Effects of the absence of procollagen C-endopeptidase enhancer-2 on myocardial collagen accumulation in chronic pressure overload

Abstract

Cardiac interstitial fibrillar collagen accumulation, such as that associated with chronic pressure overload (PO), has been shown to impair left ventricular diastolic function. Therefore, insight into cellular mechanisms that mediate excessive collagen deposition in the myocardium is pivotal to this important area of research. Collagen is secreted as a soluble procollagen molecule with NH(2)- and COOH (C)-terminal propeptides. Cleavage of these propeptides is required for collagen incorporation to insoluble collagen fibrils. The C-procollagen proteinase, bone morphogenic protein 1, cleaves the C-propeptide of procollagen. Procollagen C-endopeptidase enhancer (PCOLCE) 2, an enhancer of bone morphogenic protein-1 activity in vitro, is expressed at high levels in the myocardium. However, whether the absence of PCOLCE2 affects collagen content at baseline or after PO induced by transverse aortic constriction (TAC) has never been examined. Accordingly, in vivo procollagen processing and deposition were examined in wild-type (WT) and PCOLCE2-null mice. No significant differences in collagen content or myocardial stiffness were detected in non-TAC (control) PCOLCE2-null versus WT mice. After TAC-induced PO, PCOLCE2-null hearts demonstrated a lesser collagen content (PCOLCE2-null TAC collagen volume fraction, 0.41% ± 0.07 vs. WT TAC, 1.2% ± 0.3) and lower muscle stiffness compared with WT PO hearts [PCOLCE2-null myocardial stiffness (β), 0.041 ± 0.002 vs. WT myocardial stiffness, 0.065 ± 0.001]. In addition, in vitro, PCOLCE2-null cardiac fibroblasts exhibited reductions in efficiency of C-propeptide cleavage, as demonstrated by increases in procollagen α1(I) and decreased levels of processed collagen α1(I) versus WT cardiac fibroblasts. Hence, PCOLCE2 is required for efficient procollagen processing and deposition of fibrillar collagen in the PO myocardium. These results support a critical role for procollagen processing in the regulation of collagen deposition in the heart.

Fig. 1.

Survival curves of wild-type (WT) and procollagen C-endopeptidase enhancer (PCOLCE2)-null transverse aortic constriction (TAC) mice. WT and PCOLCE2-null mice both demonstrated ∼50% mortality in response to pressure overload generated by 4 wk of TAC. Although higher numbers of PCOLCE2-null mice subjected to pressure overload survived to 28 days vs. WT mice, the differences did not reach statistical significance.

Fig. 2.

Histochemistry of fibrillar collagen in PCOLCE2-null hearts. Picrosirius red-stained section of left ventricle (LV) from WT (top left), WT TAC (top right), PCOLCE2-null (bottom left), and PCOLCE2-null TAC (bottom right) mice revealed apparent decreases in interstitial collagen deposition in response to TAC. Arrows indicate insoluble collagen. Bar = 20 μm.

Fig. 3.

Reductions in collagen volume fractions (CVFs) in PCOLCE2-null TAC hearts: morphological quantification of CVF from Picrosirius red-stained sections of WT (black bars) and PCOLCE2-null (white bars) LV with and without TAC demonstrated significant increases in CVF in WT TAC mice, whereas PCOLCE2-null TAC showed less of an increase in CVF vs. WT TAC hearts. No differences in CVF in control WT vs. PCOLCE2-null hearts were found. Error bars = means ± SE. *P < 0.05 vs. corresponding non-TAC control; ^P < 0.05 vs. WT TAC.

Fig. 4.

Decreased insoluble collagen incorporation in PCOLCE2-null TAC hearts. Hydroxyproline analysis of WT (black bars) and PCOLCE2-null (white bars) LV with and without TAC showed increased amounts of insoluble collagen in WT TAC LV vs. WT control, whereas no differences in insoluble collagen in PCOLCE2-null TAC vs. control LV were detected. In addition, levels of soluble collagen were significantly reduced in WT TAC vs. control WT LV, whereas there was no detectable decrease in levels of soluble collagen in PCOLCE2-null LV. No significant differences in levels of soluble collagen between WT and PCOLCE2-null control hearts were found (P = 0.09). Error bars = means ± SE. *P < 0.001 vs. corresponding control; ^P < 0.005 vs. WT TAC.

Fig. 5.

Reductions in diastolic stiffness in TAC papillary muscle of PCOLCE2-null mice. A: examples of the passive diastolic myocardial stress vs. strain curves for the 4 groups of animals studied: control WT, control PCOLCE2-null, WT TAC, and PCOLCE2-null TAC. B: values (means ± SE) of the passive stiffness constant, β, for the 4 groups of animals studied: WT (control), PCOLCE2-null (control), WT TAC, and PCOLCE2-null TAC. *P < 0.05 vs. corresponding non-TAC control, #P < 0.05 vs. WT TAC.

Fig. 6.

Levels of PCOLCE-2 are increased in TAC hearts. Top: protein extracted from control and TAC hearts were separated by SDS-PAGE and probed with anti-PCOLCE-2 antibodies. Bottom: quantification of protein bands shown in A. *P < 0.05 vs. control.

Fig. 7.

Significant reductions in procollagen processing by PCOLCE2-null cardiac fibroblasts. A: procollagen processing by primary cardiac fibroblasts isolated from WT and PCOLCE2-null mice was evaluated by Western blot analysis of proteins soluble in detergent from fibroblast cell layers (see materials and methods). Noncontiguous lanes from a single gel are shown. Procoll, procollagen; pC, C-propeptide; Coll, collagen. B: quantification of 3 separate cardiac fibroblast preparations revealed a consistent decrease in procollagen processing in the absence of PCOLCE2-null (white bars) vs. WT (black bars) cells demonstrated by increased amounts of unprocessed, procollagen α1(I)/pC collagen α1(I) and decreased amounts of processed, collagen α1(I). Pro, procollagen. Error bars = means ± SE. *P < 0.05 vs. WT procollagen α1(I); ^P < 0.05 vs. WT collagen α1(I).