very moderate exercise training; variations in stress and toxicity gene expression in rat hearts.. Rattus norvegicus

Array analysis of a set of 96 stress and toxicity genes was carried out in left ventricular cardiomyocytes from 8 control and 7 trained rats. The training protocol was very gradual and mild. Animals were sacrificed 48 hrs after the last training session in order to evaluate gene expression during the "second window of cardioprotection". Array results were validated by Real Time PCR of selected genes and Western Blot analysis of selected proteins. Plasma MDA and resistance to ischemia/reperfusion were evaluated as well. A total of 11 genes were found to be upregulated by moderate exercise, 3 to be downregulated. MDA was found to be increased in plasma of trained rats. Myocardial resistance to ischemia-reperfusion was found to be improved in trained rats with respect to controls. Results suggest that such a pattern of mild training does at the same time elicit stress responses, but also the upregulation of a number of defensive genes, that are likely to constitute the core of the adaptive mechanisms. Keywords: exercise training-related stress response and cardioprotection Overall design: Male albino Sprague-Dawley rats entered the study at 2 months of age. After one week of acclimatization, eight animals were randomly chosen for exercise training. The exercise was produced on a specific treadmill for rats, with four lanes. The training protocol was aimed at providing a gradually increasing effort to reach 50 % VO2max in 11 weeks; such workload was sustained for a further 3 wks. Animals were trained 1hr /day 3 times a week. Control animals were placed on a non-moving treadmill during the training sessions. Animals were sacrificed 48 hrs after the last training session. Rats were anesthetized (100 mg/kg ip heparinized sodium thiopental); for RNA preparation hearts were removed and immediately frozen in liquid nitrogen and stored at -80°C. According to the original design, the sample size of control and trained animals was the same (8 control and 8 trained rats), but 1 trained rat died for reasons unrelated to the training procedure. Frozen cardiac tissue (n= 8 control and 7 trained rats) was reduced to powder by means of a sterilized ceramic mortar and pestle. RNA isolation was obtained by TRIZOLTM (Invitrogen s.r.l., Milan, Italy), as suggested by the manufacturer. The absence of RNA degradation was assessed by evaluation of 28S and 18S band sharpness after denaturing electrophoresis. The absence of contaminating genomic DNA was confirmed by PCR analysis with tubulin promoter-specific primers. To control for inter-individual variation, an equivalent mass of total RNA from each subject in the group was pooled to generate the total RNA sample for probe generation. Synthesis of Biotin-16-dUTP labeled cDNA probes was obtained with the use of AmpoLabeling-LPRTM Kit (SuperArray, Bioscience Corporation, Frederick, USA). In order to minimize false signals, for each RNA pool (control or trained rats), three replicas were made , with independent labeling and hybridizations procedures, so that six arrays were prepared. The Stress and Toxicity-focused Arrays (GEArray Q SeriesTM, SuperArray) were employed. Signal was developed by a chemiluminescent method (alkaline phosphatae-conjugated streptavidin and CDP-Star®) and detected by exposing the membranes to X-ray film Kodak X-Omat AR (Sigma-Aldrich) . The images from the arrays were scanned, acquired in a PC and analyzed using the software provided by ScanAlyze ([e-mail]). Minimum spot intensity was used for automatic background subtraction. Data were then normalized with respect to the housekeeping gene RPL13a, one of the positive controls in the array. The three results obtained for each gene were averaged to give an expression score. Other evaluations reported in Summary and contributing to the interpretation of array data (myocardial morphology, ischemia-reperfusion) were carried out on additional rats that were trained and handled in the same way and at the same time as those employed for the array study.