Abstract
Sustained pacemaker function is a challenge in biological pacemaker engineering. Human cardiomyocyte progenitor cells (CMPCs) have exhibited extended survival in the heart after transplantation. We studied whether lentivirally transduced CMPCs that express the pacemaker current If (encoded by HCN4) can be used as functional gene delivery vehicle in biological pacing. Human CMPCs were isolated from fetal hearts using magnetic beads coated with Sca-1 antibody, cultured in nondifferentiating conditions, and transduced with a green fluorescent protein (GFP)- or HCN4-GFP-expressing lentivirus. A patch-clamp analysis showed a large hyperpolarization-activated, time-dependent inward current (−20 pA/pF at −140 mV, n = 14) with properties typical of If in HCN4-GFP-expressing CMPCs. Gap-junctional coupling between CMPCs and neonatal rat ventricular myocytes (NRVMs) was demonstrated by efficient dye transfer and changes in spontaneous beating activity. In organ explant cultures, the number of preparations showing spontaneous beating activity increased from 6.3% in CMPC/GFP-injected preparations to 68.2% in CMPC/HCN4-GFP-injected preparations (P < 0.05). Furthermore, in CMPC/HCN4-GFP-injected preparations, isoproterenol induced a significant reduction in cycle lengths from 648 ± 169 to 392 ± 71 ms (P < 0.05). In sum, CMPCs expressing HCN4-GFP functionally couple to NRVMs and induce physiologically controlled pacemaker activity and may therefore provide an attractive delivery platform for sustained pacemaker function.
Highlights
Biological pacing based on gene and cell therapy technologies has been the subject of intensive research, focused primarily on treating bradycardia
An in vitro analysis indicated that cardiomyocyte progenitor cells (CMPCs) were efficiently transduced by LV vectors and connexins, leading to increased spontaneous activity in cardiac myocytes coupled to HCN4expressed connexins, leading to increased spontaneous activity in cardiac myocytes coupled to overexpressing CMPCs
To demonstrate functional interaction between CMPCs and cardiomyocytes, we performed a dye transfer experiment (Figure 2C) and studied the beating rate of neonatal rat ventricular myocytes (NRVMs) monolayers co-cultured with CMPCs expressing either HCN4-green fluorescent protein (GFP) or GFP (Figure 2E)
Summary
Biological pacing based on gene and cell therapy technologies has been the subject of intensive research, focused primarily on treating bradycardia. The safety concerns surrounding LV, together with a relatively laborious production process, have delayed the use of LV vectors for in vivo gene transfer [12] These issues have been largely addressed by the design of vectors with an LV genome that is largely devoid of virulence factors and divided over several plasmids for production [13], and by careful monitoring of preclinical animal models and human patients after LV gene therapy. No relevant RCL quantities were found in animals subjected to cell therapy with LV-transduced stem cells [14,15], or in long-term clinical studies on patients undergoing gene therapy with LV [16,17] Another concern was the potential for insertional mutagenesis that could lead to neoplasia, especially in the hematopoietic system. To determine the ability of LV vectors to provide gene transfer to CMPCs, we transduced the cells with LV expressing the red fluorescent protein DsRed at varying multiplicities of infection
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