Roles Of RhoA/ROCk Signal Pathways In High Glucose-induced Cardiomyocyte Hypertrophy Of Rats

Objective: Diabetic cardiomyopathy (DC) is one of the major deathcauses in the patients with diabetes. Cardiomyocyte hypertrophy plays animportant role in the formation of DC, which is one of the major pathologicalfeatures of DC. Up to now, it is unable to prevent clinically the formation andprogression of DC. For treatment of DC, only heart failure at advanced stagewas empirically improved. Several researches show that RhoA/Rho kinase(ROCK) pathway is closely related with cardiomyocyte hypertrophy. Theobjectives of the study were to determine whether RhoA/ROCK pathway isinvolved in cardiomyocyte hypertrophy induced by high glucose in vitro, withthe objective of providing a novel strategy for the treatment of DC.Methods: Primary cultured SD rat cardiomyocytes were obtainedthrough trypsin enzymic digestion and differential attachment. Trypan bluestaining method was used to identify cardiomyocytes activity. Thecardiomyocytes identification was finished by immunocytochemical stainingfor α-SMA. The cardiomyocytes were divided into nine groups: normal group(NG, containing5.5mmol/L glucose); high glucose group (HG, containing25mmol/L glucose); high osmotic pressure group (OSM,5.5mmol/L glucose+19.5mmol/L mannitol); NG+fasudil group (50μmol/L,75μmol/L and100μmol/L); HG+fasudil group (50μmol/L,75μmol/L and100μmol/L). Thenthese cardiomyocytes were cultured for48hours. Cardiomyocyte surfaceareas were determined by image analysis; cardiomyocyte protein contentswere measured using BCA method; ANF mRNA expression was assessed byreal-time PCR; the phosphorylation of MYPT1, as a maker of ROCK activity,was evaluated by Western blot analyses.Results:1Cardiomyocyte surface areas Compared with NG group, the surface areas of cardiomyocytes in HGgroup were significantly increased (P<0.01), which was significantlysuppressed by fasudil (50μmol/L,75μmol/L and100μmol/L) in adose-dependent manner (P<0.01vs HG group); there was significantdifference in the surface areas of cardiomyocytes between NG group andHG+100μmol/L fasudil group. There was no significant difference in thesurface areas of cardiomyocytes among OSM group, NG group andNG+fasudil groups (P>0.05).2Cardiomyocyte protein contentsCompared with NG group, the protein contents of cardiomyocytes weresignificantly increased in HG group (P<0.01), which was significantlysuppressed by fasudil (50μmol/L,75μmol/L and100μmol/L) in adose-dependent manner (P<0.05vs HG group); There was no significantdifference in the protein contents of cardiomyocytes among OSM group, NGgroup and NG+fasudil groups (P>0.05).3ANF mRNA expressionCompared with NG group, exposure of cardiomyocytes to HG induced asignificant upregulation of ANF mRNA expression (P<0.01), which wassignificantly suppressed by fasudil (50μmol/L,75μmol/L and100μmol/L) in adose-dependent manner (P<0.05vs HG group); There was no significantdifference in ANF mRNA expression between HG+100μmol/L fasudil groupand NG group (P>0.05); There was no significant difference in ANF mRNAexpression among OSM group, NG group and NG+fasudil groups (P>0.05).4Phosphorylation of MYPT1Exposure of cardiomyocytes to HG significantly increased thephosphorylation of MYPT1(P<0.05vs NG group). Fasudil (50μmol/L,75μmol/L and100μmol/L) inhibited HG-induced increase in phosphorylationof MYPT1(P<0.05vs HG group). There was no significant difference inphosphorylation of MYPT1among OSM group, NG group and NG+fasudilgroups (P>0.05). Conclusions:1High glucose can induce cardiomyocyte hypertrophy.2RhoA/ROCK signal pathways may be involved in the process of highglucose-induced cardiomyocyte hypertrophy. ROCK inhibitor, Fasudil, caninhibit high glucose-induced cardiomyocyte hypertrophy in a dose-dependentmanner.