Abstract 1445: Exchange of C-Terminal Truncated Troponin I into Human Cardiomyocytes Preserves Force Responses to Beta-Adrenergic Stimulation and Increased Preload
The specific and selective proteolysis of cardiac troponin I (cTnI) has been proposed to play a key role in human myocardial ischemic disease. In the present study myofilament responsiveness to beta-adrenergic stimulation and to increased preload (Frank-Starling mechanism) were investigated in human cardiomyocytes containing truncated cTnI. The predominant human cTnI degradation product (cTnI1–192) and full length cTnI (cTnIFL) were expressed in E.coli, purified, reconstituted with the other cardiac troponin subunits and subsequently exchanged into human cardiomyocytes, isolated from healthy donor hearts. Maximal isometric force, passive force and Ca2+-sensitivity were measured in exchanged cardiomyocytes before and after protein kinase A treatment and at different sarcomere lengths. Protein analysis by 1D-SDS gel electrophoresis, Western Immunoblotting and 3 dimensional imaging microscopy revealed that approximately 50% of endogenous cTnI was homogeneously replaced by cTnI1–192 in exchanged cardiomyocytes. Maximal (Fmax) and passive (Fpas) tension were not altered by cTnI1–192, while myofilament Ca2+-sensitivity was increased in cardiomyocytes containing cTnI1–192 compared to cells with cTnIFL (ΔpCa50 was 0.24 and 0.27 at sarcomere lengths of 1.9 and 2.2 μm, respectively; Table⇓). Increasing sarcomere length from 1.9 to 2.2 μm increased all force characteristics to a similar extent in both groups (Table⇓). In addition, the PKA-induced shift in Ca2+-sensitivity was not different between cTnIFL and cTnI1–192 exchanged cardiomyocytes (ΔpCa50 amounted to 0.08 and 0.09, respectively). In conclusion, PKA-mediated desensitization and sarcomere length dependent increases in force-generating capacity of myofilaments are preserved in cTnI1–192 exchanged cardiomyocytes, indicating that cTnI degradation in human myocardium does not affect beta-adrenergic and preload-dependent responses.