Chronic Pressure Overload and Ovariectomy Alters the Regulation of Cardiac Fibrosis in a Chamber‐Specific Manner in Yucatan Mini‐Swine
Authors: Kelly Shannon C. , Thorne Pamela K. , Ivey Jan R. , Emter Craig A. 
Published: May 2022

Cardiac fibrosis, a hallmark feature of heart failure with preserved ejection fraction (HFpEF), is increased in postmenopausal women and may occur due to imbalances in the regulation of collagen metabolism. Our goal was to evaluate the effect of the loss of sex hormones via ovariectomy (OVX) on the development of aortic‐banding (AB) induced heart failure and cardiac fibrosis in 4 groups of female Yucatan mini‐swine: control (N=7); OVX (N=6); AB (N=7); and AB‐OVX (N=7). A 2x2 ANOVA (AB X OVX) was used to determine significance at the p < 0.05 and 0.10 levels. Biochemical evaluation of total collagen demonstrated an increase due to OVX (main effect) in the left ventricle (LV) independent of AB. In contrast, total collagen was increased due to AB (main effect) in the right ventricle (RV) independent of OVX. These results led to the hypothesis that collagen is differentially regulated in a chamber‐specific manner due to imbalances in its metabolism. Collagen I and III mRNA levels were evaluated using qRT‐PCR. Collagen I mRNA was increased by AB (main effect) in both the LV and the RV. Collagen III mRNA levels were not altered in the LV, but were decreased due to OVX (main effect) in the RV. Collagen I and III protein levels, evaluated via western blot, were not significantly different in the LV, demonstrating a disconnect whereby changes to collagen I mRNA did not directly translate to alterations in protein. In the RV, collagen I protein levels were increased due to AB (main effect), while collagen III protein levels were unaltered. Thus, in contrast to the LV, a direct relationship between mRNA and protein was observed in the RV for collagen I. Given alterations to mRNA did not always match changes in protein level, collagen degradation was evaluated. Collagen degradation is regulated by matrix metalloproteinases (MMPs), which degrade collagen, and tissue inhibitors of matrix metalloproteinases (TIMPs), which inhibit MMPs. MMP‐2 and ‐9 activity/abundance was evaluated with gelatin zymography, MMP‐14 activity was evaluated via a fluorogenic substrate assay, and TIMP2 protein was evaluated through western blot. In the LV, there was a significant interaction for MMP‐2 and MMP‐9 whereby activity/abundance due to AB was dependent upon the presence of sex hormones. This interaction was not observed in the RV. MMP‐14 activity was decreased due to AB (main effect) in the LV, but not altered in the RV. TIMP2 protein levels were decreased due to OVX (main effect) in the LV, with no differences observed in the RV. In conclusion, these results demonstrate chamber‐specific differences in the regulation of cardiac fibrosis including: 1) An imbalance in collagen metabolism in the LV consisting of collagen mRNA levels not directly translating to protein, with dysregulation of MMP/TIMP protein and activity levels resulting from both OVX and AB; and 2) in the RV, an increase in collagen I mRNA and protein due to AB with no alterations to bioregulators of collagen degradation. These findings demonstrate important differences in the regulation of fibrosis due to the loss of sex hormones and chronic pressure overload between the LV and RV that may be critical to understanding the pathophysiology of HFpEF in postmenopausal women.