The Role Of Stress PKCs On The Transition From Cardiac Hypertrophy To Heart Failure

Chronic hypertension results in compensatory cardiac hypertrophy, which will thereby enhance cardiac contractility and reduce the ventricular wall-stress. And cardiac output will consequently increase to some degree. Excessive cardiac hypertrophy will finally weaken cardiac performance during long-term over-load blood pressure. In the transition from compensatory cardiac hypertrophy to cardiac dysfunction, there might be some pivotal nodes which can be regulated to slow down or even inverse this process.Protein kinase C (PKC) is a group of closely related serine-threonine protein kinases, extensively demonstrated to be involved in a variety of chronic cardiac diseases as well as in precondition and acute cardiac injuries. This study aimed to observe the roles of stress PKCs on contractile function of hypertrophic heart in transverse abdominal aortic constriction (TAC) rats, so as to demonstrate that PKC isoform contributed as a pivotal node during the transition from cardiac hypertrophy to heart failure.Fourty eight male Sprague-Dawley rats were randomly divided into control and TAC groups. After 1w, 8w, 16w, and 20w, working-heart perfusion system and Millar probe were applied to monitor cardiac function indexes. Immunofluorescent cytochemistry, confocal analysis and other methods such as Western Blot and in vivo siRNA transfection were also applied to detect the relationship bwteen stress PKC activation and cardiac function. The results are as follows.1. TAC model induced different levels of cardiac hypertrophy and CO decrease.The effect of long-term hypertension played an important role in the transition from hypertrophy to heart failure.We applied isolated working-perfusion to test the cardiac function of the TAC rats and the control. Under the preload of 10 mmHg and the afterload of 60 mmHg, there was no significant difference of cardiac functions between 1w or 8w TAC and the control groups in the working heart mode. With 100 nm PMA perfusion, TAC rats showed a slight decrease in CO and IHR. The cardiac function remarkably up-regulated in 16w TAC rats compared with its control group. After PMA perfusion for 10 min, CO and IHR decreased significantly in 16w TAC rats. Heart function had been conversed into decompensation with a decreased CO but increased IHR, which resulted in SV shrink in 20w TAC rats. After 100 nM PMA perfusion for 10 min, 20w TAC rats suffered from a more serious cardiac dysfunction compared with 16w TAC rats. The LVEDP increased and -dP/dt_max decreased more after PMA perfusion, which showed an evidence of diastolic dysfunction.2. Long-term TAC rats suffered a serious impairment of both contraction and relaxation, with a more severe damage of diastole function at a high heart rate.The results and analysis of working-heart perfusion showed that long-term TAC rats suffered a serious impairment of both contraction and relaxation, with a more severe damage of diastole function.Under the combination with the pacing of 300 beats/min, 360 beats/min and 420 beats/min and 100 nM PMA perfusion for 10 min,CO slightly increased and then significantly decreased associated with a higher heart rate in 16w TAC rats. The slight increase of CO at 360 beats/min whould be induced by FDAR. At an even higher HR, the cardiac cycle markedly shortened, and cardiac diastole was damaged more severly.3. PKCαtranslocation increased in cardiomyocytes of TAC rats, which implied that PKCαmay play an important role in the transition from cardiac hypertrophy to heart dysfunction.Translocation of PKCs from cytoplasm to plasmalemma has been regarded as the symbol of activation. In the present study, immunofluorescent cytochemistry and confocal analysis were applied to observe the translocation of PKCs. The results showed that no significant differences were observed in the translocation of PKCβ, PKCδor PKCε. But, distinguished differences showed up in PKCαtranslocation. Without PMA, cardiomyocytes showed augmented immunofluorescence density in both 16w and 20w TAC rats compared with their synchronous control groups. With PMA incubation, the PKCαtranslocation increased even more in TAC group.4. Changes of the expression of PKC isoforms and proteins associated with Ca²⁺ handling in the myocarium of TAC rats.Western Blot was applied to test the expression of PKC isoforms and proteins associated with Ca²⁺ handling in the myocarium. We observed that there was an increase in protein expressions of PKCα, PKCβⅠ,PKCεand PKCδin TAC group compared to their synchronous control group. But PKCαshowed a significant translocation from cytoplasm to sarcolemma. The above conclusion guided us into further research of PKCαand some relative protein expression.We tested some vital protein expressions related with excitation-contraction coupling. There was no significant difference in the expression of LTCC. The expressions of PLB and phosphorylation of PLB at Ser16 decreased in the myocardium of 16w and 20w TAC hearts, but no significant change of PLB-Thr17 phosphorylation level. And the SERCA2αactivity was weakened in TAC group.PKCαactivation may inderectly depresses the activity of SERCA2α, thus Ca²⁺ storage in sarcoplasmic reticulum reduces, which induces a reduction of Ca²⁺ released from sarcoplasmic reticulum and causes cardiac dysfunction. There must be intermedial node between PKCαtranslocation and PLB-Ser16 phosphorylation decrease. Some reports show that translocalization of PKCαpermits upregulation of PP-1 activity through direct phosphorylation of I-1. An increase in PP-1 activity reduces PLB phosphorylation, promoting greater inhibition of SERCA2αand diminished sarcoplasmic reticulum Ca²⁺ loading and release, which was shown in transgenic animals model, but not in isolated working heart from surgical model as our study did.In order to explore the underlying mechanism, this study had tested in-vivo siRNA transfection. Until now, we have tried to establish the right tranfection methods, to get the right specific siRNA sequence, and to achieve a success in experimental in-vivo siRNA injetion. The next step to do is to continue to apply the in-vivo transfetion reagent into TAC rats, and monitor the relative indices such as CO and protein expression.