ATPase Activity Of Myofibrils Research Articles

Drug discovery for heart failure targeting myosin-binding protein C

Cardiac MyBP-C (cMyBP-C) interacts with actin and myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces the binding of cMyBP-C to actin and myosin, is often decreased in patients with heart failure (HF) and is cardioprotective in model systems of HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic its phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL) and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethylrhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized the specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish the feasibility of the discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.

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.

P4465Acute Cardio-Selective Functional Modulation via a Small-Molecule Direct Myosin-Attenuator (MYK-581) Preserves Diaphragmatic Function in vivo: Comparison versus Disopyramide

Abstract Background Hypertrophic cardiomyopathy (HCM) is a heritable cardiac disease characterized by hyper-contractility, hindered relaxation, and impaired exercise capacity. Conventional negative inotropes can alleviate enhanced inotropy in HCM, but do not improve ventricular filling and have the potential to further decrease exercise capacity due to their off/on-target systemic effects. Recently, a novel small molecule cardiac-myosin attenuator, Mavacamten, has been shown to normalize hyper-contractility in the setting of HCM. This study evaluated and compared the in vivo functional selectivity of MYK-581, a mavacamten analog, with those of disopyramide (DISO), a commonly-used negative inotrope. Methods In vivo, the acute cardiac (left ventricular pressures) and neuro-muscular (force of diaphragmatic and skeletal muscle contractions) responses to MYK-581 (up to 0.5 mg/kg IV) and DISO (up to 10 mg/kg IV) were simultaneously evaluated using anesthetized and mechanically-ventilated Sprague-Dawley rats; diaphragmatic (both intrinsic and phrenic nerve stimulated) and skeletal contractions (stimulated quadriceps) were measured via strain gauges. Results MYK-581 decreased indices of systolic function (dP/dtmax: −32±2% and vmax: −14±2%, P<0.05 vs. pre-dose) and increased LV EDV (+9±2%, P<0.05), while preserving EDP (6±1 to 6±1 mmHg), suggesting improved ventricular distensibility. Despite these marked cardiac effects, MYK-581 preserved both diaphragmatic (23.7±2.0 to 23.3±2.2 g) and skeletal in situ function (14.9±1.4 to 13.9±0.8 g). In contrast, DISO at matched levels of negative inotropy (dP/dtmax: −25±2% and vmax: −19±2%, P<0.05 vs. pre-dose) depressed diaphragmatic force (−16±3%, 23.8±1.9 to 19.8±1.2 g, P<0.05). Finally, the cardiac selectivity of MYK-581 was confirmed in kinetic experiments evaluating the Ca2+-activated ATPase activity of both cardiac and diaphragmatic myofibrils. Conclusions Direct myosin modulation with MYK-581, a mavacamten analog, is characterized in vivo by reductions in systolic function with preserved filling pressures and improved LV compliance. Moreover, this cardiovascular profile was devoid in vivo of diaphragmatic/skeletal off-target effects that could further hinder exercise capacity in patients with HCM.

Effect of trehalose on Lateolabrax japonicus myofibrillar protein during frozen storage

The effect of trehalose on the denaturation of weever (Lateolabrax japonicus) myofibrillar protein during frozen storage at −18°C for 90d was investigated. Trehalose (2.5–10g dry weight) was added to 100g of myofibrillar protein, and changes in the Ca2+-adenylpyrophosphatase (ATPase) activity, solubility, sulfhydryl content, and unfrozen water content of myofibrils were examined during frozen storage. Ca2+-ATPase activity and myofibrillar protein solubility decreased gradually during frozen storage at −18°C upon addition of trehalose. By contrast, Ca2+-ATPase activity and myofibrillar protein solubility in the control group dropped drastically during the first 45d of storage and then further decreased gradually for up to 90d of storage, indicating a biphasic denaturation pattern. Trehalose addition significantly increased sulfhydryl and unfrozen water contents in the myofibrillar protein of the treatment groups compared with that of the control group (p<0.05) during frozen storage at −18°C.

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.

The Ca2+/Mg2+ sites of troponin C modulate crossbridge-mediated thin filament activation in cardiac myofibrils

The Ca2+/Mg2+ sites (III and IV) located in the C-terminal domain of cardiac troponin C (cTnC) have been generally considered to play a purely structural role in keeping the cTnC bound to the thin filament. However, several lines of evidence, including the discovery of cardiomyopathy-associated mutations in the C-domain, have raised the possibility that these sites may have a more complex role in contractile regulation. To explore this possibility, the ATPase activity of rat cardiac myofibrils was assayed under conditions in which no Ca2+ was bound to the N-terminal regulatory Ca2+-binding site (site II). Myosin-S1 was treated with N-ethylmaleimide to create strong-binding myosin heads (NEM-S1), which could activate the cardiac thin filament in the absence of Ca2+. NEM-S1 activation was assayed at pCa 8.0 to 6.5 and in the presence of either 1mM or 30μM free Mg2+. ATPase activity was maximal when sites III and IV were occupied by Mg2+ and it steadily declined as Ca2+ displaced Mg2+. The data suggest that in the absence of Ca2+ at site II strong-binding myosin crossbridges cause the opening of more active sites on the thin filament if the C-domain is occupied by Mg2+ rather than Ca2+. This finding could be relevant to the contraction–relaxation kinetics of cardiac muscle. As Ca2+ dissociates from site II of cTnC during the early relaxing phase of the cardiac cycle, residual Ca2+ bound at sites III and IV might facilitate the switching off of the thin filament and the detachment of crossbridges from actin.

BIOCHEMICAL AND PHYSICOCHEMICAL PROPERTIES OF ACTOMYOSIN AND MYOFIBRILS FROM FROZEN STORED FLOUNDER (PARALICHTHYS PATAGONICUS) FILLETS

ABSTRACT The biochemical and physicochemical properties of myofibrils and actomyosin (AM) from frozen stored fillets of flounder were investigated. Solubility of proteins increased after freezing and decreased during frozen storage. No significant (P < 0.05) changes were detected in both Mg2+ (Ca2+) and Mg2+ Ethylene glycol tetraacetic acid (EGTA) Adenosinetriphosphatase (ATPase) activities of myofibrils due to freezing and frozen storage. Freezing produced a significant (P < 0.05) increase in Mg2+ Ca2+ and Mg2+(EGTA) and Mg2+ATPase activities of AM. The (Ca2+) ATPase activity remained unchanged. All the ATPases activities of AM decreased during frozen storage. The changes in the enzymatic activities of AM during storage were accompanied by a decrease in the reduced viscosity of the protein. No signals of proteolysis were detected in the profiles of SDS-Polyacrilamyde Gel Electrophoresis 10% gels of both myofibrils and AM. A significant decrease (P < 0.05) in the relative percentage of myosin and myosin/actin ratio was observed at the end of storage. These results suggest that denaturation–aggregation of myosin occurs at the end of storage in frozen stored fillets. PRACTICAL APPLICATIONS Flounder (Paralichthys patagonicus) is an important species of fish in South American waters. In recent years, catches in Argentina greatly increased. The fishing is commonly called “fine fishing” because of the high quality of the meat and its high price in the market. It is processed and exported as frozen interfoliated fillets and consumed as fresh fish in the internal market. Only a few reports on changes in biochemical and physicochemical properties of both AM and myofibrils from iced flounder are available. However, there is a lack of information on the behavior of myofibrillar proteins from flounder (P. patagonicus) during freezing and frozen storage. This information could be very important to establish technological actions to obtain optimal quality in frozen flounder.

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.

Functional aberration of myofibrils by cardiomyopathy-causing mutations in the coiled-coil region of the troponin-core domain

Two cardiomyopathy-causing mutations, E244D and K247R, in human cardiac troponin T (TnT) are located in the coiled-coil region of the Tn-core domain. To elucidate effects of mutations in this region on the regulatory function of Tn, we measured Ca 2+-dependent ATPase activity of myofibrils containing various mutants of TnT at these residues. The results confirmed that the mutant E244D increases the maximum ATPase activity without changing the Ca 2+-sensitivity. The mutant K247R was shown for the first time to have the effect similar to the mutant E244D. Furthermore, various TnT mutants (E244D, E244M, E244A, E244K, K247R, K247E, and K247A) showed various effects on the maximum ATPase activity while the Ca 2+-sensitivity was unchanged. Molecular dynamics simulations of the Tn-core containing these TnT mutants suggested that the hydrogen-bond network formed by the side chains of neighboring residues around residues 244 and 247 is important for Tn to function properly.

Crossbridge-mediated Activation of Rabbit Skeletal Muscle Myofibrillar ATPase: a Role for the Calcium Binding Domains of Troponin C

Activation of the thin filament in striated muscle is a cooperative process requiring both the binding of Ca2+ to troponin C (TnC) and the binding of myosin crossbridges to actin. The aim of this study was to assess the role of TnC domains in the crossbridge-mediated activation of rabbit skeletal muscle myofibrils in the absence of Ca2+. Activation of myofibrillar ATPase was produced by addition of varying concentrations of myosin S1 modified by N-ethylmaleimide (NEM-S1), which facilitates crossbridge cycling by forcing tropomyosin into the open position on the filament. Comparisons were made of native myofibrils, myofibrils from which TnC was extracted, and myofibrils reconstituted with either a TnC mutant (TnC48-82) in which Ca2+ activation was blocked by a disulfide bond in the N-terminal domain (Grabarek, et al, Nature, 345:132,1990) or a proteolytic fragment of TnC (TR2C) lacking the N-terminal Ca2+- binding domain (Grabarek, et al, J. Biol. Chem. 265:13121, 1981). The ATPase activity of native myofibrils was increased ∼170% by the addition of NEM-S1 (2-4μM). Following extraction of TnC the addition of the same concentrations of NEM-S1 produced ∼60% activation. In both cases higher concentrations of NEM-S1 produced no further increase in activation. With the addition of either TnC48-82 or TR2C the degree of activation was higher (70-100%), but required higher NEM-S1 concentrations (4-8μM). These results suggest that both domains of TnC play a role in facilitating optimal crossbridge-mediated activation of the thin filament, presumably by providing alternative binding sites for troponin I.

Functional Impact of TnT Mutations Responsible for Hypertrophic Cardiomyopathy on Tn-Exchanged Single Myofibrils

Hypertrophic Cardiomyopathy (HCM) is an autosomal dominant cardiac disease resulting from mutations in genes encoding contractile proteins, including troponin (Tn; the complex of TnT, TnI and TnC). From measurements of ATPase activity of myofibrils, into which TnT mutants were exchanged, we showed that two HCM-associated mutations of TnT, E244D and K247R, increase the maximum ATPase activity without any change in Ca2+ sensitivity (Matsumoto et al., BSJ 2007, 1P150). It is, however, still not clear if the mutations have direct effects on the mechanical properties of muscle because ATPase activity and tension development are not necessarily coupled. We thus examined the effects of the mutations on maximal isometric tension and kinetics of Tn-exchanged myofibrils. Measurements were made in single myofibrils activated and relaxed by a fast solution switching device, with a dead time < 10ms (Tesi et al., Biophys. J., 2002, 83, 2142). Maximal isometric tension values of the mutant TnT exchanged myofibrils were significantly higher (E244D) or tended to be higher (K247R) than those of the wild type TnT exchanged myofibrils while Ca2+ sensitivity was unchanged. These results indicate that the effects of the mutations on myofibril mechanics are directly coupled with the effects on the ATPase activity. Measurements of the rate constants of force generation showed that the mutant TnT exchanged myofibrils had similar kinetics as the wild type TnT exchanged myofibrils. The result suggests that kinetics of cross bridge transitions are not influenced by these mutants.

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

Determination of the big head carp myofibrillar ( Aristichthys nobilis) adenosine triphosphatase activity by ion chromatography

A method for the determination of adenosine triphosphatase (ATPase) activity of myofibrils of big head carp by using ion chromatography was introduced. Adenosine triphosphate (ATP), adenosine diphosphate (ADP) and orthophosphate (Pi) were separated completely. Recoveries for ATP, ADP and Pi were 98 ± 5%, 97 ± 4% and 98 ± 5%, respectively. Pi liberated from ATP during reaction was monitored by ion chromatography using the suggested method. This method was applicable to the determination of myofibrils ATPase activity for quick quality evaluation of surimi.

Myosin heavy chain isoform composition influences the susceptibility of actin-activated S1 ATPase and myofibrillar ATPase to pH inactivation

The objectives of this study were to determine the influence of pH and MyHC isoforms on myofibrillar and actin-activated myosin subfragment 1 (S1) ATPase activity and the protective effect of actin. Red (RST) semitendinosus and white (WST) semitendinosus myofibrils were incubated at pH 7, 6, or 5.5 with 0 or 2 mM ATP. RST and WST S1 isolates were incubated at pH 7, 6, or 5.5 in the presence or absence of actin. Maximum calcium-activated myofibrillar and actin-activated S1-ATPase activity were then assayed at pH 7. Incubation of myofibrils with ATP caused ATPase activity of myofibrils to decrease ( p < 0.05) with the pH of the incubation. RST myofibrils maintained a higher ( p < 0.0001) relative activity than WST myofibrils after incubation at pH 6 with ATP. Myofibrils incubated without ATP exhibited higher ( p < 0.001) activities than those incubated with ATP following pH 5.5 treatments. WST myofibrils had a lower ( p < 0.05) relative activity than RST following incubation at pH 5.5 without ATP. S1 ATPase activities decreased ( p < 0.05) with incubation pH in WST samples, but not in RST samples. WST S1 activity was higher ( p < 0.01) in samples exposed to pH 6 and 5.5 with actin bound compared to those incubated without actin. RST S1 exhibited a higher ( p < 0.01) relative activity than WST samples following pH 5.5 treatment with bound actin. These data show that low pH inactivates myofibrils by altering actin-activated S1 ATPase. Furthermore, these results suggest that muscles with high proportions of fast fibers are more susceptible to pH inactivation of ATPase activity and that the protective effect of actin binding to myosin is less in fast fibers.

Myosin heavy chain isoforms influence myofibrillar ATPase activity under simulated postmortem pH, calcium, and temperature conditions

The pH and Ca 2+ sensitivity of myofibrillar ATPase activity plays an integral role in regulating postmortem muscle ATP utilization and likely paces postmortem glycolysis. The objective of this study was to determine the influence of pH and Ca 2+ concentration on the ATPase activity of myofibrils from red semitendinosus (RST) and white semitendinosus (WST) porcine muscles. Myofibrillar ATPase was measured at 39 °C over a pH range 5–7.5 and a [Ca 2+] range pCa 4–9 (10 −4–10 −9M). At maximum Ca 2+-dependent activation (pCa 4), RST myofibrils had lower ( p<0.0001) ATPase activity than WST myofibrils. This maximum activity of myofibrils from both muscle regions was not influenced from pH 7.5 to 6.5, declined between pH 6.5 and 5.75 (Hill coefficient, n H=2.7–3.4; pH at half maximum activity, pH 50=5.97) and was near zero at pH 5.5. At pH 7, pCa-activity relationships showed that RST required less Ca 2+ for half-maximum activation (higher pCa 50; 6.50) than WST myofibrils (pCa 50=6.35) but had no difference in n H. At pH 7, both RST and WST myofibrils had maximum Ca 2+-dependent, actin-activated ATPase activity at pCa ⩽6 and Ca 2+-independent myosin ATPase activity at pCa ⩾6.75. pCa-activity relationships at different pH levels indicated that pCa 50 decreased with pH from pH 6.5 to 6.125 in both RST and WST myofibrils. At pH <5.75, [Ca 2+] did not influence ATPase activity in RST or WST myofibrils. These data show that myofibrils with predominantly fast MyHC (WST) have a higher actin-activated myosin ATPase activity than myofibrils with primarily slow MyHC isoforms (RST) at Ca 2+ concentrations and pH values characteristic of postmortem muscle.

At Physiological Temperatures the ATPase Rates of Shortening Soleus and Psoas Myofibrils Are Similar

We obtained the temperature dependences of the adenosine triphosphatase (ATPase) activities (calcium-activated and relaxed) of myofibrils from a slow muscle, which we compared with those from a fast muscle. We chose rabbit soleus and psoas because their myosin heavy chains are almost pure: isoforms I and IIX, respectively. The Arrhenius plots of the ATPases are linear (4–35°C) with energies of activation for soleus myofibrils 155 kJ mol −1 (activated) and 78 kJ mol −1 (relaxed). With psoas myofibrils, the energies of activation were 71 kJ mol −1 (activated) and 60 kJ mol −1 (relaxed). When extrapolated to 42°C the ATPase rates of the two types of myofibril were identical: 50 s −1 (activated) and 0.23 s −1 (relaxed). Whereas with psoas myofibrils the K m for adenosine triphosphate (activated ATPase) is relatively insensitive to temperature, that for soleus myofibrils increased from 0.3 μM at 4°C to 66.5 μM at 35°C. Our results illustrate the importance of temperature when comparing the mechanochemical coupling in different types of muscle. We discuss the problem of how to reconcile the similarity of the myofibrillar ATPase rates at physiological temperatures with their different mechanical properties.

Biochemical Properties of Myofibrils from Frozen Longissimus Dorsi Muscle of Three Lamb Genotypes

The protein composition and biochemical properties of myofibrils from fresh and frozen stored (−30 °C) for 1 or 120 days ovine longissimus dorsi muscle were studied by SDS-PAGE and ATPase activity assays. Muscles from two ovine ‘double-purpose’ (wool and meat production) breeds (Romney Marsh and Corriedale), and from one ‘meat production’בdouble-purpose’ crossbreed (Ile de France×Romney Marsh) were compared. Myofibrils from unfrozen postrigor muscles showed similar SDS-PAGE patterns. Independently of the genotypes the troponin I (Tn-I) band gave a doublet after freezing and thawing. In addition, a significant (P<0.01) increase in the relative areas of both 30- and 32-kDa bands was observed. The genotypes differed with regard to Mg2+- and Ca2+-ATPase activities of myofibrils during frozen storage of the muscles. Differential Scanning Calorimetry (DSC) studies demonstrated that the whole ovine muscle had similar thermogram profiles than those previously reported for bovine muscle. However, the Tmax corresponding to the first endothermal transitions in the muscle of two ovine genotypes showed a trend to higher values compared to those formerly reported for bovine muscles. Only myofibrils from Corriedale breed were affected by frozen storage.