Study On The Mechanism And Intervention Effect Of Metabolic Remodeling Of Failing Heart By Adjusting Substrate Metabolism

Background and ObjectivesHeart is a high energy-consuming organ, and myocardial cells need enormous and sustained energy to keep its normal function. Recent study revealed that myocardial metabolism will alter greatly after chronic heart failure (CHF), resulting insufficient of adenosine triphosphate adenosine (ATP)and leaving the heart in a state of"energy hungry". Mitochondria are organelles of energy metabolism, and it is gradually revealed that CHF might accompanied by mitochondria dysfunction. Mitochondria dysfunction maybe play a pivotal role in myocardial metabolism disorder in CHF, so it is important to investigate the biological change of mitochondria in CHF. Uncoupling protein 2 (UCP2) is a proton transporter protein located in the inner membrane of mitochondria, and when it is activated, can leads to proton leakage, uncoupling of oxidative phosphorylation, and decrease of ATP production. Thus the activation of UCP2 is unfavorable to the energy metabolism of heart in CHF. It has been found that the expression of UCP2 increases in CHF and is related to the decreased myocardial performance. However, the reason and the significance of increased UCP2 expression in CHF need further investigation.The sympathetic system activity and the level of catecholamine increase in CHF, which in turn can lead to increment of plasma free fatty acid (FFA). Excess FFA is detrimental to failed myocardium by causing mitochondrial oxidative stress by participating inβoxidation. Also, the expression of UCP2 is closed related to the concentration of plasma FFA, and it has been found that high plasma FFA can lead to increasing expression of UCP2 in skeletal muscle. Large-scale epidemiological investigation showed that high FFA levels were significantly related to sudden cardiac death. Restriction of the utilization of FFA and enhancement of the utilization of glucose, such as by reducing plasma level of FFA through application of insulin and/or glucose, measures of anti-sympathetic activity, and application of trimetazindine, an inhibitor of FFA oxidation, will improve the ischemia state of myocardium. Under the direction of such concept, trimetazidine have been used clinically for treatment of ischemic cardiomyopathy and achieved good results. Clinical studies show that long-term use of trimetazidine can improve cardiac function in patients with heart failure, improve the left ventricular function, and can improve energy metabolism of myocardium. We speculate this effect may relate to the restriction of use of the FFA, but the underlying mechanisms remain unclear and need further study.To provide a new theoretical basis and treatment targets for the prevention and treatment of CHF, research work were developed in two levels, namely cell culture in vitro and animal models, to investigate the alteration of metabolism and oxidation respiration function of mitochondria, and the role of FFA treatment targets in CHF development as well as after medicine intervention.Methods1. CHF model were developed in rats by constriction of abdominal aorta, and sham operation and normal rats served as control. Twenty weeks later, the rats were sacrificed and the following items were examined. (1) The Left ventricular end diastolic diameter (LVEDd), end-diastolic interventricular septum dimensions(IVSTd) and left ventricular posterior wall dimensions(LVPWd) were measured by echocardiography, and then ejection fraction were calculated. (2) Hemodynamics and CHF parameters including heart rate (HR), LVEDP and±dp/ dtmax were measured, and then mean aortic pressure were calculated. (3) To observe the pathological changes of failed myocardium by Hematoxylin-eosin staining and transmission electron microscopy. (4) Mitochondria were isolated from ventricle of failed heart by density gradient centrifugation. Then mitochondrial oxidative respiratory function was measured by Clark oxygen electrode, the content of adenine nucleotide pools were measured by high performance liquid chromatography (HPLC), and the mitochondrial membrane potential (MMP) was detected by rhodamine 123method. (5) The concentration of FFA and the expression level of UCP2 were measured, and then the correlation analysis were made between FFA concentration and UCP2 expression level, and between ATP content and UCP2 expression level.2. Cardiomyocytes were isolated and cultured, and then different concentrations of FFA were added to the culture medium to observe the effect of FFA on the expression of FFA and the apoptosis of cardiomyocytes at different time points (6h, 12h and 24h). The expression of UCP2 was examined by RT-PCR and Western-blot, and the apoptosis-related protein bax and bcl-2 were monitored by Tunnel method and Western-blot.3. MicroRNA interference adenovirus vector of UCP2 was constructed and used to transfect cultured cardiomyocytes to observe the effect of down-regulation of UCP2 expression on apoptosis rates of cardiomyocytes, the content of ATP, ADP and AMP in cardiomyocytes, and the expression of UCP2, PPARα, PPARγ, bax and bcl-2. Empty vector and saline served as control and all culture medium containing 2mmol/l FFA. 4. To observe the effect of PPAR on the expression of UCP2, 5 mM MK886 (a specificantagonist of PPAR-a) and 10 mM GW9662 (a specific antagonist of PPAR-γ) was added to the culture medium of cardiomyocytes and then 2mmol/l FFAs were added. The expression of UCP2 in cardiomyocytes was then examined by Western blot.5. Twenty weeks after the operation when CHF were successfully introduced, the rats of CHF were randomly divided into three groups: trimetazidine treated group (group T), acipimox treated group (group A), untreated group (group HF) and sham operation group (group SH). Then the following parameters were examined: (1) LVEDd,IVSTd,LVPWd and EF evaluated by echocardiography.(2) The hemodynamics and heart failure parameters including HR, LVEDP,±dp/dtmax and MAP were measured. (3) The mitochondrial oxidative respiratory function was measured by Clark oxygen electrode and the content of adenine nucleotide pools were measured by high performance liquid chromatography (HPLC). (4) Plasma FFA and blood glucose concentrations were measured. (5) The expression of UCP2 in mitochondria was detected by PT-PCR and Western blot analysis.Results1. Twenty weeks after the ligation, echocardiography examination showed that the heart were enlarged, and left ventricular ejection fraction and±dp/dtmax of group SH20w decreased in group SH20w when compared with control groups. Also, the results of hemodynamics examination showed that LVEDP and MAP increased, and±dp/ dtmax decreased in group SH20w when compared with control groups. Together, these data indicating that CHF model could be successfully introduced in rats by abdominal aorta constriction method.2. The content of ATP, ADP, AMP, Pcr, the level of MMP and ST3 were lower in HF20w rats than in control rats (P<0.05). The level of ST4 were higher in HF20w rats than in control rats, but no significant difference exist among groups (P>0.05).3. The concentration of plasma FFA were 1.7 times higher in in HF20w rats than in control rats (P<0.05), and the expression of UCP2 at mRNA and protein levels were significant higher in HF20w rats than in control rats (P<0.05). Correlation analysis showed myocardial ATP content and UCP2 protein expression was significantly negatively correlated (r = -0.929), and cardiac UCP2 expression and serum FFA was significantly positvely correlated (r = 0.89).4. Six hours after treatment of rat cardiomyocytes with FFAs (2 mmol/L), the mRNA and protein levels of UCP2 increased significantly when compared with the control group, reaching a peak at 24 hours after treatment. Incubating cells with FFAs for 12 hours at concentrations of 1, 2, or 4 mmol/L significantly increased the expression of UCP2 compared with the control group.5. suppressed the expression of UCP2 using RNAi. The inhibitory effect of siRNA on UCP2 expression was evident 24 hours after transfection, when the level of UCP2 protein had decreased by 78%. No apparent effect on UCP2expression in control group. When culture in the medium containing 2mmol/l FFAs for 12h,the content of ATP and ADP in cardiomyocytes were lower than control cells, and decreased further 24 hours after the adding of FFAs. The content of ATP increased significantly in cardiomyocytes cultured in 2mmol/l FFAs and SiRNA treatment than in cardiomyocytes cultured in 2mmol/l FFAs only.6. When cells were treated with FFAs for 6 hours, the number of apoptotic cells did not increase significantly when compared with the untreated control group. However, at 12 and 24 hours after treatment, the number of apoptotic cells significantly increased significantly when compared with the control group. Furthermore, incubating cells with FFAs for 24 hours at concentrations of 1, 2, or 4 mmol/L resulted in a significant increase of apoptosis cells when compared with the control group. After 12- and 24-hour culture in medium containing 2mM FFAs, an increase in bax and a reduction in bcl-2 were observed when compared with control cells. Also, the bcl-2/bax ratio was clearly reduced from 2.041 to 0.48. RNAi-mediated knockdown of UCP2 attenuated FFA-induced apoptosis in cardiomyocyte. Western blot analysis showed that UCP2 knockdown prevented bax increase and bcl-2 reduction. The bcl-2/bax ratio went from 0.48 to 1.63 after treatment with FFAs plus UCP2 knockdown.7. Incubating cells with 2 mmol/l FFAs for 12 or 24 hours did not change the level of both PPAR-a and PPAR-γexpression. Pretreated adult rat cardiomyocytes with MK886 (5 mM) blocked FFA-induced UCP2 expression at both the mRNA and protein levels, whereas GW9662 had no effect.8.Echocardiography were performed eight weeks after medicine intervention, and the results showed that EF improved in rats of group T than those in group HF and group A. When compared with group HF and group A, the ST3 and Pcr level in group T ameliorated significantly (P<0.05), and the content of ATP and ADP increased too in group F. There was no significant change in ST4 level in each group.9. Eight weeks after medicine intervention, the concentration of plasma FFA in group A reduced significantly, while the concentration in group HF and gourp T were still higher than that in group SH. The level of glucose in group HF were higher than that in group SH, while were significant lower than that in group A. When the mRNA and protein expression level of UCP2 were concerned, group T and group A were significantly lower than group HF (P<0.05); and there were no significant difference among other groups (P>0.05).Conclusions1. Metabolic remolding such as decrease of the content of ATP and Pcr, alteration of respiratory function and reduction of MMP in mitochondria take place in chronic failed myocardium. Insufficient energy supply and increasing consume is the possible cause of reduction of cardiac function.2. The expression of UCP2 in chronic failed myocardium increase and is correlated with the reduction of ATP content in mitochondria, and inhibition of UCP2 expression in cardiomyocytes can reduce the extent of ATP reduction inducing by FFA. The increased expression of UCP2 is one of the mechanisms of insufficient ATP production in CHF.3. UCP2 plays a role in FFA-induced apoptosis of adult rat cardiomyocytes, and knockdown of UCP2 expression attenuates FFA-induced apoptosis in these cells. Also, FFA induction of UCP2 expression involved PPAR-a, but not PPAR-γ.4. Trimetazidine can promote energy production of mitochondria by ameliorating respiratory function of mitochondria, inhibiting the expression UCP2 and the lekeage of proton in mitochondria, which can in turn improve the energy metabolism of myocardium. To transfer the metabolic substrate of myocardium from FFA to glucose, improve the function of mitochondria and optimize the substrate metabolism is the mechanism of protection effect of trimetazidine on cardiac function.5. Acipimox can ameliorate oxidation respiration function of mitochondria, decrease UCP2 expression level, but can not increase ATP production, thus showing no obvious effect to improve cardiac function. This may lie in the reduction of glucose level in myocardiocyte at the same time. These data indicating increment of glucose consumption that and reduction of FFA are needed simultaneously to optimize the metabolism in failed myocardium.