Age-dependent alterations in fibrillar collagen content and myocardial diastolic function: role of SPARC in post-synthetic procollagen processing

Abstract

Advanced age, independent of concurrent cardiovascular disease, can be associated with increased extracellular matrix (ECM) fibrillar collagen content and abnormal diastolic function. However, the mechanisms causing this left ventricular (LV) remodeling remain incompletely defined. We hypothesized that one determinant of age-dependent remodeling is a change in the extent to which newly synthesized procollagen is processed into mature collagen fibrils. We further hypothesized that secreted protein acidic and rich in cysteine (SPARC) plays a key role in the changes in post-synthetic procollagen processing that occur in the aged myocardium. Young (3 mo old) and old (18-24 mo old) wild-type (WT) and SPARC-null mice were studied. LV collagen content was measured histologically by collagen volume fraction, collagen composition was measured by hydroxyproline assay as soluble collagen (1 M NaCl extractable) versus insoluble collagen (mature cross-linked), and collagen morphological structure was examined by scanning electron microscopy. SPARC expression was measured by immunoblot analysis. LV and myocardial structure and function were assessed using echocardiographic and papillary muscle experiments. In WT mice, advanced age increased SPARC expression, myocardial diastolic stiffness, fibrillar collagen content, and insoluble collagen. In SPARC-null mice, advanced age also increased myocardial diastolic stiffness, fibrillar collagen content, and insoluble collagen but significantly less than those seen in WT old mice. As a result, insoluble collagen and myocardial diastolic stiffness were lower in old SPARC-null mice (1.36 +/- 0.08 mg hydroxyproline/g dry wt and 0.04 +/- 0.005) than in old WT mice (1.70 +/- 0.10 mg hydroxyproline/g dry wt and 0.07 +/- 0.005, P < 0.05). In conclusion, the absence of SPARC reduced age-dependent alterations in ECM fibrillar collagen and diastolic function. These data support the hypothesis that SPARC plays a key role in post-synthetic procollagen processing and contributes to the increase in collagen content found in the aged myocardium.

Fig. 1.

Increased levels of secreted protein acidic and rich in cysteine (SPARC) in the aged myocardium. A: immunoblots showing SPARC abundance in the left ventricular (LV) myocardium. B: optical density analysis was used to semiquantitate SPARC abundance. Young mice were 3 mo of age; old mice were 18–24 mo of age. Wild-type (WT) mice were from the C57Bl6/SV129 background, and SPARC-null mice were created using targeted gene insertion to prevent the expression of SPARC. *P < 0.05 vs. young WT mice.

Fig. 2.

Altered collagen fiber morphology in hearts from old SPARC-null mice. Collagen fibers were visualized using picrosirius red staining and polarized light microscopy. Scale bar = 20 μm.

Fig. 3.

Reduced collagen content in hearts from old SPARC-null mice. The absence of SPARC decreased age-dependent increases in collagen content. Collagen content was examined using picrosirius red staining and light microscopy to quantify the collagen volume fraction.

Fig. 4.

Changes in collagen composition in hearts from old SPARC-null mice. The effects of the absence of SPARC on age-dependent changes in collagen composition are shown. Collagen composition was examined by measuring NaCl-insoluble collagen versus NaCl-soluble collagen by hydroxyproline quantification.

Fig. 5.

Distinct collagen fibril morphology in hearts from old SPARC-null compared with WT mice. The morphological structure of collagen was qualitatively examined using scanning electron microscopy. Scale bar = 10 μm.

Fig. 6.

Reductions in diastolic Stiffness in aged papillary muscle of SPARC-null mice. A: examples of passive diastolic myocardial stress-vs.-strain curves for the four groups of animals studied. B: mean ± SE values of the passive stiffness constant for the four groups of animals studied.