Attenuation of length dependence of calcium activation in myofilaments of transgenic mouse hearts expressing slow skeletal troponin I.

We compared sarcomere length (SL) dependence of the Ca2+-force relation of detergent-extracted bundles of fibres dissected from the left ventricle of wild-type (WT) and transgenic mouse hearts expressing slow skeletal troponin I (ssTnI-TG). Fibre bundles from the hearts of the ssTnI-TG demonstrated a complete replacement of the cardiac troponin I (cTnI) by ssTnI. Compared to WT controls, ssTnI-TG fibre bundles were more sensitive to Ca2+ at both short SL (1.9 +/- 0.1 micrometer) and long SL (2.3 +/- 0.1 micrometer). However, compared to WT controls, the increase in Ca2+ sensitivity (change in half-maximally activating free Ca2+; DeltaEC50) associated with the increase in SL was significantly blunted in the ssTnI-TG myofilaments. Agents that sensitize the myofilaments to Ca2+ by promoting the actin-myosin reaction (EMD 57033 and CGP-48506) significantly reduced the length-dependent DeltaEC50 for Ca2+ activation, when SL in WT myofilaments was increased from 1.9 to 2.3 micrometer. Exposure of myofilaments to calmidazolium (CDZ), which binds to cTnC and increases its affinity for Ca2+, sensitized force developed by WT myofilaments to Ca2+ at SL 1.9 micrometer and desensitized the WT myofilaments at SL 2.3 micrometer. There were no significant effects of CDZ on ssTnI-TG myofilaments at either SL. Our results indicate that length-dependent Ca2+ activation is modified by specific changes in thin filament proteins and by agents that promote the actin-myosin interaction. Thus, these in vitro results provide a basis for using these models to test the relative significance of the length dependence of activation in situ.

The light chain binding domain of expressed smooth muscle heavy meromyosin acts as a mechanical lever.

Structural data led to the proposal that the molecular motor myosin moves actin by a swinging of the light chain binding domain, or "neck." To test the hypothesis that the neck functions as a mechanical lever, smooth muscle heavy meromyosin (HMM) mutants were expressed with shorter or longer necks by either deleting or adding light chain binding sites. The mutant HMMs were characterized kinetically and mechanically, with emphasis on measurements of unitary displacements and forces in the laser trap assay. Two shorter necked constructs had smaller unitary step sizes and moved actin more slowly than WT HMM in the motility assay. A longer necked construct that contained an additional essential light chain binding site exhibited a 1.4-fold increase in the unitary step size compared with its control. Kinetic changes were also observed with several of the constructs. The mutant lacking a neck produced force at a somewhat reduced level, while the force exerted by the giraffe construct was higher than control. The single molecule displacement and force data support the hypothesis that the neck functions as a rigid lever, with the fulcrum for movement and force located at a point within the motor domain.

R403Q and L908V mutant beta-cardiac myosin from patients with familial hypertrophic cardiomyopathy exhibit enhanced mechanical performance at the single molecule level.

Familial hypertrophic cardiomyopathy (FHC) is a disease of the sarcomere. In the beta-myosin heavy chain gene, which codes for the mechanical enzyme myosin, greater than 40 point mutations have been found that are causal for this disease. We have studied the effect of two mutations, the R403Q and L908V, on myosin molecular mechanics. In the in vitro motility assay, the mutant myosins produced a 30% greater velocity of actin filament movement (v(actin)). At the single molecule level, v(actin) approximately d/t(on), where d is the myosin unitary step displacement and t(on) is the step duration. Laser trap studies were performed at 10 microM MgATP to estimate d and t(on) for the normal and mutant myosin molecules. The increase in v(actin) can be explained by a significant decrease in the average t(on)'s in both the R403Q and L908V mutants (approximately 30 ms) compared to controls (approximately 40 ms), while d was not different for all myosins tested (approximately 7 nm). Thus the mutations affect the kinetics of the cross-bridge cycle without any effect on myosin's inherent motion and force generating capacity. Based on these studies, the primary signal for the hypertrophic response appears to be an apparent gain in function of the individual mutant myosin molecules.

Tropomyosin directly modulates actomyosin mechanical performance at the level of a single actin filament.

Muscle contraction is the result of myosin cross-bridges (XBs) cyclically interacting with the actin-containing thin filament. This interaction is modulated by the thin filament regulatory proteins, troponin and tropomyosin (Tm). With the use of an in vitro motility assay, the role of Tm in myosin's ability to generate force and motion was assessed. At saturating myosin surface densities, Tm had no effect on thin filament velocity. However, below 50% myosin saturation, a significant reduction in actin-Tm filament velocity was observed, with complete inhibition of movement occurring at 12. 5% of saturating surface densities. Under similar conditions, actin filaments alone demonstrated no reduction in velocity. The effect of Tm on force generation was assessed at the level of a single thin filament. In the absence of Tm, isometric force was a linear function of the density of myosin on the motility surface. At 50% myosin surface saturation, the presence of Tm resulted in a 2-fold enhancement of force relative to actin alone. However, no further potentiation of force was observed with Tm at saturating myosin surface densities. These results indicate that, in the presence of Tm, the strong binding of myosin cooperatively activates the thin filament. The inhibition of velocity at low myosin densities and the potentiation of force at higher myosin densities suggest that Tm can directly modulate the kinetics of a single myosin XB and the recruitment of a population of XBs, respectively. At saturating myosin conditions, Tm does not appear to affect the recruitment or the kinetics of myosin XBs.

Kinetic differences at the single molecule level account for the functional diversity of rabbit cardiac myosin isoforms.

1. Cardiac V3 myosin generates slower actin filament velocities and higher average isometric forces (in an in vitro motility assay) when compared with the V1 isoform. 2. To account for differences in V1 and V3 force and motion generation at the molecular level, we characterized the mechanics and kinetics of single V1 and V3 myosin molecules using a dual laser trap setup. 3. No differences in either unitary displacement (approximately 7 nm) or force (approximately 0.8 pN) were observed between isoforms; however, the duration of unitary displacement events was significantly longer for the V3 isoform at MgATP concentrations > 10 microM. 4. Our results were interpreted on the basis of a cross-bridge model in which displacement event durations were determined by the rates of MgADP release from, and MgATP binding to, myosin. 5. We propose that the release rate of MgADP from V3 myosin is half that of V1 myosin without any difference in their rates of MgATP binding; thus, kinetic differences between the two cardiac myosin isoforms are sufficient to account for their functional diversity.

Correlation between myofilament response to Ca2+ and altered dynamics of contraction and relaxation in transgenic cardiac cells that express beta-tropomyosin.

We compared the dynamics of the contraction and relaxation of single myocytes isolated from nontransgenic (NTG) mouse hearts and from transgenic (TG-beta-Tm) mouse hearts that overexpress the skeletal isoform of tropomyosin (Tm). Compared with NTG controls, TG-beta-Tm myocytes showed significantly reduced maximal rates of contraction and relaxation with no change in the extent of shortening. This result indicated that the depression in contraction dynamics determined in TG-beta-Tm isolated hearts is intrinsic to the cells. To further investigate the effect of Tm isoform switching on myofilament activity and regulation, we measured myofilament force and ATPase rate as functions of pCa (-log of [Ca2+]). Compared with controls, force generated by myofilaments from TG-beta-Tm hearts and myofibrillar ATPase activity were both more sensitive to Ca2+. However, the shift in pCa50 (half-maximally activating pCa) caused by changing sarcomere length from 1.8 to 2.4 microm was not significantly different between NTG and TG-beta-Tm fiber preparations. To test directly whether isoform switching affected the economy of contraction, force versus ATPase rate relationships were measured in detergent-extracted fiber bundles. In both NTG and TG-beta-Tm preparations, force and ATPase rate were linear and identically correlated, which indicated that crossbridge turnover was unaffected by Tm isoform switching. However, detergent extracted fibers from TG-beta-Tm demonstrated significantly less maximum tension and ATPase activity than NTG controls. Our results provide the first evidence that the Tm isoform population modulates the dynamics of contraction and relaxation of single myocytes by a mechanism that does not alter the rate-limiting step of crossbridge detachment. Our results also indicate that differences in sarcomere-length dependence of activation between cardiac and skeletal muscle are not likely due to differences in the isoform population of Tm.

Molecular mechanisms regulating the myofilament response to Ca2+: implications of mutations causal for familial hypertrophic cardiomyopathy.

In this chapter we consider a current perception of the molecular mechanisms controlling myofilament activation with emphasis on alterations that may occur in familial hypertrophic cardiomyopathy (FHC). FHC is a sarcomeric disease (100) with an autosomal dominant pattern of heritability (27, 51). There is a substantial body of evidence implicating missense mutations in the beta-MHC gene as causal for the development of this disease. Recently, mutations in genes of two thin filament regulatory proteins, cardiac troponin T(cTnT) and alpha-tropomyosin (alpha-Tm), have also been linked to FHC. The commonality among the functional consequences of these mutations remains an important question. This review discusses how these pathological mutations may impact the activation process by disrupting critical structure function relations in both the thick and thin filaments.

E-1020, a water soluble imidazopyridine, has direct effects on Ca(2+)-dependent force and ATP hydrolysis of canine and bovine cardiac myofilaments.

E-1020 is a cardiotonic agent that acts as a cyclic-AMP phosphodiesterase inhibitor but also may have actions which alter myofilament response to Ca2+. To identify direct actions of E-1020 on cardiac contractile proteins, effects of E-1020 on myofibrillar Ca2+ dependent MgATPase and force generation in chemically skinned fiber bundles were measured. In bovine cardiac myofibrils, E-1020 (100 microM) significantly increased myofilament Ca2+ sensitivity and Ca(2+)-dependent ATPase activity at submaximal pCa values. At pCa 6.75, E-1020 significantly increased ATPase activity in bovine (10-100 microM) and canine (1-100 microM) cardiac myofibrils but had no effect on rat cardiac myofibrils. Moreover, in one population of canine ventricular fiber bundles, E-1020 (0.01-10 microM) significantly increased isometric tension at pCa 6.5 and 6.0, whereas in another population of bundles E-1020 had no effect on tension. In no case was resting (pCa 8.0) or maximal tension (pCa 4.5) increased by E-1020. Measurements of Ca2+ binding to canine ventricular skinned fiber preparations demonstrated that E-1020 does not alter the affinity of myofilament troponin C for Ca2+. We conclude that part of the mechanism by which E-1020 acts as an inotropic agent may involve alterations in the responsiveness of contractile proteins to Ca2+. The lack of effect of E-1020 on some preparations may be dependent on isoform populations of myofilament proteins.

Exchange of beta- for alpha-tropomyosin in hearts of transgenic mice induces changes in thin filament response to Ca2+, strong cross-bridge binding, and protein phosphorylation.

Despite its potential as a key determinant of the functional state of striated muscle, the impact of tropomyosin (Tm) isoform switching on mammalian myofilament activation and regulation in the intact lattice remains unclear. Using a transgenic approach to specifically exchange beta-Tm for the native alpha-Tm in mouse hearts, we have been able to uncover novel functions of Tm isoform switching in the heart. The myofilaments containing beta-Tm demonstrated an increase in the activation of the thin filament by strongly bound cross-bridges, an increase in Ca2+ sensitivity of steady state force, and a decrease in the rightward shift of the Ca2+-force relation induced by cAMP-dependent phosphorylation. Our results are the first to demonstrate the specific effects of Tm isoform switching on mammalian thin filament activation in the intact lattice and suggest an important role for Tm in modulation of myofilament activity by phosphorylation of troponin.

CGP-48506 increases contractility of ventricular myocytes and myofilaments by effects on actin-myosin reaction.

We measured the effects of the benzodiazocine derivative, CGP-48506 (5-methyl-6-phenyl-1,3,5,6-tetrahydro-3,6-methano-1, 5-benzodiazocine-2,4-dione), on contraction of intact myocytes and permeabilized fibers of rat ventricular muscle. CGP-48506 is unique in that it is able to sensitize cardiac myofilaments to Ca2+, but unlike all other agents in this class, it is not an inhibitor of type III phosphodiesterase. When added to isolated intact myocytes, CGP-48506 significantly increased the amplitude of cell shortening with little or no change in the Ca2+ transient, as determined by the fluorescence ratio of fura 2. The late phase of the relation between fura 2 ratio and cell length was shifted to the left in the presence of CGP-48506. CGP-48506 also induced a relatively small decrease in diastolic length. However, compared with the thiadiazinone EMD-57033, CGP-48506 had a much smaller effect on diastolic length at concentrations in which there was a bigger inotropic effect. When added to solutions bathing detergent-extracted (skinned) fiber bundles, CGP-48506 increased maximum force. CGP-48506 also increased submaximal force and shifted the pGa-force relation to the left. However, compared with EMD-57033, there was less of an effect of CGP-48506 on force at relatively high pCa values. CGP-48506 did not alter Ca2+ binding to myofilament troponin C. CGP-48506 was able to reverse inhibition of contraction induced by butanedione monoxime both in intact cells and in skinned fiber bundles. Our results indicate that CGP-48506, like EMD-57033, is a positive inotropic agent working through a direct effect downstream from troponin C. CGP-48506, however, appears to have a unique mechanism resulting in less effect on diastolic function.

Mutagenesis of cardiac troponin I. Role of the unique NH2-terminal peptide in myofilament activation.

Phosphorylation of Ser residues in the NH2-terminal extension unique to cardiac troponin I (cTnI) is known to occur through protein kinase A and to alter myofilament Ca2+ activation (Robertson, S. P., Johnson, J. D., Holroyde, M. J., Kranias, E. G., Potter, J. D., and Solaro, R. J. (1982) J. Biol. Chem. 257, 260-263). Yet, how the NH2-terminal extension may itself affect thin filament Ca2+ signaling is unknown. To approach this question we have used molecular cloning, mutagenesis, and bacterial synthesis of a full-length cTnI and a truncated mutant (cTnI/NH2) missing the 32 amino acids. Using reconstituted preparations we could show no differences between cTnI and cTnI/NH2 either in inhibition of actomyosin ATPase activity, in Ca(2+)-reversible inhibitory activity, or in the relation between pCa and Ca2+ binding to the regulatory site of cTnC at either pH 7.0 or 6.5. There were also no significant differences at either pH in the pCa-MgATPase activity relation of myofibrils into which the various species of TnI has been exchanged. Our results indicate: 1) that phosphorylation most likely induces a new state of TnI activity rather than altering an intrinsic effect of the NH2-terminal peptide on Ca2+ activation; and 2) that domains outside the NH2-terminal extension are important with regard to differences in effects of acidic pH on Ca2+ activation on cardiac and skeletal myofilaments.

Slowly exchanging calcium binding sites unique to cardiac/slow muscle troponin C.

Evidence is presented for the existence of slowly exchanging Ca2(+)-binding sites in troponin C (CTNC) of cardiac and slow twitch skeletal muscles. These sites were revealed in the course of experiments aimed at measuring the Ca2(+)-binding properties of TNC in the myofilament lattice. 45Ca bound to chemically skinned muscle fibers or myofibrils of cardiac and soleus muscles was eluted by EGTA in a two-exponential timecourse with a slow phase of a rate constant of about 2 x 10(-4)/s. The slow phase was not found in skinned fiber or myofibrils of psoas, a fast skeletal muscle. However, skinned psoas fibers in which the native TNC was replaced by CTNC exhibited the slow 45Ca elution characteristic of soleus and cardiac preparations, indicating that the slowly-exchanging sites are located in CTNC. These sites are tentatively identified as the Ca2(+)-Mg2+ sites of CTNC on the basis that the slow phase was observed under conditions known to restrict Ca2+ binding to the Ca2(+)-Mg2+ sites.