Massive Accumulation of Myofibroblasts in the Critical Isthmus Is Associated With Ventricular Tachycardia Inducibility in Post-Infarct Swine Heart

Abstract

Objectives: In this study the authors determined the extent of cellular infiltration and dispersion, and regional vascularization in electrophysiologically (EP) defined zones in post-myocardial infarction (MI) swine ventricle.

Background: The critical isthmus (CI) in post-MI re-entrant ventricular tachycardia (VT) is a target for catheter ablation. In vitro evidence suggests that myofibroblasts (MFB) within the scar border zone (BZ) may increase the susceptibility to slow conduction and VT, but whether this occurs in vivo remains unproven.

Methods: Six weeks after mid-left anterior descending coronary artery occlusion, EP catheter-based mapping was used to assess susceptibility to VT induction. EP data were correlated with detailed cellular profiling of ventricular zones using immunohistochemistry and spatial distribution analysis of cardiomyocytes, fibroblasts, MFB, and vascularization.

Results: In pigs with induced sustained monomorphic VT (mean cycle length: 353 ± 89 ms; n = 6) the area of scar that consisted of the BZ (i.e., between the normal and the low-voltage area identified by substrate mapping) was greater in VT-inducible hearts (iVT) than in noninducible hearts (non-VT) (p < 0.05). Scar in iVT hearts was characterized by MFB accumulation in the CI (>100 times that in normal myocardium and >5 times higher than that in the BZ in non-VT hearts) and by a 1.7-fold increase in blood vessel density within the dense scar region extending towards the CI. Sites of local abnormal ventricular activity potentials exhibited cellularity and vascularization that were intermediate to the CI in iVT and BZ in non-VT hearts.

Conclusions: The authors reported the first cellular analysis of the VT CI following an EP-based zonal analysis of iVT and non-VT hearts in pigs post-MI. The data suggested that VT susceptibility was defined by a remarkable number of MFB in the VT CI, which appeared to bridge the few remaining dispersed clusters of cardiomyocytes. These findings define the cellular substrate for the proarrhythmic slow conduction pathway.

Keywords: BZ, border zone; CI, critical isthmus; CM, cardiomyocytes; ECM, extracellular matrix; EP, electrophysiology; FB, fibroblasts; IHC, immunohistochemistry; LAD, left anterior descending; LAVA, local abnormal ventricular activity; MFB, myofibroblasts; MI, myocardial infarction; MRI, magnetic resonance imaging; VT; VT, ventricular tachycardia; Vim, vimentin; border zone; cTnT, cardiac troponin T; critical isthmus; iVT, inducible ventricular tachycardia; myocardial infarction; myofibroblasts; pig; vWF, von Willebrand factor; α-SMA, α-smooth muscle actin.

Graphical abstract

Figure 1

Electroanatomic Histological Overlay (A) Epicardial markers (a, b, c) were sutured at the corresponding location on the (B) 3-dimensional electroanatomic map to identify functionally distinct electrophysiologically defined zones (see Methods). Purple dots, late and/or local abnormal ventricular activity; black and green dots, poor and good pace maps with <11 of 12 and ≥11 of 12 electrocardiographic leads matching the ventricular tachycardia, respectively.

Figure 2

Histological Assessments of Zones 1 to 4 in Post–Myocardial Infarction Ventricles Ventricular sections were stained for cardiomyocytes (CM) (cardiac troponin T–positive) (brown; left panels), myofibroblasts (MFB) (α-smooth muscle actin–positive/von Willebrand factor–negative [α-SMA⁺/vWF⁻) (red), and endothelium and/or blood vessels (α-SMA⁺/vWF⁺) (co-incident red and black; middle panels), and fibroblasts (FB) (α-SMA⁻/vimentin-positive) (black; right panels). Light green and picromethylblue were used to visualize cellularity and connective tissue and/or the extracellular matrix, respectively (Online Appendix). Boxes outlined in white (0.15 mm²) are magnified in the middle and right panels. iVT = inducible ventricular tachycardia; VT = ventricular tachycardia.

Figure 3

Zonal Analysis of Cellularity and Vascularization in iVT and Non-VT Hearts (A) The relative proportion of MFB, FB, and vessels in zones 1 to 4 in iVT and non-VT hearts. The diameter of the circle is directly proportional to the cumulative number of cells per square millimeter. Because CM were analyzed using a different method (percentage of area coverage), these data are omitted here. Quantification of (B) CM, (C) blood vessels, (D) FB, and (E) MFB in zones 1 to 4 in iVT (black) and non-VT (gray) hearts. *p < 0.05. Data are from 5 and 4 iVT and non-VT hearts, respectively (4 to 7 sections for each zone per heart). All parameters in zones 1 to 3 are significantly different from normal myocardium (zone 4) in iVT and non-VT hearts, and from hearts from sham-operated control pigs (p < 0.05). Sham data are from 2 pigs (8 to 10 sections for each zone per pig). ND = not determined due to the absence of zone 2 in non-VT hearts; other abbreviations as in Figure 2.

Figure 4

Cellular Distribution and MFB Alignment Does Not Correlate With VT Inducibility (A) The spatial distribution of CM, MFB, FB, and blood vessels in zones 2 and 3 in iVT hearts and zone 3 in non-VT hearts was calculated (Online Figure 3). iVT (n = 5) and non-VT hearts (n = 4) and data are from the following number of sections: zone 2 iVT , zone 3 iVT , and zone 3 nonVT . *p < 0.05. (B) Conduits of directionally oriented MFB (arrow) in representative sections of zone 2 (iVT) and zone 3 (iVT and non-VT). (C) Relative axial alignment of MFB. Number of experiments is given in (A). ECM = extracelluar matrix; other abbreviations as in Figure 2.

Figure 5

Late/LAVA Potentials Exhibit Cellular Profile Intermediate Between CI and BZ Regions (A) Substrate map of late and/or LAVA sites (pink dots) located within the superior aspect of the border zone (BZ). (B) The corresponding surface electrocardiographic leads (I, II and III) in sinus rhythm and local bipolar electrograms at 3 of these LAVA sites (left, middle, right panels). Arrows indicate late and/or LAVA potentials. Analysis of the abundances of (C) CM, (D) blood vessels, (E) MFB, and (F) FB in late and/or LAVA potential sites compared with zones 2 (VT critical isthmus [CI]) and 3 (BZ regions with poor pace map) in iVT hearts (data from Figure 3B to 3E). Seventeen sections obtained from 3 iVT hearts (pigs 4, 11, and 16) were analyzed. The single incidence of late and/or LAVA potentials in a non-VT heart (pig 6) was not analyzed. *p < 0.05. Abbreviations as Figures 1 and 2.