac, yet not intramuscular, injection. Finally, we defined the minimal MHC enhancer/promoter sequences required for specific and robust in vivo expression in the context of a rAAV vector. For the first time we describe a panel of rAAV vectors capable of long term cardiac specific expression of intracellular and secreted proteins.     

Targeted inactivation of Galpha(i) does not alter cardiac function or beta-adrenergic sensitivity

Inhibitory Galpha(i) protein increases in the myocardium during hypertrophy and has been associated with beta-adrenergic receptor (beta-AR) desensitization, contractile dysfunction, and progression of cardiac disease. The role of Galpha(i) proteins in mediating basal cardiac function and beta-AR response in nonpathological myocardium, however, is uncertain. Transgenic mice with targeted inactivation of Galpha(i2) or Galpha(i3) were examined for in vivo cardiac function with the use of conscious echocardiography and for ex vivo cardiac response to inotropic stimulation with the use of Langendorff blood-perfused isolated hearts and adult ventricular cardiomyocytes. Echocardiography revealed that percent fractional shortening and heart rate were similar among wild-type, Galpha(i2)-null, and Galpha(i3)-null mice. Comparable baseline diastolic and contractile performance was also observed in isolated hearts and isolated ventricular myocytes from wild-type mice and mice lacking Galpha(i) proteins. Isoproterenol infusion enhanced diastolic and contractile performance to a similar degree in wild-type, Galpha(i2)-null, and Galpha(i3)-null mice. These data demonstrate no observable role for inhibitory G proteins in mediating basal cardiac function or sensitivity to beta-AR stimulation in nonpathological myocardium.     

Heat stress aggravates viral myocarditis in mice

While a beneficial effect of hyperthermia on viral infection has been hypothesized, there are no data on viral myocarditis in vivo. To investigate whether hyperthermia might attenuate the course or severity of viral myocarditis, we studied the pathological changes in a murine model of viral myocarditis. C3H mice were inoculated i.p. with the encephalomyocarditis virus (500 pfu). They were anesthetized and heated to a body temperature of 42.5+/-0.2 degrees C for 30 min. The latter was performed 4 hr before (n=28, HB) or 4 hr after (n=28, HA) the viral inoculation; results were compared with nonheated, infected controls (n=30, Cont). Cardiac viral titers were recorded on day 3, and the body weight (BW), heart weight (HW) and pathological changes were recorded on days 5 and 10. The incidence of spontaneous mortality on day 10 was significantly higher in the HA group (all deaths occurring by day 7 post-inoculation) as compared with the HB (35%) or Cont (18%) groups. Viral titers in the HA group (n=4) were significantly (P<0.05) higher than those in the Cont (n=7) or HB (n=7) groups (4.11+/-0.54 vs 3.01+/-0.44 and 3.23+/-0.45 LogTCID50/mg, respectively). On day 5, the HW, the BW/HW ratio, and the severity of myocardial necrosis were all significantly higher in the HA than in the Cont and HB groups. To confirm the effect of hyperthermia on the expression of heart shock protein (HSP), immunohistochemical staining was done in the virus-infected hearts. The nucleus and cytoplasm of the injured myocardium in the HA group strongly expressed HSP70, whereas the HB and Cont groups were negative for this protein. In conclusion, induction of hyperthermia after viral inoculation aggravated the viral-induced myocardial necrosis and increased the mortality rate in a murine model of viral myocarditis and induced myocardial heat shock protein 70.     

Cardiac mTOR protects the heart against ischemia-reperfusion injury

Cardiac mammalian target of rapamycin (mTOR) is necessary and sufficient to prevent cardiac dysfunction in pathological hypertrophy. However, the role of cardiac mTOR in heart failure after ischemic injury remains undefined. To address this question, we used transgenic (Tg) mice with cardiac-specific overexpression of mTOR (mTOR-Tg mice) to study ischemia-reperfusion (I/R) injury in two animal models: 1) in vivo I/R injury with transient coronary artery ligation and 2) ex vivo I/R injury in Langendorff-perfused hearts with transient global ischemia. At 28 days after I/R, mortality was lower in mTOR-Tg mice than littermate control mice [wild-type (WT) mice]. Echocardiography and MRI demonstrated that global cardiac function in mTOR-Tg mice was preserved, whereas WT mice exhibited significant cardiac dysfunction. Masson's trichrome staining showed that 28 days after I/R, the area of interstitial fibrosis was smaller in mTOR-Tg mice compared with WT mice, suggesting that adverse left ventricular remodeling is inhibited in mTOR-Tg mice. In the ex vivo I/R model, mTOR-Tg hearts demonstrated improved functional recovery compared with WT hearts. Perfusion with Evans blue after ex vivo I/R yielded less staining in mTOR-Tg hearts than WT hearts, indicating that mTOR overexpression inhibited necrosis during I/R injury. Expression of proinflammatory cytokines, including IL-6 and TNF-α, in mTOR-Tg hearts was lower than in WT hearts. Consistent with this, IL-6 in the effluent post-I/R injury was lower in mTOR-Tg hearts than in WT hearts. These findings suggest that cardiac mTOR overexpression in the heart is sufficient to provide substantial cardioprotection against I/R injury and suppress the inflammatory response.     

Critical role of extracellular heat shock cognate protein 70 in the myocardial inflammatory response and cardiac dysfunction after global ischemia-reperfusion

Previous studies showed that Toll-like receptor 4 (TLR4) modulates the myocardial inflammatory response to ischemia-reperfusion injury, and we recently found that cytokines link TLR4 to postischemic cardiac dysfunction. Although TLR4 can be activated in cultured cells by endogenous agents including heat shock protein 70, how it is activated during myocardial ischemia-reperfusion is unknown. In the present study, we examined 1) whether heat shock cognate protein 70 (HSC70), which is constitutively expressed in the myocardium, is released during ischemia-reperfusion; 2) whether extracellular HSC70 induces the myocardial inflammatory response and modulates cardiac function; and 3) whether HSC70 exerts these effects via TLR4. We subjected isolated mouse hearts to global ischemia-reperfusion via the Langendorff technique. Immunoblotting and immunostaining detected the release of HSC70 from the myocardium during reperfusion. Treatment with an antibody specific to HSC70 suppressed myocardial cytokine expression and improved cardiac functional recovery after ischemia-reperfusion. Recombinant HSC70 induced NF-kappaB activation and cytokine expression and depressed myocardial contractility in a TLR4-dependent manner. These effects required the substrate-binding domain of HSC70. Fluorescence resonance energy transfer analysis of isolated macrophages demonstrated that extracellular HSC70 interacts with TLR4. Therefore, this study demonstrates for the first time that 1) the myocardium releases HSC70 during ischemia-reperfusion, 2) extracellular HSC70 contributes to the postischemic myocardial inflammatory response and to cardiac dysfunction, 3) HSC70 exerts these effects through a TLR4-dependent mechanism, and 4) the substrate-binding domain of HSC70 is required to induce these effects. Thus extracellular HSC70 plays a critical role in regulating the myocardial innate immune response and cardiac function after ischemia-reperfusion.