Volume overload induces autophagic degradation of procollagen in cardiac fibroblasts

Abstract

In a pure volume overloaded (VO) heart, interstitial collagen loss is degraded by matrix metalloproteinases (MMPs) that leads to left ventricular (LV) dilatation and heart failure. Cardiac fibroblasts are the primary source of extracellular matrix proteins that connect cardiomyocytes. The goal of this study was to determine how VO affects intracellular procollagen in cardiac fibroblasts. Using the aortocaval fistula (ACF) model in Sprague-Dawley rats, we demonstrate that cardiac fibroblasts isolated from 4 and 12 wk ACF animals have decreased intracellular procollagen I compared to the fibroblasts from age-matched shams. The reduction of procollagen I is associated with increased autophagy as demonstrated by increased autophagic vacuoles and LC3-II expression. To test the relationship between autophagy and procollagen degradation, we treated adult cardiac fibroblasts with either an autophagy inducer, rapamycin, or an inhibitor, wortmannin, and found that procollagen I protein levels were decreased in fibroblasts treated with rapamycin and elevated in wortmannin-treated cells. In addition, we demonstrated that VO induces oxidative stresses in cardiac fibroblasts from 4 and 12 wk ACF rats. Treatment of cultured cardiac fibroblasts with an oxidative stress-inducing agent (DMNQ) induces autophagy and intracellular procollagen I and fibronectin degradation, which is reversed by wortmannin but not by the global MMP inhibitor (PD166793). Mechanical stretch of cardiac fibroblasts also induces oxidative stress and autophagic degradation of procollagen I and fibronectin. Our results suggest that in addition to the well-known effects of MMPs on extracellular collagen degradation in VO, there is a concurrent degradation of intracellular procollagen and fibronectin mediated by oxidative stress-induced autophagy in cardiac fibroblasts.

Keywords: Autophagy; Cardiac fibroblast; Intracellular procollagen; Matrix metalloproteinase; Oxidative stress; Volume overload.

Fig. 1. Cardiac fibroblasts isolated from 4 wk ACF rats have an increased level of autophagy and decreased procollagen I

(A) Cardiac fibroblasts are isolated from 4 wk sham and ACF rats. TEM analysis demonstrates an increase of autophagic vacuoles. (B) Cardiac fibroblast expression of procollagen I and an autophagic marker (LC3-II) in 4 wk sham and ACF rats are shown by immunoblotting with GAPDH as loading control. (C) Quantification of procollagen I and LC3-II in sham and ACF rats. (D) Fold changes of fibroblast collagen I mRNA level from 4 wk sham and ACF rats. n = 6. *P<0.05 vs. age-matched shams. Nuc: nucleus; av: autophagic vacuoles.

Fig. 2. Cardiac fibroblasts isolated from 12 wk ACF rats have an increased level of autophagy and decreased procollagen I

(A) TEM of cardiac fibroblasts isolated from 12 wk sham and ACF rats demonstrates a marked increase of autophagic vacuoles. (B) Representative Western blot images of procollagen I and LC3. (C) Quantification of procollagen I and the LC3-II in 12 wk sham and ACF rats. (D) Fold changes of fibroblast collagen I mRNA level from sham and ACF rats. n = 6. *P<0.05 vs. age-matched shams. Nuc: nucleus; av: autophagic vacuoles.

Fig. 3. Procollagen I is decreased by autophagy enhancer (rapamycin) and increased by autophagy inhibitor (wortmannin)

Cardiac fibroblasts isolated from adult rats are cultured in 6-well plates before 0.5 μM rapamycin or 50 nM wortmannin treatments for 24 h. (A) Rapamycin increases the formation of autophagic vacuoles as labeled by Cyto-ID (green). (B) Representative western blot images showing rapamycin treatment increases the LC3-II expression and decreases procollagen I protein in fibroblasts while wortmannin treatment has opposite effects. (C) Quantification of LC3-II, procollagen I protein and (D) collagen I mRNA after rapamycin or wortmannin treatments. n = 5, *P<0.05 vs. control (Ctrl).

Fig. 4. Oxidative stress is increased in isolated cardiac fibroblasts from 4 and 12 wk ACF rats

Analysis of ROS by 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (CM-DCF), a fluorescent marker of oxidative stress, in isolated fibroblasts from 4 and 12 wk ACF rat hearts indicates a significant increase in ROS in ACF rats. n = 80 cells/group. *P<0.05 vs. age-matched shams.

Fig 5. Autophagic degradation of procollagen is induced by DMNQ

Cardiac fibroblasts are isolated from adult normal rat and cultured in 6-well tissue culture plates followed by treatments with DMNQ (5 μM) and an MMP inhibitor (PD166793, 100 μM) or an autophagy inhibitor (wortmannin, 50 nM) for 16 h. (A) Representative images of immunoblotting show an increased expression of LC3-II and decreased level of intracellular procollagen I and fibronectin by DMNQ that are reversed by autophagy inhibition not MMP inhibition. (B-D). Quantitative results of LC3-II, procollagen I and fibronectin protein expression, respectively. n = 5. *P<0.05 vs. untreated control (Ctrl).

Fig. 6. Mechanical stretch results in increased autophagy and decreased procollagen I and fibronectin in isolated cardiac fibroblasts

Normal rat cardiac fibroblasts are cultured in BioFlex culture plates and then subjected to 24 h, 20% cyclic stretch (1 Hz). (A) TEM demonstrates the formation of perinuclear autophagic vacuoles in isolated fibroblasts after stretch. Nuc: nucleus, av: autophgic vacuoles. (B) Western blot demonstrates an increase in LC3-II and decrease in procollagen I and fibronectin in stretched versus unstretched control cells. n = 5, *P<0.05 vs. control (Ctrl). (C) Quantitative RT-PCR shows no significant changes in collagen I and fibronectin mRNA after stretch. (D) There is a 5-fold increase in ROS production after stretch as demonstrated by CM-DCF staining.

Fig. 7. ACF induces interstitial collagen loss and MMP-2 activation in cardiac fibroblasts

(A) Interstitial collagen in LV of 4 and 12 wk sham and ACF rats are stained with PASR and (B) the collagen volume percentages quantified as described in the Materials and methods. (C) MMP-2 activity in isolated cardiac fibroblasts is analyzed by gel zymography. n = 6. *P<0.05 vs. age-matched shams