Differential Receptor‐Mediated Inducible Gene Expression in Post Infarction Cardiac Fibroblasts is Altered by Intrinsic Exercise Phenotype
Authors: Alsahly Musaad B. , Zakari Madaniah O. , Koch Lauren G. , Britton Steven L. , Nguyen Hilary M. , Fisher-Wellman Kelsey , Lust Robert M. , Katwa Laxmansa C. 
Published: April 2020

Background

Ischemic heart disease is a major cause of morbidity. Regular active exercise is therapeutic after myocardial infarction, but up to 70% of individual exercise capacity is due to an intrinsic genetic component. Intrinsic capacity can be studied using high (HCR) and low (LCR) aerobic running capacity rat strains. The phenotypes differ by more than 5 fold in sedentary average running distance and time. The HCR rats have been characterized as “disease resistant”, while the LCRs are characterized as “disease prone”. In the heart, cardiac fibroblasts play a critical role in healing post myocardial infarction, but little is known about the effect of intrinsic aerobic capacity on the inducible, receptor‐mediated fibroblast gene expression pathways following ischemia reperfusion.

Methods

On each study day, one HCR/LCR pair (n = 8 each) was anesthetized and hearts were rapidly excised. The hearts were immediately flushed with iced hyperkalemic, hyperosmotic, cardioplegia solution, and subjected to cold global ischemic arrest (80 min). Following arrest, the hearts underwent warm reperfusion (120 min) using a Langendorff style perfusion system. Following reperfusion, the heart was weighed and the LV was isolated. A mid ventricular ring was obtained to estimate infarction size (TTC), and part of the remaining tissue (~150 mg) was transferred to homogenation buffer on ice for subsequent isolation of fibroblasts. Cardiac fibroblasts from the LV of HCRs and LCR rats were isolated, and cultured in 6 well plates. Different wells were treated with BMP7, Angiotensin or both, with or without receptor antagonists (BMP2R, noggin; AngR, Losartan). After conditioning, RNA was isolated from the cultured fibroblasts, and RT‐PCR was used to determine baseline gene expression for Collagen I and III, MMP2, TIMP1, BMPR2, ATR1, SMAD5, and Bag3 in each phenotype.

Results

Overall, Collagen I gene expression was significantly higher in LCRs more than HCRs, but the activating pathways were phenotype dependent. Treating with Ang II induced gene expression for collagen I in LCR fibroblasts but not HCR, and was completely blocked with Losartan. By Contrast, treating with BMP7 induced gene expression of collagen I in HCR fibroblasts but not LCRs, and was completely blocked by noggin. Treating the fibroblasts with a combination of receptor agonists confirmed the phenotypic selectivity of the inducible pathways. Inhibiting BMP7 using noggin brought collagen I gene expression back to the control level with no changes in LCRs.

Conclusion

The Angiotensin receptor pathway is responsive only in post‐ischemic LCR cardiac fibroblasts, and the BMP receptor pathway is responsive only in post‐ischemic HCR cardiac fibroblasts. How the intrinsic aerobic phenotype governs these pathways is unknown, but could shed new light on individuals who develop post‐infraction fibrosis despite therapeutic dosing with Angiotensin pathway antagonists.