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

Abstract

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.

Figure 1. SDS-PAGE of myofibrillar protein expression in wild-type (WT) and transgenic (Tg) hearts

Left and right lanes show the protein content of myofibrils obtained from hearts of WT (left) and Tg (right) mice. MHC, myosin heavy chain; TnT, troponin T; Tm, tropomyosin; cTnI, cardiac troponin I; ssTnI, slow skeletal muscle troponin I; MLC-1, myosin light chain 1.

Figure 2. Ca²⁺-force relations of wild-type (WT) and transgenic (TG) skinned fibre bundles over-expressing ssTnI at sarcomere lengths (SL) of 1.9 and 2.3 μm

The half-maximal activating Ca²⁺ concentration (EC₅₀) for WT skinned myofilaments was 3.20 ± 0.24 μm at SL 1.9 μm (n = 28, ▪) and 2.40 ± 0.16 μm at SL 2.3 μm (n = 25, •); for TG skinned myofilaments, the EC₅₀ was 1.53 ± 0.08 μm at SL 1.9 μm (n = 28, □) and 1.25 ± 0.09 μm at SL 2.3 μm (n = 25, ○) (P < 0.05).

Figure 3. The effect of 3 μm EMD 57033 on Ca²⁺-force relations of WT and TG skinned myofilaments at SL 1.9 μm and SL 2.3 μm

A, WT controls, EC₅₀ 3.20 ± 0.24 μm at SL 1.9 μm (n = 28, ▪) and 2.40 ± 0.16 μm at SL 2.3 μm (n = 25, •). WT + EMD, EC₅₀ 1.69 ± 0.33 μm at SL 1.9 μm (n = 5, □) and 1.58 ± 0.24 μm at SL 2.3 μm (n = 6, ○). B, TG controls, EC₅₀ 1.53 ± 0.08 μm at SL 1.9 μm (n = 28, □) and 1.25 ± 0.09 μm at SL 2.3 μm (n = 25, ○). TG + EMD, EC₅₀ 1.08 ± 0.05 μm at SL 1.9 μm (n = 6, ▴) and 1.00 ± 0.14 μm at SL 2.3 μm (n = 7, *).

Figure 4. The effect of 10 μm CGP-48506 on Ca²⁺-force relations of WT and TG skinned myofilaments at SL 1.9 μm and SL 2.3 μm

A, WT controls, EC₅₀ 3.20 ± 0.24 μm at SL 1.9 μm (n = 28, ▪) and 2.40 ± 0.16 μm at SL 2.3 μm (n = 25, •). WT + CGP, EC₅₀ 1.18 ± 0.22 μm at SL 1.9 μm (n = 7, □) and 1.05 ± 0.18 μm at SL 2.3 μm (n = 5, ○). B, TG controls, EC₅₀ 1.53 ± 0.08 μm at SL 1.9 μm (n = 28, □) and 1.25 ± 0.09 μm at SL 2.3 μm (n = 25, ○). TG + CGP, EC₅₀ 0.46 ± 0.13 μm at SL 1.9 μm (n = 4, *) and 0.39 ± 0.06 μm at SL 2.3 μm (n = 7, ⋄).

Figure 5. The effect of 20 μm calmidazolium (CDZ) on Ca²⁺-force relations of WT and TG skinned myofilaments at SL 1.9 μm and SL 2.3 μm

A, WT controls, EC₅₀ 3.20 ± 0.24 μm at SL 1.9 μm (n = 28, ▪) and 2.40 ± 0.16 μm at SL 2.3 μm (n = 25, •). WT + CDZ, EC₅₀ 2.89 ± 0.08 μm at SL 1.9 μm (n = 6, □) and 3.02 ± 0.07 μm at SL 2.3 μm (n = 4, ○). B, TG controls, EC₅₀ 1.53 ± 0.08 μm at SL 1.9 μm (n = 25, ▪) and 1.25 ± 0.09 μm at SL 2.3 μm (n = 20, •). TG + CDZ, EC₅₀ 1.38 ± 0.04 μm at SL 1.9 μm (n = 4, □) and 1.38 ± 0.14 μm at SL 2.3 μm (n = 5, ○).

Figure 6. Theoretical representation of the predictive association between length-dependent activation and the slope of the ventricular volume-end-systolic pressure (ESP) relation

The blue data points and lines represent volume-ESP and Ca²⁺-force relations for the WT hearts demonstrating normal length-dependent activation. The red data points and lines represent the volume-ESP and myofilament Ca²⁺-force relations for ssTnI-TG hearts, and illustrate that blunting of length-dependent activation may reduce the slope of the volume-ESP relation.