Time course of right ventricular pressure-overload induced myocardial fibrosis: relationship to changes in fibroblast postsynthetic procollagen processing

Abstract

Myocardial fibrillar collagen is considered an important determinant of increased ventricular stiffness in pressure-overload (PO)-induced cardiac hypertrophy. Chronic PO was created in feline right ventricles (RV) by pulmonary artery banding (PAB) to define the time course of changes in fibrillar collagen content after PO using a nonrodent model and to determine whether this time course was dependent on changes in fibroblast function. Total, soluble, and insoluble collagen (hydroxyproline), collagen volume fraction (CVF), and RV end-diastolic pressure were assessed 2 days and 1, 2, 4, and 10 wk following PAB. Fibroblast function was assessed by quantitating the product of postsynthetic processing, insoluble collagen, and levels of SPARC (secreted protein acidic and rich in cysteine), a protein that affects procollagen processing. RV hypertrophic growth was complete 2 wk after PAB. Changes in RV collagen content did not follow the same time course. Two weeks after PAB, there were elevations in total collagen (control RV: 8.84 ± 1.03 mg/g vs. 2-wk PAB: 11.50 ± 0.78 mg/g); however, increased insoluble fibrillar collagen, as measured by CVF, was not detected until 4 wk after PAB (control RV CVF: 1.39 ± 0.25% vs. 4-wk PAB: 4.18 ± 0.87%). RV end-diastolic pressure was unchanged at 2 wk, but increased until 4 wk after PAB. RV fibroblasts isolated after 2-wk PAB had no changes in either insoluble collagen or SPARC expression; however, increases in insoluble collagen and in levels of SPARC were detected in RV fibroblasts from 4-wk PAB. Therefore, the time course of PO-induced RV hypertrophy differs significantly from myocardial fibrosis and diastolic dysfunction. These temporal differences appear dependent on changes in fibroblast function.

Fig. 1.

Time course of increase in right ventricle (RV) mass normalized to tibia length (TL) and collagen volume fraction (CVF) following placement of pulmonary artery band (PAB). The increase in RV mass was rapid and reached a plateau over 2 wk and then remained constant. By contrast, there was an increase in CVF that was delayed until 4 wk after PAB and then was progressive through 10 wk. Values are means ± SE; n, no. of animals contributing to each time point. *P < 0.05 vs. RV Mass/TL wk 1. #P < 0.05 vs. CVF 2 wk. $P < 0.05 vs. CVF 4 wk.

Fig. 2.

Quantification of levels of total hydroxyproline in RV and left ventricular (LV) tissue from control (n = 9) and PAB cats at 2 days (n = 6), 1 wk (n = 5), 2 wk (n = 11), 4 wk (n = 9), and 10 wk (n = 6) after banding is shown. A significant increase in amounts of total hydroxyproline was detected in 2, 4, and 10 wk in the RV samples from the PAB cats compared with both RV from the control cats and the LV from control and PAB cats. Values are means ± SE. *P < 0.05 vs. RV control.

Fig. 3.

Insoluble fibrillar collagen fibers visualized with polarized light from picrosirius red (PSR)-stained tissue sections taken from the RV of control (A) and RV of cats with PAB for 2 days (B), 2 wk (C), 4 wk (D), and 10 wk (E). Interstitial insoluble fibrillar collagen fibers appeared green/yellow under polarized light, were composed of insoluble fully processed collagen, and were quantified as CVF. The quantity and distribution at 2 days (B) or 2 wk (C) after banding were not significantly changed compared with control RV (A). At 4 and 10 wk, an increase in volume fraction of collagen fibers was evident (white arrows, D and E). Thicker collagen fibers in D and E are indicated with white arrows. Bar = 50 μm.

Fig. 4.

Morphometric quantification of CVF from PSR-stained tissue sections. An increase in the CVF was noted at 4 and 10 wk after PAB. Values are means ± SE. *P < 0.05 vs. LV control. #P < 0.05 vs. RV 2 wk after PAB. $P < 0.05 vs. RV 4 wk after PAB.

Fig. 5.

Insoluble collagen deposition by RV PAB fibroblasts from control and 2-wk and 4-wk PAB. Fibroblasts isolated from 4-wk PAB, but not 2-wk PAB, demonstrated significantly greater insoluble collagen deposition vs. control. Values from RV PAB fibroblasts were normalized to that of LV same-animal control for each experiment. Values are means ± SE. *P < 0.05 vs. control.

Fig. 6.

A: representative Western blots of SPARC (secreted protein acidic and rich in cysteine) from fibroblast cultures. Actin is shown as loading control. Noncontiguous lanes from the same gel are shown. B: quantification of SPARC expression in RV fibroblasts normalized to levels in LV fibroblasts isolated from normal (open bars) and 4-wk PAB (solid bars) animals. Values are means ± SE. *P < 0.05. C: representative Western blots of SPARC expression in RV and LV myocardial tissue from 2-wk PAB, 4-wk PAB, and control animals. p85 subunit of phosphatidylinositol 3-kinase is shown as loading control. Fibroblast function, measured by quantifying SPARC abundance in primary fibroblast culture and RV myocardial tissue, was not significantly changed at 2 wk after PAB compared with control cats, but was significantly increased after 4 wk of PAB vs. control. Levels of SPARC were increased in 4-wk PAB RV fibroblasts (A and B) and in PAB RV myocardial tissue (C) vs. control fibroblasts and tissue.