Myofilament Level Research Articles

Cardiac troponin T mutations: correlation between the type of mutation and the nature of myofilament dysfunction in transgenic mice.

1. The heterogenic nature of familial hypertrophic cardiomyopathy (FHC) in humans suggests a link between the type of mutation and the nature of patho-physiological alterations in cardiac myocytes. Exactly how FHC-associated mutations in cardiac troponin T (cTnT) lead to impaired cardiac function is unclear. 2. We measured steady-state isometric force and ATPase activity in detergent-skinned cardiac fibre bundles from three transgenic (TG) mouse hearts in which 50, 92 and 6 % of the native cTnT was replaced by the wild type (WT) cTnT, R92Q mutant cTnT (R92Q) and the C-terminal deletion mutant of cTnT (cTnT(DEL)), respectively. 3. Normalized pCa-tension relationships of R92Q and cTnT(DEL) fibres demonstrated a significant increase in sensitivity to Ca2+ at short (2.0 microm) and long (2.3 microm) sarcomere lengths (SL). At short SL, the pCa50 values, representing the midpoint of the pCa-tension relationship, were 5.69 +/- 0.01, 5.96 +/- 0.01 and 5.81 +/- 0.01 for WT, R92Q and cTnT(DEL) fibres, respectively. At long SL, the pCa50 values were 5.81 +/- 0.01, 6.08 +/- 0.01 and 5.95 +/- 0.01 for WT, R92Q and cTnT(DEL) fibres, respectively. 4. The difference in pCa required for half-maximal activation (DeltapCa50) at short and long SL was 0.12 +/- 0.01 for the R92Q (92 %) TG fibres, which is significantly less than the previously reported DeltapCa50 value of 0.29 +/- 0.02 for R92Q (67 %) TG fibres. 5. At short SL, Ca2+-activated maximal tension in both R92Q and cTnT(DEL) fibres decreased significantly (24 and 21 %, respectively; P < 0.005), with no corresponding decrease in Ca2+-activated maximal ATPase activity. Therefore, at short SL, the tension cost in R92Q and cTnT(DEL) fibres increased by 35 and 29 %, respectively (P < 0.001). 6. The fibre bundles reconstituted with the recombinant mutant cTnT(DEL) protein developed only 37 % of the Ca2+-activated maximal force developed by recombinant WT cTnT reconstituted fibre bundles, with no apparent changes in Ca2+ sensitivity. 7. Our data indicate that an important mutation-linked effect on cardiac function is the result of an inefficient use of ATP at the myofilament level. Furthermore, the extent of the mutation-induced dysfunction depends not only on the nature of the mutation, but also on the concentration of the mutant protein in the sarcomere.

Maintained contractile reserve in a transgenic mouse model of myocardial stunning.

Cardiac excitation-contraction (E-C) coupling is impaired at the myofilament level in the reversible postischemic dysfunction known as "stunned" myocardium. We characterized tension development and calcium cycling in intact isolated trabeculae from transgenic (TG) mice expressing the major proteolytic degradation fragment of troponin I (TnI) found in stunned myocardium (TnI(1-193)) and determined the ATPase activity of myofibrils extracted from TG and non-TG mouse hearts. The phenotype of these mice at baseline recapitulates that of stunning. Here, we address the question of whether contractile reserve is preserved in these mice, as it is in genuine stunned myocardium. During twitch contractions, calcium cycling was normal, whereas tension was greatly reduced, compared with non-TG controls. A decrease in maximum Ca2+-activated tension and Ca2+ desensitization of the myofilaments accounted for this contractile dysfunction. The decrease in maximum tension was paralleled by an equivalent decrease in maximum Ca2+-activated myofibrillar ATPase activity. Exposure to high calcium or isoproterenol recruited a sizable contractile reserve in TG muscles, which was proportionately similar to that in control muscles but scaled downward in amplitude. These results suggest that calcium regulatory pathways and beta-adrenergic signal transduction remain intact in isolated trabeculae from stunned TG mice, further recapitulating key features of genuine stunned myocardium.

Brief rapid pacing depresses contractile function via Ca(2+)/PKC-dependent signaling in cat ventricular myocytes.

The purpose of this study is to determine the effects of brief rapid pacing (RP; approximately 200-240 beats/min for approximately 5 min) on contractile function in ventricular myocytes. RP was followed by a sustained inhibition of peak systolic cell shortening (-44 +/- 4%) that was not due to changes in diastolic cell length, membrane voltage, or L-type Ca(2+) current (I(Ca,L)). During RP, baseline and peak intracellular Ca(2+) concentration ([Ca(2+)](i)) increased markedly. After RP, Ca(2+) transients were similar to control. The effects of RP on cell shortening were not prevented by 1 microM calpain inhibitor I, 25 microM L-N(5)-(1-iminoethyl)-orthinthine, or 100 microM N(G)-monomethyl-L-arginine. However, RP-induced inhibition of cell shortening was prevented by lowering extracellular [Ca(2+)] (0.5 mM) during RP or exposure to chelerythrine (2-4 microM), a protein kinase C (PKC) inhibitor, or LY379196 (30 nM), a selective inhibitor of PKC-beta. Exposure to phorbol ester (200 nM phorbol 12-myristate 13-acetate) inhibited cell shortening (-46 +/- 7%). Western blots indicated that cat myocytes express PKC-alpha, -delta, and -epsilon as well as PKC-beta. These findings suggest that brief RP of ventricular myocytes depresses contractility at the myofilament level via Ca(2+)/PKC-dependent signaling. These findings may provide insight into the mechanisms of contractile dysfunction that follow paroxysmal tachyarrhythmias.

The contractile mechanism as an approach to building left ventricular pump models.

The aim was to construct a model linking a simplified interpretation of the contractile process at the myofilament level to the mechanical behaviour of the left ventricle to improve the ability of elastic-resistive models to represent the pumping response of the left ventricle. The mechanical model, consisting of an elastic component connected in series with a contractile component and an elastic component parallel to both the series elastic and contractile components, is able to develop pressure by the binding of a structural substance T to an excitatory substance C, the behaviour of which is a simplification of miofibrillar Ca2+ kinetics. Theoretically, the model was validated for its ability to reproduce by computer simulation, experiments that described the pumping properties of the left ventricle--namely, elasticity, resistivity, deactivating and positive effect of ejection, and the behaviour of intracellular Ca2+. Experimentally, the model was tested to fit intraventricular pressure (P(t)) and volume (V(t)) of single ejective beats in nine open chest dogs fitted with a pressure microtransducer to measure intraventricular P(t) and an aortic flowprobe to measure ventricular outflow and calculate V(t). Parameters were estimated up to maximum negative dP/dt adjusting P(t) or V(t) data of the ejective beats, and the goodness of the fit was evaluated through the root mean square error normalised with respect to the corresponding mean P(t) or V(t) in the fitting interval (NE). Descriptive validation of the model showed that the mean NE for the ejective P(t) fit was 0.03(SD 0.005) and for the V(t) fit 0.014(0.003). Predictive validation of P(t) and V(t) data of beats with partial occlusion of the aorta was performed up to end ejection, with parameters estimated from the P(t) or V(t) fit of the preceding ejective beat. Results gave a mean NE equal to 0.05(0.02) for predicted P(t) and 0.02(0.007) for predicted V(t), from either source of estimated parameters. Explanative validation showed that all the estimated parameters were in the same range used in simulation and that derived indexes [isovolumic maximum pressure (Pmax) = 166(13) mm Hg, time to maximum pressure (TPmax) = 0.186(0.012) s and the slope of the end systolic pressure volume relation (Emax) = 5.45(1.5) mm Hg.ml-1] were within reported experimental values. Finally, the model responded to increased inotropic state [dobutamine (5-35 micrograms.kg-1.min-1)] causing the estimated Pmax and Emax to increase by 33% and 25%, respectively, and TPmax to decrease by 10%. This model represented an improvement over previous pump models because (1) the model was able to represent behaviours other than purely elastic-resistive ones, such as the deactivation and positive effect of ejection; (2) left ventricular properties were the response of model behaviour and not constitutive elements of its structure; and (3) it adequately fulfilled model validation procedures.

Calcium-activated force in a turkey model of spontaneous dilated cardiomyopathy: Adaptive changes in thin myofilament Ca 2+ regulation with resultant implications on contractile performance

Using saponin skinned fibers, we investigated whether decreased myofilament calcium responsiveness and contractile activation may in part contribute to heart failure in an animal model of idiopathic spontaneous cardiomyopathy (SCM). We addressed the question as to whether there are adaptive changes at the level of the thin myofilaments in turkey poults with SCM. The calcium concentration ([Ca 2+]) required for 50% activation ([Ca 2+] 50%) was 0.80±0.12 μ m ( n=12) vs. 0.76±0.08 μ m ( n=12) and the Hill coefficient was 1.98±0.20 ( n=12) vs. 2.14±0.38 ( n=12) for control and SCM muscles respectively. Maximal Ca 2+-activated force was not different between control fibers and fibers from failing hearts (3.83±0.88 g/mm 2 vs. 3.65±0.39 g/mm 2). These data indicate there are no differences in calcium-activation between fibers from control and failing myocardium. The effects of caffeine, an agent that increases myofilament Ca 2+ sensitivity, were also studied. Addition of 10 m m caffeine resulted in a 0.06 pCa unit leftward shift of the force-pCa relationship in control hearts and 0.14 pCa units in SCM hearts. Caffeine (30 m m) increased force by 26±2.1% ( n=7) in control fibers and 44.5±8.7% ( n=8) in myopathic fibers at a pCa of 6.0. The increased responsiveness of muscles from failing hearts to caffeine indicates adaptive changes at the level of the thin myofilaments. Addition of dibutyryl-3′,5′-cyclic-Adenosine Monophosphate (D-cAMP) resulted in a 0.21 pCa rightward shift on the calcium axis to higher intracelluar calcium concentrations in control myocardium and 0.38 pCa units in SCM failing myocardium. The muscles were also sinusoidally oscillated at frequencies ranging between 0.01 and 100 Hz. In this analysis the frequency at which dynamic stiffness is minimum is taken as a measure of cross-bridge cycling rate. In control muscles, the frequency of minimum stiffness (f min) was 1.20±0.11 ( n=4) whereas it was 0.71±0.08 Hz ( n=4) in myopathic muscles. The addition of 10 μ m D-cAMP shifted f min from 1.20±0.11 Hz to 1.68±0.09 Hz (Δ= 0.48±0.06) in control fibers whereas in SCM fibers it caused greater shift of f min from 0.71±0.08 Hz to 1.73±0.08 Hz (Δ= 1.02±0.07). This differential effect of D-cAMP indicates adaptive changes at the level of the myofilaments. Our studies indicate that adaptive changes at the level of the thin myofilaments can potentially alter the “apparent” calcium-force relationship in failing myocardium and contractile performance, and that failing myocardium has the potential to generate similar levels of force as normal non-failing myocardium.

Modulation of calcium-activation in control and pressure-overload hypertrophied ferret hearts: Effect of DPI 201-106 on myofilament calcium responsiveness

We examined the relationship between free Ca 2+ and developed force in chemically skinned fibers from control and pressure-overload hypertrophied (POH) hearts of ferrets in the absence and presence of DPI 201-106 (4-([3-(4-diphenyl-methyl-1-piperazinyl)-2-hydroxypropoxy]-1H-indole-2-carbonitrile), a positive inotropic and negative lusitropic agent. Force production in both control and hypertrophied fibers increased with Ca 2+ concentration ([Ca 2+]) over a range from 10 −7 m to 10 −4 m, and did not differ significantly in response to Ca 2+ under isometric conditions. The [Ca 2+] required for half-maximal activation ([Ca 2+] 50%) was estimated to be 1.84 × 10 −6 m in control muscles and 1.76 × 10 −6 m in POH muscles. The maximal Ca 2+-activated force was significantly higher in the POH group (3.68 ± 0.27 g/mm 2) as compared to the control muscles (2.41 ± 0.56 g/mm 2). A DPI concentration of 10 −6 m shifted the force-pCa relation leftward by 0.13–0.18 pCa units in the control hearts, and by 0.40–0.45 pCa units in the hypertrophied hearts. In the concentration range between 10 −8 m and 10 −5 m, DPI induced a concentration dependent increase in force production that reached about 40% in the hypertrophied hearts and only 18% in the control hearts at pCa 6. The influence of DPI on the myofibrillar Ca 2+ binding may be due to the effect of the drug on the troponin T-tropomyosin complex. In view of our results, we propose that hypertrophied hearts may demonstrate an adaptational change in the contractile proteins at the level of the thin myofilaments. This adaptational change may result in altered contractile performance and response to agents that potentially act at the level of the myofilaments. Further, alteration at the level of the myofilaments may not be detected by standard force-[Ca 2+] relationships.