Abstract 2426: Cardiac Fibroblasts Strongly Affect Cardiac Action Potential Propagation by Paracrine Rather Than Coupling Mechanisms
We systematically studied in vitro contact-mediated and paracrine actions of neonatal rat cardiac fibroblasts (CFs) negative for α-smooth muscle actin on neonatal rat cardiomyocytes (CMs). Three independent, 2–4 day long, cell contact assays were used:
Over 1500 individual micropatterned CM-CM, CM-CF, and CF-CF cell pairs with same shape, size, and cell contact length (20–100μm);
100 μm wide, 1 cm long CM strands with a central bridge of variable length (≥150 μm) made of CFs; and
confluent anisotropic CM monolayers covered at 80–100% density with CFs.
For paracrine studies, CMs were incubated for 24h with serum-free media conditioned by CFs for 24h. We found:
All CM-CM pairs, but only 0–2% of CF-CF and 3.8–9.6% of CM-CF pairs, exhibited Cx43 and N-cadherin staining at cell contacts;
CFs always created conduction block in CM strands for bridges as small as 150 μm (microscopic optical mapping);
CFs expressed connexin-45 but not connexin-43 or connexin-40 (immunostaining, westerns), weakly functionally coupled to CMs (FRAP), and without significant depolarization of CM resting potential (sharp microelectrodes) slowed cardiac conduction to only 75% of control, without altering action potential duration (APD), maximum capture rate (MCR), anisotropy ratio, or incidence of spontaneous beating of CMs (macroscopic optical mapping).
In contrast, the CF paracrine factors produced a 52% reduction in CM conduction velocity, a 217% APD prolongation, a 64% MCR decrease, a 21% increase in membrane resting potential, and an 80% decrease of action potential upstroke velocity. No CF proliferation, CM apoptosis, or decreased connexin-43 expression, phosphorylation, and function were found in conditioned CMs. The expression of the fast sodium, inward rectifying potassium, and transient outward potassium channels in conditioned CMs were respectively reduced 3.8-fold, 6.6-fold, and to undetectable levels (qRT-PCR). No electrophysiological changes were observed from media conditioned by CFs in the presence of CMs suggesting balanced paracrine cross-talk. Taken together, paracrine rather than contact-mediated effects may be a primary mechanism by which the excessive presence of CFs in the heart could adversely affect function of surrounding CMs.
This research has received full or partial funding support from the American Heart Association, National Center.