| Objectives: Previously, we identified that ANP32A expressed in cardiomyocyteand ANP32A knockdown in cardiomyocyte suppressed doxorubicin-inducedapoptosis. MAPKs play a crucial role in doxorubicin-induced cardiomyocyteapoptosis. We hypothesized that ANP32A knockdown suppress doxorubicin-inducedcardiomyocyte apoptosis via MAPKs．Methods: Cardiomyocytes were isolated, cultured and identified by cTnI.Cardiomyocytes were divided into six groups: CMs group, DOX group, NC-LVgroup, NC-LV+DOX group, Anp group and Anp+DOX group. While at the sametime we transfected lentivirus-scrRNAi into the NC-LV group, NC-LV+DOX groupand lentivirus-ANP32A/GV115-RNAi into the Anp group, Anp+DOX group ofcardiomyocytes. After72hours later, we observed the transfect efficiency byfluorescent microscope, and then we treated the DOX group, NC-LV+DOX group,Anp+DOX group of cardiomyocytes with doxorubicin(1μmol/L) in order to constructthe apoptosis model of cardiomyocytes. After12hours later, we examined theapoptotic rates by AnnexinV-FITC detection kit. Cardiomyocytes apoptosis wereexamined which was determined by the apoptotic index of cardiomyocytes to explorethe effect of ANP32A knockdown on doxorubicin-induced apoptosis. Intracellularsignal molecules, such as ERK1/2, phosphorylated ERK1/2, JNK, phosphorylatedJNK, P38MAPKs, phosphorylated P38MAPKs protein expression of the CMs group,DOX group, Anp group, Anp+DOX group were detected by western blot. Thedifference among the experimental groups was compared by unpaired t-test orone-way ANOVA used for statistical evaluation of the data.Results: Primary cultured cardiomyocytes in vitro showed pleomorphism, suchas polygon, spindle and so on. Some of the cardiomyocytes formed cell clusters andradial concentric circles in shape, beating asynchronously. The beating frequency ofcardiomyocytes was approximately from60to100times per minute.Immunofluorescene showed that more than95%of the cardiomyocytes were positivefor cTnI which is significant marker of the cardiomyocyte and was in accordance with cardiomyocytes. After the transfection of lentivirus-scrRNAi andlentivirus-ANP32A knockdown/GV115-RNAi into the cardiomyocytes72hours later,we observed that more than90%and80%of the cardiomyocytes expressed greenfluorescent protein(GFP) by fluorescent microscope. Comparing cardiomyocytesapoptosis rates between the DOX group and the CMs group[(31.94±1.24)%vs(3.62±0.53)%,(p<0.01)] with the method of AnnexinV-FITC indicated that themodel of doxorubicin-induced cardiomyocyte apoptosis had been successful;Comparing apoptosis rates between the NC-LV group and the CMsgroup[(4.53±0.61)%vs (3.62±0.53)%,(p>0.05)]showed that the cardiotoxicityof lentivirus had not affected analysis of the results; Comparing the apoptosis ratesbetween Anp+DOX group and the DOX group[(16.62±2.07)%vs(31.94±1.24)%,(p<0.05)]showed that ANP32A knockdown could suppresseddoxorubicin-induced apoptosis. There were no obvious difference between thep-ERK1/2/ERK1/2of CMs group and the DOX group(p>0.05) which showed thatDOX group did not cause the change of the activity of ERK1/2．The ratio ofp-JNK/JNK between DOX group and CMs group increased about1.5times(p<0.05)which showed that upregulating the activity of JNK in DOX group could induce thecardiomyocyte apoptosis; the ratio of p-P38/P38MAPKs between DOX group andCMs group increased0.9times(p<0.05) which showed that upregulating the activityof P38MAPKs in DOX group could induce the cardiomyocyte apoptosis. The ratio ofp-JNK/JNK between Anp+DOX group and DOX group decreased1.43times (p<0.05)which showed that ANP32A knockdown via deregulating the activity of JNK could suppresscardiomyocyte apoptosis. The ratio of p-P38/P38between Anp+DOX group and DOXgroup decreased0.83times (p<0.05) which showed that ANP32A knockdown viaderegulating the activity of P38MAPKs could suppress cardiomyocyte apoptosis.Conclusion: ANP32A knockdown suppressed doxorubicin-inducedcardiomyocyte apoptosis via deregulating JNK/P38MAPKs activity. |
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