Phosphorylation of cardiac troponin-I (Tn-I) by protein kinase A (PKA) reduces myofilament Ca²⁺ responsiveness and contributes to the myocardial effects of β-adrenoceptor stimulation, in particular acceleration of relaxation. However, the contribution of Tn-I phosphorylation to β-mediated changes in systolic and diastolic function of the whole heart remain unclear. We investigated the effects of isoprenaline (Iso, 10 nM) on contractile function of Langendorff-perfused hearts isolated from male transgenic mice (TG) over-expressing the slow skeletal isoform of Tn-I (lacking PKA phosphorylation sites) and non-transgenic (NTG), male littermate controls (n ▓ge│ 6 per group). Mice were killed by an overdose of sodium pentobarbitone (Sagatal, 120 mg kg^-1 I.P.). Hearts were perfused at constant coronary perfusion pressure (75 mmHg) and isovolumic left ventricular pressure (LVP) was measured at varying LV volumes using an intraventricular balloon connected to a pressure transducer. Data are expressed as means ± standard error and compared using unpaired t tests unless otherwise stated. In NTG hearts Iso had pronounced positive inotropic and lusitropic effects, as indicated by an increase in the maximum rates of rise (dP/dt_max) and fall (dP/dt_min) of the LV pressure trace, respectively. For example, at 25 ml balloon volume, Iso increased LV dP/dt_max from 7663 ± 1019 to 14860 ± 1524 mmHg s^-1 (P < 0.05) and LV dP/dt_min from -5812 ± 768 to -9997 ± 695 mmHg s^-1 (P < 0.05). The facilitated relaxation was also apparent as a reduction in minimum diastolic pressure (min DP) at all LV volumes (P < 0.05 by repeated measures ANOVA). In contrast, in TG hearts, Iso had no significant effect on LV dP/dt_min: -4890 ± 682 and -7084 ± 1022 mmHg s^-1 for control and Iso, respectively, at 25 ml balloon volume) or on the min DP-volume relationship (P > 0.05 by repeated measures ANOVA). Furthermore, end-diastolic pressure was significantly greater in TG compared with NTG hearts in the presence of Iso (e.g. a difference of 12 mmHg at 25 ml volume, P < 0.05). Surprisingly, although Iso also increased LV dP/dt_max in TG hearts (i.e. from 7563 ± 1045 to 12 146 ± 1865 mmHg s^-1 at 25 ml balloon volume, P < 0.05), the positive inotropic effect appeared to be blunted compared with the response in NTG hearts. These results indicate that phosphorylation of cardiac Tn-I is essential for both the relaxant response and increased diastolic compliance induced by Iso in the mouse heart. The unexpected blunting of the inotropic effect of Iso in TG hearts may be secondary to impairment of relaxation and diastolic responses.

This work was supported by the British Heart Foundation.

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