Introduction 1 9 8 11 20 29 18 14 17 5 19 6 28 36 39 8 20 26 26 27 We hypothesize that the stretch-related process through which cTnI can be released from viable cardiomyocytes is mediated by integrin stimulation. The purpose of the present study is therefore to investigate whether (1) RGD-induced integrin stimulation causes release of intact and/or fragmented cTnI from cardiomyocytes in the absence of necrotic cell death, (2) integrin stimulation is associated with MMP2 activation and MMP2-related cTnI degradation, and (3) cTnI release in the absence of necrosis differs from the release of cTnI in the presence of necrosis. Materials and methods Primary cultures of neonatal cardiomyocytes 24 6 2 v v Experimental protocol for integrin stimulation 4 2 4 2 3 d GRGDS SDGRG GRGDS Troponin-I ELISA 13 \documentclass[12pt]{minimal}
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\begin{document}$$ {\left[ {{{\text{TnI}}_{{{\text{medium}}}} } \mathord{\left/  {\vphantom {{{\text{TnI}}_{{{\text{medium}}}} } {{\left( {{\text{TnI}}_{{{\text{medium}}}}  + {\text{TnI}}_{{{\text{cells}}}} } \right)}}}} \right.  \kern-\nulldelimiterspace} {{\left( {{\text{TnI}}_{{{\text{medium}}}}  + {\text{TnI}}_{{{\text{cells}}}} } \right)}}} \right]}*100\%  $$\end{document} Western blot analysis Protein extracts were size-fractionated on NuPage Novex 12% Bis–Tris gels (Invitrogen) and transferred to Hybond PVDF membranes (Amersham Biosciences, Roosendaal, The Netherlands). Non-specific binding sites were blocked using blocking solution consisting of 20 g/l ECL advance blocking agent (Amersham Biosciences) in TBS-Tween (10 mmol/l Tris–HCl, 150 mmol/l NaCl, 0.05% Tween pH 8.0). Membranes were incubated for 1 h with the anti-TnI antibody (K83085G, Biodesign, Saco, ME, USA), which detects various cTnI degradation products. After four washes in TBS-Tween, membranes were incubated with horseradish peroxidase-labeled secondary antibody (rabbit anti-goat IgG, Santa Cruz Biotechnology, Heidelberg, Germany). Chemiluminescence was induced by ECL advance detection reagent (Amersham Biosciences) and detected by exposure to Hyperfilm ECL (Amersham Biosciences). LDH activity assay 41 Nuclear staining with propidium iodide GRGDS SDGRG In vitro degradation of purified cTnI by active MMP2 o Zymography GRGDS SDGRG 12 33 Experimental protocol for necrotic cell death n Statistics t p Results Effect of integrin stimulation on release of cTnI 1 2 GRGDS GRGDS p GRGDS p 1 SDGRG GRGDS 1 2 Fig. 1 a n GRGDS n n n SDGRG n b n GRGDS n SDGRG n p # p GRGDS $ p GRGDS ‡ p GRGDS § p SDGRG  Fig. 2 left right GRGDS a b SDGRG c d e f  Effect of integrin stimulation on cell viability 1 25 GRGDS 1 SDGRG GRGDS p n GRGDS n p SDGRG n GRGDS p GRGDS Effect of integrin stimulation on MMP2 activity 3 2 4 Fig. 3 o  Fig. 4 left right n GRGDS n SDGRG n  4 GRGDS SDGRG 4 GRGDS 4 GRGDS SDGRG p 4 Release of cTnI from necrotic cardiomyocytes GRGDS n n 2 2 Discussion GRGDS 2 18 15 20 15 17 21 6 32 GRGDS GRGDS 25 SDGRG 6 7 34 30 39 4 22 35 38 37 GRGDS 31 40 GRGDS SDGRG GRGDS 3 2 GRGDS GRGDS 3 10 16 23 Conclusions The present study demonstrates that viable cardiomyocytes release cTnI as an intact protein by a stretch-related mechanism mediated by integrins. This finding may possibly explain why, in several pathological conditions, plasma cTnI levels are elevated in the absence of myocardial necrosis.