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 ϩ -sensitive current (31.1Ϯ3.8 pA/pF at Ϫ150 mV) activating in the diastolic potential range with reversal potential of Ϫ37.5Ϯ1.0 mV, confirming the expressed current as I f -like. The expressed current responded to isoproterenol with an 11-mV positive shift in activation.

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... 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 93Ϯ16 bpm when hMSCs were transfected with control plasmid (expressing only EGFP) and 161Ϯ4 bpm when hMSCs were expressing both EGFPϩmHCN2 (PϽ0.05). 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 (45Ϯ1 bpm, 2 of right-sided origin and 2 of left). In the EGFPϩmHCN2 group, 5 of 6 animals developed spontaneous rhythms of left-sided origin (rateϭ61Ϯ5 bpm; PϽ0.05). 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. (Circ Res. 2004;94:952-959.)