G_q-activated fibroblasts induce cardiomyocyte action potential prolongation and automaticity in a Three-Dimensional microtissue environment

Cardiac fibroblasts (CFs) are known to regulate cardiomyocyte (CM) function in vivo and in two-dimensional in vitro cultures. This study examined the effect of CF activation on the regulation of CM electrical activity in a three-dimensional (3-D) microtissue environment. Using a scaffold-free 3-D platform with interspersed neonatal rat ventricular CMs and CFs, G_q-mediated signaling was selectively enhanced in CFs by Gα_q- adenoviral infection before coseeding with CMs in nonadhesive hydrogels. After 3 days, the microtissues were analyzed by signaling assay, histological staining, quantitative PCR, Western blots, optical mapping with voltage-or Ca²⁺-sensitive dyes, and microelectrode recordings of CF resting membrane potential (RMP_CF). Enhanced G_q signaling in CFs increased microtissue size and profibrotic and prohypertrophic markers. Expression of constitutively active Gα_q in CFs prolonged CM action potential duration (by 33%) and rise time (by 31%), prolonged Ca²⁺ transient duration (by 98%) and rise time (by 65%), and caused abnormal electrical activity based on depolarization-induced automaticity. Constitutive G_q activation in CFs also depolarized RMPCF from-33 to -20 mV and increased connexin 43 and connexin 45 expression. Computational modeling confers that elevated RMPCF and increased cell-cell coupling between CMs and CFs in a 3-D environment could lead to automaticity. In conclusion, our data demonstrate that CF activation alone is capable of altering action potential and Ca²⁺ transient characteristics of CMs, leading to proarrhythmic electrical activity. Our results also emphasize the importance of a 3-D environment where cell-cell interactions are prevalent, underscoring that CF activation in 3-D tissue plays a significant role in modulating CM electrophysiology and arrhythmias.