Recreating Cardiac Biological Pacemakers In Vitro

Objective: To recreating a cardiac biological pacemaker based on bone marrow-derived mesenchymal stem cells (MSCs) which transduced with hyperpolarization- activated cyclic nucleotide-gated cation channel gene subtype 4 (HCN4); And to study the impact of changed connexin subtype by overexpression with connexin45 in MSCs on automaticity of biological pacemaker.Methods:1) Neonatal rat ventricular myocytes were dissociated and cultivated as previously described; The purity and viability of myocytes were tested; Then spontaneous action potentials of myocardial cells was recorded by the patch-clamp technique.2) The shuttle plasmid containing HCN4 gene (pCDH1-GFP-HCN4) was built by gene cloning; pCDH1-GFP-HCN4 was packaged into lentiviral particles; Lentiviral particles containing HCN4 was transfected into rat MSCs; Then HCN4+MSCs had been constructed; MSCs transfected with lentiviral particles only containing GFP gene was defined as HCN4-MSCs; HCN4 expression in MSCs was conducted by RT-PCR, and observation of GFP fluorescence under fluorescence microscope. If current in HCN4+MSCs was recorded by Voltage-clamp.3) HCN4+MSCs and neonatal rat ventricular myocytes were cocultured to build biological pacemaker model; Where HCN4-MSCs and myocytes cocultured was defined as " HCN4-MSCs control group"; To evaluate automaticity of biological pacemaker, spontaneously beat frequency of myocytes was counted, and spontaneous action potentials of myocytes was record by current patch-clamp. Immunostaining of CX43 between myocytes and MSCs was conducted with CX43 Monoclonal antibody. Carbenoxolone (200 microM), a gap junction channel blocker, was added into the model to determine the impact of gap junction channel on biological pacemaker model.4) And to construct MSCs stable overexpression with CX45, pDsRED2-N1-RFP/ Gja7 containing CX45 gene(named as Gja7, as well) was built by means of gene cloning, then transfected into MSCs with the help of Liposomes 2000. A stable cell line CX45+MSCs was acquired with the stress of G418; MSCs transfected with empty plasmid pDsRED2-N1-RFP were defined as CX45-MSCs. To test the transcription of CX45 gene in MSCs,RT-PCR was carried out; To verify the connexins in MSCs had been changed, double immunostaining was conducted with a cocktail of monoclonal antibody CX43 and CX45 in CX45+MSCs.5) To recreate a biological pacemaker based on changed connexins in MSCs, CX45+MSCs was transduced with HCN4 gene. As a result, HCN4+CX45+MSCs was constructed. Then HCN4+CX45+MSCs were cocultured with neonatal rat ventricular myocytes to recreate a biological pacemaker; As a control, CX45-MSCs transduced with HCN4 were cocultured with myocytes simultaneously. To evaluate automaticity of biological pacemaker as described above. Double immunostaining of CX43 and CX45 between heterologous cell pairs was conducted with a cocktail of CX43 and CX45 monoclonal antibody. Carbenoxolone was added into the model to determine the impact of gap junction channel on transformed biological pacemaker model .Results:1) The survival rate of neonatal rat ventricular myocytes was about 80% just after the digestion. 85% myocytes beating spontaneously at the frequency of about 80 beating per minutes(bpm) in the fifth day. The purity of myocytes was greatly reduced after 9 days. Spontaneous action potentials outbreak with the mean rate of 84±12 bpm and a irregular cycle in neonatal rat ventricular myocytes. Threshold potential for action potentials of myocytes was about-60mV. 2) Restriction enzyme digestion and gene sequencing confirmed HCN4 gene had been successfully built into the shuttle plasmid pCDH1-GFP-HCN4. Titer of lentiviral vector lentiv-GFP/HCN4 packaged with pCDH1-GFP-HCN4 was 3×108TU/mL. The transfection efficiency for HCN4 by the means of lentiviral vector was 70% after 5 days. RT-PCR confirmed the transcription of HCN4 gene in MSCs. High level time- and voltage-dependent inward hyperpolarization current that was sensitive to 4mmol/L CsCl was detected in HCN4+MSCs, confirming that HCN4 acted as If channels in MSCs.3) The effects of genetically modified MSCs on cardiomyocyte excitability were determined in MSCs cocultured with neonatal rat ventricular myocytes. Counted spontaneously beating frequency in myocytes coclutured with HCN4+MSCs was greatly faster than in which myocytes coclutured with HCN4-MSCs. Recorded action potential in myocytes demonstrated that myocytes coclutured with HCN4+MSCs was characterized with a more regular and faster beating frequency(129±11 bpm vs. 82±8 bpm, n=5), a lower Maximal diastolic potential (MDP)(-66±6mV vs.-87±4mV,P<0.05 )than in which myocytes coclutured with HCN4-MSCs. Threshold potential for action potential in all myocytes was -60mV. Immunostaining manifested that CX43 was detected in adjacent membrane between heterologous cell pairs. Addition of carbenoxolone (200 microM), a gap junction channel blocker, to the model lowered spontaneous activity of myocytes in biological pacemaker model.4) Agarose gel electrophoresis for PCR products and gene sequencing confirmed CX45 gene (Gja7) was successfully constructed with pDsRED2-N1-RFP which containing RFP gene. The transfection efficiency for CX45 by the virtue of liposomes 2000 was 60% after 48h. Concentration of G418 for screening CX45+MSCs was 50μg/ml; RT-PCR confirmed the transcription of CX45 gene in CX45+MSCs. Double Immunostaining with CX43 and CX45 antibodies illuminated that CX45 and CX43 colocalized in membrane of CX45+MSCs.5) When connexins in membrane of MSCs was transformed with CX45, a biological pacemaker based on HCN4+CX45+MSCs was characterized with elevated automaticity. Outbreak frequency for spontaneously action potential in biological pacemakers based on HCN4+CX45+MSCs was faster than in control group(147±9 bpm vs. 123±8 bpm, P<0.05).The accelerated VDD in phase 4 mainly contributed to the elevated beating frequency. Double immunostaining manifested that CX43 and CX45 were simultaneously immune-detected in adjacent membrane between heterologous cell pairs. Administration of carbenoxolone to the model lowered spontaneous activity of myocytes in both group to a level identical with native neonatal rat ventricular myocytes.Conclusions: Our findings demonstrate that myocytes cocultured with MSCs modified with HCN4 can act as a cardiac biological pacemaker model in vitro. Overexpression of CX45 will elevate automaticity of cardiac biological pacemakers based on HCN4 modified MSCs in vitro.