Effects of Cardiotrophin-1 on Adriamycin-Induced Apoptosis in H9c2 Cardiomyoblasts

BACKGROUND AND OBJECTIVES: Adriamycin (doxorubicin, ADR) is a highly effective anti-neoplastic drug, but its clinical use is limited by its adverse side effects on the heart. Cardiotrophin (CT-1), a potent cardiac survival factor, is capable of inhibiting apoptosis in cardiac myocytes. The aim of this study was to investigate the cyto-protective effects of CT-1 against ADR-induced apoptosis in vitro. MATERIALS AND METHODS: We determined a reasonable ADR concentration for inducing cell death by utilizing a cell survival test performed in a dose-dependent manner. To determine the requirements for apoptosis in ADR-treated cardiac myocytes (H9c2 cells), we examined the effect of CT-1 on survival and apoptotic changes using a cell counting kit (CCK), RT-PCR, and Western blotting. RESULTS: In analyzing cell survival as determined by CCK, ADR-induced cell death was found to occur in a dose-dependent manner (50% death at 24 hours after 2 micrometer of ADR), and ADR was shown to decrease procaspase-3. On RT-PCR, expression of Bax-alpha mRNA increased and Bcl-2 decreased during the 24 hours after ADR treatment. Consequently, the ratio of Bax-alpha/Bcl-2 mRNA peaked at 24 hours after ADR treatment. In contrast, CT-1 effectively attenuated the ADR-induced cell death in a dose-dependent manner. The changes in Bax-alpha and Bcl-2 mRNA expression after ADR treatment were reversed by CT-1 (1 ng/mL) treatment. The protein levels of procaspase-3 decreased after ADR treatment, an effect which was reversed by CT-1 treatment. Akt phosphorylation was also increased by CT-1, demonstrating that CT-1 inhibited apoptosis induced by ADR. CONCLUSION: These data demonstrated that ADR-induced apoptosis of cardiomyocytes can be prevented by CT-1; therefore, it may be possible to use CT-1 as a cardioprotective agent during ADR chemotherapy in patients with cancer.

Myocardial injury occurs earlier than myocardial inflammation in acute experimental viral myocarditis

Endomyocardial biopsy often fails to show myocardial inflammation for patients with clinically suspected myocarditis. The serum isoforms of troponin T (cTnT) level is a very sensitive marker of myocardial injury and it is elevated even in the absence of myocardial inflammation. We investigated the correlations for myocardial injury, virus titers and inflammation in acute viral infection. Using the murine coxsackievirus group B3 (CVB3) myocarditis model, the histopathologic findings and virus titers in mouse hearts were compared with the serum cTnT levels measured by ELISA at various time points. Viable virus titers in the hearts peaked at 3 days after infection (8.22+/-0.13 log10 PFU/100 mg of heart); they decreased at day 7 and no viable virus was detected from day 14. Myocardial inflammation was minimal at day 3, peaked at day 7 and markedly decreased at day 14. The individual serum TnT levels were significantly increased at day 3 (7.37+/-1.46 ng/ml), persisted to day 7 (0.73+/-0.08 ng/ml), and normalized at day 14. Serum cTnT levels were correlatable with virus titers in the heart (r=0.744, P <0.01), but the serum cTnT levels were not correlated with the degrees of inflammation. Using the less myocarditic strain of CVB3, similar relationships were observed between the changes for the serum cTnT levels and the heart virus titers. During the course of viral infection, myocardial injury precedes the pathologic evidence of inflammation, and the elevated cTnT levels provide evidence of myocardial injury even in the absence of any histologic findings of myocarditis.

Change of Serum Cardiac Troponin T and Fetal Troponin T Isoform in Rats with Adriamycin-induced Cardiac Injury

BACKGROUND AND OBJECTIVES: Cardiac troponin T (cTnT) has been used as a very sensitive marker of cardiac injury caused by ischaemia, myocarditis, and cardiomyopathy. After cardiac injury, the fetal cTnT isoform expression in the heart and serum cTnT increases. To investigate the increased levels of serum cTnT, and the expression of fetal cTnT isoform in the heart, that can predict myocardial injury, we measured serum cTnT levels and the fetal cTnT isoform expression at various time points during the early phase of myocardial toxicity induced by adriamycin (ADR) in rat. MATERIALS AND METHODS: Male Sprague-Dawley rats were injected, intraperitoneally, with ADR (5 mg/kg) twice a week for 2 weeks. Control rats were injected with saline. Serum cTnT levels were measured by ELISA. The ratio of fetal/adult (F/A) cTnT isoform expression (%) was semi-quantified by RT-PCR using total RNA from frozen hearts. RESULTS: Serum cTnT levels did not increase by 1 week after ADR injection, but increased significantly after 2 weeks. The ratio of F/A cTnT in the heart significantly increased from day 1, peaked at 1 week and persisted until the end of 2 week. CONCLUSION: The expression of the fetal cTnT isoform occurred from 1 day after ADR injection when the serum cTnT levels were still normal. Although the serum cTnT level is a very sensitive, and an early marker, of cardiac damages, the fetal cTnT isoform expression in the endomyocardial biopsy specimen may be a more sensitive and an earlier marker in the ADR-induced myocardial damage.