Alteration Of δ-PKC Expression And Late Cardioprotection Of Exercise Preconditioning Against Myocardial Injury Induced By Exhaustive Exercise

Objective: Ischemic preconditioning (IP) is the exposure of myocardialtissue to brief, repeated periods of vascular occlusion in order to render themyocardium resistant to the deleterious effects of prolonged episodes ofischemia or reperfusion. The protection involves with an endogenousmechanism and can be induced by different modes including exercisepreconditioning (EP), which has been associated with an early and latephase protection. Evidence has shown different EP protocols exertcardioprotection against myocardial ischemia/hypoxia. In sport medicine,the myocardial injury induced by exercise should not be ignored, theexhausted exercise may have a potential to induce the myocardialischemia/hypoxia injury, and it is not clear whether EP can exert acardioprotective effect on the myocardial injury induced by exhaustedexercise. As a cellular mediator, protein kinase C (PKC) is a family ofisozymes consisting of12isozymes. Most studies were focused on isozymesε-PKC, δ-PKC and α-PKC, and ε-PKC and α-PKC have been proposed toplay a vital role in IPC, whereas the role of δ-PKC is still ambiguous. Thedifferent mode of preconditioning may have a different mechanism ofprotection, and the alteration of δ-PKC expression and its role in EP stillneed investigations. Because the late window of protection lasts for a longtime and is more valuable to cardioproteciton, we investigated the latecardioprotective effect of EP against myocardial injury induced byexhausted exercise as well as the δ-PKC expression in late phase of EP,using a single bout of interval running on the treadmill as the EP protocol.Methods:120SD rats were randomly assigned to4experimental groups:C, EP, EE and EP+EE. Rats in group EP were subjected to a single bout ofinterval exercise. Rats in group EP+EE were subjected to exhaustedexercise24h after a single bout of interval exercise. The myocardial injurywas evaluated quantitatively in terms of the serum cardiac troponin I (cTnI)levels, the myocardial ischemia/hypoxia area and the integral opticaldensity (IOD) of haematoxylin-basic fuchsin-picric acid (HBFP) staining,qualitatively in terms of the myocardial ultrastructure and HE staining. At the same time, the distribution of δ-PKC in cardiomyocytes was shown byimmunohistochemistry, the positive area and IOD were calculated using theImage-Pro Plus software, the concentration of δ-PKC in cardiomyocyteswas measured by western bloting, and the expression of δ-PKC mRNA wasevaluated by the real-time fluorescence quantitative PCR.Results:(1) Compared with group EE, the exercise capability of rats ingroup EP+EE were significantly increased with longer running time anddistance during exhausted exercise (P<0.05).(2) Serum cTnI levels werefound to be normal in group C and EP, while were significantly elevated ingroup EE and EP+EE (P<0.05). Compared with group EE, serum cTnIlevels in group EP+EE were significantly decreased (P<0.05).(3)Cardiomyocytes in group C and EP showed a normal structure under HEstaining, and no ischemic/hypoxic area was found under HBFP staining;Cardiomyocytes in group EE showed a damaged structure under HE staining,and large ischemic/hypoxic area was found under HBFP staining;Cardiomyocytes in group EP+EE showed a mild extent change in structureunder HE staining, and decreased ischemic/hypoxic area was found underHBFP staining (P<0.05).(4) Cardiomyocytes in group C and EP showed anormal ultrastructure; Cardiomyocytes in group EE showed a damagedultrastructure with swollen mitochondria and disorganized myofibrils;Cardiomyocytes in group EP+EE showed a less extent change inultrastructure.(5) The immunohistochemical staining of δ-PKC in group Cwas brown grain-shaped and scattered across the cytoplasm and membrane;Staining was increased in group EP, while decreased in group EE,sometimes with membrane translocation; No membrane translocation wasfound in group EP+EE, the staining of which was similar to those describedin group C. The immunohistochemical staining of P-δ-PKC was distributedacross the cytoplasm and membrane, small brown grain-shaped and blotted,and no significant difference was found in groupEP+EE, and a increasedstaining in group EP and a decreased staining in group EE. Themorphometric analysis showed the positive area and IOD of δ-PKCimmunohistochemical staining in group C were1210±423μm~2and393882±68774, respectively. The values in group EP were1978±917μm~2and492410±96825, respectively. The values in group EP were higher thanthose in group C (P<0.05), the values in group EE decreased significantly(P<0.05), and were527±201μm~2and234246±35476, respectively.Compared with group EE, the values in group EP+EE increasedsignificantly (P<0.05), and were1087±767μm~2and319542±74211, respectively. The positive area and IOD of P-δ-PKC immunohistochemicalstaining in group C and EP+EE were218±28μm~2and55332±1840,178±34μm~2and50475±1414， respectively. There is no difference amongtwo groups. Compared with group C, the values in group EP increasedsignificantly (P<0.05), and were279±37μm~2and68270±1572. Comparedwith group C, the values in group EE decreased significantly (P<0.05), andwere146±63μm~2and44606±2229, respectively.(6) The quantitativedetermine by Western bloting showed that, compared with group C（0.66±0.12）, the expression of δ-PKC was increased in group EP(0.76±0.16), but not significantly. Significantly decreased in group EE(0.26±0.06),(P<0.05). The expression in group EP+EE (0.60±0.10) waslower than that of group C, but not significantly, and significantly higherthan that of group EE (P<0.05). The expression of P-δ-PKC in group C(0.83±0.08) and EP+EE (0.61±0.12) had no difference, while the expressionin group EP (0.92±0.06) was significantly increased (P<0.05), theexpression in group EE (0.41±0.13) was significantly decreased (P<0.05).(7) δ-PKC mRNA content in cardiomyocytes was1.49±0.73in group C and2.33±1.32in group EP, the value in group EP was higher than that in groupC, but not significantly. Compared with group C, δ-PKC mRNA content ingroup EE (1.46±0.89) had no change and the value in group EP+EE(1.19±0.45) was decreased, but not significantly.Conclusion:(1) A single bout of interval running on the treadmill can beused to prepare an EP protocol, and the exhausted exercise can induce anacute myocardial injury in rats. Exercise preconditioning provdie latecardioprotection against myocardial injury induced by exhaustive exerciseused to evaluate (2) After exhausted exercise, the expression of δ-PKC inthe myocardium was decreased, exercise preconditioning can attenuate theδ-PKC reduction in the myocardium during exhausted exercise in the latephase, exercise preconditioning may increase δ-PKC expression which isresponsible for the injury in cardiomyocytes.(3) EP has no influence on theexpression of δ-PKC mRNA, the regulation of δ-PKC in EP is mainly basedon the protein level.