Repetitive Preload Elevation in Swine with Chronic Coronary Artery Disease Promotes Pro‐Fibrotic Gene Expression and Transmural Fibrosis in Remote Non‐Ischemic Myocardium

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ObjectiveUsing implantable telemetry, we recently found that transient elevations in preload are common in swine with multi‐vessel coronary artery disease (CAD), presumably as a result of intermittent demand‐induced regional myocardial ischemia. Based on prior evidence that repetitive preload elevation can lead to interstitial fibrosis and myocardial stiffening in the absence of ischemia, the present study was designed to compare temporal changes in pro‐fibrotic gene expression and transmural variability in interstitial fibrosis in ischemic vs. non‐ischemic areas of the left ventricle (LV) in a porcine model of chronic CAD.MethodsA total of 20 juvenile swine were instrumented with fixed 1.5 mm constrictors on the proximal left anterior descending (LAD) and left circumflex (LCx) coronary arteries and followed for up to 3‐months, during which coronary stenosis severity progressed to >90 % (LAD: 93±2 %; LCx: 91±3 %). Expression of fibronectin (FN1) and lysyl oxidase like‐2 (LOXL‐2) was assessed in ischemic (LAD) and non‐ischemic (RCA) regions of the LV via quantitative PCR 1‐month (n=4) or 3‐months (n=8) after instrumentation and compared to normal controls (n=8). Post‐mortem quantification of interstitial fibrosis was performed on picrosirius red‐stained tissue samples from ischemic and non‐ischemic regions (n=16), with an automated classifier‐based approach to compare regional transmural variability in collagen content to that of normal controls (n=11). Flash‐frozen tissue samples from a sub‐set of animals with CAD (n=4) and normal controls (n=4) were subjected to uniaxial tensile testing to assess passive subendocardial stiffness.ResultsOne month after instrumentation, FN1 expression was 2.5±0.9‐fold higher than control in the ischemic region and 2.7±0.7‐fold higher than control in the non‐ischemic region of the LV (both p<0.05). LOXL2 expression was also significantly higher in the ischemic (2.2 ± 0.6‐fold higher than control) and non‐ischemic (2.4 ± 0.3‐fold higher than control) regions of the LV at this timepoint. Two months later, pro‐fibrotic gene expression had normalized but a significant elevation in interstitial collagen content was observed throughout the LV of animals with CAD (Figure; left panel). Interestingly, subendocardial fibrosis was particularly prominent in the non‐ischemic RCA territory, which coincided with a significant increase in subendocardial passive stiffness in this region (Figure; right panel).ConclusionsIn a porcine model of chronic CAD, repetitive preload elevation is associated with transient changes in pro‐fibrotic gene expression that ultimately lead to transmural fibrosis that is particularly prominent in non‐ischemic areas of the LV. Regional transmural variability in collagen content coincides with changes in the passive stiffness of non‐ischemic myocardium, suggesting that repetitive preload elevation may contribute to fibrosis‐mediated diastolic dysfunction in patients with chronic CAD.