Myofibroblasts Induce Ectopic Activity in Cardiac Tissue
Focal ectopic activity in cardiac tissue is a key factor in the initiation and perpetuation of tachyarrhythmias. Because myofibroblasts as present in fibrotic remodeled myocardia and infarct scars depolarize cardiomyocytes by heterocellular electrotonic interactions via gap junctions in vitro, we investigated using strands of cultured ventricular cardiomyocytes coated with myofibroblasts, whether this interaction might give rise to depolarization-induced abnormal automaticity. Whereas uncoated cardiomyocyte strands were invariably quiescent, myofibroblasts induced synchronized spontaneous activity in a density dependent manner. Activations appeared at spatial myofibroblast densities >15.7% and involved more than 80% of the preparations at myofibroblast densities of 50%. Spontaneous activity was based on depolarization-induced automaticity as evidenced by: (1) suppression of activity by the sarcolemmal KATP channel opener P-1075; (2) induction of activity in current-clamped single cardiomyocytes undergoing depolarization to potentials similar to those induced by myofibroblasts in cardiomyocyte strands; and (3) induction of spontaneous activity in cardiomyocyte strands coated with connexin 43 transfected Hela cells but not with communication deficient HeLa wild-type cells. Apart from unveiling the mechanism underlying the hallmark of monolayer cultures of cardiomyocytes, ie, spontaneous electromechanical activity, these findings open the perspective that myofibroblasts present in structurally remodeled myocardia following pressure overload and infarction might contribute to arrhythmogenesis by induction of ectopic activity.
Structural remodeling of the myocardium during pressure overload and following infarction is typically accompanied by the appearance of interstitial myofibroblasts which contribute to cardiac fibrosis by excessive secretion of extracellular matrix proteins.1 The resulting collagenous septa contribute to arrhythmogenesis in structurally remodeled hearts by inducing discontinuous slow conduction.2 More recently, studies in vitro demonstrated that myofibroblasts can directly induce arrhythmogenic slow conduction following establishment of heterocellular gap junctional coupling with cardiomyocytes.3 This slowing of conduction is the result of a decrease in inward currents secondary to the partial depolarization of the cardiomyocytes by the less polarized myofibroblasts. Because partial depolarization of cardiac tissue has previously been shown to induce abnormal automaticity,4 we investigated in the present study whether heterocellular electrotonic interactions between myofibroblasts and cardiomyocytes might precipitate spontaneous ectopic activity.
Materials and Methods
The effects of myofibroblasts on cardiac excitability were investigated in patterned growth strands of neonatal rat ventricular cardiomyocytes using optical recording of transmembrane voltage, immunocytochemistry and patch clamp recording techniques. Detailed descriptions of the materials and methods used are available in the online data supplement at http://circres.ahajournals.org.
The hypothesis that myofibroblasts might generate abnormal automaticity in cardiac tissue was investigated in patterned growth strands of neonatal rat ventricular cardiomyocytes (Figure 1). Whereas control preparations were invariably quiescent (n=102; Figure 1A,C), coating of the strands with increasing numbers of myofibroblasts (25 to 950 cells/mm2) elicited spontaneous electrical activity in 54.2% of the preparations with an average frequency of 64.4±21.7 min−1 (n=548; Figure 1B,C). In contrast, control cardiomyocyte strands cocultured with myofibroblast in a noncontact configuration remained quiescent indicating that induction of spontaneous activity was not dependent on conditioning of the medium by paracrine activity of myofibroblasts (n=333; Figure 1C).5 Spontaneous activations started preferentially at the lateral ends of the strands (83%; Figure 1D) which can be explained by favorable source-to-load conditions offered by these sites. The likelihood of occurrence of synchronized spontaneous activity showed a strong dependence on the density of myofibroblasts (Figure 1E). Whereas preparations were quiescent at myofibroblast densities <15.7%, the percentage of active preparations above this threshold rose to more than 80% at myofibroblast densities of 50%. In parallel, and similar to a recent observation of a decline of activation frequencies in single isolated cardiomyocytes coupled to fibroblasts,6 the frequency of spontaneous activations declined from ≈ 80 min−1 to ≈ 45 min−1 with increasing myofibroblast density.
Mechanistically, previous findings showing that myofibroblasts depolarize cardiomyocytes in a cell density dependent manner suggested that myofibroblast induce ectopic activity by ways of depolarization-induced automaticity.3,4 Accordingly, we investigated in isolated cardiomyocytes whether the range of membrane depolarizations observed in myofibroblast coated cardiomyocytes is sufficient by itself to induce spontaneous activity. As shown by the patch clamp experiments in Figure 2A, single cultured cardiomyocytes undergoing stepwise depolarizations during injection of 30 s long current pulses of increasing amplitude exhibited depolarization-induced automaticity appearing at membrane potentials less negative than −65 mV. With further depolarization, the frequency of spontaneous activations transiently increased to reach a peak at - 56 mV. In a total of 15 cardiomyocytes (Figure 2B; red symbols), spontaneous activity appeared at maximal diastolic potentials (MDPs) less negative than −67.4 mV with >90% of the cells being active at ≈ −55 mV. This voltage dependence is highly similar to that estimated for myofibroblast coated cardiomyocyte strands (Figure 2B; black symbols) which suggests that myofibroblast induced depolarization of cardiomyocyte strands is a major mechanism underlying abnormal automaticity. In further support of this hypothesis, increasing the degree of membrane polarization of myofibroblast coated cardiomyocyte strands by superfusion with the surface sarcolemmal KATP channel opener P-1075 (10 μmol/L, n=43) invariably stopped spontaneous activity. In contrast to the similarities regarding appearance of spontaneous activity as a function of MDP, current-clamped cardiomyocytes showed a biphasic dependence of the frequency of spontaneous activations on MDP (Figure 2C; red symbols) whereas myofibroblast coated cardiomyocyte strands showed a linear decay (Figure 2C; black symbols). This difference likely reflects the circumstance that, in contrast to the frequency response of a given individual cell stepped through different MDPs during patch clamp experiments, the prevailing frequency of multicellular strand preparations is the result of the selection of the fastest pacemaking region capable of driving the load at a given MDP.
If spontaneous activity is the result of a partial depolarization of cardiomyocytes by electrotonically coupled myofibroblasts, it should be possible to evoke the same response by coupling cardiomyocytes to other cell types which have an inherently depolarized membrane potential and establish heterocellular gap junctional coupling with cardiomyocytes. This hypothesis was investigated by coating strands of cardiomyocytes with communication deficient HeLa wild-type cells (HeLawt; resting potentials ≈ -40 mV)7 and HeLa cells transfected with connexin 43 (HeLaCx43) which are able to establish functional heterocellular gap junctional coupling with cardiomyocytes.8 As shown in Figure 3, coating of cardiomyocyte strands with HeLawt cells failed to induce automaticity even though cell densities reached nearly 100% (n=203). In contrast, coating of cardiomyocyte strands with HeLaCx43 cells elicited spontaneous electrical activity at cell densities >39.5% with maximal effects (≈ 80% spontaneously active strands) observed at densities of 80%. Average activation rates (58.4±26.2 min−1, n=233) showed no significant dependence on HeLaCx43 cell density (data not shown). As for the case of myofibroblasts, activations started preferentially at the lateral ends of the strands (89%).
Abnormal electrical activity based on depolarization-induced automaticity is a well established arrhythmogenic mechanism thought to occur, eg, in the context of injury currents flowing across the border zones of acute infarcts.9 Whereas this type of ectopic activity is dependent on spatial heterogeneities of electrophysiological properties within homocellular networks of cardiomyocytes, the results of the present study demonstrate that depolarization-induced automaticity can similarly be induced by heterocellular electrotonic interactions between cardiomyocytes and communication competent but less polarized cells like myofibroblasts and HeLaCx43 cells. For monolayer cultures of cardiomyocytes, these findings demonstrate that spontaneous activity is not, as commonly assumed, due to a culture-dependent dedifferentiation of cardiomyocyte toward a spontaneously active immature phenotype, but is the specific result of electrotonic interactions with a sufficient number of myofibroblasts. As a consequence, the interpretation of beat rate changes in these preparations following specific experimental interventions need to take into account the possibility that observed effects are not necessarily cardiomyocyte-related but might occur secondary to a modification of the electrophysiology of myofibroblasts. In regard to intact cardiac tissue, the findings of this study open the perspective that contact regions between cardiomyocytes and sufficiently large numbers of myofibroblasts as occurring in the borderzone of healing infarcts10 a few days after the acute event or in the fibrotic working myocardium11 might give rise to arrhythmogenic ectopic activity. Proof of this hypothesis is pending confirmation that myofibroblasts in vivo retain an electrophysiological phenotype similar to that in culture and that their capacity to depolarize adjacent cardiomyocytes is sufficient to induce ectopic activity. Finally, the findings suggest that transplantation of communication competent cells exhibiting a reduced resting membrane potential like undifferentiated human mesenchymal stem cells12 might bear the potential to induce ectopic activity independent on their ability to produce regenerative activity.
Sources of Funding
This study was supported by the Swiss National Science Foundation (3100AO-105916 to S.R.).
Original received July 23, 2007; revision received September 4, 2007; accepted September 5, 2007.
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