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.     

Replacement of troponin-I in slow-twitch skeletal muscle alters the effects of the calcium sensitizer EMD 53998

 We extracted troponin-I (TnI) from skinned rat and rabbit soleus muscle fibres using a modification of the method described by Strauss et al. (FEBS Lett 310:229–234, 1992) for replacement of TnI in cardiac preparations. Incubation of soleus muscle fibres with 10 mmol/l vanadate virtually completely abolished the Ca²⁺dependence of force. Immunoblot analysis revealed that more than 80% of TnI had been extracted from the preparations. The Ca²⁺dependence of force was restored by incubation with a complex of cardiac TnI (cTnI) and troponin-C (cTnC). We examined the effects of the Ca²⁺-sensitizing compound EMD 53998 on isometric tension in native porcine cardiac and rabbit soleus skinned fibres as well as soleus in which the endogenous slow skeletal TnI (ssTnI) had been replaced by cTnI (soleus–cTnI). It was found that 10 μmol/l EMD 53998 in native soleus increased maximum Ca²⁺-activated force to 120±1.4% of control. In soleus–cTnI fibres, maximum force was increased to only 105±0.9%, which was similar to the effect observed in cardiac muscle (108±0.6%). In cardiac muscle, 10 μmol/l EMD 53998 induced a leftward shift of the pCa-tension relation by 0.65 log units. In native soleus, ΔpCa was only 0.40. Again, the effect of EMD 53998 on soleus–cTnI (ΔpCa=0.56) more closely resembled the response found in cardiac muscle than that observed in native soleus muscle. The apparent TnI-isoform dependence of the effects elicited by EMD 53998 suggests that its actions are modulated by the regulatory proteins of the thin filament. Received: 15 December 1997 / Accepted: 16 March 1998     

Inhibition of TnI-TnC interaction and contraction of skinned muscle fibres by the synthetic peptide TnI [104–115]

Circular dichroism was used to study the induction of helix in TnC or TnI-TnC by the TnI peptide [104–115] at various Ca²⁺ concentrations. The increase in negative ellipticity and pCa²⁺ values for the peptide-TnC complex, indicates that binding of the peptide to TnC, induces a small helical conformational change in TnC. This results in an increase in the Ca²⁺ binding constant and the pCa₅₀ value required to induce 50% of Ca²⁺-dependent helix in TnC. The introduction of the peptide to a preformed mixture of TnI-TnC resulted in an increase in negative ellipticity and a decrease in the pCa₅₀ and the apparent Ca²⁺ binding constant towards the values obtained for the TnI peptide-TnC complex and away from those of TnI-TnC. This demonstrates that the TnI peptide can successfully compete with TnI for TnC and thereby inhibit the TnI-TnC interaction. The addition of the TnI peptide to skinned rabbit psoas or porcine cardiac fibres resulted in the inhibition of the force development and a decrease in the pCa₅₀ values required for 50% Ca²⁺ activation. The magnitude of the inhibition of tension development and the shift in the Ca²⁺ sensitivity for skinned cardiac muscle fibres was approximately half that observed with skeletal muscle fibres. In view of the CD findings, these skinned fibre results can be accounted for by the peptide inhibiting the TnI interaction with TnC. However, it is possible that the TnI peptide also has a direct inhibitory effect on TM-actin. Mastoparan, another TnC binding peptide, also inhibited the tension development in skinned skeletal and cardiac muscle fibres, but was much less efficient than the TnI peptide.Abbreviations used Boc N-tert-butyloxycarbonyl - Tn troponin - TM tropomyosin - TnI troponin I - TnC troponin C - HPLC high performance liquid chromatography - DIEA diisopropylethylamine - TFA trifluoroacetic acid - DCC dicyclohexylcarbodiimide - 2-Cl-Z 2-chlorobenzyloxycarbonyl - Tos 4-toluenesulfonyl - EGTA ethylene glycol bis(-amino ethyl ether) N,N,N,N-tetraacctic acid - DTE 1,2-ethanedithiol - ME -mercaptoethanol - S1 myosin subfragment 1 - acto-S1 actin and myosin subfragment This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie, and by grants from the Medical Research Council of Canada, the Alberta Heart Foundation and a studentship (J. V. E.) from the Alberta Heritage Foundation for Medical Research     

Fluorescence changes on contractile activation in TnCDANZ labeled skinned rabbit psoas fibers

The increase in fluorescence of dansylaziridine (DANZ) labeled troponin C (TnC_DANZ) substituted into skinned rabbit psoas fibers was determined as a function of the pCa. The fluorescence data are expressed as the ratio of two wavelength bands, one that sees the fluorescence of TnC_DANZ, and one that sees background fluorescence and scatter. The percent TnC replaced with TnC_DANZ was varied between 10 and 50% and, the fibers were randomly stretched, at the start of each experiment, between 10 and 50%. A large ratio increase accompanies increase in [Ca²⁺]. The pCa/force data are best fit by the Hill equation but the pCa/ratio data are best fit by a model in which Ca²⁺ binds in two phases. The position of the force curve on the pCa axis varies little between fibers, in contrast to that of the ratio or Δ-fluorescence curve. In accord with previous reports the Δ-fluorescence can be left of the force on the pCa axis (type I) or superimpose in part on the force (type II). Not described previously, we find curves in which the second phase of the ratio cross-over the pCa/force curve. This type III relationship is found only in fibers less than 3 weeks postmuscle harvest. We propose that the first, relatively invariant, phase of the biphasic pCa/ratio curve accompanies Ca²⁺ binding to either of the two low affinity sites on TnC_DANZ as it does for TnC in solution. The second, highly cooperative, phase of the ratio curve that accompanies muscle contraction and enhanced Ca²⁺ binding is initiated when sufficient Ca²⁺ is bound to overcome inhibitory systems. Loose coupling between the initial Ca²⁺ binding and the cooperative switch point may account for much of the variation in the shape and position of the pCa/ratio curve. There is evidence that, in the overlap zone, weakly attached myosin cross-bridges enhance cooperation between the regulatory units of the thin filaments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cross-bridge movement in rat slow skeletal muscle as a function of calcium concentration

A single fibre bundle from rat soleus muscle was chemically skinned with saponin and the transfer of myosin heads from the thick filaments to the thin filaments at a sarcomere length of 2.4 μm was measured as a function of Ca²⁺ concentration using an x-ray diffraction method at 4–7 °C. In the relaxed state, the 1,0 spacing was 42.08 nm. The spacing showed no significant decrease when the Ca²⁺ concentration was below the threshold (−log₁₀ [Ca²⁺] or pCa 5.8). No significant transfer of the myosin heads occurred when the Ca²⁺concentration was below the threshold (pCa 5.8). When the muscle was maximally activated at pCa 4.4, the spacing decreased to 40.35 nm. During the maximum isometric contraction at pCa 4.4, 54.9 ± 6.5% (±SE of the mean) of the myosin heads were transferred to the thin filaments. The transfer of the myosin heads was approximately proportional to relative tension. These results suggest that myosin heads of both fast-twitch and slow-twitch skeletal muscles transferred on the common movement as a function of Ca²⁺ concentration. Received: 1 December 1995/Received after revision and accepted: 20 May 1996     

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     

Modulation of sarcoplasmic reticulum Ca2+-release channels by caffeine,Ca2+, and Mg2+ in skinned myocardial fibers of fetal and adult rats

Ryanodine causes depression of the caffeine-induced tension transient (ryanodine depression) in skinned muscle fibers, because it blocks the sarcoplasmic reticulum (SR) Ca²⁺-release channels [Su, J. Y. (1988) Pflügers Arch 411:132–136, 371–377; (1992) Pflügers Arch 421:1–6]. This study was performed to examine the sensitivity of SR Ca²⁺-release channels to ryanodine in fetal compared to adult myocardium and to investigate the influence of Ca²⁺, caffeine, and Mg²⁺ on ryanodine depression in skinned fibers. Ryanodine (0.3 nM–1 M) caused a dose-dependent depression in skinned myocardial fibers of the rat, and the fetal fibers (IC₅₀74 nM) were 26-fold less sensitive than those of the adult (IC₅₀2.9 nM). The depression induced by 0.1 M or 1 M ryanodine was a function of [caffeine], or [Ca²⁺] (pCa<6.0), which was potentiated by caffeine, and an inverse function of [Mg²⁺]. At pCa>8.0 plus 25 mM caffeine, a 20% ryanodine depression was observed in both the fetal and adult fibers, indicating independence from Ca²⁺. Ryanodine depression in skinned fibers of the fetus was less affected than that seen in the adult by pCa_i, [caffeine]_i, or 25 mM caffeine plus pCa_i or plus pMg_i (IC₅₀pCa 4.5 versus 5.1; caffeine 12.7 mM versus 2 mM; pCa 6.7 versus 7.3; and pMg 3.9 versus 3.3 respectively). The results show that the SR Ca²⁺-release channel in both fetal and adult myocardium is modulated by Ca²⁺, caffeine, and Mg²⁺. It is concluded that less ryanodine depression seen in the skinned fibers of the fetus, indicating a relatively insensitive SR Ca²⁺-release channel, could contribute to the resistance of intact myocardium to ryanodine.     

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.     

Characterisation of a mutant of barnacle troponin C lacking Ca2+-binding sites at positions II and IV

 This study investigates a mutant barnacle troponin C (TnC) protein (BTnC2–4-) in which the Ca²⁺-binding sites (sites II and IV) have been rendered non-functional. Eliminating Ca²⁺ binding at both Ca²⁺-binding sites of barnacle TnC did not prevent the incorporation of BTnC2–4- into TnC-depleted myofibrillar bundles, although, as expected, the mutant was not able to effect muscle regulation. We conclude that the Mg²⁺ involved in stabilising the interaction between TnC and TnI in the barnacle must bind at a separate location to the Ca²⁺-binding sites. Competition experiments between BTnC2–4- and wild-type barnacle TnC (BTnCWT) or the native isoform BTnC₂ indicate that BTnC2–4- has an approximately fourfold higher affinity for barnacle TnI than BTnCWT but a lower affinity for TnI compared to BTnC₂. These results indicate that disabling sites II and IV changes the affinity of BTnC2–4- for TnC-denuded barnacle myofibrils, altering the stability of the bond formed between TnC and the thin filament. Received: 30 September 1998 / Received after revision: 12 February 1999 / Accepted: 15 February 1999     

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     

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.     

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.     

Spectrofluorometric analysis of length-dependent conformational changes in cardiac troponin C

Length modulation of cardiac muscle is manifested in the Frank–Starling relation of the heart. Recently, it has been shown that length-dependent changes in SH reactivity of cardiac troponin C (cTnC) occurred in association with cross-bridge attachment and Ca²⁺. However, the presence of two SH groups (Cys-35 and Cys-84) in the regulatory region of cTnC complicates efforts to detect conformational changes. In this study skinned porcine cardiac fibers were reacted with 7-diethylamino-3-[4′maleimidylphenyl]-4-methylcoumarin (CPM). Alkaline urea gel electrophoresis, along with protein elution, was used to isolate filament bound cTnC. Analysis of fluorescence measurement showed that there is a Ca²⁺-increased fluorescence for CPM-labeled cTnC in long fibers (sarcomere length = 2.2 ∼ 2.5 μm) but not in short fibers (sarcomere length = 1.6 ∼ 1.8 μm). In addition, the labeled cTnC was measured for the fluorescence decrease over time by adding a non-fluorescence energy acceptor, 4-dimethylaminophenylazophenyl-4′maleimide (DABMI), in the presence and absence of Ca²⁺. Fluorescence quenching by DABMI is not affected by Ca²⁺ in long fibers but it is significantly increased in short fibers. However, the fibers maintained in the relaxed state with 5 mM MgATP and 1 mM Vanadate showed no length effect on the CPM-labeled cTnC in terms of the Ca²⁺-mediated changes in fluorescence spectrum and in fluorescence quenching by DABMI. All together, our results suggest that the relative reactivities of Cys-35 and Cys-84 vary with sarcomere length. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of troponin-C interactions in skinned barnacle muscle: comparison with troponin-C from rabbit striated muscle

Previously it was shown that when troponin-C (TnC) is extracted from barnacle myofibrillar bundles they lose their Ca2+ sensitivity, which can be restored by adding back barnacle TnC (either isoform, BTnC1 or BTnC2). Thus barnacle muscle shows thin filament regulation, as does rabbit psoas skeletal muscle. In this paper we comp are the interactions of barnacle and rabbit fast muscle TnC in their respective muscles. We demonstrate that muscle fibres from the giant barnacle, Balanus nubilus, contain about 186 μm kg−1 muscle tissue of BTnC1 plus BTnC2 compared to about 91 μm kg−1 of TnC in rabbit psoas muscle fibres. Extraction of BTnC is achieved using similar low ionic strength, low divalent ion Ca2+-low Mg2+ conditions which are required for TnC extraction in rabbit psoas skinned muscle fibres; extraction was prevented by 1 mm Mg2+. Full reconstitution of Ca2+-sensitivity was achieved by adding back BTnC (1 + 2, or 2). Reconstitution of barnacle muscle with rabbit fast skeletal TnC (RTnC) was more complex, with partial recovery of Ca2+-sensitivity with reconstitution in the presence of 3 mm Mg2+ and more fully with reconstitution in the presence of activating Ca2+ (pCa 4.0). This suggests that the barnacle TnC-TnI (troponin I) recognition sites may be more complex than in rabbit because the barnacle sites appear to have at least two different conformations or types, in which one recognizes RTnC in the presence of Mg2+ and the other only in the presence of Ca2+ and Mg2+. This is consistent with the presence of several TnI isoforms in barnacle striated myofibrils. RTnC has two C-terminal Ca2+-Mg2+ binding sites that are thought to be involved in the Mg2+-sensitive binding of RTnC in rabbit muscle, yet it has been suggested that this site in barnacle muscle does not bind Mg2+, even though Mg2+ stabilizes BTnC binding in barnacle muscle. Consistent with this stabilizing action of Mg2+, using fluorescent probes IAANS or IAE on isolated BTnC2 we demonstrate that BTnC2 binds both Ca2+ and Mg2+, but the data do not suggest direct competition. Consistent with the C-terminal sites on BTnC being Ca2+-specific, BTnC1+2 could only reconstitute low levels of force (about 1/3) in TnC-extracted rabbit skinned muscle fibers in the presence of pCa 4.0 (not just Mg2+) and only at low ionic strengths (0.09 m). Ca2+-activation of contraction was further examined using fluorescently labelled BTnC2 (labelled with IANBD) incorporated into skinned barnacle myofibrillar bundles. Maximal Ca2+ binding produced structural changes in BTnC which resulted in a 45% decrease in the fluorescence compared to the value at pCa 9.2. The magnitude of the fluoresence decrease paralleled the increase in force with increas ing Ca2+. The Hill fits to the data gave pCa1/2 and n of 5.61 ± 0.02 and 2.06 ± 0.12 for force, and 5.52 ± 0.02 and 1.88 ± 0.10 for fluoresence. Removing MgATP to induce rigor in the fibre decreased BTnC2-NBD fluorescence only about 11%, but the addition of Ca2+ in rigor further decreased the fluorescence to a slightly larger extent than under maximal Ca2+ activating conditions. These fluorescence changes are qualitatively similar to the fluorescence enhancement seen with Ca2+-activation and rigor with RTnCDanz exchanged into rabbit psoas skinned muscle fibres. The data support a similar model for Ca2+-activation of force in barnacle muscle and in rabbit psoas skeletal muscle fibres This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of thapsigargin and cyclopiazonic acid on sarcoplasmic reticulum Ca2+ uptake,spontaneous force oscillations and myofilament Ca2+ sensitivity in skinned rat ventricular trabeculae

Thapsigargin (TG) and cyclopiazonic acid (CPA) have been reported to be potent inhibitors of the sarcoplasmic reticulum (SR) Ca²⁺ uptake in isolated SR vesicles and cells. We have examined the effect of TG and CPA on (1) the Ca²⁺ uptake by the SR in saponin-skinned rat ventricular trabeculae, using the amplitude of the caffeine-induced contraction to estimate the Ca²⁺ content loaded into the SR, (2) the spontaneous Ca²⁺ oscillations at pCa 6.6 using force oscillation as the indicator, and (3) the myofilament Ca²⁺ sensitivity in Triton X-100-treated preparations. Inhibition of Ca²⁺ loading by TG and CPA increased with time of exposure to the inhibitor over 18–24 min. TG and CPA produced half inhibition of Ca²⁺ loading at 34.9 and 35.7 μM respectively, when 18–24 min were allowed for diffusion. The spontaneous force oscillations were more sensitive to the inhibitors: 10 μM TG and 30 μM CPA both abolished the oscillations in this time. The myofilament Ca²⁺ sensitivity was not affected by 10 and 300 μM TG or CPA. The results show that the concentrations of TG and CPA necessary to inhibit the SR Ca²⁺ uptake of skinned ventricular trabeculae are much higher than the reported values for single intact myocytes. One reason for this may be slow diffusion of the inhibitors into the multicellular trabecula preparation. Received: 28 July 1995/Received after revision: 11 December 1995/Accepted: 18 December 1995     

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.     

Effects of glycine and proline on the calcium activation properties of skinned muscle fibre segments from crayfish and rat

The effects of the polar amino acid glycine (20 mmol l⁻¹) and the non-polar amino acid proline (20 mmol l⁻¹) on Ca²⁺-activated contraction have been examined in four types of striated muscle fibres. Single fibres dissected from the claw muscle of a crustacean (long- and short-sarcomere) and the hindlimb muscles of the rat (slow-twitch from soleus and fast-twitch from extensor digitorum longus) were activated in matched solutions that either contained the amino acid (‘test’) or not (‘control’). The steady-state force produced in these solutions was used to determine the relation between force production and pCa (−log₁₀[Ca²⁺]). The results show that in the concentrations used, glycine and proline had only small effects on the maximum Ca²⁺-activated force, pCa corresponding to 10, 50 and 90% maximum force (pCa₁₀, pCa₅₀, pCa₉₀, respectively) or on the slope of the force-pCa curves in the four different fibre types. The relative lack of effects of glycine and proline on contractile activation would confer a distinct physiological advantage to force production of muscle of Cherax, where the concentrations of glycine and proline vary considerably. Finally, the results show that glycine and proline may be useful to balance control solutions when the effects of other amino acids or zwitterions on contractile activation are examined. This revised version was published online in July 2006 with corrections to the Cover Date.     

Modulation of the ryanodine receptor sarcoplasmic reticular Ca2+ channel in skinned fibers of fast- and slow-twitch skeletal muscles from rabbits

This study was performed to compare skinned fibers from rabbit adductor magnus (AM) and soleus (SL) muscles with regard to the influence of caffeine, Ca²⁺ and Mg²⁺ on the depressive effects of ryanodine (RYA) on the caffeine-induced tension transients. Single skinned fibers were immersed in solution: to load Ca²⁺ into, and release Ca²⁺ from the SR (a load-release cycle). Three cycles were sequentially performed in each skinned fiber: (1) a control (no RYA) (2) a conditioning period in which activation was car ried out in the presence of ryanodine plus various con centrations of the modulators, i.e. caffeine, Ca²⁺ or Mg²⁺, and (3) a test (no RYA) which monitored the release activity retained after the conditioning cycle. The depressive effect of RYA was found to be a function of [ryanodine], [caffeine], or [Ca²⁺], and an inverse function of [Mg²⁺], where [caffeine] denotes concentration. The half-maximal effects of RYA in AM (5 M RYA) and SL (10 M RYA), respectively, occurred at a pCa₅₀ of 5.32 versus 5.43 without caffeine, or pCa₅₀ of 7.24 versus 6.88 and pMg₅₀ of 3.29 versus 3.61 with 25 mM caffeine, at a [caffeine] of 4.96 versus 7.29 mM, and at a [ryanodine] of 31.0 versus 101.6 M. Thus, the RYA depression in skinned muscle fibers is modulated by caffeine, Ca²⁺, and Mg²⁺ in both muscle types, and AM is at least two- to fourfold more sensitive than SL.     

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.     

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.