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. 2008 Nov 1;95(9):4469-80.
doi: 10.1529/biophysj.108.136473. Epub 2008 Jul 25.

Electrotonic myofibroblast-to-myocyte coupling increases propensity to reentrant arrhythmias in two-dimensional cardiac monolayers

Sharon Zlochiver  1 , Viviana Muñoz, Karen L Vikstrom, Steven M Taffet, Omer Berenfeld, José Jalife
Affiliations

Electrotonic myofibroblast-to-myocyte coupling increases propensity to reentrant arrhythmias in two-dimensional cardiac monolayers

Sharon Zlochiver et al. Biophys J. .

Abstract

In pathological conditions such as ischemic cardiomyopathy and heart failure, differentiation of fibroblasts into myofibroblasts may result in myocyte-fibroblast electrical coupling via gap junctions. We hypothesized that myofibroblast proliferation and increased heterocellular coupling significantly alter two-dimensional cardiac wave propagation and reentry dynamics. Co-cultures of myocytes and myofibroblasts from neonatal rat ventricles were optically mapped using a voltage-sensitive dye during pacing and sustained reentry. The myofibroblast/myocyte ratio was changed systematically, and junctional coupling of the myofibroblasts was reduced or increased using silencing RNAi or adenoviral overexpression of Cx43, respectively. Numerical simulations in two-dimensional models were used to quantify the effects of heterocellular coupling on conduction velocity (CV) and reentry dynamics. In both simulations and experiments, reentry frequency and CV diminished with larger myofibroblast/myocyte area ratios; complexity of propagation increased, resulting in wave fractionation and reentry multiplication. The relationship between CV and coupling was biphasic: an initial decrease in CV was followed by an increase as heterocellular coupling increased. Low heterocellular coupling resulted in fragmented and wavy wavefronts; at high coupling wavefronts became smoother. Heterocellular coupling alters conduction velocity, reentry stability, and complexity of wave propagation. The results provide novel insight into the mechanisms whereby electrical myocyte-myofibroblast interactions modify wave propagation and the propensity to reentrant arrhythmias.

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Figures

FIGURE 1
FIGURE 1
Quantification of areas occupied by myocytes and myofibroblasts in co-cultures. (A) Neonatal rat myocyte-myofibroblast co-culture after peroxidase stain. (Dark gray areas) cells positive for sarcomeric α-actinin (myocytes). (White areas) myofibroblasts. (B) Areas occupied by myofibroblasts were quantified using BioQuant software based on color contrast (26% of total in this example). (C) Low magnification of dish in A. (D) Computer model of dish in C.
FIGURE 2
FIGURE 2
Non-myocyte characterization. (A) Non-myocytes expressing smooth muscle actin (α-SMA) in culture. Scale bar = 50 μm. (B) Fluorescent image showing the expression of VWF (green) in 9 cells. Blue fluorescence corresponds to nuclear specific DAPI stain. Scale bar = 50 μm. (C) Western immunoblot showing DDR2 expression only in cultures containing myofibroblasts. M, myocytes; MF, myofibroblasts. (D) Fluorescent micrograph of confluent co-cultured monolayer confirming sarcomeric α-actinin antibody specificity to myocytes (left) with corresponding phase contrast image (right). (E) Fluorescent micrographs of a co-cultured preparation triple-stained for sarcomeric α-actinin (red), α-SMA (green), and DAPI nuclear probe (blue), demonstrating the specificity of the antibodies used and cellular composition of our dishes. Scale bar = 100 μm.
FIGURE 3
FIGURE 3
Optical mapping of monolayers. (A) Phase maps (top), TSPs (middle), and single pixel recordings (bottom) show increasing number of singularity points with increasing number of myofibroblasts in three experiments; myofibroblast/myocyte area ratios: 0.1, 0.5, and 0.7. (B) Rotation frequency (top, n=19) and number of PSs (bottom, n = 14) as functions of myofibroblast/myocyte area ratio.
FIGURE 4
FIGURE 4
Simulations of reentry. All panels organized as in Fig. 2. (A) Phase maps (top), TSPs (middle), and single pixel recordings (bottom) in three simulations with myofibroblast/myocyte area ratios of 0.1, 0.55, and 0.65 and myofibroblast coupling coefficient of 0.08. (B) Rotation frequency (top) and number of PSs (bottom) as functions of myofibroblast/myocyte area ratio and heterocellular coupling.
FIGURE 5
FIGURE 5
Conduction velocity at 2 Hz versus percentage of myofibroblasts. (A) Simulation results, with three levels of myofibroblast coupling coefficient. (B) Typical activation maps (myofibroblast/myocyte area ratio = 0.25, low and high myofibroblast coupling coefficients) showing fragmented wavefronts at low coupling. (C) Experimental results, n = 15; p < 0.05. (D) Typical activation maps (myofibroblast/myocyte area ratio = 0.25, with and without myofibroblast Cx43 silencer) reproduce numerical prediction.
FIGURE 6
FIGURE 6
Immunofluorescence samples showing Cx43-positive staining (green) between two myofibroblasts (A), between two myocytes (B), and between a myocyte and a myofibroblast (C and D; higher magnification of box in C). Myocyte-specific sarcomeric α-actinin in red and nuclei in blue. Scale bars, 10 μm. (E) Western blots from cardiomyocytes after transfection of Cx43 siRNA (Cx43 Sil) or a control siRNA (Ctr). Abundance of Cx43 in myocyte or fibroblast monocultures was assessed to determine preferential Cx43 silencing in fibroblasts. Duplicate immunoblot probed with anti-βactin antibody demonstrates equivalent loading of samples.
FIGURE 7
FIGURE 7
Western blots from nontreated myofibroblasts as well as myofibroblasts 24 hr after treatment with siRNA silencer or Cx43 adenovirus. Representative exposure-matched immunofluorescence images for each myofibroblast treatment condition are shown, Cx43 is stained in green, scale bars are 20 μm.
FIGURE 8
FIGURE 8
Effects of heterocellular electrical coupling during pacing in 2D monolayers and simulations. (A) Experimental CV versus myofibroblast coupling coefficient for pacing at 3 (diamonds) and 4 Hz (triangles). Three levels of myofibroblast coupling were achieved using Cx43 siRNA treated myofibroblasts (silenced), control myofibroblasts and Cx43 overexpressed myofibroblasts (overexpressed). (B) Numerical results for CV versus myofibroblast coupling coefficient for pacing at 3 and 4 Hz. All simulations and experiments were performed for a myofibroblast/myocyte area ratio of 0.25.
FIGURE 9
FIGURE 9
1D cable simulations with uniform diffuse fibroblast distribution. (Inset) cable model consisting of 500 cells. Conduction velocity was calculated from the time of impulse propagation across the middle 100 cells and plotted as a function of myofibroblast coupling coefficients for various myofibroblast/myocyte ratios.
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References

    1. Manabe, I., T. Shindo, and R. Nagai. 2002. Gene expression in fibroblasts and fibrosis: involvement in cardiac hypertrophy. Circ. Res. 9112:1103–1113. - PubMed
    1. Brown, R. D., S. K. Ambler, M. D. Mitchell, and C. S. Long. 2005. The cardiac fibroblast: therapeutic target in myocardial remodeling and failure. Annu. Rev. Pharmacol. Toxicol. 45:657–687. - PubMed
    1. de Bakker, J. M., F. J. van Capelle, M. J. Janse, S. Tasseron, J. T. Vermeulen, N. de Jonge, and J. R. Lahpor. 1993. Slow conduction in the infarcted human heart. ‘Zigzag’ course of activation. Circulation. 88:915–926. - PubMed
    1. Bian, W., and L. Tung. 2006. Structure-related initiation of reentry by rapid pacing in monolayers of cardiac cells. Circ. Res. 98:e29–e38. - PubMed
    1. Spach, M. S., and J. P. Boineau. 1997. Microfibrosis produces electrical load variations due to loss of side-to-side cell connections: a major mechanism of structural heart disease arrhythmias. Pacing Clin. Electrophysiol. 20:397–413. - PubMed

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