Roles And Mechanisms Of Recombinant Adeno-associated Virus Mediated Delivery Of MicroRNA In Doxorubicin-induced Cardiotoxicity And Diabetic Cardiomyopathy

Background and Aim The care for patients with cancer has advanced greatly over the past decades.Cardiovascular complications of cancer treatments may have a profound impact on the health of cancer patients.Doxorubicin,one of anthracycline agents,is the major culprit in chemotherapyinduced cardiotoxicity.However,the mechanisms and pertinent interventions of its specific cardiotoxicity are still unclear.A clinical study reveals that combination of doxorubicin and bevacizumab resulted in an unacceptably higher exacerbation of cardiotoxicity,which prompted a hypothesis that doxorubicin may cause cardiac dysfunction by destroying vascular homeostasis.MicroRNAs are a class of small endogenous non-coding RNAs.Mounting evidences have demonstrated that microRNAs play key roles in diverse physiological/pathological processes.MiR-320 a has been reported to be involved in regulation of vascular homeostasis abnormalities including ischemia reperfusion injury,atherosclerosis,and diabetic microvascular disease.Here we found that doxorubicin increased cardiac miR-320 a level,and then designed a series of experiments to explore the role of miR-320 a in vascular homeostasis abnormalities and doxorubicin induced cardiotoxicity.Methods and Results Firstly,animal models of doxorubicin induced cardiotoxicity were established in C57BL/6 mice.We found that the expressions of CD31 and CD34,indicators of tissue microvessel density,were markedly decreased in heart from mice treated with doxorubicin.On the other hand,significantly increased level of miR-320 a was found by real-time PCR after doxorubicin treatment,which was relative specificity in heart and endothelial cells.Secondly,recombinant adeno-associated virus(rAAV)in vivo and miR-320 a mimics/inhibitors in vitro were used to further explore the roles of miR-320 a in doxorubicin induced cardiotoxicity.Knockdown of miR-320 a not only resulted in enhanced proliferation and inhibited apoptosis in cultured endothelial cells,but also attenuated cardiac abnormalities induced by doxorubicin.On the contrary,overexpression of miR-320 a enhanced apoptosis in vitro,and aggravated vessel abnormalities in heart and subsequent cardiac dysfunction in mice.Lastly,target prediction showed that VEGF-A was a potential target of miR-320 a,which was verified by anti-Ago2 co-immunoprecipitation and Western blots.To verify the function of VEGF-A in doxorubicin induced impairment,VEGF-A was down-expressed by siRNA in cultured endothelial cells and over-expressed by rAAV in mice.As same as miR-320 a,siRNA against VEGF-A reinforced doxorubicin induced endothelial cells injury,and the negative effects of miR-320 a on vascular homeostasis and cardiac function were alleviated by VEGFA re-expression in doxorubicin treated mice.Conclusion Our observations demonstrate that miR-320 a mediates doxorubicin induced cardiotoxicity by targeting VEGF signal pathway and thus,inhibition of miR-320 a may be applied to the treatment of cardiac dysfunction induced by anthracycline.Our study also suggests that doxorubicin may induce cardiac dysfunction via disturbing of vascular homeostasis,which may be a novel theoretical ground for developing therapeutics against doxorubicin induced cardiotoxicity.Background and Aim Diabetes mellitus is becoming an epidemic in the world.Cardiovascular complication is the principal cause of morbidity and mortality among diabetic patients.As the leading burden of cardiovascular complications,diabetic cardiomyopathy is closely related to a greater risk of heart failure and myocardial ischemia.The role of metabolic derangement in diabetic cardiomyopathy has been established.Although metabolic remodeling in heart is the consequence of exposure to abnormal circulating substrates,restoring suitable systemic parameters can not achieve effective prevention and treatment of diabetic cardiomyopathy.Looking for measures to regulate the metabolic disturbance in diabetic heart has become a research hotspot.micro RNAs are indicated to be involved in metabolism.Micro RNAs are a class of small endogenous non-coding RNAs,which modulate gene expression at the posttranslational level.Emerging evidence has implicated cardiac-enriched mi RNAs in heart diseases.Mi R-30 c,a cardiac-enriched micro RNA,has been reported to be involved in regulation of lipid metabolism in liver and adipose tissue.However,whether mi R-30 c contributes to diabetic cardiomyopathy by regulating cardiac metabolism is unclear.Here we found the reduced expression of mi R-30 c in the heart of db/db mice and then designed a series of experiments to explore the role of mi R-30 c in metabolic normalities and diabetic cardiomyopathy.Methods and Results Db/db mice were used to establish animal models of diabetic cardiomyopathy.Reduced cardiac mi R-30 c expression was detected in 28-week-old db/db mice,whose cardiac alterations represented diabetic cardiomyopathy.To further explore the roles of mi R-30 c in myocardial metabolism under diabetic stimuli,micro RNA mimics/inhibitor in vitro and r AAV administration in vivo were employed to manipulate the expression of mi R-30 c.Overexpression of mi R-30 c suppressed enhanced lipid metabolism,improved glucose utilization,reduced excessive reactive oxygen species(ROS)production and myocardial lipid accumulation,and subsequently attenuated apoptosis and cardiac dysfunction of db/db mice.Similar regulatory effects of mi R-30 c on cultured H9c2 cells treated with free fatty acid(FFA)were also observed.Furthermore,PGC-1β was identified as a direct target of mi R-30 c by Ago2 co-immunoprecipitation,Western blot and Dual Luciferase assays.Moreover,si RNA against PGC-1β mimicked the effect of mi R-30 c on FFA treated H9c2 cells.At the molecular level,inhibition of PGC-1β blocked PPARα transcriptional activity,which is deeply involved in the regulation of cardiac metabolism.Conclusion Our data demonstrate the role of mi R-30 c as an actionable branch point in the cardiometabolic signal pathway,and suggest that modulation of mi R-30 c levels may provide a therapeutic approach for diabetic cardiomyopathy.