Release of cardiac troponin I from viable cardiomyocytes is mediated by integrin stimulation

Abstract

Elevated cardiac troponin-I (cTnI) levels have been demonstrated in serum of patients without acute coronary syndromes, potentially via a stretch-related process. We hypothesize that this cTnI release from viable cardiomyocytes is mediated by stimulation of stretch-responsive integrins. Cultured cardiomyocytes were treated with (1) Gly-Arg-Gly-Asp-Ser (GRGDS, n = 22) to stimulate integrins, (2) Ser-Asp-Gly-Arg-Gly (SDGRG, n = 8) that does not stimulate integrins, or (3) phosphate-buffered saline (control, n = 38). Cells and media were analyzed for intact cTnI, cTnI degradation products, and matrix metalloproteinase (MMP)-2. Cell viability was examined by assay of lactate dehydrogenase (LDH) activity and by nuclear staining with propidium iodide. GRGDS-induced integrin stimulation caused increased release of intact cTnI (9.6 +/- 3.0%) as compared to SDGRG-treated cardiomyocytes (4.5 +/- 0.8%, p < 0.001) and control (3.0 +/- 3.4%, p < 0.001). LDH release from GRGDS-treated cardiomyocytes (15.9 +/- 3.8%) equalled that from controls (15.2 +/- 2.3%, p = n.s.), indicating that the GRGDS-induced release of cTnI is not due to cell necrosis. This result was confirmed by nuclear staining with propidium iodide. Integrin stimulation increased the intracellular and extracellular MMP2 activity as compared to controls (both p < 0.05). However, despite the ability of active MMP2 to degrade cTnI in vitro, integrin stimulation in cardiomyocytes was not associated with cTnI degradation. The present study demonstrates that intact cTnI can be released from viable cardiomyocytes by stimulation of stretch-responsive integrins.

Fig. 1

a Release of cardiac troponin-I (cTnI) from cardiomyocytes incubated with PBS (control, n = 38), GRGDS (100 μg/ml, n = 5; 200 μg/ml, n = 4; 300 μg/ml, n = 22), or SDGRG (300 μg/ml, n = 8), detected by ELISA. b Release of LDH from cardiomyocytes incubated with PBS (control, n = 38), GRGDS (300 μg/ml, n = 22), or SDGRG (300 μg/ml, n = 8). (*p < 0.001 vs control, ^#p < 0.001 vs GRGDS 100, ^$p < 0.001 vs GRGDS 200, ^‡p < 0.001 vs GRGDS 300, and ^§p < 0.001 vs SDGRG 300)

Fig. 2

Western blots of intact cTnI (29 kDa) and cTnI degradation products present in cell samples (left, 2.5× diluted) and medium samples (right, 10× concentrated) of cardiomyocytes incubated with PBS (control), GRGDS (300 μg/ml, a and b), SDGRG (300 μg/ml, c and d), or sodium azide (1 mmol/l, e and f) for 24 h

Fig. 3

Western blot of intact cTnI (29 kDa) and cTnI degradation products after in vitro incubation of purified intact cTnI without and with active MMP2 and with active MMP2 in combination with a MMP2 inhibitor (o-phenanthroline) for 0, 60, and 120 min

Fig. 4

Gelatinolytic activities of MMP2 isoforms in cell samples (left, 10× concentrated) and medium samples (right, unconcentrated) of cardiomyocytes incubated with PBS (control, n = 12), GRGDS (300 μg/ml, n ≥ 9), or SDGRG (300 μg/ml, n ≥ 3) for 24 h