Abstract 16606: Cardiac Fibroblast-Restricted Enhancement of Gq Signaling Prolongs Calcium Transients and Increases Spontaneous Activity in Biomimetic Cardiac Microtissues
Introduction: Enhanced Gq signaling in response to hemodynamic stress and injury is well known to induce remodeling in both cardiac myocytes (CMs) and fibroblasts (CFs). However, the effect of CF activation and fibrosis on CMs and integrated myocardial function cannot be easily discerned from concomitant effects on CMs.
Hypothesis: Enhanced Gq signaling in CFs influences CM function in a 3D microtissue model previously developed in our lab, in which neonatal rat ventricular CMs and CFs are highly interspersed as seen in the myocardium.
Methods: CFs infected in suspension (2 hrs, 10 MOI) with empty adenovirus (Ad-Ctr) or Ad encoding wild-type (WT) or constitutively active (Q209L or QL) Gαq were co-seeded with uninfected CMs (CM:CF 1:1) into non-adhesive hydrogels at ~1200 cells per recess (400/800 μm wide/deep). After 2-4 days, we assessed tissue size by microscopy, Gq and extracellular matrix proteins proteins by Western Blot analysis, and Ca2+ transients ([Ca2+]i) with Rhod 2/AM.
Results: Overexpression of Gαq in CFs was associated with a 3-fold (WT) and 7-fold (QL) increase in total inositolphosphate formation, indicating enhanced basal phospholipase β activity. After 4 days, CM:CFQL tissues were larger in size by 30-40% and expressed more laminin and fibronectin. After 3 days, CM:CFCTR paced at 1 Hz had [Ca2+]i duration of 218±3 ms, decay of 141±2 ms, and rise time of 35±4 ms (n=55-69). In CM:CFQL, duration and decay were prolonged by 160 ms and 21 ms, resp., and rise time was decreased by 5 ms (n=10-27). In CM:CFWT with less Gq signaling, [Ca2+]i duration and decay were also prolonged but only by 30 ms and 7 ms, resp. (n=46-51). These effects were similar but less pronounced after 2 days and/or at 2 Hz. Spontaneous [Ca2+]i activity was seen in 50% of CM:CFQL, 11% of CM:CFWT, but only 2% of CM:CFCTR microtissues. The gap junction inhibitor carbenoxolone (100 μM) decreased this activity in CM:CFQL by 80% and in CM:CFWT by 15%.
Conclusion: We show that enhanced CF-restricted Gq signaling in biomimetic cardiac microtissues increases tissue size, prolongs [Ca2+]i duration and decay, and increases spontaneous Ca2+ activity. Our findings suggest that depending on the CF activation state, CFs can greatly modulate CM calcium handling and potentially influence arrhythmogenesis.
Author Disclosures: C.M. Kofron: None. T. Kim: None. M.E. King: None. E.M. Park: None. B. Choi: None. U. Mende: None.
This research has received full or partial funding support from the American Heart Association.
- © 2014 by American Heart Association, Inc.