Arrhythmogenic consequences of myofibroblast-myocyte coupling

Abstract

Aims: Fibrosis is known to promote cardiac arrhythmias by disrupting myocardial structure. Given recent evidence that myofibroblasts form gap junctions with myocytes at least in co-cultures, we investigated whether myofibroblast-myocyte coupling can promote arrhythmia triggers, such as early afterdepolarizations (EADs), by directly influencing myocyte electrophysiology.

Methods and results: Using the dynamic voltage clamp technique, patch-clamped adult rabbit ventricular myocytes were electrotonically coupled to one or multiple virtual fibroblasts or myofibroblasts programmed with eight combinations of capacitance, membrane resistance, resting membrane potential, and gap junction coupling resistance, spanning physiologically realistic ranges. Myocytes were exposed to oxidative (1 mmol/L H(2)O(2)) or ionic (2.7 mmol/L hypokalaemia) stress to induce bradycardia-dependent EADs. In the absence of myofibroblast-myocyte coupling, EADs developed during slow pacing (6 s), but were completely suppressed by faster pacing (1 s). However, in the presence of myofibroblast-myocyte coupling, EADs could no longer be suppressed by rapid pacing, especially when myofibroblast resting membrane potential was depolarized (-25 mV). Analysis of the myofibroblast-myocyte virtual gap junction currents revealed two components: an early transient-outward I(to)-like current and a late sustained current. Selective elimination of the I(to)-like component prevented EADs, whereas selective elimination of the late component did not.

Conclusion: Coupling of myocytes to myofibroblasts promotes EAD formation as a result of a mismatch in early vs. late repolarization reserve caused by the I(to)-like component of the gap junction current. These cellular and ionic mechanisms may contribute to the pro-arrhythmic risk in fibrotic hearts.

Figure 1

Induction of bradycardia-dependence of EAD by hypokalaemia or H₂O₂. A patch-clamped rabbit myocyte was superfused with Tyrode's solution containing normal 5.4 mmol/L K⁺ (A), reduced 2.7 mmol/L K⁺ (B), or 1.0 mmol/L H₂O₂ (in normal 5.4 mmol/L K⁺) (C). Both hypokalaemia and oxidative stress with H₂O₂ caused EADs (*) at long pacing cycle lengths (PCL) but not at PCL 1 s.

Figure 2

Promotion of EADs by myofibroblast–myocyte coupling. (A) Coupling a patch-clamped myocyte superfused with normal Tyrode's solution to a virtual fibroblast did not induce EADs at PCL 1 s. Superimposed traces at a faster time scale to the right illustrate that coupling lowers the AP plateau and shortens ADP. (B and C) In myocytes exposed to hypokalaemia or H₂O₂ to induce bradycardia-dependent EADs at PCL 6 s (not shown), pacing at PCL 1 s suppressed EADs completely (top traces). However, coupling the myocyte to a virtual fibroblast (C_f 6.3 pF, G_j 3.0 nS) caused EADs to reappear, which were more prominent when E_f was −25 mV (bottom traces) than −50 mV (middle traces). In addition to EADs, DADs (some triggering APs) were also frequently observed (arrows). Lower panels show superimposed traces of control vs. coupled for the E_f −50 and −25 mV cases, respectively, at a faster time scale. Note that coupling lowers the early AP plateau voltage before EAD onset.

Figure 3

Data summary of EAD reappearance at PCL 1 s induced by myocyte–myofibroblast 1:1 coupling. Patch-clamped myocytes were exposed to H₂O₂ to induce bradycardia-dependent EADs at PCL 6 s, which were then suppressed at PCL 1 s. Bars indicate the fraction of myocytes in which EADs reappeared when the myocyte was coupled to a virtual myofibroblast, for each of the eight different coupling parameter sets listed in Table 1. The total number of ventricular myocytes tested for each coupling parameter set is indicated inside each bar, with the corresponding probability of EAD reappearance shown above.

Figure 4

Higher probability of EAD induction by coupling multiple myofibroblasts to a single myocyte. A patch-clamped myocyte was exposed to H₂O₂ to induce bradycardia-dependent EADs at PCL 6 s, which were then suppressed at PCL 1 s. Coupling the myocyte (MC) to one myofibroblast (MF, with C_f 50.0 pF, G_j 3.0 nS, E_f −25 mV) depolarized the myocyte and shortened APD (middle trace), but did not cause EADs to reappear. However, when the myocyte was coupled to two myofibroblasts in parallel (effectively increasing C_f to 100 pF and G_j to 6 nS), EADs and repolarization failure ensued (lower trace).

Figure 5

Effects of E_f vs. G_f on EAD threshold. Threshold for EAD formation (see text) of the myofibroblast-coupled state was compared with that of the control myofibroblast-uncoupled state. (A) Coupling a passive myofibroblast model to a Luo-Rudy 1 myocyte AP model to simulate the same eight coupling combinations tested experimentally in Table 1 lowered EAD threshold much more prominently for high E_f–high G_f (E_f −25 mV/G_f 123 pS) cases than for low E_f–low G_f (E_f −50 mV/G_f 26 pS) cases. (B) When E_f was kept low at −50 mV for all eight parameter combinations, G_f had little effect or increased EAD threshold. (C) When E_f was held high at −25 mV for all eight parameter combinations, G_f decreased EAD threshold in all cases, indicating that high E_f is more important than high G_f at lowering EAD threshold. Numbers indicate the per cent change in EAD threshold.

Figure 6

The early I_to-like component of I_j promotes EADs. (A) A patch-clamped myocyte was exposed to 1.0 mmol/L H₂O₂ to induce bradycardia-dependent EADs at PCL 6 s, which were then suppressed at PCL 1 s (not shown). Coupling the myocyte to a virtual fibroblast (C_f6.3pF, E_f −25 mV, G_j 0.3 nS) (left upper trace) caused EADs to reappear. The virtual gap junction current I_j (left lower trace) consisted of an early transient outward I_to-like component followed by a sustained component. When coupling was allowed only during the first 100 ms of the AP, the EAD persisted (middle upper trace). However, when coupling was allowed only during the last 900 ms, the EAD disappeared (right upper trace). (B) Corresponding simulation: same protocol as in (A), but with the real myocyte replaced by the Luo-Rudy 1 ventricular AP model modified to produce bradycardia-induced EADs. EADs reappear at PCL 1 s when coupling to the fibroblast is present throughout the cardiac cycle (left) or only the first 100 ms of the AP (middle), but not when uncoupled during the first 100 ms of the AP (right). Bottom row shows expanded traces of the corresponding gap junction currents I_j.