The 2010 Mw 8.8 Maule megathrust earthquake of Central Chile, monitored by GPS.

Large earthquakes produce crustal deformation that can be quantified by geodetic measurements, allowing for the determination of the slip distribution on the fault. We used data from Global Positioning System (GPS) networks in Central Chile to infer the static deformation and the kinematics of the 2010 moment magnitude (M(w)) 8.8 Maule megathrust earthquake. From elastic modeling, we found a total rupture length of ~500 kilometers where slip (up to 15 meters) concentrated on two main asperities situated on both sides of the epicenter. We found that rupture reached shallow depths, probably extending up to the trench. Resolvable afterslip occurred in regions of low coseismic slip. The low-frequency hypocenter is relocated 40 kilometers southwest of initial estimates. Rupture propagated bilaterally at about 3.1 kilometers per second, with possible but not fully resolved velocity variations.

Ca2+ sensitivity of stunned myocardium after skinning using retrograde infusion of detergent.

Myofilament Ca2+ desensitization contributes to the contractile dysfunction of ischemic/reperfused ("stunned") myocardium. We examined the presence of reduced Ca2+ sensitivity of isometric force in chemically skinned fibers obtained from stunned myocardium using different modes of applying the detergent Triton X-100. Langendorff-perfused rat hearts underwent 20 min ischemia/20 min reperfusion, which caused a 35 +/- 3% decrease in left ventricular developed pressure, compared to continuously perfused control hearts. Stunned and control hearts were randomly allocated to two different permeabilization protocols: In group A, trabeculae were dissected and immersed in skinning solution containing 1% Triton X-100 for 20 min. Group B hearts remained fixed to the aortic cannula and skinning solution was infused retrogradely for 6 min prior to dissection of trabeculae. Extraction of cytosolic marker proteins was more complete in group-B than in group-A preparations. Group-A preparations from stunned hearts exhibited significant Ca2+ desensitization (pCa50 = 5.07 and 5.15 in stunned and control myocardium, respectively). In group B no such difference was observed, all preparations showing higher Ca2+ sensitivity and maximum force than group-A preparations (pCa50 = 5.32 in stunned versus 5.33 in control hearts). Prolonging group-A skinning to 150 min also abolished the difference in Ca2+ sensitivity between stunned and control myocardium. In conclusion, compared to a conventional protocol, skinning by perfusion results in more complete permeabilization and better preservation of myocardial contractile function. Ischemia/reperfusion at this moderate degree of contractile dysfunction induces Ca2+ desensitization at least partially by factors that can be extracted by thorough skinning.

Force responses of skinned molluscan catch muscle following photoliberation of ATP.

Isometric force responses following flash photolysis of caged-ATP were measured from skinned preparations of the catch muscle anterior byssus retractor of Mytilus (ABRM). When fibres were transferred from Ca(2+)-free to Ca(2+)-containing rigor solution (pCa < 4) the force remained low, but flash photolysis produced an extended force increase (half-time, 0.30 +/- 0.07 s, n = 6). When Ca(2+)-activated fibres were transferred to a Ca(2+)-free rigor solution, their force remained at a high level. Flash photolysis produced a rapid force decay (half-time, 0.28 +/- 0.06 s, n = 9) to about 19% of the initial Ca(2+)-activated force. In the presence of 0.5 mM MgADP, the force increase was slowed down by a factor of 3 and the force decay by a factor of 5. These effects of MgADP on crossbridge kinetics are comparable to those observed in vertebrate smooth muscle and are thought to cause "latch", a catch-like state (Fuglsang et al. J Muscle Res Cell Motil 14:666-677, 1993). They are consistent with a model implicating competition between MgADP and MgATP for the nucleotide-binding site on crossbridges. Considering the relatively fast force responses induced by caged-ATP photolysis, even in the presence of MgADP, it appears unlikely that the detachment of crossbridges from the rigor state can account for catch-related processes. In view of the low myosin ATPase under maximal activating conditions (0.6 s-1, Butler et al. Biophys J 75:1904-1914, 1998), neither crossbridge attachment nor detachment of rigor crossbridges seems to be the rate-limiting processes of the crossbridge cycle.

Expression of vascular endothelial growth factor during the development of cardiac hypertrophy in spontaneously hypertensive rats.

Left ventricular hypertrophy (LVH) is often associated with an impaired maximal coronary blood flow and increases the vulnerability of the heart tissue to ischaemia. In this study, the correlation between coronary blood flow and expression of the vascular endothelial growth factor (VEGF) mRNA was investigated. Using both haemodynamic measurements and analysis of mRNA, we have demonstrated that during development of LVH, in spontaneously hypertensive rats (SHR), an impaired maximal coronary flow at 12 weeks of age is associated with low levels of VEGF mRNA. However, in older SHR (32 weeks) with stabilised hypertrophy and a normal maximal coronary flow response, VEGF mRNA levels are increased 3-fold. These results suggest that the mechanism for the impaired flow, observed in some types of cardiac hypertrophy, might involve an inadequate growth of the coronary vessels due to insufficient activation of the VEGF gene.

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 micromol/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 micromol/l EMD 53998 induced a leftward shift of the pCa-tension relation by 0.65 log units. In native soleus, DeltapCa was only 0.40. Again, the effect of EMD 53998 on soleus-cTnI (DeltapCa=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.

Striational autoantibodies in myasthenia gravis patients recognize I-band titin epitopes.

Myasthenia gravis (MG) patients develop autoantibodies primarily against the acetylcholine receptor in the motor endplate, but also against intracellular striated muscle proteins, notably titin, the giant elastic protein of the myofibrillar cytoskeleton. Titin antibodies have previously been shown to be directed against a single epitope on the molecule, located at the A-band/I-band junction and referred to as the main immunogenic region (MIR) of titin. By using immunofluorescence microscopy on stretched single myofibrils, we now report that approximately 40% of the sera from 18 MG/thymoma patients and 8 late-onset MG patients with thymus atrophy contain antibodies that bind to a more central I-band titin region. This region consists of homologous immunoglobulin domains and is known to be differentially spliced dependent on muscle type. All patients with I-band titin antibodies also had antibodies against the MIR. Although a statistically significant correlation between the occurrence of I-band titin antibodies and MG severity was not apparent, the results could hint at an initial immunoreactivity to titin's MIR, followed by reactivity along the titin molecule in the course of the disease.

Cardiac contractility: how calcium activates the myofilaments.

In both cardiac and skeletal muscle, the force-generating molecular motors (crossbridges) are turned on by increasing the intracellular free calcium level that regulates the troponin-tropomyosin system. However, calcium activation is a two-way process in the sense that activated crossbridges also affect the troponin-tropomyosin system. Here we review the mechanism of calcium action on myofilament proteins, particularly tropomyosin, that affects both the extent and the rate of force development and hence the contractility of the myocardium. At low myoplasmic Ca2+ concentrations tropomyosin is located at the edge of the thin filament, thereby interfering with the formation of strong actin-myosin linkages (blocked state). An increase in Ca2+ activity causes an azimuthal shift of tropomyosin around the filament (by about 30 degrees), thereby increasing the probability of low-force crossbridge interaction, a process which by cooperative effects induces further tropomyosin movement (by an additional 10 degrees) which results in the open state of the filament characterized by forceful crossbridge interaction. (This mechanism may be analogous to that in ligand-gated ion channels, where ligand binding increases the open probability of the pore.) The extent of activation then depends on the free Ca2+ concentration and on the calcium sensitivity of the thin filament that may be affected by protein phosphorylation, crossbridge attachment, the troponin isoform composition of the filament, and novel calcium-sensitizing drugs that act on the contractile or regulatory proteins and thus increase the force of the heart.

Actin-titin interaction in cardiac myofibrils: probing a physiological role.

The high stiffness of relaxed cardiac myofibrils is explainable mainly by the expression of a short-length titin (connectin), the giant elastic protein of the vertebrate myofibrillar cytoskeleton. However, additional molecular features could account for this high stiffness, such as interaction between titin and actin, which has previously been reported in vitro. To probe this finding for a possible physiological significance, isolated myofibrils from rat heart were subjected to selective removal of actin filaments by a calcium-independent gelsolin fragment, and the "passive" stiffness of the specimens was recorded. Upon actin extraction, stiffness decreased by nearly 60%, and to a similar degree after high-salt extraction of thick filaments. Thus actin-titin association indeed contributes to the stiffness of resting cardiac muscle. To identify possible sites of association, we employed a combination of different techniques. Immunofluorescence microscopy revealed that actin extraction increased the extensibility of the previously stiff Z-disc-flanking titin region. Actin-titin interaction within this region was confirmed in in vitro cosedimentation assays, in which multimodule recombinant titin fragments were tested for their ability to interact with F-actin. By contrast, such assays showed no actin-titin-binding propensity for sarcomeric regions outside the Z-disc comb. Accordingly, the results of mechanical measurements demonstrated that competition with native titin by recombinant titin fragments from Z-disc-remote, I-band or A-band regions did not affect passive myofibril stiffness. These results indicate that it is actin-titin association near the Z-disc, but not along the remainder of the sarcomere, that helps to anchor the titin molecule at its N-terminus and maintain a high stiffness of the relaxed cardiac myofibril.

Exogenous caldesmon promotes relaxation of guinea-pig skinned taenia coli smooth muscles: inhibition of cooperative reattachment of latch bridges?

In smooth muscle, the state of prolonged contraction (latch state) is associated with very slow energy turnover and cycling of crossbridges that are dephosphorylated. A similar state may be reproduced in skinned fibres when the calcium-induced contraction is terminated by calcium removal with ethylenebis(oxonitrilo)tetraacetate (EGTA) and, during the slow relaxation that follows, force is maintained by dephosphorylated crossbridges that cycle slowly or not at all and may cooperatively reattach after detachment (Khromov et al. 1995, Biophys J 69:2611-2622). In guinea-pig skinned taenia coli that has been pretreated by prolonged incubation with caldesmon (5 microM), the rate of relaxation is approximately 1.6 times greater than in untreated controls, with half-times of relaxation being 1.3 and 2.1 min, respectively. In contrast, preloading the fibres with calponin does not accelerate relaxation. Preloading the fibres with caldesmon also accelerates the relaxation of skinned fibres from the state of rigor contraction when the latter is terminated by immersion into an ATP-containing relaxing solution or, in the presence of inorganic phosphate (Pi), also by flash-photolytic release of ATP from caged-ATP. Even in the latter case, relaxation is comparatively slow, possibly because of cooperative reattachment of dephosphorylated crossbridges which delays net crossbridge detachment and hence relaxation. We propose that by inhibition of cooperative reattachment caldesmon accelerates relaxation, even in the presence of Pi, and that the latch-like state of skinned fibres is supported by dephosphorylated cooperatively attaching crossbridges and may be regulated by the activity of caldesmon in the smooth muscle cell.

Dissociation of left ventricular hypertrophy, beta-myosin heavy chain gene expression, and myosin isoform switch in rats after ascending aortic stenosis.

BACKGROUND: Reexpression of the fetal beta-myosin heavy chain (beta-MHC) gene was reported to be a marker for phenotypic reprogramming and cardiac hypertrophy in rats. Recent in vitro studies strongly suggested a role of angiotensin II for phenotypic reprogramming. In the present investigation, beta-MHC gene expression was studied in an experimental model of pressure-over-load hypertrophy that is not associated with a concurrent activation of the circulating renin-angiotensin system. METHODS AND RESULTS: Hypertrophy was induced in rats by ascending aortic banding (n = 40). After 7 days, myosin contained 31% (P < .05) of the beta-MHC isoform in banded but < 5% in sham-operated animals. However, no specific elevation of beta-MHC mRNA levels was found in banded animals. In contrast, hearts of rats with abdominal aortic banding displayed a marked increase in beta-MHC mRNA levels (3-fold to 5-fold, P < .05). Both the left ventricular weight and left ventricular peak systolic pressure were significantly elevated compared with sham-operated animals (abdominal aortic banding, +13% and 164 +/- 7 mm Hg; ascending aortic banding, +27% and 191 +/- 9 mm Hg). Plasma renin activity was elevated in rats with abdominal aortic banding (2.5-fold, P < .05) but not in rats with ascending aortic banding. CONCLUSIONS: The results of the present work do not support the concept that increased beta-MHC gene expression is a general "stable late marker" of myocardial hypertrophy in rats. Our results suggest that the stimulation of the renin-angiotensin system is crucial for the activation of the beta-MHC gene.

Differential effects of a K+ channel agonist and Ca2+ antagonists on myosin light chain phosphorylation in relaxation of endothelin-1-contracted tracheal smooth muscle.

Smooth muscle contraction and relaxation are generally considered to be associated with phosphorylation and dephosphorylation of the 20-kDa regulatory myosin light chain (LC20). Thus, contractions of lamb tracheal smooth muscle induced by Bay K 8644 and relaxed by calcium channel blockers (verapamil, D-600 and nitrendipine) are accompanied by an increase and decrease, respectively, of LC20 phosphorylation. Similarly, endothelin-1 (ET-1) induces a sustained contraction, which is coupled with elevated LC20 phosphorylation and reversed by LC20 dephosphorylation after application of a potassium channel agonist (EMD 52692). In contrast, calcium channel blockers relax ET-1-induced contraction without any dephosphorylation of myosin light chains (MLC), suggesting that MLC phosphatase is inhibited in this case. Obviously, MLC dephosphorylation is not a prerequisite for smooth muscle relaxation. The variable relationship between MLC phosphorylation and force during relaxation suggests that there are mechanisms other than MLC phosphorylation that are important for regulation of contraction and relaxation in smooth muscle.

Cardiac contractility: modulation of myofibrillar calcium sensitivity by beta-adrenergic stimulation.

Under conditions of beta-adrenergic receptor stimulation, cardiac performance is enhanced. cAMP-dependent phosphorylation of proteins located in the sarcolemma, in the membrane of the sarcoplasmic reticulum (SR), and in the myofibrils of the cardiomyocytes, mediates the effects of catecholamines on the heart. Altered Ca2+ handling leads to increased levels of intracellular free Ca2+. This is mainly responsible for the enhanced contractility of the myocardium that can be observed following beta-adrenergic receptor stimulation. Phosphorylation of the thin filament regulatory protein troponin I (TnI), on the other hand, decreases the Ca2+ sensitivity of the myofilaments, which means that the Ca2+ concentration necessary for the development of half-maximal force is increased. Cardiac TnI has a 26-33 amino acid N-terminal extension that is not present in fast and slow skeletal muscle TnI isoforms. Within this segment, two adjacent serine residues can be phosphorylated by a cAMP-dependent protein kinase. Replacement of endogenous TnI by different mutants obtained using site-directed mutagenesis of one or both of the serine residues has shown that only the bis-phosphorylated form decreases the Ca2+ sensitivity. This Ca2+ desensitizing effect, together with an increased rate of Ca2+ uptake into the SR due to phosphorylation of the SR membrane protein phospholamban, is responsible for the relaxation-enhancing effect (lusitropic action) of catecholamines. The latter is an important determinant of coronary perfusion and rapid diastolic filling of the ventricles, and is also a prerequisite for the elevation of heart rate that accompanies beta-adrenergic receptor stimulation.

Towards a molecular understanding of the elasticity of titin.

Vertebrate striated muscle behaves elastically when stretched and this property is thought to reside primarily within the giant filamentous protein, titin (connectin). The elastic portion of titin comprises two distinct structural motifs, immunoglobulin (Ig) domains and the PEVK titin, which is a novel motif family rich in proline, glutamate, valine and lysine residues. The respective contributions of the titin Ig and the PEVK sequences to the elastic properties of the molecule have been unknown so far. We have measured both the passive tension in single, isolated myofibrils from cardiac and skeletal muscle and the stretch-induced translational movement of I-band titin antibody epitopes following immunofluorescent labelling of sites adjacent to the PEVK and Ig domain regions. We found that with myofibril stretch, I-band titin does not extend homogeneously. The Ig domain region lengthened predominantly during small stretch, but such lengthening did not result in measurable passive tension and might be explained by straightening, rather than by unfolding, of the Ig repeats. At moderate to extreme stretch, the main extensible region was found to be the PEVK segment whose unravelling was correlated with a steady passive tension increase. In turn, PEVK domain transition from a linearly extended to a folded state appears to be principally responsible for the elasticity of muscle fibers. Thus, the length of the PEVK sequence may determine the tissue-specificity of muscle stiffness, whereas the expression of different Ig domain motif lengths may set the characteristic slack sarcomere length of a muscle type.

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 "mu"M), 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 "mu"M and 150 "mu"M, 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.

The Ca2+ sensitizer EMD 53998 antagonizes the effect of 2,3-butanedione monoxime on skinned cardiac muscle fibres.

The effects of 2,3-butanedione monoxime (BDM) and 5-[1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydro-6-quinolyl]-6-me thy l-3,6-dihydro-2H-1,3,4-thiadiazin-2-one (EMD 53998) on cardiac muscle were studied in skinned muscle fibres from the right ventricle of the porcine heart. BDM decreases the Ca2+ sensitivity (pCa50 for 50% activation) and it exerts a dose-dependent inhibitory effect on force in troponin I (TnI)-depleted (unregulated) cardiac skinned muscle fibres (IC50 approximately 20 mM) thereby mimicking the effect of the TnI inhibitory peptide (cTnI 137-148, corresponding to the cardiac TnI inhibitory region) and that of inorganic phosphate (Pi). This inhibitory action can be antagonized by the calcium-sensitizing cardiotonic thiadiazinone derivative EMD 53998 that increases the IC50 to about 30 mM. In skinned fibres, BDM (10 mM) also increased the ratio of ATPase activity to isometric force (tension cost), whereas EMD 53998 (20 mu M) decreased it. We propose that BDM antagonizes EMD 53998 because both compounds affect the Pi release step of the crossbridge cycle in an antagonistic manner.

Reconstitution of skinned cardiac fibres with human recombinant cardiac troponin-I mutants and troponin-C.

Troponin C (TnC) could be extracted from skinned porcine cardiac muscle fibres and their Ca2+ sensitivity restored by reconstitution with recombinant human cardiac TnC. After extraction of troponin I (TnI) and TnC using the vanadate treatment method of Strauss et al. [Strauss, J. D., Zeugner, C., Van Eyk, J.E., Bletz, C., Troschka, M. and Rüegg, J.C. (1992) FEBS Lett. 310, 229-234], skinned porcine cardiac muscle fibres were reconstituted with wild-type recombinant human cardiac TnC and either wild-type cardiac TnI or several mutant isoforms of human TnI. Reconstitution with wild-type proteins restored the Ca2+ sensitivity of the tissue and phosphorylation of the TnI with the catalytic subunit of protein kinase A reduced the Ca2+ sensitivity (i.e.-log[Ca2+] for 50% of maximal force) as has been shown by others. However, reconstitution with the TnI mutant Ser-23Asp/Ser-24Asp mimicking the phosphorylated form of cardiac TnI, led to a reduced Ca2+ sensitivity compared with reconstitution with wild-type TnI, whereas the mutant Ser-23Ala/Ser-24Ala behaved as the dephosphorylated form of TnI. These data confirm the importance of negative charge in this region of the TnI molecule in altering the Ca2+ responsiveness in this system.

Ca(2+)- and GTP[gamma S]-induced translocation of the glucose transporter, GLUT-4, to the plasma membrane of permeabilized cardiomyocytes determined using a novel immunoprecipitation method.

In cardiomyocytes glucose transport is activated not only by insulin but also by contractile activity that causes translocation of the glucose transporter, GLUT-4, from intracellular vesicles to the plasma membrane. The latter effect may possibly be mediated by intracellular Ca2+, as suggested by previous studies. To investigate the role of Ca2+, we permeabilized neonatal rat myocytes with alpha-toxin and incubated them for 1 h either at a pCa (i.e.--log10 [Ca2+]) of 8 (control) or at a pCa of 5 in the presence of adenosine 5'-triphosphate (ATP). Translocation of GLUT-4 was then monitored by a novel immunoprecipitation method using a peptide antibody directed against an exofacial (extracellular) loop of GLUT-4 (residues 58-80). Incorporation of GLUT-4 into the plasmalemma was stimulated 1.8-fold by 10 microM Ca2+ and 1.7-fold by insulin (as in the case of intact cells). The insulin effect was Ca2+ independent, i.e. it was identical in the absence and presence of Ca2+ (10 microM). Guanosine 5'-O-(3-thio-triphosphate) (GTP[gamma S]), which was inactive in intact cells, also caused translocation of GLUT-4 in permeabilized cardiomyocytes. Thus, incorporation of GLUT-4 into the plasma membrane was enhanced 2.5-fold by 200 microM GTP[gamma S] in the virtual absence of Ca2+ (pCa 8) and even 3.5-fold at 10 microM free Ca2+. We conclude that an increase in intracellular Ca2+ concentration increases GLUT-4 translocation of (permeabilized) cardiomyocytes to a similar extent as do insulin and GTP[gamma S] in the absence of Ca2+, but that the effects of Ca2+ and GTP[gamma S] may be additive.

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 Ca(2+)-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 microM). The Ca(2+)-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.(ABSTRACT TRUNCATED AT 250 WORDS)