| Summary: | 碩士 === 高雄醫學院 === 醫學研究所 === 87 === Sepsis is the deleterious systemic response to severe infection. In the heart, enzyme/receptor systems have been reported to be damaged during sepsis, causing a decrease of contractility and leading to heart failure and even death. Differential performances occur in various regions of myocardium in heart failure during sepsis. However, protein kinase A (PKA) has been implicated to play a pivotal role in the regulation of signal transduction, Ca2+ handling and contractility in the heart. The subunit isoforms (RI、RII、C 、C) of PKA exhibit diverse patterns of tissue expression, intracellular distribution and different biological roles. Accordingly, the present study was to investigate the sepsis-induced changes in PKA activities and subunit expression in various regions of rat ventricle for understanding the further pathophysiologic mechanism of altered myocardial function during sepsis.
Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis were referred to those animals killed at 7~9 and 18~20h, respectively, after cecal ligation and puncture. Cardiac PKA was extracted and partially purified by acid precipitation, ammonium sulfate fractionation, and DEAE-cellulose chromatography. PKA was eluted from DEAE-cellulose column with a linear NaCl gradient. Two peaks of PKA, Type I (eluted at low ionic strength) and Type II (eluted at high ionic strength), were collected, and their activities were determined basing on the rate of incorporation of [γ-32P]-ATP into histone. PKA expression was analyzed by using Western bolt analysis.
Our results showed that PKA activities were unevenly distributed among various regions of ventricle of the control rat. Cytosolic Type I PKA activities were the lowest in septum and about a third as that as right and left ventricle while cytosolic Type II PKA activities were evenly distributed among different regions of rat ventricle. In the membrane-associated fractions, the activities of Type I PKA and Type II PKA were not significant difference among various regions in the ventricle of the control rat. During early sepsis, both cytosolic and membrane-associated PKA activities remained unchanged. During late sepsis, cytosolic Type I PKA activities were increased by 228% but membrane-associated Type I PKA were decreased by 51.4% in septum, while both cytosolic and membrane-associated Type II PKA activities remained unchanged. The results of PKA subunit expression revealed that cytosolic C subunit expression was increased by 20.50% in right ventricle during early sepsis. During late sepsis, cytosolic RI subunit expression was decreased by 14.72% and 19.74% in septum and left ventricle respectively. Cytosolic C subunit expression was increased by 26.29% in right ventricle, while membrane-associated C subunit expression was decreased by 31.09% in septum during late sepsis.
The results of the present study suggest that sepsis-induced changes of PKA activities in septum and selective alternative expression of PKA subunit among various regions of rat ventricle may contribute to the altered cardiac function during sepsis. In septum, Type I PKA activities were preferentially stimulated in cytosol and decreased in membrane-associated fractions, respectively, whereas the expression of cytosolic RI subunit and membrane-associated C subunit were decreased selectively. We suggest that (1) changes of Type I PKA activities in septum may be the predominate effect causing heart failure in sepsis；(2) during late sepsis, the increased activities of cytosolic Type I PKA in septum may due to the decreased expression of RI subunit while the decreased activities of membrane-associated Type I PKA in septum may due to the decreased expression of C subunit；(3) dysfunction of the septum may be the primary cause of heart failure during sepsis.
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