ATP Free Energy Research Articles

Interplay between ATP and hydrolysis free energy in promoting and restraining biochemical switches

In living cells, the bistable switches involving phosphorylation-dephosphorylation (PdP) cycles, operate far from thermodynamic equilibrium and consume hydrolysis free energy to perform ultrasensitive response and exhibit strong robustness against noise. Here, we focus on the distinct roles of cellular ATP levels ([ATP]) and hydrolysis free energy (Δμ) in bistable switches with different structures of reaction network. Specifically, we herein propose thermodynamically consistent models of single- and double-PdP-cycle bistable switches to study the relationship between network structures and the interplay between [ATP] and Δμ. We demonstrate that [ATP] and Δμ act cooperatively to promote the activation of switches in identical networks wherein all phosphorylations promote target production, irrespective of different feedback mechanisms. On the other hand, the competition between [ATP] and Δμ found in the nonidentical, or reversed, networks can enhance the robustness of bistability. We also discuss special network structures inducing multistability or nonmonotonic functions of Δμ. Our results gain insight into the relationship between network structures and the roles of [ATP] and Δμ in bistable switches, potentially guiding the future design of synthetic biochemical switches. Published by the American Physical Society 2024

Abstract 9199: A Novel Small Molecule Troponin Activator Increases Cardiac Contractile Function Without Negative Impact on Energetics or Diastolic Function

Background: Current heart failure (HF) therapies improve cardiac energy balance and prolong survival by unloading the failing heart without targeting the underlying problem of reduced cardiac contractility. Traditional inotropic medications, also termed calcitropes, stimulate contractility via energetically costly augmentation of calcium cycling and worsen patient survival. Myotropes are a new class of agents that activate the sarcomere directly, independent of cAMP and calcium. We hypothesize that a novel myotrope, troponin activator (TA1), increases contractility without the deleterious myocardial energetic impact of calcitrope dobutamine (DOB). Methods: We determined the pharmacodynamics of TA1 ex vivo and in vivo in anesthetized normal rats. Isolated rat hearts were perfused with incremental concentrations of TA1 or DOB to compare their effect on cardiac contractility and high-energy phosphate concentrations using 31 P NMR spectroscopy. Results: A dose dependent increase in contractility was observed in vivo by echocardiography after acute administration of TA1. In isolated hearts, increasing concentrations of TA1 and DOB similarly increased rate pressure product (RPP), which is a product of developed pressure (DevP) and heart rate (HR). At equivalent doses (DOB 120 nM, TA1 60 nM), TA1 increased DevP more and HR less compared to DOB. DOB decreased phosphocreatine (PCr) to ATP ratio and free energy of ATP hydrolysis (ΔG ~ATP ) and elevated left ventricular end-diastolic pressure (LVEDP). In contrast, the TA1 increased RPP without any effect on HR, LVEDP, PCr/ATP ratio or ΔG ~ATP . Conclusions: Here we describe a novel myotrope, TA1, that increased myocardial contractility by sensitizing the sarcomere to calcium without impairing diastolic function or depleting the cardiac energy reserve. Since energetic depletion negatively correlates with long term survival, TA1 may represent a superior alternative to traditional inotropes in HF management.

Natural variant frequencies across domains from different sarcomere proteins cross-correlate to identify inter-protein contacts associated with cardiac muscle function and disease

Coordinated sarcomere proteins produce contraction force for muscle shortening. In human ventriculum they include the cardiac myosin motor (βmys), repetitively converting ATP free energy into work, and myosin binding protein C (MYBPC3) that in complex with βmys is regulatory. Single nucleotide variants (SNVs) causing hereditary heart diseases frequently target this protein pair. The βmys/MYBPC3 complex models a regulated motor and is used here to study how the proteins couple. SNVs in βmys or MYBPC3 survey human populations worldwide. Their protein expression modifies domain structure affecting phenotype and pathogenicity outcomes. When the SNV modified domain locates to inter-protein contacts it could affect complex coordination. Domains involved, one in βmys the other in MYBPC3, form coordinated domains (co-domains). Co-domain bilateral structure implies the possibility for a shared impact from SNV modification in either domain suggesting a correlated response to a common perturbation could identify their location. Genetic divergence over human populations is proposed to perturb SNV probability coupling that is detected by cross-correlation in 2D correlation genetics (2D-CG). SNV probability data and 2D-CG identify three critical sites, two in MYBPC3 with links to several domains across the βmys motor, and, one in βmys with links to the MYBPC3 regulatory domain. MYBPC3 sites are hinges sterically enabling regulatory interactions with βmys. The βmys site is the actin binding C-loop (residues 359-377). The C-loop is a trigger for actin-activated myosin ATPase and a contraction velocity modulator. Co-domain identification implies their spatial proximity suggesting a novel approach for in vivo protein complex structure determination.

Intrinsic OXPHOS limitations underlie cellular bioenergetics in leukemia.

Currently there is great interest in targeting mitochondrial oxidative phosphorylation (OXPHOS) in cancer. However, notwithstanding the targeting of mutant dehydrogenases, nearly all hopeful 'mito-therapeutics' cannot discriminate cancerous from non-cancerous OXPHOS and thus suffer from a limited therapeutic index. Using acute myeloid leukemia (AML) as a model, herein, we leveraged an in-house diagnostic biochemical workflow to identify 'actionable' bioenergetic vulnerabilities intrinsic to cancerous mitochondria. Consistent with prior reports, AML growth and proliferation was associated with a hyper-metabolic phenotype which included increases in basal and maximal respiration. However, despite having nearly 2-fold more mitochondria per cell, clonally expanding hematopoietic stem cells, leukemic blasts, as well as chemoresistant AML were all consistently hallmarked by intrinsic OXPHOS limitations. Remarkably, by performing experiments across a physiological span of ATP free energy, we provide direct evidence that leukemic mitochondria are particularly poised to consume ATP. Relevant to AML biology, acute restoration of oxidative ATP synthesis proved highly cytotoxic to leukemic blasts, suggesting that active OXPHOS repression supports aggressive disease dissemination in AML. Together, these findings argue against ATP being the primary output of leukemic mitochondria and provide proof-of-principle that restoring, rather than disrupting, OXPHOS may represent an untapped therapeutic avenue for combatting hematological malignancy and chemoresistance.

Energy metabolism design of the striated muscle cell.

The design of the energy metabolism system in striated muscle remains a major area of investigation. Here, we review our current understanding and emerging hypotheses regarding the metabolic support of muscle contraction. Maintenance of ATP free energy, so called energy homeostasis, via mitochondrial oxidative phosphorylation is critical to sustained contractile activity, and this major design criterion is the focus of this review. Cell volume invested in mitochondria reduces the space available for generating contractile force, and this spatial balance between mitochondria acontractile elements to meet the varying sustained power demands across muscle types is another important design criterion. This is accomplished with remarkably similar mass-specific mitochondrial protein composition across muscle types, implying that it is the organization of mitochondria within the muscle cell that is critical to supporting sustained muscle function. Beyond the production of ATP, ubiquitous distribution of ATPases throughout the muscle requires rapid distribution of potential energy across these large cells. Distribution of potential energy has long been thought to occur primarily through facilitated metabolite diffusion, but recent analysis has questioned the importance of this process under normal physiological conditions. Recent structural and functional studies have supported the hypothesis that the mitochondrial reticulum provides a rapid energy distribution system via the conduction of the mitochondrial membrane potential to maintain metabolic homeostasis during contractile activity. We extensively review this aspect of the energy metabolism design contrasting it with metabolite diffusion models and how mitochondrial structure can play a role in the delivery of energy in the striated muscle.

Correction: Tissue-specific characterization of mitochondrial branched-chain keto acid oxidation using a multiplexed assay platform.

Alterations to branched-chain keto acid (BCKA) oxidation have been implicated in a wide variety of human diseases, ranging from diabetes to cancer. Although global shifts in BCKA metabolism-evident by gene transcription, metabolite profiling, and in vivo flux analyses have been documented across various pathological conditions, the underlying biochemical mechanism(s) within the mitochondrion remain largely unknown. In vitro experiments using isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across disciplines to shed valuable insight into mitochondrial-linked pathologies. That said, few studies have attempted to model in vitro BCKA oxidation in isolated organelles. The impetus for the present study stemmed from the knowledge that complete oxidation of each of the three BCKAs involves a reaction dependent upon bicarbonate and ATP, both of which are not typically included in respiration experiments. Based on this, it was hypothesized that the inclusion of exogenous bicarbonate and stimulation of respiration using physiological shifts in ATP-free energy, rather than excess ADP, would allow for maximal BCKA-supported respiratory flux in isolated mitochondria. This hypothesis was confirmed in mitochondria from several mouse tissues, including heart, liver and skeletal muscle. What follows is a thorough characterization and validation of a novel biochemical tool for investigating BCKA metabolism in isolated mitochondria.

Demographic model for inheritable cardiac disease

The cardiac muscle proteins, generating and regulating energy transduction during a heartbeat, assemble in the sarcomere into a cyclical machine repetitively translating actin relative to myosin filaments. Myosin is the motor transducing ATP free energy into actin movement against resisting force. Cardiac myosin binding protein C (mybpc3) regulates shortening velocity probably by transient N-terminus binding to actin while its C-terminus strongly binds the myosin filament. Inheritable heart disease associated mutants frequently modify these proteins involving them in disease mechanisms. Nonsynonymous single nucleotide polymorphisms (SNPs) cause single residue substitutions with independent characteristics (sequence location, residue substitution, human demographic, and allele frequency) hypothesized to decide dependent phenotype and pathogenicity characteristics in a feed-forward neural network model. Trial models train and validate on a dynamic worldwide SNP database for cardiac muscle proteins then predict phenotype and pathogenicity for any single residue substitution in myosin, mybpc3, or actin. A separate Bayesian model formulates conditional probabilities for phenotype or pathogenicity given independent SNP characteristics. Neural/Bayes forecasting tests SNP pathogenicity vs (in)dependent SNP characteristics to assess individualized disease risk and in particular to elucidate gender and human subpopulation bias in disease. Evident subpopulation bias in myosin SNP pathogenicities imply myosin normally engages multiple sarcomere proteins functionally. Consistent with this observation, mybpc3 forms a third actomyosin interaction competing with myosin essential light chain N-terminus suggesting a novel strain-dependent mechanism adapting myosin force-velocity to load dynamics. The working models, and the integral myosin/mybpc3 motor concept, portends the wider considerations involved in understanding heart disease as a systemic maladaptation.

Tissue-specific characterization of mitochondrial branched-chain keto acid oxidation using a multiplexed assay platform.

Alterations to branched-chain keto acid (BCKA) oxidation have been implicated in a wide variety of human diseases, ranging from diabetes to cancer. Although global shifts in BCKA metabolism-evident by gene transcription, metabolite profiling, and in vivo flux analyses have been documented across various pathological conditions, the underlying biochemical mechanism(s) within the mitochondrion remain largely unknown. In vitro experiments using isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across disciplines to shed valuable insight into mitochondrial-linked pathologies. That said, few studies have attempted to model in vitro BCKA oxidation in isolated organelles. The impetus for the present study stemmed from the knowledge that complete oxidation of each of the three BCKAs involves a reaction dependent upon bicarbonate and ATP, both of which are not typically included in respiration experiments. Based on this, it was hypothesized that the inclusion of exogenous bicarbonate and stimulation of respiration using physiological shifts in ATP-free energy, rather than excess ADP, would allow for maximal BCKA-supported respiratory flux in isolated mitochondria. This hypothesis was confirmed in mitochondria from several mouse tissues, including heart, liver and skeletal muscle. What follows is a thorough characterization and validation of a novel biochemical tool for investigating BCKA metabolism in isolated mitochondria.

Chronic high fat diet lowers male detrusor mitochondrial fatty acid oxidation while females are protected

IntroductionObesity increases the prevalence of bladder dysfunction in both males and females. The bladder's mucosal and detrusor smooth muscle layers are involved in bladder signaling and contraction, respectively. The underlying causes of obesity‐induced bladder dysfunction in males and females are poorly understood. Mitochondrial dysfunction is common in the heart, liver and skeletal muscle with high fat diet (HFD). This study will characterize sex differences in mitochondrial respiration in bladder mucosal and detrusor layers. Additionally, we will determine the impact of long‐term HFD on bladder mitochondrial respiration in male and female mice.MethodsMale and female mice (n=7/group, 20–24 wks) were used to assess bladder mitochondrial sex differences. A second group of male and female mice (10 wks, n=5–7/group) were fed a control (10% kcal fat) or HFD (45% kcal fat) for 24 weeks. Bladders were collected, weighed and separated into mucosal and detrusor layers. Tissues were cut into strips, permeabilized, and placed in an OROBOROS Oxygraphy‐2K to measure respiration using a modified version of the creatine kinase energetic clamp. Substrates (pyruvate/malate, glutamate, L‐octanoylcarnitine/malate, or succinate/rotenone) were added followed by increasing concentrations of phosphocreatine to allow for substrate‐specific bioenergetic analyses across a physiological span of ATP free energies. Respiratory conductance was calculated for each substrate based on the rate of change in respiration for a given increase in ATP free energy.ResultsBladder respiration and respiratory conductance in the presence of L‐octanoylcarnitine/malate in mucosal and detrusor tissue was significantly decreased in males (p<0.05). Respiration and respiratory conductance remained unchanged between sexes in the presence of pyruvate/malate, glutamate, and succinate/rotenone. Chronic HFD increased body weight and decreased the bladder to body weight ratio in both sexes (p<0.05). In HFD males, mucosal respiration and respiratory conductance were decreased in the presence of L‐octanoylcarnitine/malate (p<0.05). In contrast, pyruvate/malate, glutamate, and succinate/rotenone did not change HFD male respiration and respiratory conductance. In HFD females, respiration and respiratory conductance were unchanged in presence of pyruvate/malate, glutamate, and L‐octanoylcarnitine/malate. However, HFD female mucosal layer had decreased respiration with succinate/rotenone but no change in respiratory conductance.ConclusionsMale mice have lower baseline fatty acid metabolism within the bladder's detrusor smooth muscle layer compared to females. With chronic HFD, male fatty acid metabolism within the mucosal and detrusor muscle layers is further lowered whereas female mice are protected. Ongoing studies will determine if excess lipids accumulate within the bladder and if we can rescue fatty acid metabolism with co‐factors such as L‐carnitine or acetyl‐CoA. Understanding bladder metabolic and bioenergetic sex differences will help to create more effective treatments for bladder dysfunctionSupport or Funding InformationNIDDK DiaComp, ECU SBRP, APS UGSRFThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cardiac and skeletal actin substrates uniquely tune cardiac myosin strain-dependent mechanics.

Cardiac ventricular myosin (βmys) translates actin by transducing ATP free energy into mechanical work during muscle contraction. Unitary βmys translation of actin is the step-size. In vitro and in vivo βmys regulates contractile force and velocity autonomously by remixing three different step-sizes with adaptive stepping frequencies. Cardiac and skeletal actin isoforms have a specific 1 : 4 stoichiometry in normal adult human ventriculum. Human adults with inheritable hypertrophic cardiomyopathy (HCM) upregulate skeletal actin in ventriculum probably compensating the diseased muscle's inability to meet demand by adjusting βmys force–velocity characteristics. βmys force–velocity characteristics were compared for skeletal versus cardiac actin substrates using ensemble in vitro motility and single myosin assays. Two competing myosin strain-sensitive mechanisms regulate step-size choices dividing single βmys mechanics into low- and high-force regimes. The actin isoforms alter myosin strain-sensitive regulation such that onset of the high-force regime, where a short step-size is a large or major contributor, is offset to higher loads probably by the unique cardiac essential light chain (ELC) N-terminus/cardiac actin contact at Glu6/Ser358. It modifies βmys force–velocity by stabilizing the ELC N-terminus/cardiac actin association. Uneven onset of the high-force regime for skeletal versus cardiac actin modulates force–velocity characteristics as skeletal/cardiac actin fractional content increases in diseased muscle.

Single cardiac ventricular myosins are autonomous motors.

Myosin transduces ATP free energy into mechanical work in muscle. Cardiac muscle has dynamically wide-ranging power demands on the motor as the muscle changes modes in a heartbeat from relaxation, via auxotonic shortening, to isometric contraction. The cardiac power output modulation mechanism is explored in vitro by assessing single cardiac myosin step-size selection versus load. Transgenic mice express human ventricular essential light chain (ELC) in wild- type (WT), or hypertrophic cardiomyopathy-linked mutant forms, A57G or E143K, in a background of mouse α-cardiac myosin heavy chain. Ensemble motility and single myosin mechanical characteristics are consistent with an A57G that impairs ELC N-terminus actin binding and an E143K that impairs lever-arm stability, while both species down-shift average step-size with increasing load. Cardiac myosin in vivo down-shifts velocity/force ratio with increasing load by changed unitary step-size selections. Here, the loaded in vitro single myosin assay indicates quantitative complementarity with the in vivo mechanism. Both have two embedded regulatory transitions, one inhibiting ADP release and a second novel mechanism inhibiting actin detachment via strain on the actin-bound ELC N-terminus. Competing regulators filter unitary step-size selection to control force-velocity modulation without myosin integration into muscle. Cardiac myosin is muscle in a molecule.

Auxotonic to isometric contraction transitioning in a beating heart causes myosin step-size to down shift.

Myosin motors in cardiac ventriculum convert ATP free energy to the work of moving blood volume under pressure. The actin bound motor cyclically rotates its lever-arm/light-chain complex linking motor generated torque to the myosin filament backbone and translating actin against resisting force. Previous research showed that the unloaded in vitro motor is described with high precision by single molecule mechanical characteristics including unitary step-sizes of approximately 3, 5, and 8 nm and their relative step-frequencies of approximately 13, 50, and 37%. The 3 and 8 nm unitary step-sizes are dependent on myosin essential light chain (ELC) N-terminus actin binding. Step-size and step-frequency quantitation specifies in vitro motor function including duty-ratio, power, and strain sensitivity metrics. In vivo, motors integrated into the muscle sarcomere form the more complex and hierarchically functioning muscle machine. The goal of the research reported here is to measure single myosin step-size and step-frequency in vivo to assess how tissue integration impacts motor function.A photoactivatable GFP tags the ventriculum myosin lever-arm/light-chain complex in the beating heart of a live zebrafish embryo. Detected single GFP emission reports time-resolved myosin lever-arm orientation interpreted as step-size and step-frequency providing single myosin mechanical characteristics over the active cycle. Following step-frequency of cardiac ventriculum myosin transitioning from low to high force in relaxed to auxotonic to isometric contraction phases indicates that the imposition of resisting force during contraction causes the motor to down-shift to the 3 nm step-size accounting for >80% of all the steps in the near-isometric phase. At peak force, the ATP initiated actomyosin dissociation is the predominant strain inhibited transition in the native myosin contraction cycle. The proposed model for motor down-shifting and strain sensing involves ELC N-terminus actin binding.Overall, the approach is a unique bottom-up single molecule mechanical characterization of a hierarchically functional native muscle myosin.

In vivo myosin step-size from zebrafish skeletal muscle

Muscle myosins transduce ATP free energy into actin displacement to power contraction. In vivo, myosin side chains are modified post-translationally under native conditions, potentially impacting function. Single myosin detection provides the ‘bottom-up’ myosin characterization probing basic mechanisms without ambiguities inherent to ensemble observation. Macroscopic muscle physiological experimentation provides the definitive ‘top-down’ phenotype characterizations that are the concerns in translational medicine. In vivo single myosin detection in muscle from zebrafish embryo models for human muscle fulfils ambitions for both bottom-up and top-down experimentation. A photoactivatable green fluorescent protein (GFP)-tagged myosin light chain expressed in transgenic zebrafish skeletal muscle specifically modifies the myosin lever-arm. Strychnine induces the simultaneous contraction of the bilateral tail muscles in a live embryo, causing them to be isometric while active. Highly inclined thin illumination excites the GFP tag of single lever-arms and its super-resolution orientation is measured from an active isometric muscle over a time sequence covering many transduction cycles. Consecutive frame lever-arm angular displacement converts to step-size by its product with the estimated lever-arm length. About 17% of the active myosin steps that fall between 2 and 7 nm are implicated as powerstrokes because they are beyond displacements detected from either relaxed or ATP-depleted (rigor) muscle.

In vitro and in vivo single myosin step-sizes in striated muscle.

Myosin in muscle transduces ATP free energy into the mechanical work of moving actin. It has a motor domain transducer containing ATP and actin binding sites, and, mechanical elements coupling motor impulse to the myosin filament backbone providing transduction/mechanical-coupling. The mechanical coupler is a lever-arm stabilized by bound essential and regulatory light chains. The lever-arm rotates cyclically to impel bound filamentous actin. Linear actin displacement due to lever-arm rotation is the myosin step-size. A high-throughput quantum dot labeled actin in vitro motility assay (Qdot assay) measures motor step-size in the context of an ensemble of actomyosin interactions. The ensemble context imposes a constant velocity constraint for myosins interacting with one actin filament. In a cardiac myosin producing multiple step-sizes, a "second characterization" is step-frequency that adjusts longer step-size to lower frequency maintaining a linear actin velocity identical to that from a shorter step-size and higher frequency actomyosin cycle. The step-frequency characteristic involves and integrates myosin enzyme kinetics, mechanical strain, and other ensemble affected characteristics. The high-throughput Qdot assay suits a new paradigm calling for wide surveillance of the vast number of disease or aging relevant myosin isoforms that contrasts with the alternative model calling for exhaustive research on a tiny subset myosin forms. The zebrafish embryo assay (Z assay) performs single myosin step-size and step-frequency assaying in vivo combining single myosin mechanical and whole muscle physiological characterizations in one model organism. The Qdot and Z assays cover "bottom-up" and "top-down" assaying of myosin characteristics.

In Vivo Orientation of Single Myosin Lever Arms in Zebrafish Skeletal Muscle

Cardiac and skeletal myosin assembled in the muscle lattice power contraction by transducing ATP free energy into the mechanical work of moving actin. Myosin catalytic/lever-arm domains comprise the transduction/mechanical coupling machinery that move actin by lever-arm rotation. In vivo, myosin is crowded and constrained by the fiber lattice as side chains are mutated and otherwise modified under normal, diseased, or aging conditions that collectively define the native myosin environment. Single-myosin detection uniquely defines bottom-up characterization of myosin functionality. The marriage of in vivo and single-myosin detection to study zebrafish embryo models of human muscle disease is a multiscaled technology that allows one-to-one registration of a selected myosin molecular alteration with muscle filament-sarcomere-cell-fiber-tissue-organ- and organism level phenotypes. In vivo single-myosin lever-arm orientation was observed at superresolution using a photoactivatable-green-fluorescent-protein (PAGFP)-tagged myosin light chain expressed in zebrafish skeletal muscle. By simultaneous observation of multiphoton excitation fluorescence emission and second harmonic generation from myosin, we demonstrated tag specificity for the lever arm. Single-molecule detection used highly inclined parallel beam illumination and was verified by quantized photoactivation and photobleaching. Single-molecule emission patterns from relaxed muscle in vivo provided extensive superresolved dipole orientation constraints that were modeled using docking scenarios generated for the myosin (S1) and GFP crystal structures. The dipole orientation data provided sufficient constraints to estimate S1/GFP coordination. The S1/GFP coordination in vivo is rigid and the lever-arm orientation distribution is well-ordered in relaxed muscle. For comparison, single myosins in relaxed permeabilized porcine papillary muscle fibers indicated slightly differently oriented lever arms and rigid S1/GFP coordination. Lever arms in both muscles indicated one preferred spherical polar orientation and widely distributed azimuthal orientations relative to the fiber symmetry axis. Cardiac myosin is more radially displaced from the fiber axis. Probe rigidity implies the PAGFP tag reliably indicates cross-bridge orientation in situ and in vivo.

Ventricular myosin modifies in vitro step-size when phosphorylated

Cardiac and skeletal muscle myosins have the central role in contraction transducing ATP free energy into the mechanical work of moving actin. Myosin has a motor domain containing ATP and actin binding sites and a lever-arm that undergoes rotation impelling bound actin. The lever-arm converts torque generated in the motor into the linear displacement known as step-size. The myosin lever-arm is stabilized by bound essential and regulatory light chains (ELC and RLC). RLC phosphorylation at S15 is linked to modified lever-arm mechanical characteristics contributing to myosin filament based contraction regulation and to the response of the muscle to disease. Myosin step-size was measured using a novel quantum dot (Qdot) assay that previously confirmed a 5nm step-size for fast skeletal myosin and multiple unitary steps, most frequently 5 and 8nm, and a rare 3nm displacement for β cardiac myosin (βMys). S15 phosphorylation in βMys is now shown to change step-size distribution by advancing the 8nm step frequency. After phosphorylation, the 8nm step is the dominant myosin step-size resulting in significant gain in the average step-size. An increase in myosin step-size will increase the amount of work produced per ATPase cycle. The results indicate that RLC phosphorylation modulates work production per ATPase cycle suggesting the mechanism for contraction regulation by the myosin filament.

Skeletal Muscle Fuel Selection Occurs at the Mitochondrial Level

Mammals exponentially increase the rate of carbohydrate oxidation as exercise intensity rises, while birds combust lipid almost exclusively while flying at high percentages of aerobic capacity. The fuel oxidized by contracting muscle depends on many factors: whole-body fuel storage mass, mobilization, blood transport, cellular uptake, and substrate selection at the level of the mitochondrion. We examined the fuel preferences of mitochondria isolated from mammalian and avian locomotory muscles using two approaches. First, the influence of substrates on the kinetics of respiration (Km,ADP and Vmax) was evaluated. For all substrates and combinations, Km,ADP was generally twofold higher in avian mitochondria. Second, fuel competition between pyruvate, glutamate and/or palmitoyl-l-carnitine at three levels of ATP free energy was determined using the principle of mass balance and the measured rates of O2 consumption and metabolite accumulation/utilization. Avian mitochondria strongly spared pyruvate from oxidation when another substrate was available and fatty acid was the dominant substrate, regardless of energy state. Mammalian mitochondria exhibited some preference for fatty acid over pyruvate at lower flux (higher energy state), but exhibited a much greater tendency to select pyruvate and glutamate when available. Studies in sonicated mitochondria revealed twofold higher electron transport chain electron conductance in avian mitochondria. We conclude that substantial fuel selection occurs at the level of the mitochondrial matrix and that avian flight muscle mitochondria are particularly biased toward the selection of fatty acid, possibly by facilitating high β-oxidation flux by maintaining a more oxidized matrix.

In Vivo Orientation of Single Myosins in a Zebrafish Embryo

Cardiac and skeletal myosin is highly organized in the muscle lattice where it powers contraction by transducing ATP free energy into the mechanical work of moving actin in a mechanism known as transduction/mechanical coupling. While muscle myosin can move actin in vitro, its in vivo environment is crowded and constrained by the fiber lattice. In vivo, myosin side chains are modified during- and post-translation by mutation, phosphorylation, deamidation, and oxidation under normal, diseased, or aging conditions and all potentially impacting transduction/mechanical coupling. Single myosin detection provides highly prized bottom-up quantitative characterization of myosin that tests hypotheses without the ambiguities inherent in ensemble derived observations. The marriage of in vivo and single myosin detection to study human cardiac or skeletal muscle contraction in zebrafish embryo models is a multi-scaled technology for basic and translational research. It allows one-to-one registration of a selected myosin molecular alteration with muscle filament-sarcomere-cell-fiber-tissue-organ- and organism levels of phenotype with confidence that all interactions and modifications are appropriately contributing their impact to myosin conformation. In vivo single myosin lever-arm orientation was observed at super-resolution using a photoactivatable GFP (PAGFP) tagged myosin light chain expressed in zebrafish skeletal muscle. Imaging was aided by an innovative microfluidic design for embryo confinement. Tag specificity was demonstrated by the simultaneous observation of 2-photon fluorescence emission and second harmonic generation (SHG) from myosin. Single molecule detection used highly inclined and laminated optical sheet (HILO) illumination and was verified by quantized photoactivation or photobleaching. Single molecule emission patterns from relaxed muscle indicated a highly orientationally confined lever-arm orientation. Results demonstrate detection of single myosin orientation in vivo. The zebrafish muscle system serves as an in vivo model for human disease and aging effects on myosin. Research supported by NIH R01AR049277 and R01HL095572.

Ventricular Myosin Modifies in Vitro Step-Size When Phosphorylated

Cardiac and skeletal muscle myosins have the central role in contraction transducing ATP free energy into the mechanical work of moving actin in transduction/mechanical coupling. Inheritable cardiomyopathies are more frequently linked to myosin mutations than other sarcomeric proteins. Hereditary skeletal myopathies linked to myosin are less common. They lead to muscle weakness or affect myosin isoforms expressed during development leading to arthrogryposis syndromes. Myosin has a motor domain containing ATP and actin binding sites and light chains stabilized lever-arm that undergoes rotation impelling bound actin. The lever-arm converts torque generated in the motor into linear displacement (step-size). Relative myosin and actin filament sliding is modeled in vitro with a motility assay quantitating actin filament translation over a myosin coated surface. A novel quantum dot super-resolution in vitro motility assay confirmed a 5 nm step-size for fast skeletal myosin while β cardiac myosin (βMys) had multiple unitary steps, most frequently 5 and 8 nm, and a rare 3 nm displacement. The myosin lever-arm is stabilized by bound essential and regulatory light chains (ELC and RLC). RLC phosphorylation at S15 is linked to modified lever-arm mechanical characteristics contributing to disease and to myosin filament based contraction regulation. We have studied the effect of RLC phosphorylation on the step-size of porcine βMys. Phosphorylated βMys has ∼85% of the myosin phosphorylated. We find RLC phosphorylation causing the distribution of longest step increasing from 37% to 71%. This dramatic re-distribution of step-sizes provides significant gain in average step-size. The results indicate a mechanism for contraction regulation by step-size adaptation using post-translational modification of the myosin filament via RLC phosphorylation. Research supported by R01AR049277, R01HL095572 and the Mayo Foundation.

The Qdot-Labeled Actin Super-Resolution Motility Assay Measures Low-Duty Cycle Muscle Myosin Step Size

Myosin powers contraction in heart and skeletal muscle and is a leading target for mutations implicated in inheritable muscle diseases. During contraction, myosin transduces ATP free energy into the work of muscle shortening against resisting force. Muscle shortening involves relative sliding of myosin and actin filaments. Skeletal actin filaments were fluorescently labeled with a streptavidin conjugate quantum dot (Qdot) binding biotin-phalloidin on actin. Single Qdots were imaged in time with total internal reflection fluorescence microscopy and then spatially localized to 1-3 nm using a super-resolution algorithm as they translated with actin over a surface coated with skeletal heavy meromyosin (sHMM) or full-length β-cardiac myosin (MYH7). The average Qdot-actin velocity matches measurements with rhodamine-phalloidin-labeled actin. The sHMM Qdot-actin velocity histogram contains low-velocity events corresponding to actin translation in quantized steps of ~5 nm. The MYH7 velocity histogram has quantized steps at 3 and 8 nm in addition to 5 nm and larger compliance compared to that of sHMM depending on the MYH7 surface concentration. Low-duty cycle skeletal and cardiac myosin present challenges for a single-molecule assay because actomyosin dissociates quickly and the freely moving element diffuses away. The in vitro motility assay has modestly more actomyosin interactions, and methylcellulose inhibited diffusion to sustain the complex while preserving a subset of encounters that do not overlap in time on a single actin filament. A single myosin step is isolated in time and space and then characterized using super-resolution. The approach provides a quick, quantitative, and inexpensive step size measurement for low-duty cycle muscle myosin.