Cross-bridge Turnover Research Articles

Danicamtiv Increases Myosin Recruitment and Alters Cross-Bridge Cycling in Cardiac Muscle.

Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known that danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. Permeabilized porcine cardiac tissue and myofibrils were used for X-ray diffraction and mechanical measurements. A mouse model of genetic dilated cardiomyopathy was used to evaluate the ability of danicamtiv to correct the contractile deficient. Danicamtiv increased force and calcium sensitivity via increasing the number of myosins in the on state and slowing cross-bridge turnover. Our detailed analysis showed that inhibition of ADP release results in decreased cross-bridge turnover with cross bridges staying attached longer and prolonging myofibril relaxation. Danicamtiv corrected decreased calcium sensitivity in demembranated tissue, abnormal twitch magnitude and kinetics in intact cardiac tissue, and reduced ejection fraction in the whole organ. As demonstrated by the detailed studies of Danicamtiv, increasing myosin recruitment and altering cross-bridge cycling are 2 mechanisms to increase force and calcium sensitivity in cardiac muscle. Myosin activators such as Danicamtiv can treat the causative hypocontractile phenotype in genetic dilated cardiomyopathy.

Investigating the active contractile function of the rat paraspinal muscles reveals unique cross-bridge kinetics in the multifidus

Various aspects of paraspinal muscle anatomy, biology, and histology have been studied; however, information on paraspinal muscle contractile function is almost nonexistent, thus hindering functional interpretation of these muscles in healthy individuals and those with low back disorders. The aim of this study was to measure and compare the contractile function and force-sarcomere length properties of muscle fibers from the multifidus (MULT) and erector spinae (ES) as well as a commonly studied lower limb muscle (Extensor digitorum longus (EDL)) in the rat. Single muscle fibers (n = 77 total from 6 animals) were isolated from each of the muscles and tested to determine their active contractile function; all fibers used in the analyses were type IIB. There were no significant differences between muscles for specific force (sFo) (p = 0.11), active modulus (p = 0.63), average optimal sarcomere length (p = 0.27) or unloaded shortening velocity (Vo) (p = 0.69). However, there was a significant difference in the rate of force redevelopment (ktr) between muscles (p = < 0.0001), with MULT being significantly faster than both the EDL (p = < 0.0001) and ES (p = 0.0001) and no difference between the EDL and ES (p = 0.41). This finding suggests that multifidus has faster cross-bridge turnover kinetics when compared to other muscles (ES and EDL) when matched for fiber type. Whether the faster cross-bridge kinetics translate to a functionally significant difference in whole muscle performance needs to be studied further.

Molecular and Functional Effects of a Splice Site Mutation in the MYL2 Gene Associated with Cardioskeletal Myopathy and Early Cardiac Death in Infants.

The homozygous appearance of the intronic mutation (IVS6-1) in the MYL2 gene encoding for myosin ventricular/slow-twitch skeletal regulatory light chain (RLC) was recently linked to the development of slow skeletal muscle fiber type I hypotrophy and early cardiac death. The IVS6-1 (c403-1G>C) mutation resulted from a cryptic splice site in MYL2 causing a frameshift and replacement of the last 32 codons by 19 different amino acids in the RLC mutant protein. Infants who were IVS6-1+∕+-positive died between 4 and 6 months of age due to cardiomyopathy and heart failure. In this report we have investigated the molecular mechanism and functional consequences associated with the IVS6-1 mutation using recombinant human cardiac IVS6-1 and wild-type (WT) RLC proteins. Recombinant proteins were reconstituted into RLC-depleted porcine cardiac muscle preparations and subjected to enzymatic and functional assays. IVS6-1-RLC showed decreased binding to the myosin heavy chain (MHC) compared with WT, and IVS6-1-reconstituted myosin displayed reduced binding to actin in rigor. The IVS6-1 myosin demonstrated a significantly lower Vmax of the actin-activated myosin ATPase activity compared with WT. In stopped-flow experiments, IVS6-1 myosin showed slower kinetics of the ATP induced dissociation of the acto-myosin complex and a significantly reduced slope of the kobs-[MgATP] relationship compared to WT. In skinned porcine cardiac muscles, RLC-depleted and IVS6-1 reconstituted muscle strips displayed a significant decrease in maximal contractile force and a significantly increased Ca2+ sensitivity, both hallmarks of hypertrophic cardiomyopathy-associated mutations in MYL2. Our results showed that the amino-acid changes in IVS6-1 were sufficient to impose significant conformational alterations in the RLC protein and trigger a series of abnormal protein-protein interactions in the cardiac muscle sarcomere. Notably, the mutation disrupted the RLC-MHC interaction and the steady-state and kinetics of the acto-myosin interaction. Specifically, slower myosin cross-bridge turnover rates and slower second-order MgATP binding rates of acto-myosin interactions were observed in IVS6-1 vs. WT reconstituted cardiac preparations. Our in vitro results suggest that when placed in vivo, IVS6-1 may lead to cardiomyopathy and early death of homozygous infants by severely compromising the ability of myosin to develop contractile force and maintain normal systolic and diastolic cardiac function.

Effects of IVS6-1 Mutation in MYl2 Associated with Cardioskeletal Myopathy and Early Cardiac Death of Infants

The homozygous appearance of intronic mutation (IVS6-1) in MYL2 encoding myosin ventricular/slow skeletal myosin regulatory light chain (RLC) was recently linked to the development of slow skeletal muscle fiber type I hypotrophy. The affected infants died between 4 and 6 months of age due to dilated or hypertrophic cardiomyopathy. In this report we have investigated, the molecular mechanism and functional consequences associated with IVS6-1 using recombinant human cardiac IVS6-1-RLC reconstituted in RLC-depleted porcine cardiac muscle preparations. The results were compared with those obtained for recombinant human cardiac WT-RLC. Despite the ability of the mutant to become phosphorylated, IVS6-1 displayed a significantly lower Vmax of the actin-activated myosin ATPase activity compared with WT (0.18±0.2 s-1 vs. 0.25±0.02 s-1; n=3), indicating a slower myosin cross-bridge turnover rate of the mutant. Likewise, stopped flow kinetics of the ATP induced dissociation of the acto-myosin complex showed a significantly reduced slope of the kobs-[MgATP] relationship for IVS6-1 reconstituted myosin (4.3±0.02×105 M-1 s-1, n=5), depicting slower second-order MgATP binding rates compared with WT (5.3±0.02×105 M-1 s-1, n=5). Steady-state fluorescence binding experiments of mutant vs. WT reconstituted myosin to pyrene labeled F-actin under rigor conditions demonstrated significantly reduced binding affinity of IVS6-1 (Kd=16.7±2.8 nM, n=3) compared with WT (Kd=4.6±1.3 nM, n=3). In skinned porcine cardiac muscles, a significant decrease in maximal force was observed for IVS6-1 reconstituted fibers compared with WT (27.1±1.0 kN/m2 vs. 34.9±2.4 kN/m2, n=6). The Ca2+ sensitivity and cooperativity were significantly different in IVS6-1 (pCa50=5.66±0.01, nH=2.9±0.15) vs. WT (pCa50=5.48±0.01, nH=2.2±0.14). In conclusion, we demonstrate that IVS6-1 may lead to cardiac dysfunction by disrupting the acto-myosin interaction (steady-state and kinetics) and compromising the ability of the mutant myosin to develop contractile force.

Faster Cross-Bridge Relaxation Rates Correlate with Increased Tension Cost in Hcm with the R403Q Myh7 Mutation

The first mutation found to be associated with hypertrophic cardiomyopathy (HCM) is the R403Q mutation in the gene encoding β-myosin heavy chain. R403Q is located in the globular head of myosin (S1), responsible for actin interaction, and hence motor function of myosin. According to the 2-state model of actin-myosin interaction total cross-bridge turnover rate can be described by the sum of the cross-bridge association (fapp) and detachment (gapp) rates. Gapp is equivalent to the slow rate of cross-bridge relaxation (slow krel), which equals the energetic cost of tension generation, i.e. tension cost (expressed by ATP utilization/tension). In the present study we investigated to what extent cross-bridge kinetics in HCM with the R403Q mutation correlated with cross-bridge energetics.Left ventricular multicellular muscle strips and myofibrillar preparations were isolated from 3 HCM patients with the R403Q mutation. 8 HCM sarcomere mutation negative (HCMsmn) patients served as control. In the muscle strips force generating capacity and ATP utilization were measured to assess tension cost and in the myofibrillar preparations cross-bridge kinetics was analyzed. The fraction of mutated R403Q mRNA was analyzed using specific restriction digests and realtime-PCR. Compared to HCMsmn tension cost was higher in the muscle strips of the 3 R403Q patients which showed a positive linear correlation with relaxation kinetics in the corresponding myofibrillar preparations. Variation in cross-bridge function among the 3 R403Q patients could not be explained by differences in R403Q mRNA levels.Cross-bridge cycling efficiency was decreased in HCM due to the R403Q mutation, while cross-bridge kinetics was increased. This suggests that the apparent gain in function evident from increased kinetics leads to a loss of function with respect to cross-bridge cycling efficiency in HCM caused by the R403Q mutation.

A re-interpretation of the rate of tension redevelopment (k TR) in active muscle

A slackening to zero tension by large length release (~20%) and a restretch of active muscle fibres cause a fall and a redevelopment in tension. According to the model of Brenner (Proc Natl Acad Sci USA 85(9):3265-3269, 1988), the rate constant of tension redevelopment (k TR) is the sum of attachment and detachment rate constants, hence is limited by the fast reaction. Here we propose a model in which, after restretch, cross-bridges cycle many times by stretching series elastic elements, hence k(TR) is limited by a slow reaction. To set up this model, we made an assumption that the stepping rate (v) decreases linearly with tension (F), which is consistent with the Fenn effect. The distance traveled by a cross-bridge stretches series elastic elements with stiffness σ. With these assumptions, we set up a first order differential equation, which results in an exponential time course with the rate constant k(TR) = ση(0)ν(0)(1 - λ)/F(1), where λ = ν(1)/ν(0), η = step size, the subscript 0 indicates unloaded condition, and the subscript 1 indicate isometric condition. We demonstrate that the ATP hydrolysis rate (=[myosin head]/ν(0)) is proportionate to k(TR) as the ambient temperature is changed, and that the published data fit to this relationship well if λ = 0.28. We conclude that k(TR) is limited by the cross-bridge turnover rate; hence it represents the rate constant of the slowest reaction of the cross-bridge cycle, i.e. the ADP isomerization step before ADP is released.

Tetrahydrobiopterin improves diastolic dysfunction by reversing changes in myofilament properties

Despite the increasing prevalence of heart failure with preserved left ventricular function, there are no specific treatments, partially because the mechanism of impaired relaxation is incompletely understood. Evidence indicates that cardiac relaxation may depend on nitric oxide (NO), generated by NO synthase (NOS) requiring the co-factor tetrahydrobiopterin (BH4). Recently, we reported that hypertension-induced diastolic dysfunction was accompanied by cardiac BH4 depletion, NOS uncoupling, a depression in myofilament cross-bridge kinetics, and S-glutathionylation of myosin binding protein C (MyBP-C). We hypothesized that the mechanism by which BH4 ameliorates diastolic dysfunction is by preventing glutathionylation of MyBP-C and thus reversing changes of myofilament properties that occur during diastolic dysfunction. We used the deoxycorticosterone acetate (DOCA)-salt mouse model, which demonstrates mild hypertension, myocardial oxidative stress, and diastolic dysfunction. Mice were divided into two groups that received control diet and two groups that received BH4 supplement for 7days after developing diastolic dysfunction at post-operative day 11. Mice were assessed by echocardiography. Left ventricular papillary detergent-extracted fiber bundles were isolated for simultaneous determination of force and ATPase activity. Sarcomeric protein glutathionylation was assessed by immunoblotting. DOCA-salt mice exhibited diastolic dysfunction that was reversed after BH4 treatment. Diastolic sarcomere length (DOCA-salt 1.70±0.01 vs. DOCA-salt+BH4 1.77±0.01μm, P<0.001) and relengthening (relaxation constant, τ, DOCA-salt 0.28±0.02 vs. DOCA-salt+BH4 0.08±0.01, P<0.001) were also restored to control by BH4 treatment. pCa50 for tension increased in DOCA-salt compared to sham but reverted to sham levels after BH4 treatment. Maximum ATPase rate and tension cost (ΔATPase/ΔTension) decreased in DOCA-salt compared to sham, but increased after BH4 treatment. Cardiac MyBP-C glutathionylation increased in DOCA-salt compared to sham, but decreased with BH4 treatment. MyBP-C glutathionylation correlated with the presence of diastolic dysfunction. Our results suggest that by depressing S-glutathionylation of MyBP-C, BH4 ameliorates diastolic dysfunction by reversing a decrease in cross-bridge turnover kinetics. These data provide evidence for modulation of cardiac relaxation by post-translational modification of myofilament proteins.

Tuning the Calcium Sensitivity of Cardiac Muscle

Tuning the Calcium Sensitivity of Cardiac Muscle

The N-Terminal Extension of Cardiac Troponin T Stabilizes the Blocked State of Cardiac Thin Filament

Cardiac troponin T (cTnT) is a key component of contractile regulatory proteins. cTnT is characterized by a ∼32 amino acid N-terminal extension (NTE), the function of which remains unknown. To understand its function, we generated a transgenic (TG) mouse line that expressed a recombinant chimeric cTnT in which the NTE of mouse cTnT was removed by replacing its 1–73 residues with the corresponding 1–41 residues of mouse fast skeletal TnT. Detergent-skinned papillary muscle fibers from non-TG (NTG) and TG mouse hearts were used to measure tension, ATPase activity, Ca2+ sensitivity (pCa50) of tension, rate of tension redevelopment, dynamic muscle fiber stiffness, and maximal fiber shortening velocity at sarcomere lengths (SLs) of 1.9 and 2.3 μm. Ca2+ sensitivity increased significantly in TG fibers at both short SL (pCa50 of 5.96 vs. 5.62 in NTG fibers) and long SL (pCa50 of 6.10 vs. 5.76 in NTG fibers). Maximal cross-bridge turnover and detachment kinetics were unaltered in TG fibers. Our data suggest that the NTE constrains cardiac thin filament activation such that the transition of the thin filament from the blocked to the closed state becomes less responsive to Ca2+. Our finding has implications regarding the effect of tissue- and disease-related changes in cTnT isoforms on cardiac muscle function.

Protein kinase A changes calcium sensitivity but not crossbridge kinetics in human cardiac myofibrils

We investigated the effect of PKA treatment (1 U/ml) on the mechanical properties of isolated human cardiac myofibrils. PKA treatment was associated with significant incorporation of radiolabeled phosphate into several sarcomeric proteins including troponin I and myosin binding protein C and was also associated with a right shift in the tension-pCa relation (ΔpCa(50) = 0.2 ± 0.1). PKA treatment also caused right shifts in the pCa dependence of the rate of tension development, tension redevelopment, and the linear and exponential phases of myofibril relaxation. However, there was no change in the same measures of crossbridge turnover when expressed as a function of tension. We conclude that the changes in crossbridge kinetics as a function of calcium concentration reflect a reduced tension due to a lower calcium sensitivity and that the relationship between crossbridge kinetics and tension was unchanged, indicating no direct effect of PKA treatment on crossbridge cycling.

The HCM-Linked Ala13thr Mutation in the Cardiac Myosin Regulatory Light Chain Increases Isometric Force Production

The myosin regulatory light chain (RLC) is attached to the α-helical neck region of the myosin head, the so called lever arm, which connects the catalytic and actin binding domains with the thick filament backbone thus participating in the transmission of external forces to the myosin active site. It is understandable that mutations in the RLC associated with hypertrophic cardiomyopathy (HCM) may lead to alterations in force generation affecting cardiac muscle performance. Here, we studied the physiological consequences of an Alanine to Threonine (A13T) mutation in the N-domain of myosin RLC, found in population studies to cause HCM with a specific disease phenotype characterized by mid-ventricular obstruction. We observed an A13T-induced 30-50% increase in maximal force measured in skinned cardiac muscle fibers from transgenic Tg-A13T mice compared to control, Tg-WT and non-Tg littermates. Furthermore, a mutation-mediated 1.3-fold decrease in Vmax and a 1.5-fold increase in Km were observed in the actin-activated myosin ATPase activity compared with myosin from the healthy controls. The binding of Tg-myosin to pyrene-actin was similar for all groups of mice. No changes in the maximal myofibrillar ATPase or in the Ca2+-sensitivity were noted. The same was true for the force-pCa relationship and the mutation did not introduce any alterations in the Ca2+-sensitivity of force development. Gross morphological evaluation revealed enlarged inter-ventricular septa and left ventricles in the hearts from Tg-A13T mice, a phenotype observed in patients harboring the A13T mutation. Our results indicate that the A13T mutation may result in a hypertrophic response through abnormally increased force that may exceed the tolerance of a healthy myocardium. A decreased rate of cross-bridge turnover further demonstrates inadequate energy generation in Tg-A13T mice adding to impaired sarcomeric function. Supported by NIH-HL071778 (DSC).

Structural and Functional Gradients with Temperature in the Flight Muscle of Manduca Sexta

The force/extension curve of the flight muscle of the Hawkmoth, Manduca sexta is remarkably similar to that of mammalian cardiac muscle suggesting that it may serve as a useful model system for certain aspects of cardiac muscle structure and function ( J Exp Biol. 2004;207:2455). More recently, it was discovered that these animals maintain an astonishing thermal gradient of 8.8 C in the 5 mm distance dorsal to ventral in their dorso-longitudinal flight muscles (DLMs). Does the existence of this thermal gradient necessarily imply a functional gradient? Do these changes in function have, as their basis, changes in structure? Twitch dynamics of individual fibers within the DLM in intact animals are temperature dependent so that mechanical power output (and it's phase dependence) varies with depth in the tissue. A surprising observation was that all five sub-units in the DLM were simultaneously activated. Cooler muscles subjected to 25 hz stimulation (flight frequency) are partially fused with (likely) little crossbridge turnover. To assess the structural correlates of temperature gradients, we performed small-angle x-ray fiber diffraction measurements as a function of position along the dorsal-ventral thermal gradient in intact moth thoraces. The equatorial intensity ratio (I20/I10) in unstimulated muscle increased by ∼25% in the first 1 −1.5 mm traversing from dorsal to ventral, implying that increased temperature was associated with increased association of the myosin heads with the thin filaments presumably predisposing them towards more productive acto-myosin interaction. Interestingly, X-ray patterns from skinned muscle preparations improved with increasing temperature indicating better structural order. Together, these observations suggest that cooler, superficial muscles may act mainly as elastic energy storage, whereas warmer deeper muscles may do the bulk of the mechanical work.

Functional Effects of a Restrictive-Cardiomyopathy-Linked Cardiac Troponin I Mutation (R145W) in Transgenic Mice

The human cardiac troponin I (hcTnI) mutation R145W has been associated with restrictive cardiomyopathy. In this study, simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145W transgenic mice (Tg-R145W) were explored. Tg-R145W fibers showed an ∼ 13–16% increase in maximal Ca 2+-activated force and ATPase activity compared to hcTnI wild-type transgenic mice. The force-generating cross-bridge turnover rate ( g) and the energy cost (ATPase/force) were the same in all groups of fibers. Also, the Tg-R145W fibers showed a large increase in the Ca 2+ sensitivity of both force development and ATPase. In intact fibers, the mutation caused prolonged force and intracellular [Ca 2+] transients and increased time to peak force. Analysis of force and Ca 2+ transients showed that there was a 40% increase in peak force in Tg-R145W muscles, which was likely due to the increased Ca 2+ transient duration. The above cited results suggest that: (1) there would be an increase in resistance to ventricular filling during diastole resulting from the prolonged force and Ca 2+ transients that would result in a decrease in ventricular filling (diastolic dysfunction); and (2) there would be a large (approximately 53%) increase in force during systole, which may help to partly compensate for diastolic dysfunction. These functional results help to explain the mechanisms by which these mutations give rise to a restrictive phenotype.

Functional Analysis of a Unique Troponin C Mutation, GLY159ASP, that Causes Familial Dilated Cardiomyopathy, Studied in Explanted Heart Muscle

Familial dilated cardiomyopathy can be caused by mutations in the proteins of the muscle thin filament. In vitro, these mutations decrease Ca(2+) sensitivity and cross-bridge turnover rate, but the mutations have not been investigated in human tissue. We studied the Ca(2+)-regulatory properties of myocytes and troponin extracted from the explanted heart of a patient with inherited dilated cardiomyopathy due to the cTnC G159D mutation. Mass spectroscopy showed that the mutant cTnC was expressed approximately equimolar with wild-type cTnC. Contraction was compared in skinned ventricular myocytes from the cTnC G159D patient and nonfailing donor heart. Maximal Ca(2+)-activated force was similar in cTnC G159D and donor myocytes, but the Ca(2+) sensitivity of cTnC G159D myocytes was higher (EC(50) G159D/donor=0.60). Thin filaments reconstituted with skeletal muscle actin and human cardiac tropomyosin and troponin were studied by in vitro motility assay. Thin filaments containing the mutation had a higher Ca(2+) sensitivity (EC(50) G159D/donor=0.55 + or - 0.13), whereas the maximally activated sliding speed was unaltered. In addition, the cTnC G159D mutation blunted the change in Ca(2+) sensitivity when TnI was dephosphorylated. With wild-type troponin, Ca(2+) sensitivity was increased (EC(50) P/unP=4.7 + or - 1.9) but not with cTnC G159D troponin (EC(50) P/unP=1.2 + or - 0.1). We propose that uncoupling of the relationship between phosphorylation and Ca(2+) sensitivity could be the cause of the dilated cardiomyopathy phenotype. The differences between these data and previous in vitro results show that native phosphorylation of troponin I and troponin T and other posttranslational modifications of sarcomeric proteins strongly influence the functional effects of a mutation.

Time Course and Strain Dependence of ADP Release during Contraction of Permeabilized Skeletal Muscle Fibers

A phosphorylated, single cysteine mutant of nucleoside diphosphate kinase, labeled with N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide (P∼NDPK-IDCC), was used as a fluorescence probe for time-resolved measurement of changes in [MgADP] during contraction of single permeabilized rabbit psoas fibers. The dephosphorylation of the phosphorylated protein by MgADP occurs within the lattice environment of permeabilized fibers with a second-order rate constant at 12°C of 105 M−1 s−1. This dephosphorylation is accompanied by a change in coumarin fluorescence. We report the time course of P∼NDPK-IDCC dephosphorylation during the period of active isometric force redevelopment after quick release of fiber strain at pCa2+ of 4.5. After a rapid length decrease of 0.5% was applied to the fiber, the extra NDPK-IDCC produced during force recovery, above the value during the approximately steady state of isometric contraction, was 2.7 ± 0.6 μM and 4.7 ± 1.5 μM at 12 and 20°C, respectively. The rates of P∼NDPK-IDCC dephosphorylation during force recovery were 28 and 50 s−1 at 12 and 20°C, respectively. The time courses of isometric force and P∼NDPK-IDCC dephosphorylation were simulated using a seven-state reaction scheme. Relative isometric force was modeled by changes in the occupancy of strongly bound A.M.ADP.Pi and A.M.ADP states. A strain-sensitive A.M.ADP isomerization step was rate-limiting (3–6 s−1) in the cross-bridge turnover during isometric contraction. At 12°C, the A.M.ADP.Pi and the pre- and postisomerization A.M.ADP states comprised 56%, 38%, and 7% of the isometric force-bearing AM states, respectively. At 20°C, the force-bearing A.M.ADP.Pi state was a lower proportion of the total force-bearing states (37%), whereas the proportion of postisomerization A.M.ADP states was higher (19%). The simulations suggested that release of cross-bridge strain caused rapid depopulation of the preisomerization A.M.ADP state and transient accumulation of MgADP in the postisomerization A.M.ADP state. Hence, the strain-sensitive isomerization of A.M.ADP seems to explain the rate of change of P∼NDPK-IDCC dephosphorylation during force recovery. The temperature-dependent isometric distribution of myosin states is consistent with the previous observation of a small decrease in amplitude of the Pi transient during force recovery at 20°C and the current observation of an increase in amplitude of the ADP-sensitive NDPK-IDCC transient.

Insights into the kinetics of Ca2+-regulated contraction and relaxation from myofibril studies

Muscle contraction results from force-generating interactions between myosin cross-bridges on the thick filament and actin on the thin filament. The force-generating interactions are regulated by Ca(2+) via specialised proteins of the thin filament. It is controversial how the contractile and regulatory systems dynamically interact to determine the time course of muscle contraction and relaxation. Whereas kinetics of Ca(2+)-induced thin-filament regulation is often investigated with isolated proteins, force kinetics is usually studied in muscle fibres. The gap between studies on isolated proteins and structured fibres is now bridged by recent techniques that analyse the chemical and mechanical kinetics of small components of a muscle fibre, subcellular myofibrils isolated from skeletal and cardiac muscle. Formed of serially arranged repeating units called sarcomeres, myofibrils have a complete fully structured ensemble of contractile and Ca(2+) regulatory proteins. The small diameter of myofibrils (few micrometres) facilitates analysis of the kinetics of sarcomere contraction and relaxation induced by rapid changes of [ATP] or [Ca(2+)]. Among the processes studied on myofibrils are: (1) the Ca(2+)-regulated switch on/off of the troponin complex, (2) the chemical steps in the cross-bridge adenosine triphosphatase cycle, (3) the mechanics of force generation and (4) the length dynamics of individual sarcomeres. These studies give new insights into the kinetics of thin-filament regulation and of cross-bridge turnover, how cross-bridges transform chemical energy into mechanical work, and suggest that the cross-bridge ensembles of each half-sarcomere cooperate with each other across the half-sarcomere borders. Additionally, we now have a better understanding of muscle relaxation and its impairment in certain muscle diseases.

Fluorescence Lifetime of Actin in the Familial Hypertrophic Cardiomyopathy Transgenic Heart

Clinical studies have revealed that the D166V mutation in the ventricular myosin regulatory light chain (RLC) can cause a malignant phenotype of familial hypertrophic cardiomyopathy (FHC). It has been proposed that RLC induced FHC in the heart originates at the level of the myosin cross-bridge due to alterations in the rates of cross-bridge cycling. In this report, we examine whether the environment of an active cross-bridge in cardiac myofibrils from transgenic (Tg) mice is altered by the D166V mutation in RLC. The cross-bridge environment was monitored by tracking the fluorescence lifetime (tau) of Alexa488-phalloidin-labeled actin. The fluorescence lifetime is the average rate of decay of a fluorescent species from the excited state, which strongly depends on various environmental factors. We observed that the lifetime was high when cross-bridges were bound to actin and low when they were dissociated from it. The lifetime was measured every 50 ms from the center half of the I-band during 60 s of rigor, relaxation and contraction of muscle. We found no differences between lifetimes of Tg-WT and Tg-D166V muscle during rigor, relaxation and contraction. The duty ratio expressed as a fraction of time that cross-bridges spend attached to the thin filaments during isometric contraction was similar in Tg-WT and Tg-D166V muscles. Since independent measurements showed a large decrease in the cross-bridge turnover rate in Tg-D166V muscle compared to Tg-WT, the fact that the duty cycle remains constant suggests that the D166V mutation of RLC causes a decrease in the rate of cross-bridge attachment to actin.

Abstract 1580: Phosphorylation of Myosin Binding Protein C Contributes to the Force-Frequency Response and Beta-Adrenergic Enhancement of Cardiac Contraction

Beta-adrenergic agonists enhance cardiac function by increasing contractility, lusitropy and heart rate. We propose that phosphorylation of myosin binding protein C (cMyBP-C), a thick filament accessory protein, plays a key role in adrenergic modulation of function by regulating crossbridge interaction kinetics. To test this idea, we developed transgenic mice expressing similar levels of either cMyBP-C with Ser to Ala mutations at 3 known protein kinase A (PKA) sites (t3SA mice) or wild type cMyBP-C (tWT mice) on the cMyBP-C knock-out (KO) background. Using echocardiography, t3SA hearts were found to have concentric hypertrophy and exhibited diastolic and systolic dysfunction, but tWT hearts were normal. We recorded twitch force and intracellular Ca 2+ ([Ca 2+ ] in ) in intact papillary muscles to assess in vivo myofilament function. At 1 Hz stimulation, t3SA and tWT exhibited similar twitch forces and [Ca 2+ ] in . Increasing the frequency to 3 Hz produced a smaller force increase in t3SA but similar increases in [Ca 2+ ] in compared to tWT. t3SA myocardium exhibited a smaller gain in function with increased frequency of stimulation, i.e., a smaller increase in twitch force vs. change in [Ca 2+ ] in and smaller increases in (+dF/dt) Max , and (−dF/dt) Max . These results suggest that basal levels of phosphorylation of cMyBP-C in tWT myocardium contribute to the positive force frequency relationship in myocardium. For both t3SA and tWT, 1 μM dobutamine further increased twitch force and [Ca 2+ ] in at 3 Hz. Phosphoprotein staining showed that t3SA and tWT troponin I (TnI) phosphorylation levels were initially similar and increased to similar levels with dobutamine; therefore, dobutamine effects may involve TnI phosphorylation. These results can be explained by a frequency dependent recruitment of myofilament functional groups that involves cMyBP-C phosphorylation and further effects of PKA phosphorylation of cMyBP-C and TnI to accelerate crossbridge turnover kinetics.

Functional Consequences of the Human Cardiac Troponin I Hypertrophic Cardiomyopathy Mutation R145G in Transgenic Mice

In this study, we addressed the functional consequences of the human cardiac troponin I (hcTnI) hypertrophic cardiomyopathy R145G mutation in transgenic mice. Simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145G transgenic mice (Tg-R145G) versus hcTnI wild type transgenic mice (Tg-WT) showed a significant decrease in the maximal Ca(2+)-activated force without changes in the maximal ATPase activity and an increase in the Ca(2+) sensitivity of both ATPase and force development. No difference in the cross-bridge turnover rate was observed at the same level of cross-bridge attachment (activation state), showing that changes in Ca(2+) sensitivity were not due to changes in cross-bridge kinetics. Energy cost calculations demonstrated higher energy consumption in Tg-R145G fibers compared with Tg-WT fibers. The addition of 3 mm 2,3-butanedione monoxime at pCa 9.0 showed that there was approximately 2-4% of force generating cross-bridges attached in Tg-R145G fibers compared with less than 1.0% in Tg-WT fibers, suggesting that the mutation impairs the ability of the cardiac troponin complex to fully inhibit cross-bridge attachment under relaxing conditions. Prolonged force and intracellular [Ca(2+)] transients in electrically stimulated intact papillary muscles were observed in Tg-R145G compared with Tg-WT. These results suggest that the phenotype of hypertrophic cardiomyopathy is most likely caused by the compensatory mechanisms in the cardiovascular system that are activated by 1) higher energy cost in the heart resulting from a significant decrease in average force per cross-bridge, 2) slowed relaxation (diastolic dysfunction) caused by prolonged [Ca(2+)] and force transients, and 3) an inability of the cardiac TnI to completely inhibit activation in the absence of Ca(2+) in Tg-R145G mice.

How does genotype define phenotype? Microphysiology of a tropomyosin mutation in situ shows the limitations of reductionism

Tropomyosin is one of the components of muscle thin filaments. In concert with the troponin complex it forms the regulatory Ca2+ switch of striated muscles. The tropomyosin molecule is a parallel coiled-coil dimer of 274 amino acids which is almost 100%α-helix. Two strands of tropomyosin molecules joined end-to-end are wound around the actin double helix to form the core of the thin filament. Traditionally tropomyosin has been regarded as a passive player in muscle regulation, whose function is merely to transmit Ca2+ activation signals from troponin along the filament. However, in recent years tropomyosin mutations have been found to cause a number of clinically significant myopathies including hypertrophic cardiomyopathy, dilated cardiomyopathy, distal arthrogryposis, nemaline myopathy and congenital fibre-type disproportion. In the same way that disease-causing mutations in myosin heavy chain, myosin binding protein C and troponin T have sparked off renewed investigations of apparently well-known proteins, the discovery of mutations in tropomyosin has stimulated many new structural and biochemical studies of phenotype–genotype relationships. The emerging story is that tropomyosin is a much more interesting molecule than previously thought and it plays a very fundamental role in muscle regulation. From structural investigations it is now evident that tropomyosin is not a pure coiled-coil α-helix (Brown et al. 2001). Breaks in the heptad sequence alter the shape of tropomyosin so that it bends round the actin helix and there are also points with increased flexibility that are essential for tropomyosin to change its conformation on actin between the active and relaxed states. Binding sites for troponin T and for actin in the active and relaxed states have been identified. Thus, it should be possible to predict the effect of mutations from their location and nature. In practice, studies on the functional effect of disease-causing mutations in tropomyosin by in vitro methods do not correlate well with predictions from structure since almost any aspect of Ca2+ regulation could be altered by mutations. This should not be surprising since it is well understood that the thin filament is an integrated cooperative–allosteric system so that perturbations in any location will have a global effect on structure and function (Perry, 2003). Thus the complexity of the thin filament at present defies reductionist explanation (see, for instance, our recent study on the E40K and E54K mutations in α-tropomyosin (Mirza et al. 2007)). The proper way to study genetic disease is to study the physiology of the diseased tissue itself. This is a formidable challenge to physiologists but a recent paper in The Journal of Physiology from Ochala et al. shows that it can be done (Ochala et al. 2008). In this study, the authors obtained fresh biopsies of skeletal muscle from a patient with skeletal myopathy due to the mutation of glutamic acid 41 to lysine in β-tropomyosin and performed a comprehensive series of mechanical measurements, in vitro motility and even determined the effect of a Ca2+ sensitiser as a possible therapy. In order to do this they needed to select individual type I fibres for assay based on myosin heavy chain isoform analysis. The key findings are that crossbridge turnover speed and Ca2+ sensitivity are reduced but the relationship between force and stiffness is not affected. The interpretation of these results is that crossbridge properties are not changed but the probability of activation at intermediate Ca2+ concentrations is reduced. It is interesting to note that the observed 2- to 3-fold increase in EC50 is of the same order as that found with mutations that cause dilated cardiomyopathy; indeed a very similar mutation was found at the neighbouring amino acid (E40K in TPM1; Mirza et al. 2005). In both of these cases the mutation is remote from the putative sites where troponin binds to tropomyosin, thus emphasizing the allosteric nature of muscle regulation. Another interesting feature that could only be determined in tissue samples is that the β-tropomyosin content relative to α-tropomyosin in mutant muscle is increased from 68% to 81% and that muscle fibres are small – 28% of control fibre diameter. It is by no means clear how this multiplicity of changes is caused by the initiating mutation nor which changes are significant in the disease phenotype. Nevertheless microphysiology of this kind is most valuable in showing us the overall state of a diseased muscle, which can be quite different from that predicted from the molecular properties of tropomyosin on its own.