Granulocyte colony-stimulating factor (G-CSF) was reported to induce myocardial regeneration by promoting mobilization of bone marrow stem cells to the injured heart after myocardial infarction, but the precise mechanisms of the beneficial effects of G-CSF are not fully understood. Here we show that G-CSF acts directly on cardiomyocytes and promotes their survival after myocardial infarction. G-CSF receptor was expressed on cardiomyocytes and G-CSF activated the Jak/Stat pathway in cardiomyocytes. The G-CSF treatment did not affect initial infarct size at 3 d but improved cardiac function as early as 1 week after myocardial infarction. Moreover, the beneficial effects of G-CSF on cardiac function were reduced by delayed start of the treatment. G-CSF induced antiapoptotic proteins and inhibited apoptotic death of cardiomyocytes in the infarcted hearts. G-CSF also reduced apoptosis of endothelial cells and increased vascularization in the infarcted hearts, further protecting against ischemic injury. All these effects of G-CSF on infarcted hearts were abolished by overexpression of a dominant-negative mutant Stat3 protein in cardiomyocytes. These results suggest that G-CSF promotes survival of cardiac myocytes and prevents left ventricular remodeling after myocardial infarction through the functional communication between cardiomyocytes and noncardiomyocytes.

The signal transducer and activator of transcription (STAT) 3, a transcriptional factor downstream of several cytokines, is activated by Janus kinase families and plays a pivotal role in cardiac hypertrophy through gp130. To determine the physiological significance of STAT3 in vivo, transgenic mice with cardiac-specific overexpression of the Stat3 gene (STAT3-TG) were generated. STAT3-TG manifested myocardial hypertrophy at 12 wk of age with increased expression of the atrial natriuretic factor (ANF), ␤-myosin heavy chain (MHC), and cardiotrophin (CT)-1 genes. The animals were injected i.p. with 15 mg͞kg doxorubicin (Dox), an antineoplastic drug with restricted use because of its cardiotoxicity. The survival rates after 10 days were 25% (5͞20) for control littermates (WT), but 80% (16͞20) for STAT3-TG (P < 0.01). WT showed increased expression of ␤-MHC and ANF mRNAs in the hearts 1 day after Dox treatment; this expression peaked at 3 days, suggesting that the WT suffered from congestive heart failure. Although the expression of these mRNAs was elevated in STAT3-TG hearts before Dox treatment, no additional increase was observed after the treatment. Dox administration significantly reduced the expression of the cardiac ␣-actin and Stat3 genes in WT hearts but not in STAT3-TG. These results provide direct evidence that STAT3 transduces not only a hypertrophic signal but also a protective signal against Dox-induced cardiomyopathy by inhibiting reduction of cardiac contractile genes and inducing cardiac protective factors. Signal transducer and activator of transcription (STAT) proteins, which consist of six members, are known to play important roles in cytokine signaling pathways (1). STATs are activated by the Janus kinase (JAK) family after cytokine binding to their specific receptors (2-6). Previous studies revealed that STAT3 was important for the growth, differentiation, and cell death or survival in various kinds of cells (7)(8)(9)(10)(11). Recently, we have demonstrated that STAT3 is expressed in cardiac myocytes, immediately activated by the IL-6 family of cytokines, and plays a crucial role in generating the hypertrophic signal through gp130 (12, 13). In addition, activation of gp130 has been shown to induce bcl-xL expression by means of STAT1 in cardiac myocytes, resulting in a cytoprotective effect (14). Although recent reports provide evidence of STAT phosphorylation in a pressure-overload model (15) and an acute myocardial infarction model of rat hearts (16), the physiological function of STAT in the heart has not been clarified yet. Doxorubicin (Dox), an antitumor drug, is effective for a variety of soft and solid human malignancies, but its long-term use is limited because of the development of cardiomyopathy. Several proposals regarding the pathogenesis of Dox-induced cardiac toxicity have been made, including a generation of oxygen-free radicals (17, 18), from which damage is inhibited by free-radical scavengers, and an intercalation of the drug to DNA (19), which is not affected by free-radical sc...

Background-Mice with cardiac-specific overexpression of signal transducer and activator of transcription 3 (STAT3) are resistant to doxorubicin-induced damage. The STAT3 signal may be involved in the detoxification of reactive oxygen species (ROS). Methods and Results-The effects of leukemia inhibitory factor (LIF) or adenovirus-mediated transfection of constitutively activated STAT3 (caSTAT3) on the intracellular ROS formation induced by hypoxia/reoxygenation (H/R) were examined using rat neonatal cardiomyocytes. Either LIF treatment or caSTAT3 significantly suppressed the increase of H/R-induced ROS evaluated by 2Ј,7Ј-dichlorofluorescin diacetate fluorescence. To assess whether ROS are really involved in H/R-induced cardiomyocyte injury, the amount of creatine phosphokinase in cultured medium was examined. Both LIF treatment and caSTAT3 significantly decreased H/R-induced creatine phosphokinase release. These results indicate that the gp130/STAT3 signal protects H/R-induced cardiomyocyte injury by scavenging ROS generation. To investigate the mechanism of scavenging ROS, the effects of LIF on the induction of antioxidant enzymes were examined. LIF treatment significantly increased the expression of manganese superoxide dismutase (MnSOD) mRNA, whereas the expression of the catalase and glutathione peroxidase genes were unaffected. This induction of MnSOD mRNA expression was completely blocked by adenovirus-mediated transfection of dominant-negative STAT3. Moreover, caSTAT3 augmented MnSOD mRNA and its enzyme activity. In addition, the antisense oligodeoxyribonucleotide to MnSOD significantly inhibited both LIF and caSTAT3-mediated protective effects. Key Words: antioxidants Ⅲ hypoxia Ⅲ signal transduction I n the heart, it has been reported that reactive oxygen species (ROS) contribute to cardiac dysfunction and myocardial damage under a variety of conditions, such as ischemia-reperfusion, congestive heart failure, and doxorubicin-induced cardiomyopathy. 1-3 Recent studies have shown that gp130-mediated signals transduced both cytoprotective and hypertrophic responses in the heart. 4 The signal transducer and activator of transcription-3 (STAT3) is a key molecule downstream of gp130, which is activated under various stressful conditions, such as pressure-overload and myocardial infarction. 4 Transgenic mice with cardiac-specific overexpression of the STAT3 gene are protected against doxorubicin-induced cardiomyopathy. 5 Therefore, the activation of STAT3 might induce a protective effect against oxidative stress-induced cardiomyocyte damage by scavenging ROS. Conclusions-TheIn the present study, we explored whether the gp130/ STAT3 signal has a protective function against hypoxia/reoxygenation (H/R)-induced cardiomyocyte damage. Methods Cell Culture and H/R ExperimentsPrimary cultures of neonatal rat cardiomyocytes were prepared from the ventricles of 1-day-old Sprague-Dawley rats (Kiwa Dobutsu Wakayama, Japan), as previously described. 6 Hypoxia was created by incubating cells in an airtight Plexiglas chamber w...

Background-gp130, a signal transducer of the IL-6 -related cytokines, is expressed ubiquitously, including in the heart.The activation of gp130 in cardiac myocytes was reported to induce myocardial hypertrophy. The downstream side of gp130 consists of two distinct pathways in cardiac myocytes, one a Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, the other a mitogen-activated protein kinase (MAPK) pathway. In the present study, we examined whether the JAK/STAT pathway, especially the STAT3-mediated pathway, plays a critical role in gp130-dependent myocardial hypertrophy by transfecting wild-type and mutated-type STAT3 cDNA to cardiac myocytes. Methods and Results-We constructed three kinds of replication-defective adenovirus vectors carrying wild-type (AD/WT) or mutated-type (AD/DN) STAT3 cDNA or adenovirus vector itself (AD). Cultured murine cardiac myocytes infected with adenovirus were stimulated with leukemia inhibitory factor (LIF), and the expression of c-fos and atrial natriuretic factor (ANF) mRNAs and [ 3 H]leucine incorporation were examined. There were no significant differences in MAPK activity among the three groups. Compared with AD-transfected cardiac myocytes, induction of c-fos and ANF mRNAs and protein synthesis after LIF stimulation were significantly augmented in AD/WT-transfected cells. In contrast, induction of c-fos and ANF mRNA expression and protein synthesis were attenuated after LIF stimulation in cardiac myocytes transfected with AD/DN. Conclusions-These