What does TnCDANZ fluorescence reveal about the thin filament state?

Fluorescence of skinned psoas fibres reconstituted with the troponin C subunit labelled with the fluorescent probe dansylaziridine (TnC_DANZ) increases upon activation with Ca²⁺. This fluorescence enhancement is due to Ca²⁺ binding to the Ca²⁺-specific binding sites of TnC_DANZ and attachment of cross-bridges to the actin filament. We found that approximately 20% of the enhanced fluorescence signal derived from Ca²⁺ binding to TnC_DANZ and 80% from cross-bridge attachment during maximal activation. Furthermore we studied the effects of different cross-bridge states on TnC_DANZ fluorescence. Weakly bound, non-force-generating cross-bridge states (pCa 8, low ionic strength) and rigor cross-bridges revealed similar effects on the TnC_DANZ fluorescence. Strongly attached, force-generating states, however, increased fluorescence to the greatest extent. These results suggests a complex system of reciprocal couplings between TnC and different attached cross-bridge states. Cooling or increase of inorganic phosphate decreased isometric force but hardly decreased fluorescence, suggesting the accumulation of attached cross-bridge states with low tension output.     

The apparent rate constant for the dissociation of force generating myosin crossbridges from actin decreases during Ca2+ activation of skinned muscle fibres

Summary The effect of Ca²⁺ activation on the apparent rate constant governing the dissociation of force generating myosin cross-bridges was studied in skinned rabbit adductor magnus fibres (fast-twitch) at 21±1 °C. Simultaneous measurements of Ca²⁺-activated isometric force and ATPase activity were conducted in parallel with simultaneous measurements of DANZ-labelled troponin C (TnC_DANZ) fluorescence and isometric force in fibres whose endogenous troponin C had been partially replaced with TnC_DANZ. The Ca²⁺ activation of isometric force occurred at approximately two times higher Ca²⁺ concentration than did actomyosin ATPase activity at 2.0 mM MgATP. Since increases in both TnC_DANZ fluorescence and ATPase activity occurred over approximately the same Ca²⁺ concentration range at substantially lower concentrations of Ca²⁺ than did force, this data suggests that the TnC_DANZ fluorescence is associated with the Ca²⁺ activation of myosin crossbridge turnover (ATPase) rather than force. According to the model of Huxley (1957) and assuming the hydrolysis of one molecule of ATP per cycle of the crossbridge, the apparent rate constantg _app for the dissociation of force generating myosin crossbridges is proportional to the actomyosin ATPase/isometric force ratio. This measure ofg _app shows approximately a fivefold decrease during Ca²⁺ activation of isometric force. This change ing _app is responsible for separation of the Ca²⁺ sensitivity of the normalized ATPase activity and isometric force curves. If the MgATP concentration is reduced to 0.5 mM, the change ing _app is reduced and consequently the difference in Ca²⁺ sensitivity between normalized steady state ATPase and force is also reduced.The abbreviations used are TnC troponin C - DANZ 5-dimethylaminonapthalene-2-sulphonyl aziridine - TnC_DANZ DANZ-labelled TnC - a the number of half sarcomeres - A the cross-sectional area of the fibre - AP₅A P¹,P⁵-di(adenosine-5)pentaphosphate - EGTA ethyleneglycolbis-(betaaminoethyl ether)-N,N,N,N-tetraacetic acid - F force a muscle fibre develops - f_app apparent rate of formation of force generating myosin crossbridges - F_av the average force per myosin head - Fs steady-state fraction of cycling myosin crossbridges in the force generating state - g _app apparent rate of dissociation of force generating myosin crossbridges - L_1/2s the length of a half sarcomere - LDH lactate dehydrogenase - [M] is the concentration of myosin per fibre volume - NAD nicotinamide adenine dinucleotide - NADH reduced form of NAD - pCa -log₁₀ of the free Ca²⁺ concentration - PEP phosphenol pyruvate - PK pyruvate kinase     

Calcium binding and fluorescence measurements of dansylaziridine-labelled troponin C in reconstituted thin filaments

Summary The direct binding of Ca²⁺ to reconstituted thin filaments containing troponin C and the 5-dimethylaminonaphthalene-1-sulphonylaziridine (DANZ) fluorescent analogue of troponin C (TnC _DANZ ) was measured (25° C) at three Mg²⁺ concentrations. Biphasic Scatchard plots were found for all binding curves reflecting the binding of Ca²⁺ to high- and low-affinity sites of troponin. The binding of Ca²⁺ to the high-affinity sites had a greater sensitivity to Mg²⁺ (K _Mg=1×10⁴ m ^–1) than the low-affinity sites (K _Mg=1.2×10³ m ^–1). The fluorescence change of thin filaments reconstituted with TnC _DANZ was titrated with Ca²⁺ in the same solutions used for binding assays. The Ca²⁺-dependent fluorescence change had nearly the same sensitivity to Mg²⁺ (K _Mg=9.4×10² m ^–1) as did Ca²⁺ binding to the low-affinity sites. The Ca²⁺ concentration at the midpoint of the fluorescence change was about 0.3 log units less than at the midpoint for Ca²⁺ binding to the low-affinity sites. A similar relationship between the fluorescence change and Ca²⁺ binding to the low-affinity sites of isolated TnC _DANZ was measured (4° C). From these results the binding of Ca²⁺ to either low-affinity site is concluded to produce the fluorescence change. In comparison with the low-affinity sites of isolated troponin and troponin-tropomyosin complex, the low-affinity sites of reconstituted thin filaments were consistently lower in Ca²⁺ affinity.     

Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle

Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca²⁺-dependent regulation. Force then depended on the negative logarithm of Ca²⁺ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa₅₀, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca²⁺, but restoration of Ca²⁺ dependence required incubation with both TnI and TnC. Relaxation at low Ca²⁺ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20–150 μM), while Ca²⁺ sensitivity, i.e. the pCa₅₀, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 μM and 150 μM, respectively) were more sensitive to Ca²⁺ than those reconstituted with cTnC and cTnI (difference in pCa₅₀ approx. 0.2 units). Rabbit sTnI was cloned and expressed in Escherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G¹⁰⁴-K-F-K-R-P-P-L-R-R-V-R¹¹⁵-). The four mutants produced by substitution of T for P¹¹⁰, G for P¹¹⁰, G for L¹¹¹, and G for K¹⁰⁵ were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-S₁ ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with exogenous TnC, Ca²⁺ dependence could be restored when ^gly110sTnI, ^thr110sTnI or ^gly111sTnI was used for reconstitution. The mutant ^gly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca²⁺ concentrations and it caused an increase in Ca²⁺ sensitivity. Received: 5 October 1995/Received after revision and accepted: 1 December 1995     

Biochemical characterisation of Troponin C mutations causing hypertrophic and dilated cardiomyopathies

Cardiac muscle contraction occurs through an interaction of the myosin head with the actin filaments, a process which is regulated by the troponin complex together with tropomyosin and is Ca²⁺ dependent. Mutations in genes encoding sarcomeric proteins are a common cause of familial hypertrophic and dilated cardiomyopathies. The scope of this review is to gather information from studies regarding the in vitro characterisation of six HCM and six DCM mutations on the cardiac TnC gene and to suggest, if possible, how they may lead to dysfunction. Since TnC is the subunit responsible for Ca²⁺ binding, mutations in the TnC could possibly have a strong impact on Ca²⁺ binding affinities. Furthermore, the interactions of mutant TnCs with their binding partners could be altered. From the characterisation studies available to date, we can conclude that the HCM mutations on TnC increase significantly the Ca²⁺ sensitivity of force development or of ATPase activity, producing large pCa shifts in comparison to WT TnC. In contrast, the DCM mutations on TnC have a tendency to decrease the Ca²⁺ sensitivity of force development or of ATPase activity in comparison to WT TnC. Furthermore, the DCM mutants of TnC are not responsive to the TnI phosphorylation signal resulting in filaments that preserve their Ca²⁺ sensitivity in contrast to WT filaments that experience a decrease in Ca²⁺ sensitivity upon TnI phosphorylation.     

The troponin I: inhibitory peptide uncouples force generation and the cooperativity of contractile activation in mammalian skeletal muscle

Hodges and his colleagues identified a 12 amino acid fragment of troponin I (TnI-ip) that inhibits Ca²⁺-activated force and reduces the effectiveness Ca²⁺ as an activator. To understand the role of troponin C (TnC) in the extended cooperative interactions of thin filament activation, we compared the effect of TnI-ip with that of partial troponin TnC extraction. Both methods reduce maximal Ca²⁺-activated force and increase [Ca²⁺] required for activation. In contrast to TnC extraction, TnI-ip does not reduce the extended cooperative interactions between adjacent thin filament regulatory units as assessed by the slope of the pCa/force relationship. Additional evidence that TnI-ip does not interfere with extended cooperativity comes from studies that activate muscle by rigor crossbridges (RXBs). TnI-ip increases both the cooperativity of activation and the concentration of RXBs needed for maximal force. This shows that TnI-ip binding to TnC increases the stability of the relaxed state of the thin filament. TnI-ip, therefore, uncouples force generation from extended cooperativity in both Ca²⁺ and RXB activated muscle contraction. Because maximum force can be reduced with no change—or even an increase—in cooperativity, force-generating crossbridges do not appear to be the primary activators of cooperativity between thin filament regulatory units of skeletal muscle.     

Ca2+ sensitizing effects of EMD 53998 after troponin replacement in skinned fibres from porcine atria and ventricles

Skinned fibres from porcine ventricles exhibited a higher Ca²⁺ sensitivity (pCa₅₀, i.e. -log₁₀ Ca²⁺ concentration required for half-maximal activation, for force generation) than atrial fibres. The thiadiazinone derivative EMD 53998 increased Ca²⁺ sensitivity and Ca²⁺ efficacy in both preparations. The drug effect depended on the isoform of troponin (Tn). Using the vanadate method TnI and TnC could be partly extracted and replaced by foreign tropin or by the TnI subunit of added foreign troponins. We investigated the relationship between pCa and force development before and after replacement of TnI with foreign troponin (bovine ventricular troponin, cTn, or rabbit skeletal muscle troponin, sTn) in the presence and absence of EMD 53998. Substitution with bovine cTn increased Ca²⁺ sensitivity to a value characteristic of bovine ventricular skinned fibres (pCa₅₀=5.4) and was further increased by EMD 53998. Substitution with sTn also increased Ca²⁺ sensitivity, but subsequent addition of EMD 53998 caused little further increase in Ca²⁺ sensitivity. Following extraction of TnI with vanadate, skinned fibres contracted in a Ca²⁺-independent manner and failed to relax at a pCa of 8. Relaxation could be induced, however, by bovine ventricular TnI and rabbit skeletal muscle recombinant TnI. This relaxation could be reversed by EMD 53998 (100 M). The Ca²⁺-independent force of contracted fibres could also be depressed by a TnI inhibitory peptide, (cTnI 137–148) and, in addition, this effect was antagonized by EMD 53998. These results suggest that EMD 53998 antagonizes the inhibitory action of TnI, possibly by interfering with the interaction of the TnI inhibitory region with actin.     

Functional effects of the DCM mutant Gly159Asp Troponin C in skinned muscle fibres

We recently reported a dilated cardiomyopathy (DCM) causing mutation in a novel disease gene, TNNC1, which encodes cardiac troponin C (TnC). We have determined how this mutation, Gly159Asp, affects contractile regulation when incorporated into muscle fibres. Endogenous troponin in rabbit skinned psoas fibres was partially replaced by recombinant human cardiac troponin containing either wild-type or Gly159Asp TnC. We measured both the force–pCa relationship of these fibres and the activation rate using the caged-Ca²⁺ compound nitrophenyl-EGTA. Gly159Asp TnC had no significant effect on either the Ca²⁺ sensitivity or cooperativity of force generation when compared to wild type. However, the mutation caused a highly significant (ca. 50%) decrease in the rate of activation. This study shows that whilst not affecting the force–pCa relationship, the mutation Gly159Asp causes a significant decrease in the rate of force production and a change in the relationship between the rate of force production and generated force. In vivo, this mutation may cause both a slowing of force generation and reduction in total systolic force. This represents a novel mechanism by which a cardiomyopathy-causing mutation can affect contractility.     

Functional characterization of the two isoforms of troponin C from the arthropodBalanus nubilus

Summary Two isoforms of troponin C (BTnC₁ and BTnC₂) from the striated muscle of the arthropodBalanus nubilus Darwin (giant barnacle) have been purified (Potteret al., 1987; Collinset al., 1991). Both isoforms were present in all of the white striated muscle fibres studied but not in the red fibres. The ratio of BTnC₂ to BTnC₁ in different fibre types varied between 31 and 11. Both forms of TnC could be readily extracted from myofibrillar bundles of barnacle muscle in low ionic strength EDTA solutions, reducing force activation to <10%. Both forms either separately or together reassociated with the TnC-depleted fibres in a relaxing (LR) solution (pCa>8.0, [Mg²⁺] free=1mm, I=0.15m), and the reconstituted fibres could be subsequently activated in contraction (LA) solution (pCa=< 3.8, [Mg²⁺] free=1mm, I=0.15m,). The dissociation of BTnC 1+2 is blocked in low ionic strength solutions containing Mg²⁺ (10mm). The two isoforms of crayfish TnC (CrTnC₁ and CrTnC₂) were also found to be equivalent to the barnacle TnCs in their ability to reactivate TnC-depleted barnacle myofibrillar bundles. Similar experiments using rabbit skeletal muscle TnC (STnC) (I=0.15m) in BTnC-depleted myofibrillar bundles of barnacle showed considerable variability. STnC could associate, although weakly, with the depleted bundles in either LR or LA, and force could be partially restored. In neither situation was it as effective as either BTnC or CrTnC. Interestingly, bovine cardiac TnC (CTnC), although it did not associate at pCa>7.0, did associate and effectively activate force at pCa < 3.8, but dissociated on return to pCa>7.0 (LR). Neither barnacle TnC isoform associated with TnC-depleted skinned fibres from rabbit skeletal muscle at pCa>7.0, but did associate and activate these fibres at pCa<3.8. Once these fibres were returned to LR and then placed in LA at pCa 3.8 all BTnC-restored force was lost, indicating a dissociation of BTnC once the Ca²⁺ is lowered, as observed with CTnC in barnacle myofibrillar bundles. Finally, the inhibitory effect of BTnI on force and the absence of an effect of calmodulin, trifluoperazine or ATP--S on force were all taken as evidence for a thin filament regulated Ca²⁺ control system.     

Calcium-induced inactivation of calcium release from the sarcoplasmic reticulum of skeletal muscle

The ability of myofilament space Ca²⁺ to modulate Ca²⁺ release from the sarcoplasmic reticulum (SR) of skeletal muscle was investigated. Single fibers of the frog Rana pipiens belindieri were manually skinned (sarcolemma removed). Following a standard load and pre-incubation in varying myoplasmic Ca²⁺ concentrations, SR Ca²⁺ release was initiated by caffeine. Ca²⁺ release rates were calculated from the changes in absorbance of a Ca²⁺ sensitive dye, antipyrylazo III. An apparent dissociation constant (K _d) for dye-Ca²⁺ binding of 8000 M² was determined by comparing the buffering action of the dye with that of ethylenebis(oxonitrilo)tetraacetate (EGTA) using the contractile proteins of the skinned fiber as a measure of free Ca²⁺. This value for K _d was used in the calculation of Ca²⁺ release rates. As the myoplasmic space Ca²⁺ was increased from pCa 7.4, Ca²⁺ release rates declined sharply such that at pCa 6.9 the calculated release rate was 72±3% (mean ± SEM) of control (pCa 8.4). Further increases in myoplasmic Ca²⁺ from pCa 6.9 to pCa 6.1 did not result in a further decline in release rate. The effect of a decreased driving force on Ca²⁺ ions was investigated to determine whether it could account for the change in release rates observed. At pCa 6.9, where the greatest degree of inactivation occurred, the measured effects of a change in driving force could account for at most 40% of the observed inactivation. Varying concentrations of Ba²⁺ and Sr²⁺ in the myofilament space had no inactivating effect on the SR Ca²⁺ release rates. The ability of myofilament Ca²⁺ to inhibit SR Ca²⁺ release at concentrations normally encountered during muscle activation suggests a role for released Ca²⁺ as a modulator of the SR Ca²⁺ channel.     

Regulation of force and shortening velocity by calcium and myosin phosphorylation in chemically skinned smooth muscle

 The phosphatase inhibitor okadaic acid (OA) was used to study the relationship between [Ca²⁺], rates of phosphorylation/dephosphorylation and the mechanical properties of smooth muscle fibres. Force/velocity relationships were determined with the isotonic quick release technique in chemically skinned guinea-pig taenia coli muscles at 22° C. In the maximally thiophosphorylated muscle neither OA (10 μM) nor Ca²⁺ (increase from pCa 9.0 to pCa 4.5) influenced the force-velocity relationship. When the degree of activation was altered by varying [Ca²⁺] in the presence of 0.5 μM calmodulin, both force and the maximal shortening velocity (V _max) were altered. At pCa 5.75, at which force was about 35% of the maximal at pCa 4.5, V _max was 55% of the maximal value. When OA was introduced into fibres at pCa 6.0, force was increased from less than 5% to 100% of the maximal force obtained in pCa 4.5. The relationship between the degree of myosin light chain phosphorylation and force was similar in the two types of activation; varied [OA] at constant [Ca²⁺] and at varied [Ca²⁺]. The relation between force and V _max when the degree of activation was altered with OA was almost identical to that obtained with varied [Ca²⁺]. The results show that Ca²⁺ and OA do not influence force or V _max in the maximally phosphorylated state and suggest that the level of myosin light chain phosphorylation is the major factor determining V _max. The finding that the relationship between force and V _max was similar when activation was altered with OA and Ca²⁺ suggests, however, that alterations in the absolute rates of phosphorylation and dephosphorylation at a constant phosphorylation level do not influence the mechanical properties of the skinned smooth muscle fibres. Received: 1 December 1995 / Received after revision: 20 June 1996 / Accepted 12 July 1996     

Force-velocity relation in chemically skinned rat portal vein

Rat portal veins were chemically skinned using Triton X-100 and mounted for isometric and quick release experiments at 20°C. The skinned preparations were activated by Ca²⁺ (EGTA-buffered) in solutions containing 2 mM free-Mg²⁺ and 1 M calmodulin. Half maximal isometric force was obtained at pCa=6.2. Maximal force of the skinned preparations, at pCa=4.5, was 8.2±0.8 mN/mm² (n=6). Force-velocity relations were determined at varied Ca²⁺-concentrations. Maximal shortening velocity (V _max) was 0.10±0.01 lengths/s at pCa=4.5. At decreasing Ca²⁺-levelsV _max decreased (at pCa=6.25,V _max=0.05 l/s). At pCa =9 an increased level of free-Mg²⁺ (15mM) induces a slow and submaximal increase in tension. Force velocity relations of Mg²⁺-induced contractures were not different from those of Ca²⁺-contractures of similar magnitude (pCa=6.3). The results indicate that the degree of activation of the contractile system, as regulated by Ca²⁺ and Mg²⁺, influences the kinetic properties of the actomyosin interaction as well as the force development.     

Differential effects of arginine,glutamate and phosphoarginine on Ca<Superscript>2+</Superscript>-activation properties of muscle fibres from crayfish and rat

The effects of two amino acids, arginine which has a positively charged side-chain and glutamate which has a negatively charged side-chain on the Ca²⁺-activation properties of the contractile apparatus were examined in four structurally and functionally different types of skeletal muscle; long- and short-sarcomere fibres from the claw muscle of the yabby (a freshwater decapod crustacean), and fast- and slow-twitch fibres from limb muscles of the rat. Single skinned fibres were activated in carefully balanced solutions of different pCa (-log₁₀[Ca²⁺]) that either contained the test solute (“test”) or not (“control”). The effect of phosphoarginine, a phosphagen that bears a nett negative charge, was also compared to the effects of arginine. Results show that (i) arginine (33-36 mmol l^-1) significantly shifted the force–pCa curve by 0.08–0.13 pCa units in the direction of increased sensitivity to Ca²⁺-activated contraction in all fibre types; (ii) phosphoarginine (9–10 mmol l^-1) induced a significant shift of the force–pCa curve by 0.18–0.24 pCa units in the direction of increased sensitivity to Ca²⁺ in mammalian fast- and slow-twitch fibres, but had no significant effects on the force–pCa relation in either long- or short-sarcomere crustacean fibres; (iii) glutamate (36–40 mmol l^-1), like arginine affected the force–pCa relation of all fibre types investigated, but in the opposite direction, causing a significant decrease in the sensitivity to Ca²⁺-activated contraction by 0.08–0.19 pCa units; (iv) arginine, phosphoarginine and glutamate had little or no effect on the maximum Ca²⁺-activated force of crustacean and mammalian fibres. The results suggest that the opposing effects of glutamate and arginine are not related to simply their charge structure, but must involve complex interactions between these molecules, Ca²⁺ and the regulatory and other myofibrillar proteins.     

The contribution of the sarcoplasmic reticulum Ca2+-transport ATPase to caffeine-induced Ca2+ transients of murine skinned skeletal muscle fibres

The present study was carried out to investigate the contribution of the Ca²⁺-transport ATPase of the sarcoplasmic reticulum (SR) to caffeine-induced Ca²⁺ release in skinned skeletal muscle fibres. Chemically skinned fibres of balb-C-mouse EDL (extensor digitorum longus) were exposed for 1 min to a free Ca²⁺ concentration of 0.36 μM to load the SR with Ca²⁺. Release of Ca²⁺ from the SR was induced by 30 mM caffeine and recorded as an isometric force transient. For every preparation a pCa/force relationship was constructed, where pCa = −log₁₀ [Ca²⁺]. In a new experimental approach, we used the pCa/force relationship to transform each force transient directly into a Ca²⁺ transient. The calculated Ca²⁺ transients were fitted by a double exponential function: Y ₀ + A ₁⋅exp (−t/t ₁) + A ₂⋅exp(t/t ₂), with A ₁ < 0 < A ₂, t ₁ < t ₂ and Y ₀, A ₁, A ₂ in micromolar. Ca²⁺ transients in the presence of the SR Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA) were compared to those obtained in the absence of the drug. We found that inhibition of the SR Ca²⁺-ATPase during caffeine-induced Ca²⁺ release causes an increase in the peak Ca²⁺ concentration in comparison to the control transients. Increasing CPA concentrations prolonged the time-to-peak in a dose-dependent manner, following a Hill curve with a half-maximal value of 6.5 ± 3 μM CPA and a Hill slope of 1.1 ± 0.2, saturating at 100 μM. The effects of CPA could be simulated by an extended three-compartment model representing the SR, the myofilament space and the external bathing solution. In terms of this model, the SR Ca²⁺-ATPase influences the Ca²⁺ gradient across the SR membrane in particular during the early stages of the Ca²⁺ transient, whereas the subsequent relaxation is governed by diffusional loss of Ca²⁺ into the bathing solution. Received: 2 February 1996/Accepted: 1 April 1996     

Influence of caffeine,Ca2+, and Mg2+ on ryanodine depression of the tension transient in skinned myocardial fibers of the rabbit

Ryanodine, a blocker for Ca²⁺-release channels of the sarcoplasmic reticulum (SR Ca²⁺-release channels), induces depression of myocardial contraction in isolated intact muscle, which is consistent with depression of the caffeine-induced tension transient in skinned muscle fibers. In isolated SR, ryanodine binds to a specific receptor with high affinity, and this binding is enhanced by caffeine and increasing Ca²⁺ and decreased by increasing Mg²⁺. The aim of this study was to test the hypothesis that depression of myocardial contraction is mediated by changes in ryanodine-receptor binding properties. Accordingly, factors (caffeine, Ca²⁺, and Mg²⁺) affecting ryanodine-receptor binding properties in the isolated SR membrane were studied in skinned myocardial fibers from adult rabbits. The depression of the caffeine-induced tension transient by ryanodine (ryanodine depression) influenced by these three factors was measured. In a dose-dependent manner, increasing caffeine or Ca²⁺ concentrations enhanced the ryanodine depression. The concentrations for 50% ryanodine depression (IC₅₀) approximated 7mM for caffeine, and pCa 5.25 for Ca²⁺. When 1 M ryanodine and 25 mM caffeine were combined, ryanodine depression was independent of Ca²⁺ at low Ca²⁺ concentrations (20%–30% at pCa>8 and 7.5) and was a direct function of Ca²⁺ at higher concentrations (pCa 7.5–6.0 with IC₅₀ approx. pCa 6.75). In contrast, increasing Mg²⁺ reduced the ryanodine depression with IC₅₀ approximately equal to pMg 3.3. In conclusion, the caffeineor Ca²⁺-enhanced, and Mg²⁺-reduced ryanodine depression observed in this study is consistent with known ryanodinereceptor binding properties.     

Troponin C regulates the rate constant for the dissociation of force-generating myosin cross-bridges in cardiac muscle

It is well known that cardiac troponin C (cTnC) regulates the association of force-generating myosin cross-bridges. We report here evidence for an additional role for cTnC. This hypothesis states that Car²⁺ binds more strongly to cTnC when force-generating myosin cross-bridges are attached to actin and that removal of this bound Ca²⁺ accelerates the dissociation of force-generating myosin cross-bridges. Intact Fura-2-loaded rat papillary muscles and skinned (permeabilized) ventricular preparations were used. The preparations were mounted in the Guth Muscle Research System which is capable of measuring simultaneously fluorescence and force in response to length perturbations. All mechanical perturbations of muscle length (isotonic shortening, quick stretches and releases, and length vibrations) which cause dissociation of force-generating myosin cross-bridges during a twitch resulted in Ca²⁺ being released from troponin as judged from changes in the Ca²⁺ transients (Fura-2 (340/380) fluorescence ratio). Thus dissociation of force-generating myosin cross-bridges cause Ca²⁺ to be released from cTnC. Conversely, it would be expected that removal of strongly bound Ca²⁺ from cTnC would result in an increase in the rate of dissociation of force-generating myosin cross-bridges. To test this hypothesis actomyosin ATPase (NADH fluorescence change) and isometric force were measured in skinned cardiac preparations. The ratio of the ATPase/Force is proportional to the rate constant (g_app) for the dissociation of force-generating myosin cross-bridges. The data showed that decreasing the amount of Ca²⁺ bound to cTnC in skinned cardiac fibers caused an increase in the ratio of ATPase/Force, the rate of dissociation (g_app) of force-generating myosin cross-bridges.     

A revised method of troponin exchange in permeabilised cardiac trabeculae using vanadate: functional consequences of a HCM-causing mutation in troponin I

In order to incorporate human cardiac troponin I (TnI) and troponin C (TnC) into guinea pig skinned cardiac trabeculae, fibres were treated with vanadate to extract endogenous TnI and TnC using established protocols. After addition of human TnI and TnC force was inadequately restored and it was found that the vanadate treatment had unexpectedly also removed some troponin T. To recover Ca²⁺-sensitive force, the fibres had to be incubated with all three troponin subunits. Using this revised method, the hypertrophic cardiomyopathy-causing mutation TnI Gly203Ser had no significant effect on Ca²⁺-sensitivity of force production, contrasting with our earlier report of decreased Ca²⁺-sensitivity which was likely caused by the unexpectedly harsh effect of vanadate.     

Myosin light chain phosphorylation and the cross-bridge cycle at low substrate concentration in chemically skinned guinea pigTaenia coli

Force-velocity relations, rate of ATP turnover (J_ATP), and phosphorylation of the 20,000 D myosin light chains (LC₂₀) were measured in chemically skinned guinea pigTaenia coli. Relative LC₂₀ phosphorylation at 3.2 mM MgATP was 17% in relaxed tissues at pCa 9, and increased with force at increasing [Ca²⁺] to a maximum of 67% at pCa 4.5. Force at pCa 4.5 was dependent on the MgATP concentration with a half-maximal response at about 0.1 mM. At 0.1 mM MgATP LC₂₀ phosphorylation at pCa 4.5 was 38%. Both J_ATP and the maximal shortening velocity (V _max) were reduced in 0.1 mM MgATP, to 32% and 43%, respectively, of their values at 3.2 mM MgATP. Low-MgATP thus inhibits both LC₂₀ phosphorylation and the extent and rate of cross-bridge interaction. High levels of LC₂₀ phosphorylation, independent of Ca²⁺ and MgATP concentrations, were obtained by treatment with ATP--S. Maximal force at 3.2 mM MgATP after LC₂₀ thiophosphorylation was unchanged, whereas halfmaximal force occurred at 0.065 mM MgATP after thiophosphrylation, compared to 0.13 mM after activation by Ca²⁺. The contraction in thiophosphorylated preparations at low-MgATP (0.1 mM) was associated with submaximalV _max (60%) and J_ATP (27%). The results show that LC₂₀ phosphorylation is correlated with the degree of force development in the Ca²⁺ activated contraction, both when Ca²⁺ and MgATP concentrations are varied. The reduced force and rate of crossbridge turnover in lowMgATP are however primarily mediated by an influence of MgATP on the cross-bridge cycle, which is separate from the effect on LC₂₀ phosphorylation.     

A novel method of extraction of TnC from skeletal muscle myofibrils

 Incubation of mechanically skinned barnacle myofibrillar bundles in 10 mM orthovanadate (pH 6.6) results in the loss of Ca²⁺-dependent force generation, which reduces to 0.98±0.006% (mean ±SEM, n=25) of control levels. Analysis of myofibrillar bundles by gel electrophoresis showed that tension loss is primarily due to the extraction of troponin C (TnC) (65.4±5.04% mean ±SEM, n=5). This is a novel finding, since treating cardiac fibres with orthovanadate results in the removal of both TnC and troponin I (TnI) (28). Ca²⁺ dependence was restored to the myofibrillar bundles following reconstitution with either native isoform of barnacle TnC (BTnC₁: 78.72±12.8%, n=9, BTnC₂: 82.73±20.3%, n=3). The reversible loss of Ca²⁺-dependent tension generation following the removal and replacement of TnC indicates that the regulation of contraction in the barnacle is controlled by thin-filament regulatory proteins. Received: 30 September 1998 / Accepted: 16 December 1998     

Myofibrillar Ca2+ sensitivity of cardiomyopathic hamster hearts

We studied the Ca²⁺ responsiveness of skinned muscle fibre preparations from the right and left ventricles of normal (FIB) and genetically cardiomyopathic (Bio-To-2) Syrian hamsters. Thus, we compared the Ca²⁺/force relationships of preparations from myopathic hamsters to those of age-matched (11–16 months old) normal animals. The pCa (i.e. –log₁₀[Ca²⁺]) required for 50% force activation (Ca²⁺ sensitivity) was higher in the myopathic hamsters than in controls (pCa₅₀ values of 5.3±0.03 and 5.17±0.04, respectively); this difference might be due to an alteration in regulatory proteins. Indeed, after extraction (with vanadate) and replacement of troponin I with bovine cardiac troponin the pCa₅₀ values were similar (pCa 5.35) to those of bovine ventricular fibres. The Ca²⁺ sensitizer EMD 53998 (10 M) increased Ca²⁺ sensitivity in preparations from normal and cardiomyopathic hamsters equally, by 0.4 pCa units. Incubation of fibre bundles with the catalytic subunit of cyclicadenosine-monophosphate-dependent protein kinase decreased Ca²⁺ sensitivity, thereby normalizing the enhanced Ca²⁺ responsiveness of fibres from cardiomyopathic hamsters. It is not clear, however, whether the pathologically increased Ca²⁺ sensitivity of the hearts of aged myopathic hamsters reflects a maladaptation, or a compensatory mechanism of the failing heart.