Cardiomyocyte Progenitor Cells as a Functional Gene Delivery Vehicle for Long-Term Biological Pacing

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 I_f (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 I_f 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.

Keywords: HCN channels; cell therapy; gene therapy; pacemakers; progenitor cells.

Figure 1

Efficient transduction of cardiomyocyte progenitor cells (CMPCs) with lentivirus (LV) vectors. CMPCs were transduced with LV-DsRed at multiplicities of infection (MOIs) of 1, 2, and 10. Four days after transduction, cells were assessed by fluorescence microscopy (left) and flow cytometry (right). Histogram plots demonstrate that the proportion of DsRed-positive cells increases as a function of the MOI. Transduction efficiency is presented as mean ± standard error of the mean (SEM) (n = 3).

Figure 2

Functional coupling of CMPCs to cardiomyocytes. (A,B) Immunolabeling of connexin proteins Cx40 and Cx43. Scale bars represent 30 and 25 µm, respectively. (C) Fluorescent microscopy of dye transfer from CMPCs labeled with DiI (red) and Calcein (green) to unlabeled cardiomyocytes. Cardiomyocytes that have imported Calcein are indicated by white arrows. Scale bar represents 45 µm. (D) Immunolabeling of GFP (green) and HCN4 (red) in CMPCs transduced with LV-HCN4-GFP. Yellow indicates co-staining. Nuclei were counterstained blue with DAPI. Non-transduced CMPCs, shown in the inset, stained negative for GFP and HCN4. Scale bar represents 45 µm. (E) Average beating rates of neonatal rat ventricular myocyte (NRVM) monolayers co-cultured with CMPCs expressing GFP alone (grey) and HCN4 and GFP (red). * indicates P < 0.05.

Figure 3

Pacemaker current, I_f, in single LV-transduced CMPCs. (A) Voltage steps and typical I_f traces of CMPC/GFP and CMPC/HCN4-GFP cells. Inset: voltage pulse protocol to measure activation properties. (B) Average current-voltage (I–V) relationships of I_f of CMPC/GFP and CMPC/HCN4-GFP cells. (C) Voltage dependence of I_f activation. Solid line is the Boltzmann fit to the experimental data. (D) Typical I_f traces of CMPC/HCN4-GFP cells. Inset: voltage pulse protocol to measure deactivation properties. (E) I–V relationship of the fully-activated HCN4 current. Solid line is the linear fit to the experimental data. (F) Time constants of (de)activation. Solid line is the best fit curve to the equation τ = 1/[A1 × exp(−V/B1) + A2 × exp(V/B2)], where τ is the activation or deactivation kinetic time constant, and A1, A2, B1, and B2 are calculated fitting parameters [25].

Figure 4

I_f modulation in CMPCs expressing HCN4-GFP. (A) Effects of 10 nM Isoproterenol on voltage dependency of I_f activation. (B) Effects of 1 mM 8-Br-cAMP on voltage dependency of I_f activation and speed of I_f activation at −120 mV. (C) typical I_f traces at –120 mV in the absence and presence of 8-Br-cAMP. * indicates P < 0.05.

Figure 5

Baseline properties of organ explant cultures injected with CMPCs. (A,B) Summary data of spontaneous beating activity and conduction velocity of explants injected with CMPC/GFP and CMPC/HCN4-GFP. (C) Typical activation maps and optical action potentials measured during stimulation at 2 Hz. * indicates P < 0.05.

Figure 6

Autonomic modulation of pacemaker function in explants injected with CMPC/HCN4-GFP cells. (A) Isoproterenol (10 nM) significantly shortens cycle lengths in explants injected with CMPC/HCN4-GFP (left panel). In the presence of isoproterenol, cycle lengths are significantly shorter in explants injected with CMPC/HCN4-GFP than in CMPC/GFP-injected explants (right panel). (B) Typical activation maps and optical action potentials in CMPC/HCN4-GFP-injected explants at baseline and during superfusion with isoproterenol (two different preparations). * indicates P < 0.05.