Expression of slow skeletal troponin I in adult transgenic mouse heart muscle reduces the force decline observed during acidic conditions.

1. Acidosis in cardiac muscle is associated with a decrease in developed force. We hypothesized that slow skeletal troponin I (ssTnI), which is expressed in neonatal hearts, is responsible for the observed decreased response to acidic conditions. To test this hypothesis directly, we used adult transgenic (TG) mice that express ssTnI in the heart. Cardiac TnI (cTnI) was completely replaced by ssTnI either with a FLAG epitope introduced into the N-terminus (TG-ssTnI) or without the epitope (TG-ssTnI) in these mice. TG mice that express cTnI were also generated as a control TG line (TG-cTnI). Non-transgenic (NTG) littermates were used as controls. 2. We measured the force-calcium relationship in all four groups at pH 7.0 and pH 6.5 in detergent-extracted fibre bundles prepared from left ventricular papillary muscles. The force-calcium relationship was identical in fibre bundles from NTG and TG-cTnI mouse hearts, therefore NTG mice served as controls for TG-ssTnIand TG-ssTnI mice. Compared to NTG controls, the force generated by fibre bundles from TG mice expressing ssTnI was more sensitive to Ca(2+). The shift in EC(50) (the concentration of Ca(2+) at which half-maximal force is generated) caused by acidic pH was significantly smaller in fibre bundles isolated from TG hearts compared to those from NTG hearts. However, there was no difference in the force-calcium relationship between hearts from the TG-ssTnIand TG-ssTnI groups. 3. We also isolated papillary muscles from the right ventricle of NTG and TG mouse hearts expressing ssTnI and measured isometric force at extracellular pH 7.33 and pH 6.75. At acidic pH, after an initial decline, twitch force recovered to 60 +/- 3 % (n = 7) in NTG papillary muscles, 98 +/- 2 % (n = 5) in muscles from TG-ssTnIand 96 +/- 3 % (n = 7) in muscles from TG-ssTnI hearts. Our results indicate that TnI isoform composition plays a crucial role in the determination of myocardial force sensitivity to acidosis.

Attenuation of length dependence of calcium activation in myofilaments of transgenic mouse hearts expressing slow skeletal troponin I.

We compared sarcomere length (SL) dependence of the Ca2+-force relation of detergent-extracted bundles of fibres dissected from the left ventricle of wild-type (WT) and transgenic mouse hearts expressing slow skeletal troponin I (ssTnI-TG). Fibre bundles from the hearts of the ssTnI-TG demonstrated a complete replacement of the cardiac troponin I (cTnI) by ssTnI. Compared to WT controls, ssTnI-TG fibre bundles were more sensitive to Ca2+ at both short SL (1.9 +/- 0.1 micrometer) and long SL (2.3 +/- 0.1 micrometer). However, compared to WT controls, the increase in Ca2+ sensitivity (change in half-maximally activating free Ca2+; DeltaEC50) associated with the increase in SL was significantly blunted in the ssTnI-TG myofilaments. Agents that sensitize the myofilaments to Ca2+ by promoting the actin-myosin reaction (EMD 57033 and CGP-48506) significantly reduced the length-dependent DeltaEC50 for Ca2+ activation, when SL in WT myofilaments was increased from 1.9 to 2.3 micrometer. Exposure of myofilaments to calmidazolium (CDZ), which binds to cTnC and increases its affinity for Ca2+, sensitized force developed by WT myofilaments to Ca2+ at SL 1.9 micrometer and desensitized the WT myofilaments at SL 2.3 micrometer. There were no significant effects of CDZ on ssTnI-TG myofilaments at either SL. Our results indicate that length-dependent Ca2+ activation is modified by specific changes in thin filament proteins and by agents that promote the actin-myosin interaction. Thus, these in vitro results provide a basis for using these models to test the relative significance of the length dependence of activation in situ.