Effects Of MHCN₂ Gene Modification On Chronotropic Relevant Receptors In BMSCs

Background Arrhythmia is a common cardiovascular disease,not only affects people’s quality of life,but also constitutes the most common cause of sudden cardiac death.The implantation of electronic pacemakers for slow arrhythmias and malignant ventricular arrhythmias is an effective intervention and an important treatment.While considering physiologic function of heart and adaptability of the human body,electronic pacemaker treatment is still non-physiological pacing,and re-establish biological pacemaker of autonomous cells in a biological way the so-called "biological pacemaker" become a hot research in recent years.Some studies indicated that stem cells modified with hyperpolarization-activated cyclic nucleotide-gated（HCN）gene could generate hyperpolarization-activated cation current If。 The HCN₂ and HCN4 subtypes were most abundant in the heart sinoatrial nodules.The ideal biological pacemaker should not only have cardiac automaticity,but also have chronotropic ability to response to the regulation of sympathetic and parasympathetic like sinus node pacemaker cells.However,little is known about the chronotropic ability of stem cells modified with HCN₂ or HCN4 gene.Adrenergic receptor beta1（Adrb1）and cholinergic receptor muscarinic M2（Chrm2）,the receptors of sympathetic and parasympathetic nerves,are the molecular bases of chronotropic abilities in heart.The expression of Adrb1 and Chrm2 is a biological prerequisite for chronotropic ability.Objective To investigate the expression of chronotropic relevant receptors Adrb1 and Chrm2 in BMSCs with m HCN₂ gene modification and co-cultured with atrial myocytes. Methods PEGFP-C1-m HCN₂ recombinant plasmid was constructed in vitro by using gene splicing technique and DNA recombination technique,and then transfected mouse BMSCs.The modified BMSCs were induced to differentiate into mature functional cells under specific conditions.Primary cardiomyocytes were extracted from ICR suckling mice and cultured in 5% CO2 incubator at 37°C.BMSCs which extracted from 4-week-old male ICR mice were identified by flow cytometry and divided into four groups,the first group was BMSCs with transfection of p EGFP-C1-m HCN₂ and co-culture with atrial myocytes for 48h（TF+CO group）,the second one with only transfection for 48h（TF group）,the third one with only co-culture for 48h（CO group）,and the fourth one without transfection and co-culture for 48h（CTL group）.The green fluorescences could be observed in cells,after being transfected with p EGFP-C1-m HCN₂ successfully.Respectively extract four groups of cell proteins and RNA,the expressions of m HCN₂ channels,m RNA of Adrb1 and Chrm2 were examined by Western blot and real-time quantitative polymerase chain reaction（q RT-PCR）.Cardiac troponin I（c Tn I）could be detected by Gold Immunochromatographic Assay（GICA）.Results（1）The green fluorescence was seen in BMSCs of TF and TF+CO groups under fluorescent microscopy,it indicated that these two groups had been transfected with m HCN₂ gene,and the recombinant plasmid could reproduce,transcribe and translate in transfected cells.（2）The results of western-blot showed that HCN₂ protein successfully expressed in TF+CO and TF groups.（3）The concentration of c Tn I was tested in CO and TF+CO groups,it indicated that BMSCs co-cultured with atrial myocytes can express myocyte-specific marker troponin I.（4）The expressions of Adrb1 and Chrm2 m RNA were much higher in TF+CO and TF groups than those in CTL and CO groups,respectively（P<0.05）.Moreover,the expressions of m HCN₂ and Chrm2 m RNA in TF+CO group were significantly higher than those in TF group （P<0.05）.Conclusion BMSCs transfected with p EGFP-C1-m HCN₂ could successfully express m HCN₂ channel protein.Co-culture with atrial myocytes may help BMSCs modified with m HCN₂ gene to express myocyte-specific marker（c Tn I）and further increase the expressions of m HCN₂ channel,Adrb1 and Chrm2 These findings provide a theoretical foundation for biological basis of the stem cell-derived biologic pacemakers’ chronotropic ability and a new way for future slow antiarrhythmic treatment.