Creation of a Biological Pacemaker by Cell Fusion
As an alternative to electronic pacemakers, we explored the feasibility of converting ventricular myocytes into pacemakers by somatic cell fusion. The idea is to create chemically induced fusion between myocytes and syngeneic fibroblasts engineered to express HCN1 pacemaker channels (HCN1-fibroblasts). HCN1-fibroblasts were fused with freshly isolated guinea pig ventricular myocytes using polyethylene-glycol 1500. In vivo fused myocyte-HCN1-fibroblast cells exhibited spontaneously oscillating action potentials; the firing frequency increased with β-adrenergic stimulation. The heterokaryons created ectopic ventricular pacemaker activity in vivo at the site of cell injection. Coculture of nonfused HCN1-fibroblasts and myocytes without polyethylene-glycol 1500 revealed no evidence of dye transfer, demonstrating that the If-mediated pacemaker activity arises from heterokaryons rather than electrotonic coupling. This nonviral, non-stem cell approach enables autologous, adult somatic cell therapy to create biopacemakers.
Cardiac rhythm disorders are caused by malfunctions of impulse generation and/or conduction. Present therapies for deficient impulse generation, eg, electronic pacemakers, remain palliative. Here we continue to develop the alternative concept of biological therapy for cardiac arrhythmias; the objective is to achieve functional reengineering of cardiac tissue, so as to alter a specific electrical property of the tissue in a salutary manner. In this study, engineered cells were introduced to create a biological pacemaker in normally quiescent myocardium. A key ionic current present in sinoatrial nodal pacemaker cells, but largely absent in atrial and ventricular myocytes, is the pacemaker current, If.1 We used polyethylene glycol-induced fibroblast-myocyte fusion as a method to deliver If to myocardium to create a biological pacemaker.
Materials and Methods
For in vitro fusion experiments, HCN1-fibroblasts and myocytes were incubated in prewarmed (37°C) 40% polyethylene glycol 1500 (PEG) (Roche Applied Science, Indianapolis, Ind) in PBS for 2 to 4 minutes. Cells were rehydrated with high potassium solution (same solution as used after myocyte isolation) for 5 to 10 minutes and then washed with normal Tyrode’s solution.
An expanded Materials and Methods section is in the online data supplement at http://circres.ahajournals.org.
Guinea pig lung fibroblasts stably expressing HCN1 channels with a green fluorescence protein (GFP) reporter (HCN1-fibroblasts) and loaded with calcein acetoxymethyl ester were fused with freshly isolated guinea pig ventricular myocytes using PEG. Within 3 minutes, the HCN1-fibroblasts fused with ventricular myocytes, as verified by the sudden introduction of calcein fluorescence from the fibroblast cytoplasm into the myocyte cytoplasm (Figure 1A, left). To extend these in vitro observations, we focally injected HCN1-fibroblasts suspended in 50% PEG into the apex of guinea pig hearts. Langendorff isolation of ventricular myocytes from the site of injection revealed GFP-positive myocytes (Figure 1A, right). Such myocyte/HCN1-fibroblast heterokaryons formed by in vivo fusion displayed spontaneous action potentials with a slow phase 4 depolarization (Figure 1B, left), characteristic of pacemaker cells. Spontaneous activity was not observed in myocytes fused with control fibroblasts expressing GFP only; in such cells, action potentials could be elicited by only external electrical stimulation (Figure 1B, right). The maximum diastolic potentials of the heterokaryons formed with HCN1-fibroblasts were only modestly depolarized (−76±9 mV, n=9) relative to the resting membrane potentials of the heterokaryons formed with control fibroblasts (−80.5±2 mV, n=7). Subsequent voltage-clamp recordings with external Ba2+ (1 mmol/L) to block IK1 revealed the heterologously expressed pacemaker current, If (Figure 1C, left), which was not detectable either in ventricular myocytes alone or in myocytes fused with control fibroblasts (Figure 1C, right). Freshly isolated heterokaryons formed by in vivo fusion between myocytes and HCN1-fibroblasts expressed robust pacemaker current with a conductance of −770±7 pS/pF (n=9, Figure 1D), an If density >2-fold that reported in isolated rabbit sinoatrial nodal cells.2 The If expressed from heterokaryons exhibited normal HCN1 activation kinetics (Figure 1E, left) with a potential of half-maximal activation of −73.1±2.2 mV (Figure 1E, right). The chemically induced in vivo fusion events did not alter the main excitatory ionic current, INa, of the heterokaryons (Figure 1F; −22.1±3 nA [n=9] at −40 mV for myocytes fused with HCN1-fibroblasts versus −20.8±3 nA [n=7] for myocytes fused with GFP-alone control fibroblasts). Cell fusion should be accompanied by an increase in total cell surface area, a parameter that can be indexed by measurements of electrical capacitance. Indeed, GFP-positive heterokaryons exhibited a larger membrane capacitance than the GFP-negative myocytes (124±14 pF, n=9 and 97±8 pF, n=15, respectively, P<0.05), supporting the concept of in vivo fusion events. The increased cell capacitance, in effect, would dilute the density of the hyperpolarizing-current, IK1, by 20%. Thus, the robust If conductance, combined with the decreased IK1 conductance, drives the spontaneous pacemaking in the heterokaryons.
To investigate in vivo fusion events histologically, HCN1-fibroblasts were transduced with adenovirus expressing cytoplasmic β-galactosidase (Ad-lacZ). 5-Bromo-4-chloro-3-indolyl β-d-galactoside staining of the heart sections at the site of cell injection revealed β-galactosidase activity in the longitudinally oriented ventricular myocytes at the border between myocytes and injected HCN1-fibroblasts, as well as in some HCN1-fibroblasts that had not undergone fusion with myocytes (Figure 2, top left). Because most cardiomyocytes are multinucleated, detection of extra nuclei from HCN1-fibroblasts as evidence for cell fusion in the heterokaryons was not feasible. Instead, immunohistochemistry against β-galactosidase and myosin heavy chain colocalized the 2 proteins in regions of the myocardium (Figure 2, bottom right), indicating fusion of cytoplasm from HCN1-fibroblasts (containing β-galactosidase) and cardiomyocytes.
To examine ectopic pacemaker activity generated by the in vivo fusion events, the heart rates of guinea pigs were slowed with methacholine injection. Electrocardiograms recorded less than 1 to 22 days after HCN1-fibroblast injection revealed ectopic ventricular beats that were identical in polarity and similar in morphology to those recorded during bipolar pace-mapping of the apex in the same animal (Figure 2B, top; n=5 of 13). Occasionally, junctional escape rhythms (Figure 2B, bottom, horizontal arrows) could be overtaken by ectopic ventricular pacemaker activity. Such ectopic beats were not observed in animals injected with control fibroblasts expressing GFP only (data not shown, n=9).
β-Adrenergic stimulation is a potent physiological mechanism to accelerate physiologic cardiac pacing.3 We sought to determine whether heterokaryons formed between HCN1-fibroblasts and myocytes could respond to the β-adrenergic agonist isoproterenol. As demonstrated in Figure 3A, 1 μmol/L isoproterenol increased the spontaneous beating rate of isolated heterokaryons by 25±10% (n=4). Thus, chronotropic responsiveness is an intrinsic feature of fusion-engineered biopacemakers. Furthermore, the spontaneous action potential (AP) oscillations could be blocked by an If-specific blocker ZD7288 (Figure II in the online data supplement).
To exclude the possibility of gap-junctional coupling between fibroblasts and myocytes4 as an alternative mechanism of pacemaker activity, HCN1-fibroblasts were loaded with calcein and mixed with nonloaded myocytes. The dye did not diffuse from loaded HCN1 pulmonary fibroblasts to neighboring myocytes, indicating the absence of cell-cell coupling (Figure 3B, top). On the other hand, calcein transferred efficiently from cardiac fibroblasts to myocytes, consistent with the known ability of such fibroblasts to couple via gap junctions (Figure 3B, bottom).5 Thus, the If-mediated pacemaker activity arises from heterokaryons rather than electrotonic coupling between myocytes and fibroblasts.
PEG-induced membrane fusion events have served as a model system to study eukaryotic cell-cell fusion events6 and to deliver outward K+ currents into myocytes.7 Here, we used syngeneic fibroblasts expressing HCN1 channels as donor cells to induce spontaneous activity in normally quiescent ventricular myocytes after chemically induced cell fusion. The cell fusion biological pacemakers were functional for at least 3 weeks and as early as <1 day postinjection, as revealed by electrocardiography. Previous studies suggest that heterokaryons can maintain the nuclei from each fusion partner separately and stably for at least several months.8,9
Our approach differs conceptually from previous cell-based pacemakers, which rely on cell-cell coupling for transmission of the impulse from the introduced cells to surrounding myocardium.10 Such gap-junctional coupling may or may not be stable over time; many of the major forms of human heart disease, associated with increased arrhythmic risk, coincide with gap junction remodeling, and decreased cell-cell coupling.11 Furthermore, stem cells have been shown to proliferate and migrate once injected into myocardium.12 This may cause unpredictable patterns of pacemaker activity from regions of the heart other than the desired site. In contrast, the present approach creates a biological pacemaker specifically localized to heterokaryons formed by PEG-induced fusion. Furthermore, fibroblasts that did not undergo fusion with myocytes would not generate pacing from sites other than the site of injection because of the lack of cell-cell coupling.
We have demonstrated that the present approach is feasible, but we have yet to demonstrate consistent pacing in vivo, or long-term effectiveness in a large-animal model. Efforts to increase the efficiency of fusion events such as the use of different fusogens may increase the stability of pacing in vivo. Nevertheless, a number of limitations of previous approaches do not plague the present strategy: first, autologous cells (eg, skin fibroblasts) can be harvested and used; second, viral vectors and their complications can be avoided, as stable transduction can be achieved by routine plasmid transfection technology; third, stem cells are not required. For these reasons, the present methodology may merit exploitation in the future development of biological alternatives to device therapy.
Sources of Funding
This study was funded by a Heart Rhythm Society fellowship (to H.C.C.) and by The Donald W. Reynolds Foundation. E.M. holds the Michel Mirowski Professorship of Cardiology of the Johns Hopkins University.
E.M. owns stock and provides consulting services to Excigen Inc. H.C.C. is an employee of Excigen Inc.
This manuscript was sent to Harry A. Fozzard, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received February 14, 2007; revision received March 14, 2007; accepted March 21, 2007.
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