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Donald A Martyn

Publications and source records attributed to Donald A Martyn.

8 recordsLinked to original sources
PubMed2007

Thin-filament regulation of force redevelopment kinetics in rabbit skeletal muscle fibres.

Thin-filament regulation of isometric force redevelopment (k(tr)) was examined in rabbit psoas fibres by substituting native TnC with either cardiac TnC (cTnC), a site I-inactive skeletal TnC mutant (xsTnC), or mixtures of native purified skeletal TnC (sTnC) and a site I- and II-inactive skeletal TnC mutant (xxsTnC). Reconstituted maximal Ca(2+)-activated force (rF(max)) decreased as the fraction of sTnC in sTnC: xxsTnC mixtures was reduced, but maximal k(tr) was unaffected until rF(max) was <0.2 of pre-extracted F(max). In contrast, reconstitution with cTnC or xsTnC reduced maximal k(tr) to 0.48 and 0.44 of control (P < 0.01), respectively, with corresponding rF(max) of 0.68 +/- 0.03 and 0.25 +/- 0.02 F(max). The k(tr)-pCa relation of fibres containing sTnC: xxsTnC mixtures (rF(max) > 0.2 F(max)) was little effected, though k(tr) was slightly elevated at low Ca(2+) activation. The magnitude of the Ca(2+)-dependent increase in k(tr) was greatly reduced following cTnC or xsTnC reconstitution because k(tr) at low levels of Ca(2+) was elevated and maximal k(tr) was reduced. Solution Ca(2+) dissociation rates (k(off)) from whole Tn complexes containing sTnC (26 +/- 0.1 s(-1)), cTnC (38 +/- 0.9 s(-1)) and xsTnC (50 +/- 1.2 s(-1)) correlated with k(tr) at low Ca(2+) levels and were inversely related to rF(max). At low Ca(2+) activation, k(tr) was similarly elevated in cTnC-reconstituted fibres with ATP or when cross-bridge cycling rate was increased with 2-deoxy-ATP. Our results and model simulations indicate little or no requirement for cooperative interactions between thin-filament regulatory units in modulating k(tr) at any [Ca(2+)] and suggest Ca(2+) activation properties of individual troponin complexes may influence the apparent rate constant of cross-bridge detachment.

Actin Cytoskeleton↗
PubMed2005

Kinetics of cardiac thin-filament activation probed by fluorescence polarization of rhodamine-labeled troponin C in skinned guinea pig trabeculae.

A genetically engineered cardiac TnC mutant labeled at Cys-84 with tetramethylrhodamine-5-iodoacetamide dihydroiodide was passively exchanged for the endogenous form in skinned guinea pig trabeculae. The extent of exchange averaged nearly 70%, quantified by protein microarray of individual trabeculae. The uniformity of its distribution was verified by confocal microscopy. Fluorescence polarization, giving probe angle and its dispersion relative to the fiber long axis, was monitored simultaneously with isometric tension. Probe angle reflects underlying cTnC orientation. In steady-state experiments, rigor cross-bridges and Ca2+ with vanadate to inhibit cross-bridge formation produce a similar change in probe orientation as that observed with cycling cross-bridges (no Vi). Changes in probe angle were found at [Ca2+] well below those required to generate tension. Cross-bridges increased the Ca2+ dependence of angle change (cooperativity). Strong cross-bridge formation enhanced Ca2+ sensitivity and was required for full change in probe position. At submaximal [Ca2+], the thin filament regulatory system may act in a coordinated fashion, with the probe orientation of Ca2+-bound cTnC significantly affected by Ca2+ binding at neighboring regulatory units. The time course of the probe angle change and tension after photolytic release [Ca2+] by laser photolysis of NP-EGTA was Ca2+ sensitive and biphasic: a rapid component approximately 10 times faster than that of tension and a slower rate similar to that of tension. The fast component likely represents steps closely associated with Ca2+ binding to site II of cTnC, whereas the slow component may arise from cross-bridge feedback. These results suggest that the thin filament activation rate does not limit the tension time course in cardiac muscle.

Actin Cytoskeleton↗
PubMed2005

Length-dependent Ca(2+) activation in cardiac muscle: some remaining questions.

The steep relationship between systolic force and end diastolic volume in cardiac muscle (Frank-Starling relation) is, to a large extent, based on length-dependent changes in myofilament Ca(2+) sensitivity. How sarcomere length modulates Ca(2+) sensitivity is still a topic of active investigation. Two general themes have emerged in recent years. On the one hand, there is a large body of evidence indicating that length-dependent changes in lattice spacing determine changes in Ca(2+) sensitivity for a given set of conditions. A model has been put forward in which the number of strong-binding cross-bridges that are formed is directly related to the proximity of the myosin heads to binding sites on actin. On the other hand, there is also a body of evidence suggesting that lattice spacing and Ca(2+) sensitivity are not tightly linked and that there is a length-sensing element in the sarcomere, which can modulate actin-myosin interactions independent of changes in lattice spacing. In this review, we examine the evidence that has been cited in support of these viewpoints. Much recent progress has been based on the combination of mechanical measurements with X-ray diffraction analysis of lattice spacing and cross-bridge interaction with actin. Compelling evidence indicates that the relationship between sarcomere length and lattice spacing is influenced by the elastic properties of titin and that changes in lattice spacing directly modulate cross-bridge interactions with thin filaments. However, there is also evidence that the precise relationship between Ca(2+) sensitivity and lattice spacing can be altered by changes in protein isoform expression, protein phosphorylation, modifiers of cross-bridge kinetics, and changes in titin compliance. Hence although there is no unique relationship between Ca(2+) sensitivity and lattice spacing the evidence strongly suggests that under any given set of physiological circumstances variation in lattice spacing is the major determinant of length-dependent changes in Ca(2+) sensitivity.

Animals↗
PubMed2005

Different effects of cardiac versus skeletal muscle regulatory proteins on in vitro measures of actin filament speed and force.

Mammalian cardiac and skeletal muscle express unique isoforms of the thin filament regulatory proteins, troponin (Tn) and tropomyosin (Tm), and the significance of these different isoforms in thin filament regulation has not been clearly identified. Both in vitro and skinned cellular studies investigating the mechanism of thin filament regulation in striated muscle have often used heterogeneous mixtures of Tn, Tm and myosin isoforms, and variability in reported results might be explained by different combinations of these proteins. Here we used in vitro motility and force (microneedle) assays to investigate the influence of cardiac versus skeletal Tn and Tm isoforms on actin-heavy meromyosin (HMM) mechanics. When interacting with skeletal HMM, thin filaments reconstituted with cardiac Tn/Tm or skeletal Tn/Tm exhibited similar speed-calcium relationships and significantly increased maximum speed and force per filament length (F/l) at pCa 5 (versus unregulated actin filaments). However, augmentation of F/l was greater with skeletal regulatory proteins. Reconstitution of thin filaments with the heterogeneous combination of skeletal Tn and cardiac Tm decreased sliding speeds at all [Ca2+] relative to thin filaments with skeletal Tn/Tm. Finally, for filaments reconstituted with any heterogeneous mix of Tn and Tm isoforms, force was not potentiated over that of unregulated actin filaments. Combined the results suggest (1) that cardiac regulatory proteins limit the allosteric enhancement of force, and (2) that Tn and Tm isoform homogeneity is important when studying Ca2+ regulation of crossbridge binding and kinetics as well as mechanistic differences between cardiac and skeletal muscle.

Actins↗
PubMed2004

Response of equatorial x-ray reflections and stiffness to altered sarcomere length and myofilament lattice spacing in relaxed skinned cardiac muscle.

Low angle x-ray diffraction measurements of myofilament lattice spacing (D(1,0)) and equatorial reflection intensity ratio (I(1,1)/I(1,0)) were made in relaxed skinned cardiac trabeculae from rats. We tested the hypothesis that the degree of weak cross-bridge (Xbr) binding, which has been shown to be obligatory for force generation in skeletal muscle, is modulated by changes in lattice spacing in skinned cardiac muscle. Altered weak Xbr binding was detected both by changes in I(1,1)/I(1,0) and by measurements of chord stiffness (chord K). Both measurements showed that, similar to skeletal muscle, the probability of weak Xbr binding at 170-mM ionic strength was significantly enhanced by lowering temperature to 5 degrees C. The effects of lattice spacing on weak Xbr binding were therefore determined under these conditions. Changes in D(1,0), I(1,1)/I(1,0), and chord K by osmotic compression with dextran T500 were determined at sarcomere lengths (SL) of 2.0 and 2.35 micro m. At each SL increasing [dextran] caused D(1,0) to decrease and both I(1,1)/I(1,0) and chord K to increase, indicating increased weak Xbr binding. The results suggest that in intact cardiac muscle increasing SL and decreasing lattice spacing could lead to increased force by increasing the probability of initial weak Xbr binding.

Actin Cytoskeleton↗
PubMed2004

Cardiac length dependence of force and force redevelopment kinetics with altered cross-bridge cycling.

We examined the influence of cross-bridge cycling kinetics on the length dependence of steady-state force and the rate of force redevelopment (k(tr)) during Ca(2+)-activation at sarcomere lengths (SL) of 2.0 and 2.3 microm in skinned rat cardiac trabeculae. Cross-bridge kinetics were altered by either replacing ATP with 2-deoxy-ATP (dATP) or by reducing [ATP]. At each SL dATP increased maximal force (F(max)) and Ca(2+)-sensitivity of force (pCa(50)) and reduced the cooperativity (n(H)) of force-pCa relations, whereas reducing [ATP] to 0.5 mM (low ATP) increased pCa(50) and n(H) without changing F(max). The difference in pCa(50) between SL 2.0 and 2.3 microm (Delta pCa(50)) was comparable between ATP and dATP, but reduced with low ATP. Maximal k(tr) was elevated by dATP and reduced by low ATP. Ca(2+)-sensitivity of k(tr) increased with both dATP and low ATP and was unaffected by altered SL under all conditions. Significantly, at equivalent levels of submaximal force k(tr) was faster at short SL or increased lattice spacing. These data demonstrate that the SL dependence of force depends on cross-bridge kinetics and that the increase of force upon SL extension occurs without increasing the rate of transitions between nonforce and force-generating cross-bridge states, suggesting SL or lattice spacing may modulate preforce cross-bridge transitions.

Adenosine Triphosphate↗
PubMed2003

Familial hypertrophic cardiomyopathy mutations in troponin I (K183D, G203S, K206Q) enhance filament sliding.

A major cause of familial hypertrophic cardiomyopathy (FHC) is dominant mutations in cardiac sarcomeric genes. Linkage studies identified FHC-related mutations in the COOH terminus of cardiac troponin I (cTnI), a region with unknown function in Ca(2+) regulation of the heart. Using in vitro assays with recombinant rat troponin subunits, we tested the hypothesis that mutations K183Delta, G203S, and K206Q in cTnI affect Ca(2+) regulation. All three mutants enhanced Ca(2+) sensitivity and maximum speed (s(max)) of filament sliding of in vitro motility assays. Enhanced s(max) (pCa 5) was observed with rabbit skeletal and rat cardiac (alpha-MHC or beta-MHC) heavy meromyosin (HMM). We developed a passive exchange method for replacing endogenous cTn in permeabilized rat cardiac trabeculae. Ca(2+) sensitivity and maximum isometric force did not differ between preparations exchanged with cTn(cTnI,K206Q) or wild-type cTn. In both trabeculae and motility assays, there was no loss of inhibition at pCa 9. These results are consistent with COOH terminus of TnI modulating actomyosin kinetics during unloaded sliding, but not during isometric force generation, and implicate enhanced cross-bridge cycling in the cTnI-related pathway(s) to hypertrophy.

Actin Cytoskeleton↗