Cystatin C increases in cardiac injury: a role in extracellular matrix protein modulation

Abstract

Aims: Numerous lines of evidence suggest a role of oxidative stress in initiation and progression of heart failure. We identify novel pathways of oxidative stress in cardiomyocytes using proteomic technology.

Methods and results: Cardiomyocytes and cardiac fibroblasts isolated from rat hearts were treated with sublethal doses of H(2)O(2) for detection of secreted protein factors in the conditioned media by mass spectrometry-based proteomics. Comparison between the two cell types leads to the finding that H(2)O(2) caused an elevated cystatin C protein in the conditioned medium from cardiomyocytes. When cardiomyopathy was induced in mice by chronic administration of doxorubicin, elevated cystatin C protein was detected in the plasma. Myocardial ischaemia by left anterior descending coronary artery occlusion causes an increase in the level of cystatin C protein in the plasma. In myocardial tissue from the ischaemic area, an increase in cystatin C correlates with the inhibition of cathepsin B activity and accumulation of fibronectin and collagen I/III. Overexpressing cystatin C gene or exposing fibroblasts to cystatin C protein results in an inhibition of cathepsin B and accumulation of fibronectin and collagen I/III.

Conclusion: Oxidants induce elevated cystatin C production from CMCs. Cystatin C plays a role in cardiac extracellular matrix remodelling.

Figure 1

H₂O₂ treatment of cardiomyocytes causes elevation of cystatin C in conditioned medium. CMCs and CFs in 100 mm dishes were treated with 300 µM H₂O₂ for 2 h. Cells were placed in fresh DMEM containing 10% FBS for 3 days before being placed in DMEM with 0% FBS for additional 3 days to collect the conditioned medium for ESI-LC-MS/MS (A and B) or for western blot analyses (C) to detect cystatin C (20 µg protein/lane), or ProANP and NT-ANP (100 µg protein/lane) with the loading control MMP-2. In the MS/MS spectrum of cystatin C peptide detected from the conditioned media of H₂O₂-treated CMCs, bolded letters indicate the detected b and y ions matching the predicted ion mass in sequence database (A). SEQUEST Flicka information page shows the fragment of cystatin C detected by LC-MS/MS (B). Cell lysates were harvested from CMCs in 6-well plates (0.5 × 10⁶ cells/well) following 300 µM of H₂O₂ treatment for 2 h and were then cultured in fresh DMEM containing 10% FBS for indicated time for detection of cystatin C (20 µg/lane) with GAPDH as a loading control (D).

Figure 2

Dox induced elevation of cystatin C in the plasma and the myocardium. The plasma of male BL6/129SF1J mice was collected 2 weeks after final Dox injection for measurements of cystatin C protein levels (A and B). An area of Coomassie blue-stained gel corresponding to the molecular weight (Mw) of cystatin C was included to show equal plasma protein loading (A). The hearts were collected to detect levels of cystatin C protein by western blot (C and D), or cystatin C or ANP mRNA by RT–PCR (E and F) with GAPDH as a loading control (C and E). The intensities of the bands were quantified using NIH imaging J software and are presented as means ± SE (B, D, F, G). An asterisk (*) indicates P < 0.05 while double asterisks (**) indicate P < 0.01 when means from Dox treated samples are compared with that of control using Student's t-test.

Figure 3

Time-dependent induction of cystatin C in the plasma of Dox-treated mice. Male BL6/129SF1J mice treated with Dox were weighted at indicated time points (A) with the plasma (B) and whole heart tissues (C) collected at second, fourth, sixth, and ninth weeks for western blot analyses of cystatin C protein levels (100 µg protein/lane). An area of Coomassie blue staining corresponding to cystatin C Mw was shown to indicate equal plasma protein loading (B). GAPDH was used as an internal loading control for the myocardial tissues (C). The intensities of the bands were quantified using NIH imaging J software and are presented as means ± SD from indicated number (C). An asterisk (*) indicates P < 0.05 while double asterisks (**) indicates P<0.01 when means from Dox-treated samples are compared with that of control using Student's t-test.

Figure 4

Elevation of cystatin C in the plasma of mice with regional ischaemia. Male BL6/129SF1J mice were used for LAD coronary artery occlusion surgery. The plasma collected seventh day after the surgery was used for western blot analyses to detect cystatin C (100 µg protein/lane, A). An area of Coomassie blue staining corresponding to cystatin C Mw was included to show equal plasma protein loading (A). The intensities of the bands quantified by NIH imaging J software are presented as means ± SE (B). Double asterisks (**) indicate P < 0.01 when the means of mice with myocardial ischaemia were compared with that of sham-operated or control animals by Student's t-test.

Figure 5

Cystatin C, cathepsin B, and ECM proteins in the ischaemic area of the myocardium. At 24 h after LAD coronary artery occlusion surgery, the non-ischaemic (N) and ischaemic area (I) were separated for western blot analyses (60 µg protein/lane) to detect cystatin C, cathepsin B (CTB, A and B), collagen III, collagen I, and fibronectin (C and D) with vinculin as a loading control. The intensities of the bands quantified by NIH imaging J software were normalized to that of vinculin and presented as means ± SE (B and D). Cathepsin B activity was measured using the substrate of Z-Arg-Arg-AMC (B). Double asterisks (**) indicate P < 0.01, and an asterisk (*) indicates P < 0.05 when the means from the ischaemic area was compared with that of non-ischaemic area by Student's t-test.

Figure 6

Cystatin C modulates ECM proteins. CFs were seeded in 6-well plates (Falcon) at a density of 0.3 × 10⁶ cells/well for 24 h transfection with 0.5 µg/well pcDNA4-cystatin C construct. At 24 h after transfection, the cells were harvested for western blot analyses (20 µg protein/lane) with vinculin as a loading control (A) or RT–PCR with GAPDH as an internal control (B). Without transfection, CFs were starved 3 days after seeding overnight in 0.5% FBS/DMEM before treatment with 5 nM cystatin C for indicated time (C and D) or 24 h (E). Cells (C–E) or conditioned medium (E) were harvested for western blot analyses with vinculin as a loading control (C), for cathepsin B activity assay (D) or zymography (E). The means that is not significantly different from another is labelled with a common letter symbol. The means in the ‘a’ group are significantly different from means in the ‘b’ or ‘c’ group.