Effect Of Simvastatin On Myocardial Structure And Function In Mice With Viral Myocarditis

Background and objective:Clinical situations of patients with viral myocarditis are different, and the severe cases can cause congestive heart failure, Aase's syndrome, cardiogenic shock and even the death of the patient. Damage and exhaust of pumping function of heart is the main cause of the change. Up to now, there is no general agreement concerning effective therapy for viral myocarditis. Now combined therapy has been primary method. Though the therapy can improve patient prognosis, it has no effect on contractile function of the left ventricular in acute viral mycarditis. Simvastatin is a lipotropy medicine, and is one of five statins that have generally been applied to lipid lowering in the world. For the past few years, simvastatin has exhibited important actions in improvememt function of endothelial cell, anti-inflammatory, anti-oxidant, inhibiting myocardial fibrosis and hypertrophy, and the actions are independent of lipid lowering. It has been reported by Obama et al that simvastatin can lessen myocardial inflammatory reaction through anti-inflammatory, immunomodulatory and so on. But there is little document about the effect of simvastatin in the early phase of murine myocarditis, especially its effect on apoptosis of cardiomyocyte and hemodynamic of the left ventricular. This experiment will study the effect of simvastatin on myocardial structure and function in mice with viral myocarditis and try to seek a new therapy to the disease.Materials and methods:1.Materials(1)Experimental animals Sixty male BALB/c mice inbreeding (provided by Test Animal Center of School of Basic Medical Sciences in Jilin University), 4 weeks old, weighing 13 to 15 grams and healthy.(2)VirusThe CVB3 strain was provided by Department of Microbes, School of Basic Medical Sciences in Jilin University, and median tissue culture infectious dose (TCID50) of the stock is 100.(3)ReagentSimvastatin, TUNEL kit, RT PCR kit, Rabbit anti-human Fas polyclonal antibodies, S-P immunohistochemical kit, Trizol reagent, Ethidium bromide, Collagenase, Protease, CdCl2, Glibenclamide.(4)EquipmentsInverted microscope, Transmission electron microscope, ABI9700-PCR Amplification Meter, Patch clamp amplifier, Microelectrode puller, BL-420 polygraph, Hydraumatic microelectrode thruster.2.Methods:(1)Experiment groups and model preparation of VMCSixty male BALB/c mice randomized to 5 groups. One group of mice were inoculated intrapritoneally with normal saline(NS) and served as normal control (n=12, treated intragastric with NS daily for 1 wk); the remaining 4 groups were infected intrapritoneally with CVB3 and treated intragastric with NS and simvastatin at a dose of 10(low-dose), 30(middle-dose), 90mg?kg-1 (high-dose) for 1 wk(n=12 in each group, served as viral control, low-dose, middle-dose, high-dose simvastatin treatment control), respectively. On day 7 postinoculation, killing was performed by cervical dislocation.(2)Histopathological changesTissue sections from hearts were stained with hematoxylin and eosin, and examined microscopically. Semiquantitative histopathologic grading of the percentage of involved areas of cellular infiltrate (I) and necrosis (N) of the myocardium by planimetry of projected left ventricular hisological sliced was in accordance with method of literature[62]: 0, absence of infiltration or necrosis; 1=less than 25%, 2=26 to 50%, 3=51 to 75%, and 4=more than 75% of myocardium. Results are expressed as mean±SEM.(3)Apoptosis index of cardiomyocyte and content of Fas mRNA and proteinum were detected respectively by in situ end-labeled DNA(Tunel), reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.For negative controls, deoxynucleotidyl transferase was eliminated from the reaction mixture. For each myocardial specimen, tissue sections were examined microscopically and at least 500 cells were counted in a minimum of five high-power fields. The percentage of apoptotic cells was determined by means of an apoptotic index; the apoptotic index was calculated by dividing the number of positive-staining myocyte nuclei by the total number of myocyte nuclei and multiplying that value by 100.(4)ICa-L was recorded from enzymatically isolated the mice ventricular myocytes using the whole-cell patch clamp technique. levels of LCCs alpha1 subunits mRNA in myocardium were detected by RT-PCR.(5)LVSP and dp/dt of left ventricle in mice acute myocarditis were measured by 1.4 microtubular and BL-420 polygraph.(6)StatisticsStatistical software (SPSS14.0) was used for all statistical computations, and t test was used to compare with every two groups.Results:1.Effect of simvastatin on ultrastructure and apoptosis of cardiomyocyte in mice with viral myocarditis(1)General state of health and mortality①General state of healthOn the 1st day of infection, the mice in viral control group had symptom with fever, diarrhea and reduced activity; after this, some of them showed decreased spirit, bad appetite and unhairing. On the 5th day, the mice began to die. From the 5th day to the 7th day, the number of dead mice was five. The mice in simvastatin treatment control groups had a few symptoms and only two mice died.②MortalityThe deaths total ratio of mice caused by CVB3 was 14.58%(7/48), among the total, the mortality of viral group was 41.67% (5/12). The mortality of low-dose simvastatin treatment group was 16.67%(2/12). None of the animals in the normal control and middle-dose, high-dose simvastatin treatment groups died.(2)Histopathology resultUuder light microscope, mice in viral control group, the mononuclear inflammatory infiltrate was focal or diffused in myocardium, and the severe hearts revealed a large area of myocardial necrosis.Under electron microscope nucleolus of cardiomyocyte was irregular, myofibrils dissolved and disrupted, mitochondrion degenerated. The degree of inflammatory cell infiltrate and area of necrosis were significantly less in the simvastatin treatment control groups as compared with viral control group; Table1. shows the semiquantitative histopathologic grading of cellular infiltrate(I) and necrosis(N) of the myocardium in five groups.(3)Effect of simvastatin on myocardial apoptosisMyocardial apoptosis was scarce in normal control group. Apoptotic index(AI) of viral control group was (3.41±1.06)%; in low-dose, middle-dose and high-dose simvastatin treatment control groups they were (2.84±1.14, 1.93±0.89, 1.66±1.11)%, respectively, and they were lower than that in viral control group; especially in middle-dose and high-dose simvastatin treatment control groups, myocardial AI was significantly decreased (P<0.05). AI showed significant positive correlation with the levels of Fas mRNA. Simvastatin inhibited mRNA and proteinum content of Fas in a dose-dependent manner and lessen myocardial apoptosis during viral myocarditis.2.Effect of Simvastatin on LCCsICa-L enhanced from (0.31±0.06)nA in ventricular myocytes of virus groups to (0.32±0.06) nA in ventricular myocytes of simvastatin(10μmol·L-1) treatment control group (P>0.05), showed no significant difference in both groups. ICa-L enhanced from (0.25±0.04)nA of virus groups to (0.28±0.05)nA of simvastatin(30μmol·L^-1) group (P>0.05), showed no significant difference in both groups. ICa-L enhanced from (0.24±0.04)nA of virus groups to (0.31±0.06) nA of simvastatin(90μmol·L^-1) group (p<0.05), marked a significant difference in both groups.Levels of LCCs alpha1 subunits mRNA in normal control group was lower than that in viral control group (9.09±1.03 vs 192.84±11.4, P<0.05). The levels of LCCs alpha1 subunits mRNA in simvastatin(10, 30 and 90mg·kg^-1) control groups was 106.44±6.31, 25.48±2.55 and 16.35±2.34, respectively, and they were lower than that in viral control group (p<0.05); especially in simvastatin(30 and 90 mg·kg^-1 control groups, the myocardial LCCs alpha1 subunits mRNA expression was significantly decreased (P<0.05).3.Effect of simvastatin on hemodynamic of the left ventricularLVSP and +dp/dtmax of left ventricular in viral control group was much lower than that in normal control group (LVSP: 9.5±1.7vs 14.9±2.6; +dp/dtmax: 274±34 vs 353±25); LVSP and +dp/dtmax of left ventricular in simvastatin(10, 30 and 90mg·kg-1) groups were higher than that in viral control group(p<0.05); especially in simvastatin(30 and 90 mg?kg-1) control groups, LVSP and +dp/dtmax of left ventricular were significantly increased (P<0.05). Table2 shows LVSP and +dp/dtmax of left ventricular in five groups.Conclusions: 1. Animal models of acute myocarditis were successfully established in Balb/c mice infected with CVB3. Simvastatin could reduce the grade of myocardial damage of acute viral myocarditis in a dose-dependent manner.2. The myocardial AI and the expression levels of myocardial Fas mRNA and proteinum were enhanced in viral contral group; and the change was correlated with mycarditic onset, which was a significant mechanism of myocardial damage.3. Simvastatin decreased the grade of myocardial damage and AI through blocking virus-induced Fas mRNA and proteinum expression, which might exert a protective effect in myocarditis mice infected by CVB3.4. Simvastatin enhanced myocardial contractibility through increasing calcium current of single LCC; simvastatin depressed cellular calcium overload through blocking virus-induced LCCs alpha1 subunit gene expression.5. Simvastatin could improve the hemodynamics of the left ventricular in mice with viral myocarditis through decrease the grade of myocardial damage and AI, altering acviticy and structure of LCCs.