Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers
Potapova I, Plotnikov A, Lu Z, Danilo P Jr, Valiunas V, Qu J, Doronin S, Zuckerman J, Shlapakova IN, Gao J, Pan Z, Herron AJ, Robinson RB, Brink PR, Rosen MR and Cohen IS
For the following experiments, human mesenchymal stem cells (hMSC) nucleofected with mHCN2, were used: Patch clamp experiments revealed a large time-dependent inward current activation on hyperpolarizations. After extracellular application of Cs+, the reversal potential could be determined. A potential advantage of biological over electronic pacemakers is their hormonal regulation. Isoproterenol stimulation increased the current while acetylcholine had no direct effect on the time-dependent current. Neonatal rat ventricular myocytes cocultured with mHCN2 nucleofected hMSCs showed a reduced maximum diastolic potential consistent with the observed altered threshold potential. Injection of mHCN2 expressing hMSCs into canine heart led to altered idioventricular rates and the animals developed heart rhythms originating from and pace-mapped to the left ventricle at a site whose origin approximated that of the hMSC injection. Gap junctions between hMSCs and cardiac myocytes were detected by patch-clamp and microscopy analysis.
We tested the ability of human mesenchymal stem cells (hMSCs) to deliver a biological pacemaker to the heart. hMSCs transfected with a cardiac pacemaker gene, mHCN2, by electroporation expressed high levels of Cs+-sensitive current (31.1+/-3.8 pA/pF at -150 mV) activating in the diastolic potential range with reversal potential of -37.5+/-1.0 mV, confirming the expressed current as I(f)-like. The expressed current responded to isoproterenol with an 11-mV positive shift in activation. Acetylcholine had no direct effect, but in the presence of isoproterenol, shifted activation 15 mV negative. Transfected hMSCs influenced beating rate in vitro when plated onto a localized region of a coverslip and overlaid with neonatal rat ventricular myocytes. The coculture beating rate was 93+/-16 bpm when hMSCs were transfected with control plasmid (expressing only EGFP) and 161+/-4 bpm when hMSCs were expressing both EGFP+mHCN2 (P<0.05). We next injected 10(6) hMSCs transfected with either control plasmid or mHCN2 gene construct subepicardially in the canine left ventricular wall in situ. During sinus arrest, all control (EGFP) hearts had spontaneous rhythms (45+/-1 bpm, 2 of right-sided origin and 2 of left). In the EGFP+mHCN2 group, 5 of 6 animals developed spontaneous rhythms of left-sided origin (rate=61+/-5 bpm; P<0.05). Moreover, immunostaining of the injected regions demonstrated the presence of hMSCs forming gap junctions with adjacent myocytes. These findings demonstrate that genetically modified hMSCs can express functional HCN2 channels in vitro and in vivo, mimicking overexpression of HCN2 genes in cardiac myocytes, and represent a novel delivery system for pacemaker genes into the heart or other electrical syncytia.
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