P2X7 Purinergic Receptor Is Involved In Apelin-13/APJ-induced Cardiomyocyte Hypertrophy In Mouse HL-1 And Rat H9c2

Objective: Apelin-13 is the endogenous ligand of APJ.Previous studies have shown that apelin-13/APJ could promote cardiomyocyte hypertrophy and accelerate pathological myocardial hypertrophy.Our previous studies have found that apelin-13/APJ promoted cardiomyocyte hypertrophy by activating Pannexin-1-endoplasmic reticulum autophagy pathway,whereas the intermediate mechanisms regulating reticulophagy after Pannexin-1 activation has not been fully elucidated.Related studies have confirmed that the opening of Pannexin-1 half-channel mediated the release of intracellular ATP to extracellular and then activation of P2X7 purinergic receptors on the cell membrane.As an important member of P2 X purine family,P2X7 receptor was not only highly expressed in cardiovascular system,but also closely related to cardiovascular diseases such as cardiac hypertrophy.On the basis of previous studies,we intended to further explore whether P2X7 mediated apelin-13/APJ system to promote cardiomyocyte hypertrophy,thereby providing a new theoretical basis for screening anti-cardiac hypertrophy drugs with APJ as molecular target.Methods:1.Western Blot detected the expression of P2X7,FAM134 B,LC3II/GAPDH,Beclin-1,ANP and BNP in HL-1 and H9c2 cardiomyocytes.2.Small molecular RNA interference chains are designed to inhibit the expression of P2X7 and FAM134 B in HL-1 and H9c2 cardiomyocytes.3.Immunofluorescence technique observed the co-localization of endoplasmic reticulum retention protein KDEL with autophagy marker proteins LC3 B,KDEL and lysosomal marker protein CD63,and FAM134 B with autophagy marker protein LC3 B in HL-1 and H9c2 cardiomyocytes treated with apelin-13,si RNA-P2X7 and P2X7 antagonist A-8045984.Immunofluorescence technique observed the cytoskeleton size of HL-1 and H9c2 cardiomyocytes treated with apelin-13,si RNA-P2X7 and P2X7 antagonist A-804598.5.Scepter TM handheld cell counter measured the diameter and volume of HL-1 and H9c2 cardiomyocytes after treatment with apelin-13,si RNA-P2X7 and P2X7 antagonist A-804598.6.BCA protein quantitative kit detected the protein content in HL-1 and H9c2 cardiomyocytes treated with apelin-13,si RNA-P2X7 and P2X7 antagonist Amuri 804598.Results:1.Apelin-13 promoted the expression of P2X7 in mouse HL-1 atrial myocytes and rat H9c2 cardiomyocytes in a dose-dependent and time-dependent manner,while APJ receptor antagonist F13 A,P2X7antagonist A-804598 and si RNA-P2X7 significantly inhibited P2X7 expression in HL-1 and H9c2 cardiomyocytes induced by apelin-13.2.Apelin-13 promoted the expression of FAM134 B protein and autophagy marker proteins Beclin-1 and LC3II/GAPDH in mouse HL-1 cardiomyocytes in a dose-dependent and time-dependent manner,while APJ receptor antagonist F13 A,P2X7 antagonist A-804598 and si RNA-P2X7 significantly inhibited apelin-13 induced expression of FAM134 B,Beclin-1 and LC3II/GAPDH in mouse HL-1cardiomyocytes.3.Apelin-13 promoted the co-localization of endoplasmic reticulum resident protein KDEL and autophagy marker protein LC3 B in HL-1and H9c2 cardiomyocytes,while P2X7 antagonists A-804598 and si RNA-P2X7 significantly inhibited apelin-13-induced co-localization of KDEL and autophagy marker protein LC3 B in HL-1 and H9c2 cardiomyocytes.4.Apelin-13 promoted the co-localization of endoplasmic reticulum resident protein KDEL and lysosomal marker protein CD63 in HL-1and H9c2 cardiomyocytes,while P2X7 antagonists A-804598 and si RNA-P2X7 significantly inhibited apelin-13-induced co-localization of KDEL and lysosomal marker protein CD63 in HL-1 and H9c2 cardiomyocytes.5.Apelin-13 promoted the co-localization of FAM134 B and LC3 B in HL-1 and H9c2 cardiomyocytes,while P2X7 antagonists A-804598 and si RNA-P2X7 significantly inhibited the co-localization of FAM134 B and LC3 B in HL-1 and H9c2 cardiomyocytes induced by apelin-13.6.Apelin-13 promoted the expression of ANP and BNP in HL-1 and H9c2 cardiomyocytes in a dose-dependent and time-dependent manner,while APJ receptor antagonist F13 A,P2X7 antagonist A-804598 and si RNA-P2X7 significantly inhibited apelin-13 induced expression of ANP and BNP in HL-1 and H9c2 cardiomyocytes.7.siRNA-FAM134 B was transfected into HL-1 and H9c2 cardiomyocytes with Lipo3000 transfection reagent for 6 h,and then treated with apelin-13 for 24 h.It was observed that apelin-13 promoted the expression of ANP and BNP,while si RNA-FAM134 B significantly inhibited the expression of ANP and BNP induced by apelin-13.8.Scepter TM handheld cell counter and BCA protein quantitative results showed that apelin-13 promoted the increase of diameter and volume in HL-1 and H9c2 cardiomyocytes,while si RNA-P2X7 and P2X7 antagonist A-804598 inhibited the increase of cardiomyocyte diameter and volume induced by apelin-13.9.HL-1 and H9c2 cardiomyocytes were pretreated with fluorescent labeled phalloidine at 37 ℃ for 20 min.Immunofluorescence results showed that apelin-13 promoted the hypertrophy of HL-1 and H9c2 cardiomyocytes,while P2X7 inhibitors A-804598 and si RNA-P2X7 significantly inhibited apelin-13-induced cardiomyocyte hypertrophy.Conclusion: P2X7 purinergic receptor is involved in apelin-13/APJ-induced cardiomyocytes hypertrophy in HL-1 and H9c2cells...