Cardiotrophin-1 (8. Cardiotrophin-1 and Myocardial hypoxia)

CT-1 expression is augmented after hypoxic stimulation and hypoxic conditioned medium presented enhanced ability to activate STAT3 in cardiac myocytes. Thus, CT-1 might play an important role in the pathogenesis of ischemic heart disease ^Ref . It was shown that prooxidants (menadione, hydrogen peroxide) as well as chemical (CoCl2) and physiological (1% O2) hypoxia increased CT-1 as well as HIF-1alpha protein and mRNA expression in embryoid bodies, indicating that CT-1 expression is regulated by reactive oxygen species (ROS) and hypoxia. Treatment with either prooxidants or chemical hypoxia increased gp130 phosphorylation and protein expression of NADPH oxidase subunits p22-phox, p47-phox, p67-phox, as well as Nox1 and Nox4 mRNA. Consequently, inhibition of NADPH oxidase activity by diphenylen iodonium chloride (DPI) and apocynin abolished prooxidant- and chemical hypoxia-induced upregulation of CT-1. Prooxidants and chemical hypoxia activated ERK1,2, JNK and p38 as well as PI3-kinase. The proxidant- and CoCl2-mediated upregulation of CT-1 was significantly inhibited in the presence of the ERK1,2 antagonist UO126, the JNK antagonist Sp600125, the p38 antagonist SKF86002, the PI3-kinase antagonist LY294002, the Jak-2 antagonist AG490 as well as in the presence of free radical scavengers. Moreover, developing embryoid bodies derived from HIF-1alpha-/- ES cells lack cardiomyogenesis, and prooxidants as well as chemical hypoxia failed to upregulate CT-1 expression. Our results demonstrate that CT-1 expression in ES cells is regulated by ROS and HIF-1alpha and imply a crucial role of CT-1 in the survival and proliferation of ES-cell-derived cardiac cells ^Ref .

Ct-1 upregulation in the course of myocardial infarction

In a rat model of myocardial infarction. At 1, 3, 7, 14, 28 and 56 days (n=12 for each group) after ligation of a coronary artery, tissue samples were obtained from infarct tissue, the ventricular septum and the right ventricle. All animals developed large myocardial infarctions, with infarct sizes ranging from 39.8% to 50.3%. Progressive left ventricular dilatation and inadequate hypertrophy of the surviving myocardium were confirmed by echocardiography. CT-1 and gp130 mRNA levels were determined by semiquantitative reverse transcription-polymerase chain reaction using 1 or 5 microg of total RNA followed by Southern blotting. The densitometric analysis of the Southern blots revealed a significant increase in CT-1 and gp130 mRNA levels (p<0.01) compared with those of the sham-operated rats at 1, 3, 7, 14, 28 and 56 days post-infarct in the infarct area, the ventricular septum (non-infarcted area) and right ventricle. The protein levels of CT-1 and gp130, determined by Western blot analysis, were significantly increased (p<0.05) compared with those of sham-operated rats, peaked during the acute stage and declined thereafter in the three regions described above. Immunohistochemical staining showed that CT-1 and gp130-immunoreactivities were detected in cardiomyocytes and fibroblast-like cells and that the intensity of staining was increased at 7 days post-infarct compared with that in sham-operated rats. An augmented CT-1 and gp130 system thus appears to play an important role during ventricular remodeling after myocardial infarction ^Ref . Elevated CT-1 expression was observed in the infarct zone at 24 h and continued through 2, 4 and 8 weeks post-MI, compared to sham-operated animals. CT-1 induced rapid phosphorylation of Jak, Jak2, STAT1, STAT3, p42/44 MAPK and Akt in cultured adult cardiac fibroblasts. CT-1 induced cardiac fibroblast protein synthesis and proliferation. Protein and DNA synthesis were dependent on activation of JAK/STAT, MEK1/2, PI3K and Src pathways as evidenced by decreased 3H-leucine and 3H-thymidine incorporation after pretreatment with AG490, PD98059, LY294002 and genistein respectively. Furthermore, CT-1 treatment increased procollagen-1-carboxypropeptide (PICP) synthesis, a marker of mature collagen synthesis. CT-1 induced cell migration of rat cardiac fibroblasts. These results suggest that CT-1, as expressed in post-MI heart, may play an important role in infarct scar formation and ongoing remodeling of the scar. CT-1 was able to initiate each of the processes considered important in the formation of infarct scar including cardiac fibroblast migration as well as fibroblast proliferation and collagen synthesis ^Ref .

Cardiotophin-1 protects the heart from the consequences of ischemia

Twenty-four male Sprague-Dawley rats weighing approximately 310 g were subjected to left coronary artery ligation. Seven days before surgery, the rats were randomized to receive cardiotrophin-1 (treated group) or phosphate-buffered saline (control group). Recombinant rat cardiotrophin-1 (2 microg in 1 ml phosphate-buffered saline) or phosphate-buffered saline (1 ml) was administered daily via the tail vein for 7 days (n = 12 for each group). Hemodynamic parameters, apoptotic index, P53, Fas, Bax and Bcl-2 expression in myocardium were measured at 24 hours after coronary ligation.as compared with control animals, rats treated with cardiotrophin-1 had significantly higher mean arterial pressure,left ventricular systolic pressure and the maximum rate of left ventricular pressure rise or fall, and significantly lower left ventricular end-diastolic pressure. Cardiotrophin-1 pretreatment did not affect the heart rate,heart weight,body weight or the ratio of heart weight to body weight.the number of apoptotic cardiomyocytes in cardiotrophin-1 treated group was less than that in control group [(15.8+/-5.2) % vs (34.6+/-7.7) %, p<0.01]. Cardiotrophin-1 pretreatment significantly inhibited P53, Fas and Bax, and increased Bcl-2 expression in myocardium ^Ref . A recent study observed the effect of cardiotrophin-1 (CT-1) gene transfer on cardiomyocytes in a murine model of myocardial infarction. Sixty male cd-1 mice weighing approximately 40 g were used in the study. Forty mice were subjected to left coronary artery ligation and randomized to receive AdCT-1 vector (treated group) or AdLacZ vector (control group) treatment, with 20 mice for each group. AdCT-1 or Ad-LacZ vector was directly injected into the border zone of the ischemic myocardium at six sites, 10 min after ligation (10 mul/site, 2.5×10(6) PFU/100 mul). Twenty mice undergoing thoracotomy and injection of an equal volume of phosphate-buffered saline solution but not coronary ligation served as sham group. Hemodynamics, histopathology and cardiomyocyte apoptosis were detected at 2 weeks after injection. Four animals in sham, nine in control, and six in treated groups died during the experiment. The remaining 41 mice were included in the study. Mean arterial pressure, lezt ventricular systolic pressure, and the maximum rate of left ventricular pressure rise or fall were significantly higher in treated group than in control group (p < 0.01 for all), whereas left ventricular end-diastolic pressure, infarct size, the ratio of right ventricle or lung weight to body weight, and apoptotic index were significantly lower in treated group than in control group (p < 0.01 for all). The caspase-3 activation and mitochondrial cytochrome c release were also lower in treated group than in control group (p < 0.01 for each). AdCT-1 injection significantly inhibited Fas, Bax and p53, and increased CT-1 and Bcl-2 expression in myocardium. These results suggest that adCT-1 vector can be effectively transfected and continued to express bioactive CT-1 protein in myocardium. CT-1 plays an important cardioprotective effect on myocardial damage in the murine model of myocardial infarction ^Ref . Using right atrium specimens and ischemic preconditioning (PC), it was shovn that CT-1 exhibits a significant protection of the human myocardium against ischemic injury when tissue is exposed to this factor for a long period (e.g. 24 h) but not when exposed for a short period (e.g. 2 h). In addition, the protection afforded by long exposure to CT-1 is as potent or even greater than the one obtained by the second window of PC. The protection induced by CT-1 but not that induced by PC can be abolished by CT-1 antibody suggesting that their beneficial action is attained by different mechanisms ^Ref .

Cardiotrophin-1 and ischemia/reper­fusion injury

CT-1 can exert a protective effect against the damaging effects of simulated ischaemia/reo­xygenation both when added after the simulated ischaemia at reoxygenation (p<0.05 in trypan blue, TUNEL and annexin V assays) or when added prior to the simulated ischaemia (p<0.05). In both cases, these protective effects are blocked by an inhibitor of the p42/p44 MAPK pathway (p<0.05 in all assays). Thus, CT-1 can protect cardiac cells when added either prior to simulated ischaemia or at the time of reoxygenation following simulated ischaemia and these effects are dependent upon its ability to activate the p42/p44 MAPK pathway. Hence CT-1 may have therapeutic potential when added at the time of reperfusion following ischaemic damage ^Ref . Cardiac cells were exposed to hypoxia/ischaemia followed by reoxygenation/re­perfusion and CT-1 was administered either prior to hypoxia/ischaemia or at reoxygenation/re­perfusion. CT-1 has a protective effect in reducing ischaemic damage in the intact heart ex vivo as assayed by infarct size to area at risk ratio (20% compared to 35%). Similar protective effects against cell death were noted in adult cells in vitro. Both in vitro and ex vivo CT-1 can exert a protective effect when added at the time of reoxygenation/re­perfusion as well as prior to the hypoxic/ischaemic stimulus (cell death reduced from 50 to 20% in TUNEL assay, infarct size to zone at risk ratio reduced from 35 to 20%). These protective effects are blocked by an inhibitor of the p42/p44 MAPK pathway. Taken together, CT-1 can protect adult cardiac cells both in vitro and in vivo when added both prior to or after the hypoxic/ischaemic stimulus ^Ref .

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