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. 2010 Oct 15;107(8):1011-20.
doi: 10.1161/CIRCRESAHA.110.227421. Epub 2010 Aug 12.

Enhanced fibroblast-myocyte interactions in response to cardiac injury

Carolina Vasquez  1 , Poornima Mohandas, Karen L Louie, Najate Benamer, Ashwini C Bapat, Gregory E Morley
Affiliations

Enhanced fibroblast-myocyte interactions in response to cardiac injury

Carolina Vasquez et al. Circ Res. .

Abstract

Rationale: A critical event in the development of cardiac fibrosis is the transformation of fibroblasts into myofibroblasts. The electrophysiological consequences of this phenotypic switch remain largely unknown.

Objective: Determine whether fibroblast activation following cardiac injury results in a distinct electrophysiological phenotype that enhances fibroblast-myocyte interactions.

Methods and results: Neonatal rat myocyte monolayers were treated with media (CM) conditioned by fibroblasts isolated from normal (Fb) and infarcted (MI-Fb) hearts. Fb and MI-Fb were also plated on top of myocyte monolayers at 3 densities. Cultures were optically mapped after CM treatment or fibroblast plating to obtain conduction velocity and action potential duration (APD(70)). Intercellular communication and connexin43 expression levels were assessed. Membrane properties of Fb and MI-Fb were evaluated using patch clamp techniques. MI-Fb CM treatment decreased conduction velocity (11.1%) compared to untreated myocyte cultures. APD(70) was reduced by MI-Fb CM treatment compared to homocellular myocyte culture (9.4%) and Fb CM treatment (6.4%). In heterocellular cultures, MI-Fb conduction velocities were different from Fb at all densities (+29.8%, -23.0%, and -16.7% at 200, 400, and 600 cells/mm(2), respectively). APD(70) was reduced (9.6%) in MI-Fb compared to Fb cultures at 200 cells/mm(2). MI-Fb had more hyperpolarized resting membrane potentials and increased outward current densities. Connexin43 was elevated (134%) in MI-Fb compared to Fb. Intercellular coupling evaluated with gap fluorescence recovery after photobleaching was higher between myocytes and MI-Fb compared to Fb.

Conclusions: These data demonstrate cardiac injury results in significant electrophysiological changes that enhance fibroblast-myocyte interactions and could contribute to the greater incidence of arrhythmias observed in fibrotic hearts.

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Figures

Figure 1
Figure 1. Conduction properties of myocyte monolayers treated with media conditioned by fibroblasts from normal (Fb CM) and infarcted (MI-Fb CM) hearts
A, Representative activation map from an untreated myocyte monolayer (Myo). Scale bar is 1 mm, lines are 10 ms isochrones. B–C, Representative activation maps from myocyte monolayers treated with Fb CM and MI-Fb CM, respectively. D, Average conduction velocity (CV) for Myo (n=66), Fb CM (n=75) and MI-Fb CM (n=63) treated monolayers.
Figure 2
Figure 2. Action potential duration (APD) of myocyte monolayers treated with media conditioned by fibroblasts from normal (Fb CM) and infarcted (MI-Fb CM) hearts
A, Superimposed representative optical action potentials recorded from an untreated myocyte monolayer (Myo) and myocyte monolayers treated with Fb CM and MI-Fb CM. B, Average APD70 for Myo (n=66), Fb CM (n=75) and MI-Fb CM (n=63) treated monolayers.
Figure 3
Figure 3. Conduction properties of heterocellular cultures of myocytes and fibroblasts from normal (Fb) and infarcted (MI-Fb) hearts
A–C, Representative activation maps from heterocellular cultures of myocytes and Fb plated on top. Fb were plated at 200, 400 and 600 cells/mm2, respectively. Scale bar is 1 mm, lines are 10 ms isochrones. D–F, Representative activation maps from heterocellular cultures of myocytes and MI-Fb plated on top. MI-Fb were plated at 200, 400 and 600 cells/mm2, respectively. G, Average conduction velocity (CV) of the heterocellular cultures for different fibroblast plating densities. Dotted line corresponds to average CV of homocellular myocyte monolayers (Myo). p values correspond to significant differences between Fb and MI-Fb at the same density. n = 35, 68, 65 for Fb; 36, 46, 28 for MI-Fb.
Figure 4
Figure 4. Action potential duration (APD) of heterocellular cultures of myocytes and fibroblasts from normal (Fb) and infarcted (MI-Fb) hearts
A, Superimposed representative optical action potentials recorded from heterocellular cultures with Fb and MIFb plated on top at 200, 400 and 600 cells/mm2. Scale bar is 100 ms. B, Average APD70 of the heterocellular cultures for different fibroblast plating densities. Dotted line corresponds to average APD70 of homocellular myocyte monolayers (Myo). p value corresponds to significant difference between Fb and MI-Fb at the same density. n = 35, 68, 65 for Fb; 36, 46, 28 for MI-Fb.
Figure 5
Figure 5. Membrane currents recorded from fibroblasts isolated from normal (Fb) and infarcted (MI-Fb) hearts
A–B, Representative currents recorded with whole cell patch clamp techniques from a single Fb and MI-Fb, respectively. Membrane voltage (Vm) was clamped at −50 mV and stepped from −130 to 30 mV in 10 mV increments. C, Average membrane current density (Im) recorded from Fb (n=5) and MI-Fb (n=7). p<0.05 for MI-Fb compared to Fb for Vm greater than −70 mV.
Figure 6
Figure 6. Cx43 expression in fibroblasts isolated from normal (Fb) and infarcted (MIFb) hearts
A–B, Immunostaining of Cx43 in heterocellular cultures of myocytes (m) and Fb or MI-Fb (f), respectively. Green is α-SMA, red is Cx43, arrowheads indicate Cx43 staining at cell contact areas. Scale bar is 20 μm. C, Representative immunoblot showing expression of Cx43 in cultured Fb and MI-Fb. GAPDH was used as a loading control. D, Representative immunoblot showing expression of Cx43 in freshly isolated Fb and MI-Fb. E, Quantification of immunoblots from a total of 6 Fb and 6 MI-Fb cultured samples. Cx43 expression levels were normalized to Fb. F, Quantification of immunoblots from a total of 10 Fb and 10 MI-Fb freshly isolated samples. Cx43 expression levels were normalized to Fb
Figure 7
Figure 7. Intercellular coupling between myocytes and fibroblasts from normal (Fb) and infarcted (MI-Fb) hearts
A, micrograph of an Fb monolayer with a myocyte plated on top. Myocyte is indicated by the dotted lines. Scale bar is 25 μm. B–C, heterocellular myocyte and Fb culture loaded with calcein-AM before and after myocyte photobleaching, respectively. Dotted line indicates the bleached area. D, Micrograph of an MI-Fb monolayer with a myocyte plated on top. E–F, heterocellular myocyte and MI-Fb culture loaded with calcein-AM before and after myocyte photobleaching. G, Average fluorescence recovery curves of myocytes plated on top of Fb and MI-Fb monolayers. H, Average permeability constants (k) from myocytes plated on top of Fb and MI-Fb monolayers under control conditions, with 200 μmol/L carbenoxolone (CBX) and after CBX washout. n = 20, 10 and 11 for Fb, n = 24, 14, and 13 for MI-Fb.
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