University of Heidelberg (2006) Proc Physiol Soc 4, C2
Oral Communications: Stephanie Reis1, Christoph Littwitz2, Lutz Pott2, Kornelia Jaquet1
1. St Josef-Hospital, Herz- und Kreislaufzentrum der Ruhr-Universität Bochum, Bochum, Germany. 2. Zelluläre Physiologie, Ruhr-Universität Bochum, Bochum, Germany.
We investigated the effects of amino acid exchanges in cardiac troponin I (cTnI), known to induce familial hypertrophic cardiomyopathy (FHC), on the contractility of adult cardiomyocytes isolated post mortem from rat left ventricles. The extent and rate of sarcomer shortening as well as its relaxation rate are analysed in response to β1/2-, β1- and β2-adrenergic stimulation. Contractions were induced by bipolar external stimuli (0.4 ms, 40 V). The stimulation protocol was 0.5, 3.0, 1.0, 2.0 and 1.5 Hz with 20 stimuli per frequency interrupted by a 30 s stimulation pause. Adenovirus driven expression of human cTnI-wt in adult rat myocytes, monitored by the expression of GFP (green fluorescent protein), does not alter the parameters listed above in comparison to non-infected myocytes. The localisation of human cTnI in the sarcomeres is controlled with anti-human cTnI monoclonal antibodies, not recognizing rat cTnI. As expected, the extent and rate of shortening as well as the relaxation rate of infected and non-infected cardiomyocytes are enhanced in response to isoproterenol (1 µM) alone and the β2-adrenergic receptor (AR) blocker ICI 118,551 (50 nM) added in addition to isoproterenol. Similar effects, but less pronounced, are obtained upon incubation with isoproterenol and the β1-AR blocker CGP-20712A (300 nM). The amino acid exchange R145G in the inhibitory region of cTnI reduced the rate of shortening (1.56 ± 0.35 to 0.52 ± 0.13 µm/s, n = 10, 2 Hz) and relaxation (0.94 ± 0.30 to 0.23 ± 0.08 µm/s, n = 10, 2 Hz) significantly (P ≤ 0.0007, unpaired Student’s t test) in non-AR stimulated ventricle cells. Upon stimulation via β1/2-, β1- and β2-AR both parameters were enhanced as expected. However, in comparison to cells with TnI-wt rates of shortening and relaxation were reduced upon stimulation of β1/2- (1.69 ± 0.22 to 1.14 ± 0.22 µm/s for shortening rate and 2.17 ± 0.59 to 1.29 ± 0.44 µm/s for relaxation rate, n = 10, 2 Hz, P ≤ 0.0074) and β2-AR (1.89 ± 0.48 to 1.23 ± 0.23 µm/s for shortening rate and 1.56 ± 0.45 to 0.94 ± 0.34 µm/s for relaxation rate, n = 10, 2 Hz, P ≤ 0.0027), but not in ventricle cells stimulated via β1-AR (2.18 ± 0.65 to 2.25 ± 0.27 µm/s for shortening rate and 2.10 ± 0.82 to 1.45 ± 0.65 µm/s for relaxation rate, n = 10, 2 Hz, P ≤ 0.1138). Changes in the extent of shortening due to the mutation were only observed in cardiomyocytes which were not stimulated via β-AR depending on the stimulation frequency. The fact that there are no differences between non-infected and hcTnI-wt infected cardiomyocytes but between hcTnI-wt and hcTnI-R145G infected cardiomyocytes depending either on the stimulation frequency or on the method of receptor stimulation is a further indicator that the subunit is incorporated into the rat troponin complex at the right position.
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Where applicable, experiments conform with Society ethical requirements.