| Background:Cystatin C (CysC) is a novel marker of renal function which may replace creatinine and urea nitrogen. Recent data suggested that CysC may be a early marker of the endpoint events of cardiovascular diaseses(including of nonfatal myocardial infarction and heart failure, etc.) and CysC was strongly associated with left ventricular remodeling and heart dysfunction, especially in the subgroups of old person or essential hypertension. Some studys on rats showed that Cats/CysC mRNA and protein expression were upregulated at the stage of myocardial hypertrophy, but CysC expression was absolutely or relatively downregulated at the stage of heart failure while Cats expression kept on rising. All of those showed that CysC was not only a biomarker, but also a participator in the left ventricular remodeling and heart failure. So this investigation tried to explore the effect of the CysC over-expression in SHR myocardium on the left ventricular remodeling and cardiac function.Methods:1. Animal models and experimental design:63 spontaneously hypertensive rats were randomly assigned to Ad-CysC overexpressed group,Ad-Null group and Control group.21 Wistar rats were selected for reference group. The rats were killed in batches at 0 days before intramyocardial injections and 2 days,4weeks after the transfection. Blood pressures,weights and left ventricular mass were measured, and then left ventricular mass index (LVMI) was calculated. The cardiac apex tissue (about 1/2 of ventricle) was stored in-80℃for qRT-PCR. The other tissue was fixated in 4% neutrality phosphate paraformaldehyde liquid for 48 hours and then was imbedded with paraffin for immunohistochemical staining.2. TransfectionGuiding by ultrasound (PHILIPS IE33, S12-5 ultrasonic probe), 100ul Ad-CC or Ad-nul (5.36X1010 pfu/ml) was injected into the anterior wall and lateral wall of ventricle. The PBS was injected in the same way in the control group.3. EchocardiographyApparatus:PHILIPS IE33, S12-5 ultrasound detector (5MHz frequency). LVEDD,LVFS and LVEF were measured at the left ventricular papillary muscle echo section to evaluate the cardiac structure and heart function.4. Quantitative reverse transcription PCR (qRT-PCR)Total RNA was extracted by Trizol. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was used to detecte the relative mRNA expression. CysC specific primers were designed by Primer5.0, beta-actin for reference.5. Immunohistochemical stainingImmunohistochemistry staining was used to detect the protein expression of CysC and CatK. The average of IOD were detected to show the difference of the groups.6. Masson stainingMasson staining was used to test the collagen in this study. And CVF was calculated to show the difference of the groups.Results: 1. Three rats died in the experiment, including two in Ad-CC group and one in Ad-null group.2. In the Ad-CC group, CysC expression was upregulated on the second day after transfection (P value<0.001), and it still had statistically significance in the fourth week after transfection(P value＜0.05). There was no difference between N and C group. Immunohistochemical staining showed the same changes. Compared with the CatK expression in W group, the CatK expression in all SHR groups was upregulated, but there was no difference within groups. Compared with CysC expression in W group, CysC expression in N and C groups were downregulated.3. Compared with C group, Ad-CC group showed significant difference (All of P value< 0.05) about LVMI,LVEDD and LVFS 4weeks after transfection. CVF increased, but no statistically significance (P=0.076). There was no significant difference between N and C group. Compared with W group, all the SHR groups showed significant difference (All of P value< 0.01) about LVMI,LVEDD and LVFS.Conclusion:1. Intromyocardial injection guiding by ultrasound was successful and may reduce the mortality of experimental rats.2. The expression of CysC in myocardium of SHR was downregulated at the stage of chronic heart failure, while the expression of CatK was upregulated. So it suggested that there was an association between the banlance of Cats and CysC and heart function.3. The upregulated expression of CysC in the myocardium may keep down the left ventricular remodeling and heart failure progresses at the stage of chronic heart failure in SHR. Key Words:Cystatin C;overexpressing;left ventricular remodeling; spontaneously hypertensive rats. |
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