Theaflavin 3,3’-digallate Reverses The Downregulation Of Connexin 43 And Autophagy Induced By High Glucose Via AMPK Activation

Diabetes mellitus is the seventh leading cause of mortality around the world.In the past three decades,the number of diabetic patients has more than doubled.Diabetic patients are more prone to develop cardiac arrhythmia compared with normal subjects.The higher rate of cardiac arrhythmia in diabetic patients is tightly related to the impaired gap junction and autophagy.The well balanced physiological activity of heart needs normal electric coupling and electric conduction in cardiomyocytes,and it is based on the normal expression and distribution of connexins.In the ventricular cardiomyocytes,connexin 43(Cx43)is the main connexin while cornexin 40 and cormexin 37 also exist.Cx43 is considered to be the major connexin for maintaining electrical functioning in the ventricular cardiomyocytes.It’s found that Cx43 is downregulated in neonatal rat cardiomyocytes induced by high glucose treatment in the previous studies.In vivo studies,Cx43 changes according to STZ dose and model phase.For example,Cx43 expression is decreased in rat cardiomyocytes 3 weeks after streptozocin(STZ)injection.However,Cx43 is elevated after successful modeling of type Ⅰ diabetic rats which may be influenced by metabolites produced by long-term high glucose stimulation.Actually,the changes of the expression and distribution of Cx43 are influenced by a lot of factors during the course of diabetes mellitus and actually the precise mechanism is still unclear.Therefore,to exclude the confounding factors,this experiment mainly discusses the influence of high glucose in cardiomyocytes and in rats of three week STZ injection.Theaflavin is a derivative of benzotropolones in the process of tea fermentation.Theaflavins are produced by catechins in the catalytic process of polyphenol oxidase,mainly including four types,theaflavins(TF)and its galloyl esters,TF 3-O-gallate(TFZa),TF 3’-O-gallate(TF2b),and TF 3,3’-digallate(TF3).TF3 is the major component of black tea extract and has the strongest antioxidative capacity.Previous studies have shown TF3 protects cardiomyocytes from lipotoxicity and reperfusion injury.Moreover,TF3 is proven to attenuate the injury of angiotensin Ⅱ(Ang Ⅱ)and endothelin in the cardiovascular system.However,the role of TF3 in the diabetes mellitus is still unclear.In this study,first we investigated the impact of high glucose on the gap junction and autophagy in neonatal rat cardiomyocytes,subsequently the role of TF3 was discussed.Moreover,the regulatory mechanism of TF3 was discussed.Finally,the role of TF3 was validated in vivo.Part 1:The Impact of Theaflavin 3,3’-digallate on connexins and Autophagy Induced by High Glucose in cardiomyocytesObjectiveIn this study,we investigated the impact of high glucose on connexins(Cx)and autophagy,and the role of TF3 in the modulation of connexins and autophagy was discussed.MethodsNeonatal rat cardiomyocytes(NRCM)were isolated from 1-2 newborn SD rats,and incubated under high glucose condition(33mM,72h).Meanwhile,the effects of theaflavin 3,35-digallate(TF3)on connexins(Cx43,Cx37,Cx40)and autophagy related proteins(LC3-Ⅱ,Beclinl)were measured by western blot.The function of gap-junctional intercellular communication(GJIC)was measured by a Lucifer yellow/rhodamine-dextran(LY/Rhd)dye transfer assay.The autophagic dots in NRCMs transfected with mCherry-GFP-LC3 adenoviruses were counted under the confocal microscope.Adenosine monophosphate-activated protein kinase(AMPK)activity was measured according to the protein expression of phosphorylated AMPK and AMPK by western blot.To investigate the role of AMPK in modulation of connexins and autophagy,AMPK was activated with aminoimidazole-4-carboxamide-1-p-D-ribofuranoside(AICAR)or inhibited by AMPKa siRNA to explore the role of AMPK in the modulation of connexin 43(Cx43)and autophagy.Besides,autophagy was activated with rapamycin or inhibited with 3-methyladenine(3-MA)and bafilomycin A1(baf A1)to observe the impact of autophagy on the expression of connexins.Finally,autophagy was observed in cardiomyocytes transfected with Cx43 siRNA.Results Cx43 and autophagy were significantly downregulated by high glucose treatment while addition of TF3 dose-and time-dependently upregulated the inhibition of Cx43 and autophagy by high glucose.On the other hand,Cx40 and Cx37 were not significantly altered.Moreover,TF3 reverses the inhibiton of functioning of gap junction and increased counts of autophagic dots induced by high glucose treatment.High glucose inhibited the activity of AMPK while TF3 significantly reversed the inhibition.AMPKa siRNA abrogated the protection of TF3,while AICAR showed similar results compared to the TF3 treatment.Meanwhile,autophagy activation caused decreased Cx43,while co-treatment with baf A1 enhanced Cx43 expression further compared with the TF3 treatment alone under high glucose.Knockdown of Cx43 expression by siCx43 didn,t change autophagic activity significantly.ConclusionsTF3 Reversed the Downregulation of Connexin 43 and Autophagy Induced by High Glucose via AMPK Activation in Cardiomyocytes.Inhibition of autophagy might be protective by preserving Cx43 expression in NRCMs stimulated by high glucose.Part 2:The role of theaflavins on connexins in streptozocin induced type Ⅰ diabetic ratsObjectiveThe mechanism of theaflaivns in the rats in modulating connexins expression is investigated.MethodsType 1 diabetes mellitus was induced by tail vein injection of streptozocin(STZ,55 mg/kg).Theaflavins were given via intragastric gavage(10mg/kg).The cardiac function of rats were assessed by echocardiography.Cx43 and adenosine monophosphate-activated protein kinase(AMPK)expression was examined by western blot.The distribution of Cx43 was investigated by immunohistochemistry.ResultsThe cardiac function didn’t vary significantly after 3 weeks of type Ⅰ diabetes mellitus induction.Cx43,phosphorylated AMPK and AMPK expression were significantly downregulated in Ieft ventricular cardiomyocytes in type 1 diabetic rats.The distribution of Cx43 was also disturbed.Theaflaivns reversed the downregulation of Cx43 and AMPK activity and restored the abnormal distribution of Cx43.ConclusionTheaflavins reversed the downregulation of AMPK activity and Cx43 expression and restored the abnormal distribution of Cx43.