Effects Of UCP2 On Inhibiting Cardiomyocyte Hypertrophy Induced By IS

Cardiovascular disease(CVD) is the most common complication and the leading cause of mortality in patients with chronic kidney disease(CKD). With the improvement of dialysis technology, uremia patients have longer survival time, while the time heart exposed to uremia have also extended accordingly. Myocardial hypertrophy, the prominent pathological feature of uremic cardiomyopathy, is important cause of heart failure and sudden death. However, the definite pathogenesis has not been completely elucidated. It has been suggested lots of factors including hypertension, anemia and volume load involved in the pathogenesis of Left ventricular hypertrophy(LVH) in CKD. Increasing evidences suggest that it is not possible to control the progression of LVH via controlling blood pressure and lowering volume load. It is shown that the persistent high level of uremic toxin may cause increasing morbidity about CVD on CKD. So it is very important to investigate the pathogenesis of uremic cardiomyopathy at cellular and molecular levels, in order to find preventive methods for clinical instructions.Indoxyl sulfate is one of a number of protein bound uremic toxin that accumulate in patients with CKD. Current conventional hemodialysis is ineffective at removing this toxin. In CKD patients, the total IS concentrations vary and can be as high as 500μM, whereas in healthy subjects concentrations are extremely low(0.1-2.39 μM). Indoxyl sulfate has been shown to accelerate the progression of CKD and CVD. A previous clinical study showed that a high serum level of IS was associated with cardiovascular mortality in CKD patients. Reports have showed the prevalence of LVH combining stage 3 and 4 is high. Interestingly, serum IS was reported to be significantly increased in stage 3 CKD in type 2 diabetic nephropathy, which indicate there are a closely connection between the level of indoxyl sulfate and left ventricular hypertrophy in CKD patients. According to new research, Indoxyl sulfate may induce cardiomyocyte hypertrophy through enhancing oxidative stress, but the definite pathogenesis has not been completely elucidated.Uncoupling proteins belong to a family of mitochondrial carrier proteins that are present in the mitochondrial inner membrane, can modulate the mitochondrial membrane potential, involve in cell energy metabolism. So far, some studies found UCP2 overexpression may protect a wide array of cell types from apoptosis and the cytoprotective effect of UCP2 is likely based on reduction of mitochondrial ROS generation. According to the current study, it is shown that UCP2 play an important role in energy metabolism and against oxidative stress damage. However, this conclusion result from study about metabolic disease, while there are few reports about the effect UCP2 on inhibiting cardiomyocyte hypertrophy induced by IS. It has been reported that IS may deteriorate uremic cardiomyopathy through enhancing oxidative stress, so we speculate UCP2 may play an important role in uremic cardiomyopathy induced by IS.AMP-activated protein kinase(AMPK), an evolutionarily conserved serine/threonine protein kinase, is considered a major metabolic regulator at both the cellular and whole-body levels. Study found that AMPK is an upstream regulatory protein of uncoupling protein 2, can reduce energy consumption, against oxidative stress, and has the potential cardiovascular protection function. Recently, several studies indicate that AICAR and metformin can inhibit the progression of cardiomyocyte hypertrophy by activating AMPK. In consequence, we consider that AMPK may protect cardiomyocyte against hypertrophy through upregulating UCP2.In the present study, in order to explore the effect of UCP2 on cardiomyocyte hypertrophy and its mechanisms, by using the methods of lentivirus transfection, fluorescent staining, Western blot, real time polymerase chain reaction(Real time PCR). We designed and carried out the following experiments:(1) Firstly we verified weather IS can induce ROS generation and accelerate hypertrophy in cardiomyocyte.(2)Establishing a UCP2 overexpression cell model, identify the effects of UCP2 overexpression on cardiomyocyte hypertrophy induced by IS.(3) And confirm the role of AMPK in IS increasing ROS generation and down regulating UCP2, explore the effects of UCP2 on cardiomyocyte hypertrophy induced by IS and its mechanisms. Results and conclusions are summarized as follows:IS induced excessive ROS production and hypertrophy in cardiomyocytes: Compared with the control group, ROS production was significantly increased in a dose dependent manner after IS treatment for 6 hours. The mRNA expression of ANF, BNP and β-MHC was increased obviously in cardiomyocytes after being cultured with 500 mM IS for 48 hours, accompanied by the rate of protein synthesis promoted and the cells size enlarged. These results suggest that IS could enhance oxidative stress and induce hypertrophy in cardiomyocytes.IS inhibited UCP2 expression in cardiomyocytes: A gradual decrease of UCP2 expression was observed after being treated with 500 μM IS for different time. Meanwhile, cardiomyocytes were incubated with different concentrations IS only for 1 h, the protein expression of UCP2 was also significantly suppressed. These findings suggest that IS inhibits UCP2 expression in a dose and time-dependent manner.UCP2 overexpression inhibited IS induced ROS production: Firstly, We used a recombinant lentivirus containing the full-length UCP2 cDNA to induce UCP2 overexpression in cardiomyocytes, and observed the effects of UCP2 overexpression on ROS production induced by IS. We found that no significant difference in ROS production was observed between the IS and IS + Vehicle groups. However, IS-induced mitochondrial ROS production was suppressed in the presence of UCP2 overexpression these results indicate that IS increases mitochondrial ROS generation by suppressing UCP2 expression in cardiomyocytes.AMPK activation attenuated IS induced the decrease of UCP2 expression and ROS production in cardiomyocytes: The expression of phosphorylated AMPK was inhibited by 500 μM IS treatment in a time-dependent manner. Meanwhile, pretreatment with AICAR, an AMPK activator, reversed the down-regulation of UCP2 induced by IS. Moreover, similar to the effects of NAC, perincubated with AICAR before IS treatment obviously diminished ROS production in cardiomyocytes. These results suggest that AMPK activation attenuates IS-induced the down-regulation of UCP2 and ROS generation in cardiomyocytes, IS may induced cardiomyocyte hypertrophy by inhibiting the AMPK/ UCP2 signaling and the enhancement of oxidative stress.AMPK activation and UCP2 overexpression attenuated IS induced cardiomyocyte hypertrophy: In order to explore the effect of AMPK activation and UCP2 overexpression on IS induced cardiomyocyte hypertrophy, we observed the variation of cell size, the rate of protein synthesis and the expression of ANF 、 BNP and β-MHC mRNA. LeUCP2 remarkably inhibited IS induced increases in hypertrophy marker. These results indicated that both UCP2 overexpression and AMPK activation attenuated IS induced cardiomyocyte hypertrophy.In conclusion, the present study demonstrates that IS may induced cardiomyocyte hypertrophy by inhibiting the AMPK/UCP2 signaling and the enhancement of oxidative stress, whereas both UCP2 overexpression and AMPK activation attenuated IS induced cardiomyocyte hypertrophy. IS induced cardiomyocyte hypertrophy may be related to the AMPK/UCP2/ROS signaling. Although our study is limited to vitro experiments, it may provide potential therapeutic targets for uremic cardiomyopathy.