Studies On The Protein Interaction Of Cardiac Hypertrophy-related Candidate Gene RMND5A

The cardiovascular research has been focused on the field of pathogenesis of cardiac hypertrophy. In recent years, the research of cardiac hypertrophy has made some progress, but its molecular mechanisms are far from clarified. Therefore, study at molecular mechanism of cardiac hypertrophy is of significance not only for clarifying the pathogenesis of cardiac hypertrophy at molecular level, but also for the use of molecular genetic diagnosis and interventions to treat heart disease ultimately.In the pre-work in our laboratory, the human gene RMND5A has been cloned from heart cDNA library, and the rabbit polyclonal antibody of RMND5A was prepared. And RMND5A is also found to relate to TGF-P signaling pathway. It is known that cardiac hypertrophy is closely related to TGF-P that is upregulated in a variety of cardiac hypertrophy disease. Using phenylephrine (PE) to stimulate myocardial hypertrophy of the mouse, using FBS to stimulate cardiac primary myocytes of rat, and with heart tissue of the patient with hypertrophic heart disease, it is found that RMND5A was upregulated. The above results showed that RMND5A and cardiac hypertrophya are closely related. Previous studies also showed that RMND5A cooperating with Smads protein family from TGF-β signaling pathway led to cardiac hypertrophy, and by using co-immunoprecipitation, it is proved that RMND5A and Smadl, Smad2, Smad3 and Smad5 from TGF-P signaling pathway have no interaction, but have weak interaction with Smad8.On the basis of above research results, in the studies the full-length of the RMND5A gene was firstly cloned. After bioinformatics analysis, the five protein domains were subcloned into the pCMV-Myc plasmid. Then the pCMV-Myc plasmids were, respectively, co-transfected with pCMV-Tag2B-Smad6 or pCMV-Tag2B-Smad7 in HEK293 cells. The immune co-precipitation experiments results showed that RMND5A and RMND5AK1, RMND5AK2 can interact with Smad6. RMND5A and its subclones interact with Smad7, with a trend that the shortest fragment gave theweakest interaction.The proteins were extracted from mice heart tissue with PE-induced cardiac hypertrophy, human heart tissue with congenital cardiac hypertrophy, and those from normal human heart and normal mouse heart. Using antibodies of RMND5A, Smad6 and Smad7 were used for the interaction of endogenous co-immune precipitation experiment. The results proved that RMND5A interacted with Smad6 and Smad7, respectively, and this interaction is more obvious in the hypertrophy heart.The above results displayed RMND5A interacted with Smad6, Smad7, respectively, in vitro and in vivo. TGF-β signaling pathway regulates cardiac hypertrophy. RMND5A might inhibit cardiac hypertrophy through the TGF-β pathway member Smad6 and Smad7, indicating that RMND5A regulates cardiac hypertrophy through TGF-β signal pathway. The study provides a basis for understanding the molecular mechanism of cardiac hypertrophy and the further curing human hypertrophic cardiomyopathy.