The aim was to examine the expression of ubiquitin (Ub), 27 kDa heat shock protein (hsp27), and hsp60 MRNA in normal and briefly ischaemic and reperfused porcine myocardium: Methods: The left anterior descending coronary artery was occluded for two periods of 10 min separated by 30 min of reperfusion. After the second occlusion the myocardium was reperfused up to 210 min. Tissue from ischaemic, ischaemic-reperfused, and non-ischaemic regions of the heart was analysed by northern and slot blot hybridisation and nuclear run-on transcription assays employing radiolabelled cDNA probes for Ub, hsp27, and hsp60, as well as by western blot using monoclonal antibodies recognising Ub protein conjugates and antiserum recognising hsp27. Results: Systolic wall thickening was significantly decreased at 30 min reperfusion after both occlusions and remained depressed at longer periods of reperfusion. Using northern blot hybridisations, several mRNAs encoding Ub, 0.9 kb mRNA encoding hsp27, and 2.2 kb mRNA encoding hsp60 were detected in sham operated, non-ischaemic, and ischaemic myocardial tissues. Densitometric analysis of northern and slot blot hybridisation signals showed significant increase of basal tissue levels of Ub mRNA in stunned regions only during the 30 min of the second reperfusion period. Increased levels of hsp27 mRNA in stunned tissue were already noted at the first ischaemic period and were sustained compared to control during the subsequent periods of reperfusion. Changes in hsp60 mRNA tissue levels were not observed during ischaemia and subsequent reperfusions. Transcription of the Ub and hsp27 genes was increased during 30 and 120 min of the second reperfusion period. The transient enhancement of tissue levels of Ub mRNA was associated with temporary formation of new Ub-protein conjugates. However, the increased synthesis of mRNA encoding hsp27 was not followed by changes of hsp27 protein content in myocardial tissue. Conclusions: The findings support the hypothesis that molecular damage occurs in stunned myocardium; however, the target molecules remain to be recognised.
