Myofibril ATPase Research Articles

Cardiac Troponin Activator CK-963 Increases Cardiac Contractility in Rats.

Novel cardiac troponin activators were identified using a high throughput cardiac myofibril ATPase assay and confirmed using a series of biochemical and biophysical assays. HTS hit 2 increased rat cardiomyocyte fractional shortening without increasing intracellular calcium concentrations, and the biological target of 1 and 2 was determined to be the cardiac thin filament. Subsequent optimization to increase solubility and remove PDE-3 inhibition led to the discovery of CK-963 and enabled pharmacological evaluation of cardiac troponin activation without the competing effects of PDE-3 inhibition. Rat echocardiography studies using CK-963 demonstrated concentration-dependent increases in cardiac fractional shortening up to 95%. Isothermal calorimetry studies confirmed a direct interaction between CK-963 and a cardiac troponin chimera with a dissociation constant of 11.5 ± 3.2 μM. These results provide evidence that direct activation of cardiac troponin without the confounding effects of PDE-3 inhibition may provide benefit for patients with cardiovascular conditions where contractility is reduced.

Myosin S2 stabilizer peptide decreases recovery rates of contraction.

A peptide, called the Stabilizer, designed to increase the energy required to unzip the myosin S2 coiled coil was tested on myofibrils, skinned trabeculae, and live cardiomyocytes. The Stabilizer bound to myosin S2 with submicromolar affinity and positive cooperativity measured by both TIRF and MST, which suggests a modification in the myosin S2 structure. Myofibril ATPase activity was activated at pCa 9 and inhibited at pCa 4.5, consistent with an inhibition of actin-activated ATPase activity. In vitro motility assays of actin sliding velocity were partially depressed in the presence of the Stabilizer. Both force development rates and regulatory light chain orientation (measured by fluorescence polarization) were also slowed by the Stabilizer following a slack-re-stretch maneuver in skinned trabeculae. To test perturbations of living cardiomyocytes, the Stabilizer peptide was chemically esterified with a cell penetrating molecule to facilitate translocation across the cell membrane and removal of the targeting molecule once inside the cell. Confocal microscopy imaging showed sarcomere labeling with fluorescently tagged targeted Stabilzer in live cardiomyocytes. The Stabilizer decreased sarcomere length shortening in living cardiomyocytes. These data are consistent with a reduced contractility by the Stabilizer, which may result from the reinforcement of the myosin S2 coiled coil structure mitigating myosin's flexibility and probability of cross-bridge formation. (Supported by NIH/NHLBI R01HL149164.)

The effect of small molecule myosin modulators on ATP turnover in pig cardiac HMM using stopped flow spectroscopy.

Myosin modulators are a novel class of pharmaceutical agents designed to treat patients with cardiomyopathies by directly modulating cardiac myosin function in the sarcomere. Compounds including omecamtiv mecarbil (OM), danicamtiv (Dani), and deoxy-ATP (dATP) have previously been shown to increase myofibril ATPase activity while mavacamten (Mava) reduced ATPase activity. In the absence of actin, ATPase activity is a combination of the direct effects of nucleotide binding and the equilibrium between the high activity (DRX) and low activity (SRX) states of myosin. In this study, we investigate how these small molecules affect the single turnover kinetics of pig cardiac heavy meromyosin (pcHMM) in the absence of actin. pcHMM bound to fluorescent mant.ATP or mant.dATP is rapidly mixed with a high concentration of unlabeled ATP. The rate constant for replacement of mant.ADP by ATP defines the turnover of mant.ATP. Preliminary titration experiments demonstrate that OM, Dani, and Mava all inhibit ATP turnover with an IC50 of 0.59 µM, 3.5 µM, and 0.276 µM, respectively. 100% dATP increases the ATP turnover by 100%. These experiments indicate that each myosin modulator differentially alters cardiac HMM activity. We will discuss how each modulator affects ATP turnover by a direct effect on catalytic activity vs an effect on the amount of HMM in the super-relaxed population.

Accessibility of myofilament cysteines and effects on ATPase depend on the activation state during exposure to oxidants.

Signaling by reactive oxygen species has emerged as a major physiological process. Due to its high metabolic rate, striated muscle is especially subject to oxidative stress, and there are multiple examples in cardiac and skeletal muscle where oxidative stress modulates contractile function. Here we assessed the potential of cysteine oxidation as a mechanism for modulating contractile function in skeletal and cardiac muscle. Analyzing the cysteine content of the myofilament proteins in striated muscle, we found that cysteine residues are relatively rare, but are very similar between different muscle types and different vertebrate species. To refine this list of cysteines to those that may modulate function, we estimated the accessibility of oxidants to cysteine residues using protein crystal structures, and then sharpened these estimates using fluorescent labeling of cysteines in cardiac and skeletal myofibrils. We demonstrate that cysteine accessibility to oxidants and ATPase rates depend on the contractile state in which preparations are exposed. Oxidant exposure of skeletal and cardiac myofibrils in relaxing solution exposes myosin cysteines not accessible in rigor solution, and these modifications correspond to a decrease in maximum ATPase. Oxidant exposure under rigor conditions produces modifications that increase basal ATPase and calcium sensitivity in ventricular myofibrils, but these effects were muted in fast twitch muscle. These experiments reveal how structural and sequence variations can lead to divergent effects from oxidants in different muscle types.

Myosin denaturation in heated myofibrils of scallop adductor muscle

The thermal inactivation of Ca2+ ATPase of scallop myofibrils (0.1 M KCl, pH 7.5) was found to be unaffected by the presence of Ca2+. Monomeric myosin content and salt solubility decreased much faster than Ca2+ ATPase inactivation in both Ca and EDTA media, which was well explained by faster denaturation of the rod portion than subfragment-1 of myosin. In contrast, when the myofibrils were heated at 0.5 M KCl, a slow decrease in salt solubility was observed, which was also explained by slow denaturation of the rod portion of myosin. Myofibrils from scallop smooth muscle showed the same denaturation pattern as those from adductor muscle. These results show that mollusk myosin is not always stabilized by Ca2+.

Protein kinase C depresses cardiac myocyte power output and attenuates myofilament responses induced by protein kinase A

Following activation by G-protein-coupled receptor agonists, protein kinase C (PKC) modulates cardiac myocyte function by phosphorylation of intracellular targets including myofilament proteins cardiac troponin I (cTnI) and cardiac myosin binding protein C (cMyBP-C). Since PKC phosphorylation has been shown to decrease myofibril ATPase activity, we hypothesized that PKC phosphorylation of cTnI and cMyBP-C will lower myocyte power output and, in addition, attenuate the elevation in power in response to protein kinase A (PKA)-mediated phosphorylation. We compared isometric force and power generating capacity of rat skinned cardiac myocytes before and after treatment with the catalytic subunit of PKC. PKC increased phosphorylation levels of cMyBP-C and cTnI and decreased both maximal Ca(2+) activated force and Ca(2+) sensitivity of force. Moreover, during submaximal Ca(2+) activations PKC decreased power output by 62%, which arose from both the fall in force and slower loaded shortening velocities since depressed power persisted even when force levels were matched before and after PKC. In addition, PKC blunted the phosphorylation of cTnI by PKA, reduced PKA-induced spontaneous oscillatory contractions, and diminished PKA-mediated elevations in myocyte power. To test whether altered thin filament function plays an essential role in these contractile changes we investigated the effects of chronic cTnI pseudo-phosphorylation on myofilament function using myocyte preparations from transgenic animals in which either only PKA phosphorylation sites (Ser-23/Ser-24) (PP) or both PKA and PKC phosphorylation sites (Ser-23/Ser-24/Ser-43/Ser-45/T-144) (All-P) were replaced with aspartic acid. Cardiac myocytes from All-P transgenic mice exhibited reductions in maximal force, Ca(2+) sensitivity of force, and power. Similarly diminished power generating capacity was observed in hearts from All-P mice as determined by in situ pressure-volume measurements. These results imply that PKC-mediated phosphorylation of cTnI plays a dominant role in depressing contractility, and, thus, increased PKC isozyme activity may contribute to maladaptive behavior exhibited during the progression to heart failure.

Perfluorooctanoic acid induces developmental cardiotoxicity in chicken embryos and hatchlings

Perfluorooctanoic acid (PFOA) is a widespread environmental contaminant that is detectable in serum of the general U.S. population. PFOA is a known developmental toxicant that induces mortality in mammalian embryos and is thought to induce toxicity via interaction with the peroxisome proliferator activated receptor alpha (PPARα). As the cardiovascular system is crucial for embryonic survival, PFOA-induced effects on the heart may partially explain embryonic mortality. To assess impacts of PFOA exposure on the developing heart in an avian model, we used histopathology and immunohistochemical staining for myosin to assess morphological alterations in 19-day-old chicken embryo hearts after PFOA exposure. Additionally, echocardiography and cardiac myofibril ATPase activity assays were used to assess functional alterations in 1-day-old hatchling chickens following developmental PFOA exposure. Overall thinning and thinning of a dense layer of myosin in the right ventricular wall were observed in PFOA-exposed chicken embryo hearts. Alteration of multiple cardiac structural and functional parameters, including left ventricular wall thickness, left ventricular volume, heart rate, stroke volume, and ejection fraction were detected with echocardiography in the exposed hatchling chickens. Assessment of ATPase activity indicated that the ratio of cardiac myofibril calcium-independent ATPase activity to calcium-dependent ATPase activity was not affected, which suggests that developmental PFOA exposure may not affect cardiac energetics. In summary, structural and functional characteristics of the heart appear to be developmental targets of PFOA, possibly at the level of cardiomyocytes. Additional studies will investigate mechanisms of PFOA-induced developmental cardiotoxicity.

Ca2+ Sensitization of Myocardial Force and Actomyosin ATPase by (D-Ala2, Met5) Enkephalinamide

The presence of proenkephalin mRNA and proenkephalin peptides in cardiac muscle cells suggests the local production of enkephalins in the myocardium. Yet, the effects of these peptides on the function of the contractile proteins are unknown. The effects of (D-Ala2, Met5) enkephalinamide (DALA) on the activity of the actin stimulated Ca, Mg-myosin ATPase in myofibrils and on the contractility and the activity of the related actomyosin ATPase of chemically skinned muscle fibres from pig myocardium were studied. In this article, it is shown that the myofibrillar actomyosin ATPase as well as the contractility and the actomyosin ATPase in skinned fibres are sensitized to Ca2+ ions by DALA. 10(-11) -10(-6) mol/l DALA decrease the effective concentration of Ca2+ stimulating the myofibrillar ATPase activity by 50% (EC50) from 4.0.10(-5) to 1.5.10(-5) mol/l (p < 0.05). The magnesium dependent myosin ATPase activity at low Ca2+ concentration (10(-9) mol/l) is increased. The EC50 values of Ca2+ for both force development and the related actomyosin ATPase activity of skinned fibres are decreased by DALA (10(-11) -10(-5) mol/l) from 2.5.10(-6) to 2.0.10(-6) mol/l (contractions; p < 0.01) and from 2.0.10(-6) to 1.3.10(-6) mol/l (ATPase activity; p < 0.01). The tension cost (ATPase/tension) of the fibres is unchanged by DALA. In conclusion, the results demonstrate a Ca2+ sensitization of the contractile proteins by low concentrations of DALA, indicating a direct regulatory involvement of enkephalins in the regulation of myocardial contractility. These results correspond with the positive inotropic effects of enkephalins in isolated heart muscle cells.

Effect of calcium ion on the thermal denaturation of subfragment-1 and rod regions of squid myosin upon the heating of myofibrils

Thermal inactivation of Ca2+ ATPase of squid myofibrils was significantly suppressed in the presence of Ca2+. Monomeric myosin content decreased much faster than Ca2+ ATPase inactivation in Ca medium, which was well explained by fast rod denaturation. In contrast, rod denaturation was slower than S-1 in EDTA medium. The decrease in monomeric myosin content was explained by faster S-1 denaturation. Comparing the S-1 and rod denaturation rates at a fixed temperature, it was concluded that S-1 denaturation was suppressed by Ca2+ whereas the rod denaturation was not. An unfolding experiment with isolated myosin rod confirmed that there was no stabilizing effect of Ca2+ on myosin rod. It was concluded that significant stabilization of the S-1 portion by Ca2+ generated the apparently different myosin denaturation patterns in the two media.

Two Mechanisms For Increasing Muscle Calcium Sensitivity by ROS

Cardiac muscle is sensitive to reactive oxygen species (ROS). Most measurements of calcium sensitivity following ROS exposure have shown a decrease, but others have measured no change or even an increase. One difference between studies was the activation state when ROS was applied. We therefore sought to characterize ROS-induced changes in myofilament proteins and their functional effects under different conditions of [Ca] and [ATP]. First, we identified all reactive cysteines in myofilament proteins by exposing myofibrils to a fluorescent maleimide probe under varying calcium concentrations, followed by SDS-PAGE. Only cysteines in troponin C had reactivity modulated by [Ca]. To find functional effects, we measured myofibril ATPase after exposure to 100uM DTDP in solutions at pCa 4.0 or 9.0. Ca sensitivity increased only when DTDP had been added at pCa 4.0. Next, we compared the reactivity of cysteines in myofibrils exposed to DTDP under rigor or relaxing conditions. Rigor conditions decreased reactivity of myosin cysteines but increased reactivity of actin cysteines, compared to relaxing conditions. We then measured ATPase rates in myofibrils exposed to DTDP under rigor or relaxing solutions. Exposure to DTDP in relaxing solution decreased both the maximum ATPase rate and the calcium sensitivity compared to myofibrils not exposed to DTDP (control). In contrast, when DTDP was exposed in rigor solution the minimum ATPase rate was increased from control, and there was an increase in calcium sensitivity. In conclusion, we found two activation dependent mechanisms to increase calcium sensitivity: the first calcium-dependent and specific to troponin C, the second dependent on whether myosin was bound to actin.

Differences between Cardiac and Skeletal Troponin Interaction with the Thin Filament Probed by Troponin Exchange in Skeletal Myofibrils

Troponin (Tn) is the calcium-sensing protein of the thin filament. Although cardiac troponin (cTn) and skeletal troponin (sTn) accomplish the same function, their subunit interactions within Tn and with actin-tropomyosin are different. To further characterize these differences, myofibril ATPase activity as a function of pCa and labeled Tn exchange in rigor myofibrils was used to estimate Tn dissociation rates from the nonoverlap and overlap region as a function of pCa. Measurement of ATPase activity showed that skeletal myofibrils containing >96% cTn had a higher pCa 9 ATPase activity than, but similar pCa 4 activity to, sTn-containing myofibrils. Analysis of the pCa–ATPase activity relation showed that cTn myofibrils were more calcium sensitive but less cooperative (pCa50 = 6.14, nH = 1.46) than sTn myofibrils (pCa50 = 5.90, nH = 3.36). The time course of labeled Tn exchange at pCa 9 and 4 were quite different between cTn and sTn. The apparent cTn dissociation rates were ∼2–10-fold faster than sTn under all the conditions studied. The apparent dissociation rates for cTn were 5 × 10−3 min−1, 150 × 10−3 min−1, and 260 × 10−3 min−1, whereas for sTn they were 0.6 × 10−3 min−1, 88 × 10−3 min−1, and 68 × 10−3 min−1 for the nonoverlap region at pCa 9, nonoverlap region at pCa 4, and overlap region at pCa 4, respectively. Normalization of the apparent dissociation rates gives 1:30:50 for cTn compared with 1:150:110 for sTn (nonoverlap at pCa 9:nonoverlap at pCa 4:overlap at pCa 4) suggesting that calcium has a smaller influence, whereas strong cross-bridges have a larger influence on cTn dissociation compared with sTn. The higher cTn dissociation rate in the nonoverlap region and ATPase activity at pCa 9 suggest that it gives a less off or inactive thin filament. Analysis of the intensity ratio (after a short time of exchange) as a function of pCa showed that cTn had greater calcium sensitivity but lower cooperativity than sTn. In addition, the magnitude of the change in intensity ratio going from pCa 9 to 4 was less for cTn than sTn. These data suggest that the influence of calcium on cTn exchange is less than sTn even though calcium can activate ATPase activity to a similar extent in cTn compared with sTn myofibrils. This may be explained partially by cTn being less off or inactive at pCa 9. Modeling of the intensity profiles obtained after Tn exchange at pCa 5.8 suggest that the profiles are best explained by a model that includes a long-range cross-bridge effect that grades with distance from the rigor cross-bridge for both cTn and sTn.

Ionic Strength Influence on Myofibril Thin Filament State Probed by Troponin Exchange

Activation of striated muscle contraction is a cooperative process initiated by calcium binding to troponin C (TnC). This is a solid-phase signal transduction process involving 3 states of thin filament: a blocked (B) state preventing myosin interaction, a closed (C) state allowing weak myosin interaction, and an fully activated open or M state. Here we evaluated the influence of ionic strength on Tn dissociation in relationship to myofibril thin filament state. The data we obtained showed that ionic strength had a significant influence on Tn dissociation with low ionic strength increasing Tn dissociation rate from non-overlap region (B or C state) and high ionic strength increasing Tn dissociation rate from overlap region (M state) of myofibril thin filament in both absence and presence of calcium. When ionic strength was decreased to ≤ 50 mM, Tn dissociation from non-overlap region became even faster than overlap region in the absence of calcium, suggesting that actin-tropomyosin in the non-overlap region actually changed from the inhibited B state to the C state at low ionic strength. Also, low ionic strength increased myofibril ATPase activity in the absence of calcium and the calcium-sensitivity of ATPase activity. Based on this and our current studies, we propose a refined kinetic scheme for Tn dissociation from myofibril thin filament, which suggests that the regulatory dissociation of TnI from actin-tropomyosin and association to TnC is not necessarily calcium coupled. Low ionic strength favors the B to C state equilibrium towards the C state (Head et al., Eur. J. Biochem, 227: 694) by favoring TnI dissociation from actin and possibly association with TnC in the absence of calcium. This well explains reported inconsistence between ATPase activity and S1 binding to actin under low ionic strength.

Cardiac Myosin Activator CK-1316719 Increases MyoFibril ATPase Activity and Myocyte Contractility in a Rat Model of Heart Failure

Cardiac Myosin Activator CK-1316719 Increases MyoFibril ATPase Activity and Myocyte Contractility in a Rat Model of Heart Failure

The effect of Mg2+ on cardiac muscle function: is CaATP the substrate for priming myofibril cross-bridge formation and Ca2+ reuptake by the sarcoplasmic reticulum?

Kinetics are established for the activation of the myofibril from the relaxed state [Smith, Dixon, Kirschenlohr, Grace, Metcalfe and Vandenberg (2000) Biochem. J. 346, 393–402]. These require two troponin Ca2+-binding sites, one for each myosin head, to act as a single unit in initial cross-bridge formation. This defines the first, or activating, ATPase reaction, as distinct from the further activity of the enzyme that continues when a cross-bridge to actin is already established. The pairing of myosin heads to act as one unit suggests a possible alternating mechanism for muscle action. A large positive inotropic (contraction-intensifying) effect of loading the Mg2+ chelator citrate, via its acetoxymethyl ester, into the heart has confirmed the competitive inhibition of the Ca2+ activation by Mg2+, previously seen in vitro. In the absence of a recognized second Ca2+-binding site on the myofibril, with appropriate binding properties, the bound ATP is proposed as the second activating Ca2+-binding site. As ATP, free or bound to protein, can bind either Mg2+ or Ca2+, this leads to competitive inhibition by Mg2+. Published physico-chemical studies on skeletal muscle have shown that CaATP is potentially a more effective substrate than MgATP for cross-bridge formation. The above considerations allow calculation of the observed variation of fractional activation by Ca2+ as a function of [Mg2+] and in turn reveal simple Michaelis–Menten kinetics for the activation of the ATPase by sub-millimolar [Mg2+]. Furthermore the ability of bound ATP to bind either cation, and the much better promotion of cross-bridge formation by CaATP binding, give rise to the observed variation of the Hill coefficient for Ca2+ activation with altered [Mg2+]. The inclusion of CaADP within the initiating cross-bridge and replacement by MgADP during the second cycle is consistent with the observed fall in the rate of the myofibril ATPase that occurs after two phosphates are released. The similarity of the kinetics of the cardiac sarcoplasmic reticulum ATPase to those of the myofibril, in particular the positive co-operativity of both Mg2+ inhibition and Ca2+ activation, leads to the conclusion that this ATPase also has an initiation step that utilizes CaATP. The first-order activation by sub-millimolar [Mg2+], similar to that of the myofibril, may be explained by Mg2+ involvement in the phosphate-release step of the ATPase. The inhibition of both the myofibril and sarcoplasmic reticulum Ca2+-transporting ATPases by Mg2+ offers an explanation for the specific requirement for phosphocreatine (PCr) for full activity of both enzymes in situ and its effect on their apparent affinities for ATP. This explanation is based on the slow diffusion of Mg2+ within the myofibril and on the contrast of PCr with both ATP and phosphoenolpyruvate, in that PCr does not bind Mg2+ under physiological conditions, whereas both the other two bind it more tightly than the products of their hydrolysis do. The switch to supply of energy by diffusion of MgATP into the myofibril when depletion of PCr raises [ATP]/[PCr] greatly, e.g. during anoxia, results in a local [Mg2+] increase, which inhibits the ATPase. It is possible that mechanisms similar to those described above occur in skeletal muscle but the Ca2+ co-operativity involved would be masked by the presence of two Ca2+-binding sites on each troponin.

The effect of Mg2+ on cardiac muscle function: Is CaATP the substrate for priming myofibril cross-bridge formation and Ca2+ reuptake by the sarcoplasmic reticulum?

Kinetics are established for the activation of the myofibril from the relaxed state [Smith, Dixon, Kirschenlohr, Grace, Metcalfe and Vandenberg (2000) Biochem. J. 346, 393-402]. These require two troponin Ca2+-binding sites, one for each myosin head, to act as a single unit in initial cross-bridge formation. This defines the first, or activating, ATPase reaction, as distinct from the further activity of the enzyme that continues when a cross-bridge to actin is already established. The pairing of myosin heads to act as one unit suggests a possible alternating mechanism for muscle action. A large positive inotropic (contraction-intensifying) effect of loading the Mg2+ chelator citrate, via its acetoxymethyl ester, into the heart has confirmed the competitive inhibition of the Ca2+ activation by Mg2+, previously seen in vitro. In the absence of a recognized second Ca2+ binding site on the myofibril, with appropriate binding properties, the bound ATP is proposed as the second activating Ca2+-binding site. As ATP, free or bound to protein, can bind either Mg2+ or Ca2+, this leads to competitive inhibition by Mg2+. Published physico-chemical studies on skeletal muscle have shown that CaATP is potentially a more effective substrate than MgATP for cross-bridge formation. The above considerations allow calculation of the observed variation of fractional activation by Ca2+ as a function of [Mg2+] and in turn reveal simple Michaelis-Menten kinetics for the activation of the ATPase by sub-millimolar [Mg2+]. Furthermore the ability of bound ATP to bind either cation, and the much better promotion of cross-bridge formation by CaATP binding, give rise to the observed variation of the Hill coefficient for Ca2+ activation with altered [Mg2+]. The inclusion of CaADP within the initiating cross-bridge and replacement by MgADP during the second cycle is consistent with the observed fall in the rate of the myofibril ATPase that occurs after two phosphates are released. The similarity of the kinetics of the cardiac sarcoplasmic reticulum ATPase to those of the myofibril, in particular the positive co-operativity of both Mg2+ inhibition and Ca2+ activation, leads to the conclusion that this ATPase also has an initiation step that utilizes CaATP. The first-order activation by sub-millimolar [Mg2+], similar to that of the myofibril, may be explained by Mg2+ involvement in the phosphate-release step of the ATPase. The inhibition of both the myofibril and sarcoplasmic reticulum Ca2+ transporting ATPases by Mg2+ offers an explanation for the specific requirement for phosphocreatine (PCr) for full activity of both enzymes in situ and its effect on their apparent affinities for ATP. This explanation is based on the slow diffusion of Mg2+ within the myofibril and on the contrast of PCr with both ATP and phosphoenolpyruvate, in that PCr does not bind Mg2+ under physiological conditions, whereas both the other two bind it more tightly than the products of their hydrolysis do. The switch to supply of energy by diffusion of MgATP into the myofibril when depletion of PCr raises [ATP]/[PCr] greatly, e.g. during anoxia, results in a local [Mg2+] increase, which inhibits the ATPase. It is possible that mechanisms similar to those described above occur in skeletal muscle but the Ca2+ co-operativity involved would be masked by the presence of two Ca2+ binding sites on each troponin.

Probing the coupling of Ca2+ and rigor activation of rabbit psoas myofibrillar ATPase with ethylene glycol.

We have exploited solvent perturbation to probe the coupling of Ca2+ and rigor activation of the ATPase of myofibrils from rabbit psoas. Three techniques were used: overall myofibrillar ATPases by the rapid-flow quench method; kinetics of the interaction of ATP with myofibrils by fluorescence stopped-flow; and myofibrillar shortening by optical microscopy. Because of its extensive use with muscle systems, ranging from myosin subfragment-1 to muscle fibres, we chose 40% ethylene glycol as the relaxing agent. At 4 degrees C, the glycol had little effect on the myofibrillar ATPase at low [Ca2+], but at high [Ca2+] the activity was reduced 50-fold, close to the level found under relaxing conditions, and there was no shortening. However, the ATPase of chemically cross-linked myofibrils (permanently activated even without Ca2+) was reduced only 3-4-fold. The lesser reduction of the ATPase of permanently activated myofibrils was also observed in single turnover experiments in which activation occurs by a few heads in the rigor state activating the remaining heads. The addition of ADP, which also promotes strong head-thin filament interactions, also activated the ATPase but only in the presence of Ca2+. Further experiments revealed that in 40% ethylene glycol, Ca2+ does initiate shortening but only with the aid of strong interactions and at temperatures above 15 degrees C. This confirms that in the organized and intact myofibril, Ca2+ and rigor activation are coupled, as proposed previously for regulated actomyosin subfragment-1.

The effect of a thiadiazinone derived Ca 2+ sensitizer on the responsiveness of Mg 2+-ATPase to Ca 2+ in myofibrils isolated from stunned and nonstunned porcine and human myocardium

Previously, we showed, in an in situ porcine model, that the thiadiazinone derivative [+]EMD 60263, a putative Ca 2+ sensitizer with minimal phosphodiesterase III inhibitory properties, increased contractility more profoundly in stunned than in nonstunned myocardium. The aim of the present investigation was to study the mechanism of action by determining the in vitro effects of [+]EMD 60263 on the Ca 2+ responsiveness of the Mg 2+-dependent ATPases of myofibrils and sarcoplasmic reticulum membrane vesicles, isolated from normal ventricle of swine and hypertrophic septum of cardiomyopathic patients. Contamination of the myofibrils with sarcoplasmic reticulum membranes was excluded by testing the effect of the sarcoplasmic reticulum Ca 2+-pumping ATPase inhibitor thapsigargin. The plasma concentrations at which [+]EMD 60263 exerted its inotropic effect in the in situ porcine model were found to be submicromolar. [+]EMD 60263 stimulated concentration-dependently (1–10 μM) the submaximally activated Mg 2+-ATPase (at pCa 6.1) of pig heart myofibrils. [+]EMD 60263 (10 μM) shifted the pCa 50 of porcine myofibrillar Ca 2+-stimulated, Mg 2+-dependent ATPase from 6.00 ± 0.05 to 6.67 ± 0.05, whereas the [−]enantiomer EMD 60264 had no significant effect. Although the effect was much less at 1 and 3 μM, [+]EMD 60263 (10 μM) also stimulated maximal myofibrillar Mg 2+-ATPase activity. The Hill coefficient, reflecting the steepness of the fitted pCa Mg 2+ -ATPase curve at half-maximal activation, was not affected by [+]EMD 60263 (10 μM). [+]EMD 60263 (10 μM) had no effect on sarcoplasmic reticulum Ca 2+-stimulated, Mg 2+-dependent ATPase from swine heart. The thiadiazinone derivative [+]EMD 57033 (10 μM), but not its [−]enantiomer EMD 57439, had similar, although less potent, effects on pig heart myofibrillar Mg 2+-ATPase activity as compared to [+]EMD 60263. [+]EMD 60263 (3 μM) produced a significantly larger leftward shift of the pCa 2+ Mg 2+ -ATPase activity curve of myofibrils isolated from the stunned compared to the adjacent nonstunned myocardium (ΔpCa 50s caused by the presence of [+]EMD 60263 amounted to +0.57 ± 0.04 and +0.42 ± 0.05, respectively) in the in situ porcine model. The effects of [+]EMD 60263 on myofibrillar Mg 2+-ATPase of hypertrophic human heart were identical to those observed with porcine heart myofibrils. The results indicate that the positive inotropic action of [+]EMD 60263 observed in the in situ porcine model of stunned myocardium may be primarily due to myofilament sensitization to Ca 2+, and that this compound may have a similar action on diseased human myocardium.

Cardiomyopathy in transgenic myf5 mice.

To explore the compatibility of skeletal and cardiac programs of gene expression, transgenic mice that express a skeletal muscle myogenic regulator, bmyf5, in the heart were analyzed. These mice develop a severe cardiomyopathy and exhibit a significantly shorter life span than do their nontransgenic littermates. The transgene was expressed from day 7.5 post coitum forward, resulting in activation of skeletal muscle genes not normally seen in the myocardium. Cardiac pathology was not apparent at midgestation but was evident by day 2 of postnatal life, and by 42 days, hearts exhibited multifocal interstitial inflammation, fibrosis, cellular hypertrophy, and occasional myocyte degeneration. All four chambers of the heart were enlarged to varying degrees, with the atria demonstrating the most significant hypertrophy (>100% in 42-day-old mice). The transgene and several skeletal muscle-specific genes were expressed only in patchy areas of the heart in heterozygous mice. However, molecular markers of hypertrophy (such as alpha-skeletal actin and atrial myosin light chain- 1) were expressed with a wider distribution, suggesting that their induction was secondary to the expression of the transgene, In older (28-week-old) mice, lung weights were also significantly increased, consistent with congestive heart failure. The life span of bmyf5 mice was significantly shortened, with an average life span of 109 days, compared with at least a twofold longer life expectancy for nontransgenic littermates. Expression of the transgene was associated with an increase in Ca2+-stimulated myofibrillar ATPase in myofibrils obtained from the left ventricles of 42-day-old bmyf5 mice. Myocardial bmyf5 expression therefore induces a program of skeletal muscle gene expression that results in progressive cardiomyopathy that may be due to incompatibility of heart and skeletal muscle structural proteins.

Mechanochemical coupling in muscle: attempts to measure simultaneously shortening and ATPase rates in myofibrils

We studied the ATPase of shortening myofibrils at 4 degrees C by the rapid flow quench method. The progress curve has three phases: a P(i) burst, a fast linear phase kF of duration tB, and a deceleration to a slow kS. We propose that kF is the ATPase of myofibrils shortening under zero external load; at tB shortening and ATPase rates are reduced by passive resistance. The total ATP consumed during the rapid shortening is ATPc. Our purpose was to obtain information on the myofibrillar shortening velocity from their ATPase progress curves. We tested tB as an indicator of shortening velocity by determining the effects of different probes upon it and the other ATPase parameters. The dependence of tB upon the initial sarcomere length was linear, giving a shortening velocity close to that of muscle fibres (Vo). The Km of ATP was larger for tB than for kF, as found with fibers for Vo and their ATPase. ADP and 2,3-butanedione monoxime, but not P(i), inhibited tB to the same extent as Vo. The delta H for tB and Vo were similar. ATPc was independent of the sarcomere length, implying that the more the myofibrils shorten, the less ATP expended per myosin head per micron shortened. We propose that tB can be used as an indicator for myofibrillar shortening velocities.

Covalent Binding of Peptides to the N-terminal Hydrophobic Region of Cardiac Troponin C Has Limited Effects on Function

Exposure of an N-terminal hydrophobic region in troponin C is thought to be important for the regulation of contraction in striated muscle. To test this hypothesis, single Cys residues were engineered at positions 45, 81, 84, or 85 in the N-terminal hydrophobic region of cardiac troponin C (cTnC) to provide specific sites for attachment of blocking groups. A synthetic peptide, Ac-Val-Arg-Ala-Ile-Gly-Lys-Leu-Ser-Ser, or biotin was coupled to these Cys residues, and the covalent adducts were tested for activity in TnC-extracted myofibrils. Covalent modification of cTnC(C45) had no effect on maximal myofibril ATPase activity. Greatly decreased myofibril ATPase activity (70-80% inhibited) resulted when the peptide was conjugated to Cys-81 in cTnC(C81), while a lesser degree of inhibition (10-25% inhibited) resulted from covalent modification of cTnC(C84) and cTnC(C85). Inhibition was not due to an altered affinity of the cTnC(C81)/peptide conjugate for the myofibrils, and the Ca2+ dependence of ATPase activity was essentially identical to the unmodified protein. Thus, a subregion of the N-terminal hydrophobic region in cTnC is sensitive to disruption, while other regions are less important or can adapt to rather bulky blocking groups. The data suggest that Ca(2+)-sensitizing drugs may bind to the N-terminal hydrophobic region on cTnC but not interfere with transmission of the Ca2+ signal.