Experimental Study Of PUMA Mediated Apoptosis Of Cardiomyocytes Induced By Hypoxia/Reoxygenation

Cardiomyocyte loss induced by apoptosis is an important pathological characteristic of cardiac ischemia reperfusion injury. Finding the critical proapaptotic proteins and clarifing the mechanisms of the signal transduction will contribute to revealing the molecular mechanisms of cardiac ischemia reperfusion injury(IRI), searching drug targets and developing new therapeutic strategies.p53 up-regulated modulator of apoptosis (PUMA) is considered as one of the most potent killers among the Bcl-2 homology 3-only subgroup of Bcl-2 family members. It triggers apoptosis through its BH3 domain which can bind and antagonize all known anti-apoptotic Bcl-2 family proteins to indirectly activate Bax and or Bak which induce mitochondria dysfunction and activate caspase cascade. PUMA is a critical mediator of p53- dependent and–independent apoptosis induced by a wide variety of stimuli.In the present study,we used neonatal rat cardiomyocyte hypoxia/reoxygenation (H/R) model to simulate in vivo cardiac ischemia/reperfusion injury. First, we investigated whether the pro-apoptotic protein PUMA is mediated cardiomyocyte apoptosis induced by H/R? And then further investigated whether interfering with PUMA can significantly improved the tolerance of cardiomyocyte to H/R injury by preventing apoptosis?Part I The Effect and Significance of PUMA in Hypoxia/Reoxygenation InjuryObjective:To investigated the effect and significance of PUMA in H/R injury by the H/R model of primary cultured neonatal rat cardiomyocytes .Methods:The primary cultured of cardiomyocytes were randomly divided into 5 groups (n=3).The group as follows:①Control group;②H/R 2h group;③H/R 4h group;④H/R 6h group;⑤H/R 12h group. LDH activity was determined by Automatic Biochemical Analyzer, Cell apoptosis was determined by Flow Cytometry Analyzer, Cell viability was determined by MTT. Then RT-PCR and Western blot was used to detect the mRNA and protein of PUMA, Bax and Bcl-2. At last Spectrophotometer was used to test caspase-3 activity.Results:1.H/R induce to apoptosis of cardiomyocytesH/R could increase Caspase3 activity and apoptosis rate . The rate of apoptosis (23.44±4.16)% and Caspase3 activity (4.57±0.48) of the H/R 12h group were significantly higher than those of control group(P <0.05).2. H/R induces PUMA and apoptosis-related protein Bax, Bcl-2 expression in isolated cardiomyocytes.RT-PCR image analysis showed: PUMA mRNA levels were elevated at 2h (0.45±0.05), peaked at 6h (0.76±0.06), continued to 12h (0.71±0.07) was still higher than the control group (0.29±0.02)(P<0.05); Bax mRNA levels were elevated at 2h (0.63±0.07), peaked at 6h (0.96±0.09) and continued to 12h (0.79±0.09) were still higher than the control group(0.28±0.05)(P<0.05); Bcl-2 mRNA levels were decreased after 2h of hypoxia(0.82±0.07) and bottomed at 12h (0.47±0.05)(P<0.05).Western Blot image analysis showed: There were very few PUMA protein expression in normal cardiomyocytes (0.08±0.01). PUMA protein levels were significant upregulated after 4h of hypoxia (0.33±0.04), and peaked at 12h (0.72±0.07)significant different from Control group (P<0.05); Compared with the Control group (0.28±0.04) , Bax protein levels were significant upregulated after 2h of hypoxia (0.49±0.05), and further increased at 6h (0.92±0.08), peaked at 12h (0.97±0.10)(P<0.05); Bcl-2 protein levels were decreased after 2h of hypoxia (0.56±0.06), bottomed out at 12h (0.26±0.02) (P<0.05).Conclusions:H/R induce to apoptosis of cardiomyocytes. Very few expression of PUMA protein was detected in normal cardiomyocytes. However, PUMA expression was upregulated significantly upon H/R of cardiomyocytes .The characteristics of PUMA expression and extent of Caiordmyocytes injury, apoptosis rate, Caspase-3 activity and Bax expression were positively correlated.Part II The Effect of PUMA-Specific SiRNA on Caiordmyocytes Hypoxia/Reoxygenation InjuryObjective:To investigated whether interfering with PUMA can significantly improved the tolerance of cardiomyocyte to H/R injury by preventing apoptosis?Methods:The primary neonatal rat cardiomyocytes were randomly divided into 4 groups(n=3). The group as follows:①Control group;②H/R 6h group;③scrambled siRNA+ H/R 6h group (si-SCR);④si-PUMA + H/R 6h group (si-PUMA). We established H / R 6h injury model of cardiomyocytes with siRNA interference cardiomyocyte for 36 h. LDH activity, Cell viability , Cell apoptosis, PUMA and Bax, Bcl-2 expression , caspase-3 activity were detected , respectively.Results:1. 50nmol/L of si-PUMA could specifically and efficiently decreased the PUMA expression in caiordmyocytes. PUMA mRNA levels were significantly reduced in si-PUMA group (0.10±0.002) significant different from H/R 6h group(1.06±0.08) (P <0.05); PUMA protein levels were significantly decreased in si-PUMA group (0.33±0.04) compared with those of H/R 6h group (0.84±0.09) (P<0.05).2. The Cell viability was obviously increased in si-PUMA group (75.3±4.29)% compared with the H/R 6h group( 60.7±6.84)% (P<0.05); the levels of LDH activity in si-PUMA group (802±55) U/L were significantly lower than that of the H/R 6h group(1237±107) U/L (P<0.05); the rate of apoptosis in si-PUMA group (14.16±4.02)% was significantly lower than that of the H/R 6h group(23.96±5.02)% (P <0.05)3. Bax protein levels showed obviously decreased in si-PUMA group (0.64±0.05) compared with that of the H/R 6h group(1.06±0.07) (P<0.05); Bcl-2 protein levels showed obviously increased in si-PUMA group (0.68±0.05) compared with that of the H/R 6h group(0.52±0.06) (P<0.05); the Caspase3 activity in si-PUMA group (2.97±0.25) was significantly lower than that of the H/R 6h group(4.62±0.34) (P<0.05).Conclusions:It was found that Chemical synthesis of PUMA-specific siRNA can be efficiently transfected into caiordmyocytes with LipofectamineTM 2000. Down regulated PUMA expression can significantly improved the tolerance of cardiomyocyte to H/R injury by preventing apoptosis . si-PUMA may be reduced cardiomyocyte apoptosis by down regulated of Bax ,Caspase3 but up regulated of Bcl-2 to achieve the purpose of myocardial protection.In the present study, we have identified the PUMA as an essential mediator of cardiomyocyte death in response to I/R injury. Down regulated PUMA expression can significantly improved the tolerance of cardiomyocyte to H/R injury by preventing apoptosis .PUMA, therefore, appears to be a novel therapeutic target in cardiac IRI.