Study Of Differentiation Of Rat MSCs With MHCN4 Gene Into Spontaneous Beating Cardiac Pacemaker Like Cells In Vitro

Background and objection:Sick sinus syndrome is a familiar decease, and it can produce severe damage to patients. But its pathological mechanism is not clear. There still have no very effective prevent and treatment methods till now. Current main treatment method is implantation of artificial electric cardiac pacemaker. But they are not optimal. Among their shortcomings are limited battery life, the need for permanent catheter implantation into the heart, and lack of response to autonomic neurohumors. For these reasons, ideal pacemaker should be the"biological pacemaker"considering physiologic function of heart and adaptability of human body. In recent years, several approaches have been attempted to provide biological pacemaker function by transplanting mesenchymal stem cells (MSCs) to animal hearts. Because of the short duration of pacing function, the low heart variation and the unclear function of the grafted cell, so these approaches aren't optimal. The results of these studies also showed we haven't found the optimal seed cell for transplant in order to fabricate biological pacemakers. So, it is important and exigent to search the optimal seed cells for biological pacemakers.Lots of study revealed that spontaneous depolarization of sino-atrial cell included many kinds of transmembrane ionic currents. the Funny Current(If)was the most important one. The family of If channels have 4 members, HCN1-HCN4. HCN4 is a optimal biological pacemaker target gene because it is essential for modulation If and maintenance of electrophysiologic function of spontaneous cell in SAN. In order to study the possibility of rat MSCs with mHCN4 gene acting as seed cells for biological pacemakers therapy, we adopted the mHCN4 as the target gene of biological pacemaker, utilized the platform of rat MSCs and obtained the rat MSCs that had stably high expression of mHCN4 through transfection with the mHCN4 retrovirus vector (pMSCV-mHCN4-EGFP) constructed successfully by our research group recently, and expected to gain the spontaneous cardiac pacemaker-like cells acting as the seed cells for biological pacemakers therapy.Methods1.Rat MSCs were transfected by mHCN4 retrovirus vector (pMSCV-mHCN4-EGFP) and the control vector (pMSCV-EGFP) respectively, and the cells stably expressing the gene of mHCN4 were selected by puromysin. The expression of GFP was observed by fluorescence microscope, and the expression of mHCN4 was detected by immunocytochemistry.2. The kinetics characters of If channels were detected by whole-cell patch clamp, in order to determine the mHCN4 channels expression in rat MSCs transfected with pMSCV-mHCN4-EGFP.3. Rat MSCs with mHCN4 gene were induced in vitro. The dynamic morphologic changes were observed with light microscope.4. The ultrastructure of rat MSCs with mHCN4 gene was observed by electronic microscope. The expressions ofα-actin, cTnT and Cx43 were detected by immunocytochemistry. Action potential was recorded by whole-cell patch clamp.5. The gap junction between rat MSCs with mHCN4 gene and cultured neonata atrial cells in coculture was observed by electronic microscope. Gap junctional intercellular communication (GJIC) was detected by fluorescence recovery after photobleaching (FRAP).Result1. Rat MSCs were transfected with (pMSCV-mHCN4-EGFP) successfully, and the cells stably expressing the gene of mHCN4 were selected by puromysin.2. A hyperpolarization-activated inward current was recorded by whole-cell patch clamp. The current had obvious time and voltage dependences, and it also was high sensitive to extrocelluar Cs+. Meanwhile, this current coule not be observed in control group under the same conditions. The mHCN4 channel current's half-maximal activation was -98.2±5.7 mV,the active potential was about -62.8 mV;the reversal potential was -22.5±5.2 mV;Time constants of activation was 451±61 ms under -140mV。 3. After cultured in DMEM (10%FBS) about 10days, the spontaneous contraction cells were observed by light microscope in mHCN4-transfeted rat MSCs. The cells are stick-like, and some one looked like mytubes. The frequency of contraction ranged from88 to 318 per minute, the average is 202±72.9 per minute. The spontaneous beating persisted for only 2-3days, vacuoles were observed at the part of contraction. But no spontaneous contraction cells appeared in the GFP group cell under same culture conditions.4. The action potential with configuration like sino-atrial node cells was recorded in spontaneous contraction cells by wheole-cell patch clamp. Maximum diastolic potential was 50.9±4.8mV, average swing of action potential is 62.9±5.2mV.5. The spontaneous contraction cells not only expressed mHCN4 channel, but also expressedα-actin and cTnT, which specially express in cardiomyocytes. Myofilaments were observed in the spontaneous contraction cells by electronic microscope, but their alignment was intricate. No sarcomere was observed. The ultrastructure of spontaneous contraction cells is same to that of neonatal rat SAN cells.6. The expression of Cx43was observed in rat MSCs with mHCN4 gene, (obvious stronger than that of rat MSCs in GEP group and no-transfection rat MSCs), and was same to that of neonata atrial cells.7. Under the transmission electron microscope, we found adjacent cells in rat MSCs with mHCN4 gene and neonata atrial cells in coculture often had intimately apposed interdigitating cell membranes. Occasional cells were identified with electron-dense membrane structures suggestive of gap junctions.8. By the ERAP, we found the average fluorescence revover rate of mHCN4-transfeted rat MSCs and cocultured with cultured neonata atrial cells was obvious higher than that of GFP-transfeted rat MSCs and cocultured with neonata atrial cells, and same to that of cultured neonata atrial cells. It showed there is the function gap junction between mHCN4-transfeted rat MSCs and neonata atrial cells.Conclusions1. Rat MSCs can stably expressing the gene of mHCN4 by transfection with mHCN4 retrovirus vector.2. Rat MSCs transfeced with mHCN4 gene can occur If. It shows them have the electrophysiological ability of cardiac pacemaker cells. 3. Rat MSCs with mHCN4 gene can different into spontaneous beating cardic pacmaker cells in vitro.4. Rat MSCs with mHCN4 gene can express Cx43, and form function gop junction with neonata atrial cells in cocultured.