Animals ; Endothelium, Vascular ; metabolism ; ultrastructure ; Environmental Exposure ; Male ; Rats ; Rats, Sprague-Dawley ; Sound ; Vascular Endothelial Growth Factor A ; metabolism

AIMTo investigate the changes in the expression of four kinds of calcium regulatory proteins mRNA on the isolated ischemia/ reperfusion (IR) hearts.

METHODSThe rat hearts were divided into two groups: control group and IR group which received 45 min exposure to Krebs-Henseleit solution after 15 min zero-flow global ischemia. The indexes of left ventricular function, such as LVDP, +dp/dt(max), -dp/dt(max), and an arrhythmia scoring system were compared between the two groups. The messenger ribonucleic acid (mRNA) amount of sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA), phospholamban (PLB), inositol 1,4,5-trisphosphate receptor2 (IP3R2) and ryanodine receptor2 (RyR2) was measured by reverse transcription-polymerase chain reaction (RT-PCR) and normalized to the mRNA levels of beta-actin.

RESULTSIn the IR group, LVDP, +dp/dt(max) and -dp/dt(min) of the isolated hearts were depressed and the high rate of arrhythmias occurred during reperfusion. The levels of SERCA, IP3R2, RyR2 mRNA were lower in the IR isolated hearts group than those in the control group, while there was no difference in the level of phospholamban.

CONCLUSIONThese data suggest that myocardial ischemia/reperfusion can induce the depression of cardiac performance and an increased risk of arrhythmias, concomitant with the decrease in SERCA, IP3R2, RyR2 mRNA steady state levels.

Animals ; Myocardial Reperfusion Injury ; metabolism ; Myocardium ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; genetics ; metabolism

OBJECTIVETo study the role of sirtuin 1 (SIRT1) in Fas ligand (FasL) expression regulation during vascular lesion formation and to elucidate the potential mechanisms.

METHODSSIRT1 and FasL protein levels were detected by Western blotting in either mouse arteries extract or the whole rat aortic vascular smooth muscle cell (VSMC) lysate. Smooth muscle cell (SMC)-specific human SIRT1 transgenic (Tg) C57BL/6 mice and their littermate wild-type (WT) controls underwent complete carotid artery ligation (ligation groups) or the ligation-excluded operation (sham groups). The carotid arteries were collected 1 day after operation. Reverse transcription-polymerase chain reaction was performed to detect the mRNA levels of SIRT1 and FasL. Luciferase reporter assays were performed to detect the effect of WT-SIRT1, a dominant-negative form of SIRT1 (SIRT1H363Y), and GATA-6 on the promoter activity of FasL. Flow cytometry assay was applied to measure the hypodiploid DNA content of VSMC so as to monitor cellular apoptosis.

RESULTSSIRT1 was expressed in both rat aortic VSMCs and mouse arteries. Forced SIRT1 expression increased FasL expression both in injured mouse carotid arteries 1 day after ligation (P<0.001) and VSMCs treated with serum (P<0.05 at the transcriptional level, P<0.001 at the protein level). No notable apoptosis was observed. Furthermore, transcription factor GATA-6 increased the promoter activity of FasL (P<0.001). The induction of FasL promoter activity by GATA-6 was enhanced by WT-SIRT1 (P<0.001), while SIRT1H363Y significantly relieved the enhancing effect of WT-SIRT1 on GATA-6 (P<0.001).

CONCLUSIONSOverexpression of SIRT1 up-regulates FasL expression in both flow-restricted mouse carotid arteries and serum-stimulated VSMCs. The transcription factor GATA-6 participates in the transcriptional regulation of FasL expression by SIRT1.

Animals ; Apoptosis ; Carotid Arteries ; physiology ; Fas Ligand Protein ; genetics ; GATA6 Transcription Factor ; physiology ; Male ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Myocytes, Smooth Muscle ; metabolism ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Sirtuin 1 ; physiology ; Up-Regulation