Investigation of thin filament near-neighbour regulatory unit interactions during force development in skinned cardiac and skeletal muscle

Abstract

Ca(2+)-dependent activation of striated muscle involves cooperative interactions of cross-bridges and thin filament regulatory proteins. We investigated how interactions between individual structural regulatory units (RUs; 1 tropomyosin, 1 troponin, 7 actins) influence the level and rate of demembranated (skinned) cardiac muscle force development by exchanging native cardiac troponin (cTn) with different ratio mixtures of wild-type (WT) cTn and cTn containing WT cardiac troponin T/I + cardiac troponin C (cTnC) D65A (a site II inactive cTnC mutant). Maximal Ca(2+)-activated force (F(max)) increased in less than a linear manner with WT cTn. This contrasts with results we obtained previously in skeletal fibres (using sTnC D28A, D65A) where F(max) increased in a greater than linear manner with WT sTnC, and suggests that Ca(2+) binding to each functional Tn activates < 7 actins of a structural regulatory unit in cardiac muscle and > 7 actins in skeletal muscle. The Ca(2+) sensitivity of force and rate of force redevelopment (k(tr)) was leftward shifted by 0.1-0.2 -log [Ca(2+)] (pCa) units as WT cTn content was increased, but the slope of the force-pCa relation and maximal k(tr) were unaffected by loss of near-neighbour RU interactions. Cross-bridge inhibition (with butanedione monoxime) or augmentation (with 2 deoxy-ATP) had no greater effect in cardiac muscle with disruption of near-neighbour RU interactions, in contrast to skeletal muscle fibres where the effect was enhanced. The rate of Ca(2+) dissociation was found to be > 2-fold faster from whole cardiac Tn compared with skeletal Tn. Together the data suggest that in cardiac (as opposed to skeletal) muscle, Ca(2+) binding to individual Tn complexes is insufficient to completely activate their corresponding RUs, making thin filament activation level more dependent on concomitant Ca(2+) binding at neighbouring Tn sites and/or crossbridge feedback effects on Ca(2+) binding affinity.

Figure 2. Determination of sarcomere protein stoicheometry

A, silver-stained SDS-PAGE for non-exchanged (lanes 1 and 2) and cTn-exchanged (lanes 4 and 5) trabeculae. Lane 3 is used as a marker lane for protein identification and contains (from top to bottom) actin, troponin (Tn) T, TnI, TnC, myosin light chain (MLC) 1 and MLC2. B, densitometric analysis of TnI, MLC 1 and MLC 2. Values are normalized to the percentage of actin to account for any variability in loading conditions. The data demonstrate similar levels of TnI, MLC1 and MLC2 for non-exchanged and cTn-exchanged trabeculae.

Figure 3. Analysis of cardiac troponin (cTn) I phosphorylation for cTn complexes and a pre-exchanged rat trabecula

A, top part of panel shows a Coommassie blue-stained SDS-PAGE of untreated cTn (lane 2), untreated xcTn (lane 3), cTn treated with PKA + PKC (lane 4), xcTn treated with PKA + PKC (lane 5), cTn treated with PP1 (lane 6) and xcTn treated with PP1 (lane 7). Lane 1 is a marker lane for verification of cTn subunit molecular weights. The bottom row is the Western blot for lanes 2–7, to determine the level of cTnI phosphorylation. Details are provided in the text (see Methods and Results). B, contains three lanes: 1, a marker lane showing 29 kDa (*) and 19 kDa (**) molecular weight standards; 2, a Coommassie blue-stained gel of an untreated rat trabecula; and 3, the Western blot for phospho-serine cTnI. C, is a densitometric analysis of the Western blot in A, with values normalized to those obtained for no treatment. The Western blot demonstrates a significant level of cTnI phosphorylation in the trabeculae used for experiments, similar to the levels in the cTn complexes used for exchanges.

Figure 1. Representative chart recordings showing the effectiveness of whole cardiac troponin (cTn) replacement and the effect of different mixtures of wild-type (WT) cTnC/cTn and non-functional (x) xcTnC/cTn on Ca²⁺-activated force development in cardiac trabeculae

A, the trabeculae in this experiment was able to produce 59.2 mN mm⁻² of force at pCa 4.5 (F_max) prior to the native cTn being replaced by cTn containing a cTnC unable to be activated by Ca²⁺. Following this replacement, F_max was 6.1 mN mm⁻² (equal to 10% of pre-replacement F_max). The non-functional cTn was then replaced by 100% functional cTn and F_max was then found to be 45.8 mm mm⁻² (equal to 77% of pre-replacement F_max). k_tr was 11.34 s⁻¹ after exchange. B, illustrates force–pCa recordings of a trabecula containing native cTnC and then after the native cTn was replaced by 100% WT cTn. F_max before replacement was 32.1 mN mm⁻², and following replacement was 27.0 mN mm⁻². C, illustrates force–pCa recordings of a trabecula containing native cTnC and then after the native cTn was replaced by a cTn mixture of 50: 50 cTn/xTn. F_max before replacement was 10.8 mN mm⁻², and following replacement was 2.7 mN mm⁻².

Figure 4. Effect of the proportion of functional cardiac troponin (cTn) on maximum force generation (pCa 4.0) in rat cardiac trabeculae (•)

For all mixture ratios > 0.25, F_max was less than proportionality between force and cTnC content (indicated by continuous line). Data from Regnier et al. (2001) for skeletal muscle containing different proportions of functional sTnC has been added to the figure (dotted line) to enable comparison.

Figure 5. Force–pCa relations of cardiac trabeculae after replacement of endogenous cardiac troponin (cTn) with different proportions of functional/non-functional cTn/xcTn

A, endogenous cTn was replaced with mixtures containing: 25: 75, cTn:xcTn (♦); 50: 50 cTn:xcTn (▪); 75: 25, cTn:xcTn (▾); and 100: 0, cTn:xcTn (•). B, replacement was with a 50: 50 mixture of cTn/xcTn for three trabeculae where the exchanged F_max was 27 ± 4% of the pre-exchanged value.

Figure 6. Effect of BDM on F_max (pCa 4.0) in skeletal and cardiac muscle for intact and isolated functional unit (FU) preparations

Force values are normalized to values obtained in the absence of BDM (dashed line) and F_max in isolated FU preparations was ∼0.2 pre-extracted F_max prior to BDM treatment.

Figure 7. Effect of deoxy-ATP (dATP) on maximal force (F_max, pCa 4.0) in skeletal (A) and cardiac muscle (B) with intact and isolated regulatory units (RUs)

Reconstituted F_max for both skeletal and cardiac preparations with isolated FUs was ∼0.2 F_max.

Figure 8. Effect of functional unit (FU) isolation on the maximum rate of force redevelopment (k_tr) (pCa 4.0; •) (A) and the rate of force redevelopment over a range of pCa values for a single example trabeculae (B)

Data from measurements made prior to extraction (▵) are added to the figure for comparison in A.

Figure 9. Comparison of the rates of Ca²⁺ dissociation from the N-terminus of cadiac and skeletal troponin at 15°C

The troponin complexes, in the presence of 30 μm Ca²⁺, were mixed rapidly with 150 μm Quin-2. Fluorescence was monitored through a 510 nm broad band-pass interference filter. The traces have been displaced vertically to allow comparison. Ca²⁺ dissociation from the C-terminus of cTn and sTn at 15°C was 0.67 ± 0.01 s⁻¹ and 0.15 ± 0.01 s⁻¹, respectively.