Myosin Isoforms Research Articles

Proteomic analysis of wild boar meat: Effect of storage method and time on muscle protein stability

Oxidation processes affect proteins from various molecular pathways and are crucial for wild boar meat quality, shelf life and human health. This study investigated the effects of different storage methods on the formation and composition of oxygen-induced protein aggregates in the muscles of European wild boar (Sus scrofa scrofa). Vacuum packaging (VAC), modified atmosphere packaging (MAP) and dry-ageing (DA) were compared over a 21-day storage period. The results showed significant differences in protein aggregation depending on the method and storage time. The most intense protein aggregation occurred in the MAP (80 % O2), while air DA (20.9 % O2) resulted in intermediate levels of protein aggregation. Crucial myofibrillar proteins involved in aggregate formation were titin, myosin isoforms (MYH1, MYH2 and MYH7) and nebulin, which were cross-linked with small sarcoplasmic enzymes, such as muscle creatine kinase, isocitrate dehydrogenase and ATPase 1. High‑oxygen storage conditions also promoted the oxidation of ATP synthase, beta-enolase 3, ADP/ATP translocase and myoglobin.

Effects of Pulsed Electromagnetic Field Treatment on Skeletal Muscle Tissue Recovery in a Rat Model of Collagenase-Induced Tendinopathy: Results from a Proteome Analysis.

Tendon disorders often result in decreased muscle function and atrophy. Pulsed Electromagnetic Fields (PEMFs) have shown potential in improving tendon fiber structure and muscle recovery. However, the molecular effects of PEMF therapy on skeletal muscle, beyond conventional metrics like MRI or markers of muscle decline, remain largely unexplored. This study investigates the metabolic and structural changes in PEMF-treated muscle tissue using proteomics in a rat model of Achilles tendinopathy induced by collagenase. Sprague Dawley rats were unilaterally induced for tendinopathy with type I collagenase injection and exposed to PEMFs for 8 h/day. Gastrocnemius extracts from untreated or PEMF-treated rats were analyzed with LC-MS/MS, and proteomics differential analysis was conducted through label-free quantitation. PEMF-treated animals exhibited decreased glycolysis and increased LDHB expression, enhancing NAD signaling and ATP production, which boosted respiratory chain activity and fatty acid beta-oxidation. Antioxidant protein levels increased, controlling ROS production. PEMF therapy restored PGC1alpha and YAP levels, decreased by tendinopathy. Additionally, myosins regulating slow-twitch fibers and proteins involved in fiber alignment and force transmission increased, supporting muscle recovery and contractile function. Our findings show that PEMF treatment modulates NAD signaling and oxidative phosphorylation, aiding muscle recovery through the upregulation of YAP and PGC1alpha and increasing slow myosin isoforms, thus speeding up physiological recovery.

Abstract We020: Myh9 plays a vital role in cardiac myofibroblast differentiation and is indispensable for cardiac repair after myocardial infarction

After myocardial infarction (MI), quiescent cardiac fibroblasts (CFs) are activated and differentiate into myofibroblasts featured by elevated expression of smooth muscle alpha-actin (SMαA, encoded by Acta2 ), which form contractile stress fibers. However, compared to the matrix formation function of cardiac myofibroblasts, much less is known about the role of their contraction in post-MI cardiac repair. In our previous study using mice lacking Acta2 in CFs, all other 5 actin isoforms were activated to compensate for the loss of Acta2 , suggesting the low feasibility of abolishing cardiac myofibroblast contraction through deleting actin genes. An alternative strategy through targeting myosin was explored. Our transcriptomic study identified Myh9 and Myh10 [encoding non-muscle myosin IIA (NM IIA) and NM IIB, respectively] as the dominant myosin isoforms in cardiac myofibroblasts. In vitro studies found that Myh9 knockout (KO) but not Myh10 KO significantly affected cardiac myofibroblast morphology, completely abolished the stress fiber formation and contractility of these cells, and reduced their proliferation and ability to stabilize the surrounding matrix. Mice lacking Myh9 in CFs have an increased post-MI cardiac rupture rate compared to WT mice regardless of sexes ( p =0.004 and 0.02 for male and female mice, respectively). In vitro and in vivo transcriptomic and proteomics analysis revealed disrupted expression of stress fiber and mesenchymal identity genes/proteins caused by Myh9 KO. Mechanistically, Myh9 KO led to an increased globular to filamentous (G/F) actin ratio. The altered actin dynamics likely trigger multiple epigenetic events, which together lead to significantly altered cardiac myofibroblast gene expression and activities beyond contraction.

Abstract We008: Hallmark Maturation of the Human Pluripotent Stem Cells-derived Cardiovascular Progenitors in Myocardial Infarcted Large Animal Model

For the goal of regenerative cardiology, cellular therapy may be used to restore injured heart muscle. The molecular mechanisms behind the engraftment and maturation of these transplanted cells, however, remain poorly understood. Thus, to obtain a comprehensive understanding of the complex transcriptional landscapes within their transplanted spatial context, we utilized spatial transcriptomics in this study. We have previously demonstrated the safety and effectiveness of transplanting our cardiovascular progenitors (CVPs) produced from laminin-221 into pig hearts that had suffered myocardial infarction (MI). Here, we undertake 10x spatial transcriptomics in chronic MI pig models at 1-, 4-, and 12 weeks post-transplantation to further our understanding of these cells in vivo. With confidence, we can distinguish between the transcripts from the pigs and the humans in the infarcted region. By using human-specific anti-Ku80 antibodies for immunohistochemical labeling of the pig tissue samples, this in silico prediction was verified. The CVPs' engraftment and longevity in the infracted region were shown by the long-term identification and staining of human cells at various time points. It is interesting to note that after 12 weeks, ribosomal and mitochondrial activity were higher than at 1 and 2 weeks. These results are consistent with the hallmarks of cardiac maturation, in which cells transit from immature fetal-like cells to mature cardiomyocytes (CMs) by switching their metabolic energy source from glucose to fatty acids. Also, by comparing the heart-specific genes, including MYH6 and MYH7, we were able to quantify the myosin isoform switches in the xenograft over time. Furthermore, to delineate the fate of the transplanted cells, we performed a ligand-receptor interaction analysis. Intriguing, we identified the midkine protein as a possible secreted human paracrine factor that enhances angiogenesis in the graft. In conclusion, we have shown the successful engraftment and maturation of human CVPs toward CMs using spatial transcriptomic technology. These data will be used to understand other aspects of regenerative cardiology such as the mechanism of VTs and in vivo cellular repair mechanisms. A shiny app atlas has been generated to share our transcriptomics data for all to explore gene expression of human transplanted cells in myocardial infarcted pig hearts.

Murf1 alters myosin replacement rates in cultured myotubes in a myosin isoform-dependent manner.

Skeletal muscle tissue increases or decreases its volume by synthesizing or degrading myofibrillar proteins. The ubiquitin-proteasome system plays a pivotal role during muscle atrophy, where muscle ring finger proteins (Murf) function as E3 ubiquitin ligases responsible for identifying and targeting substrates for degradation. Our previous study demonstrated that overexpression of Ozz, an E3 specific to embryonic myosin heavy chain (Myh3), precisely reduced the Myh3 replacement rate in the thick filaments of myotubes (E. Ichimura et al., Physiol Rep. 9:e15003, 2021). These findings strongly suggest that E3 plays a critical role in regulating myosin replacement. Here, we hypothesized that the Murf isoforms, which recognize Myhs as substrates, reduced the myosin replacement rates through the enhanced Myh degradation by Murfs. First, fluorescence recovery after a photobleaching experiment was conducted to assess whether Murf isoforms affected the GFP-Myh3 replacement. In contrast to Murf2 or Murf3 overexpression, Murf1 overexpression selectively facilitated the GFP-Myh3 myosin replacement. Next, to examine the effects of Murf1 overexpression on the replacement of myosin isoforms, Cherry-Murf1 was coexpressed with GFP-Myh1, GFP-Myh4, or GFP-Myh7 in myotubes. Intriguingly, Murf1 overexpression enhanced the myosin replacement of GFP-Myh4 but did not affect those of GFP-Myh1 or GFP-Myh7. Surprisingly, overexpression of Murf1 did not enhance the ubiquitination of proteins. These results indicate that Murf1 selectively regulated myosin replacement in a Myh isoform-dependent fashion, independent of enhanced ubiquitination. This suggests that Murf1 may have a role beyond functioning as a ubiquitin ligase E3 in thick filament myosin replacement.

<i>N</i>-terminal fragment of cardiac myosin binding protein C modulates cooperative mechanisms of the thin filament activation in the atria and ventricles

Cardiac myosin binding protein C (cMyBP-C) is one of the essential control components of the myosin cross-bridge cycle. The C-terminal part of cMyBP-C lies on the surface of the thick filament, and its N-terminal part interacts with actin, myosin, and tropomyosin, affecting both the kinetics of the ATP hydrolysis cycle and the lifetime of the cross bridge, as well as the calcium regulation of actin-myosin interaction, thereby modulating the contractile function of the myocardium. The role of cMyBP-C in atrial contraction is poorly studied. We examined the effect of the N-terminal C0-m-C2 (C0-C2) fragment of cMyBP-C on actin-myosin interaction using ventricular and atrial myosin in anin vitromotility assay. The C0-C2 fragment of cMyBP-C significantly reduced the maximum sliding velocity of thin filaments on both myosin isoforms and increased the calcium sensitivity of actin-myosin interaction. The C0-C2 fragment had different effects on the kinetics of nucleotide, ATP, and ADP exchange, increasing the affinity of ventricular myosin for ADP and decreasing the affinity of atrial myosin. The effect of the C0-C2 fragment on the activation of the thin filament depended on the myosin isoforms. Atrial myosin activates the thin filament less strongly than ventricular myosin, and the C0-C2 fragment makes these differences in myosin isoforms more pronounced.

Post-translational modifications of vertebrate striated muscle myosin heavy chains.

Post-translational modifications (PTMs) play a crucial role in regulating the function of many sarcomeric proteins, including myosin. Myosins comprise a family of motor proteins that play fundamental roles in cell motility in general and muscle contraction in particular. A myosin molecule consists of two myosin heavy chains (MyHCs) and two pairs of myosin light chains (MLCs); two MLCs are associated with the neck region of each MyHC's N-terminal head domain, while the two MyHC C-terminal tails form a coiled-coil that polymerizes with other MyHCs to form the thick filament backbone. Myosin undergoes extensive PTMs, and dysregulation of these PTMs may lead to abnormal muscle function and contribute to the development of myopathies and cardiovascular disorders. Recent studies have uncovered the significance of PTMs in regulating MyHC function and showed how these PTMs may provide additional modulation of contractile processes. Here, we discuss MyHC PTMs that have been biochemically and/or functionally studied in mammals' and rodents' striated muscle. We have identified hotspots or specific regions in three isoforms of myosin (MYH2, MYH6, and MYH7) where the prevalence of PTMs is more frequent and could potentially play a significant role in fine-tuning the activity of these proteins.

Our journey with François Gros.

Gillian Butler-Browne began working on muscle at the Institut Pasteur in the laboratory of François Gros in 1978. She characterized the expression profile of different myosin isoforms during both human and rodent development. Vincent Mouly joined this laboratory for his PhD in 1982, and defined the different populations of myoblasts appearing during development in birds and then in humans. Together, they demonstrated the impact of the limit in proliferation of the precursor cells on the regenerative capacity of human skeletal muscle, and their group developed models to evaluate the regenerative potential of skeletal muscle in vitro, measuring the telomeric erosion, and identified the involvement of a stress pathway in the proliferative arrest of muscle progenitors. A platform to produce human immortalized muscle cell lines was the successful result of this research, initiated with François Gros and W. E. Wright. The in vivo regenerative potential of human muscle cells was evaluated by injection into muscles of immunodeficient mice. Their group in collaboration with the clinical team of Professor Jean Lacau St-Guily and Professor Sophie Perié completed a successful autologous myoblast transplantation clinical trial for Oculo-pharyngeal muscular dystrophy. This common scientific career was made possible thanks to the precious and always benevolent support of François Gros.

Force and kinetics of fast and slow muscle myosin determined with a synthetic sarcomere–like nanomachine

Myosin II is the muscle molecular motor that works in two bipolar arrays in each thick filament of the striated (skeletal and cardiac) muscle, converting the chemical energy into steady force and shortening by cyclic ATP–driven interactions with the nearby actin filaments. Different isoforms of the myosin motor in the skeletal muscles account for the different functional requirements of the slow muscles (primarily responsible for the posture) and fast muscles (responsible for voluntary movements). To clarify the molecular basis of the differences, here the isoform–dependent mechanokinetic parameters underpinning the force of slow and fast muscles are defined with a unidimensional synthetic nanomachine powered by pure myosin isoforms from either slow or fast rabbit skeletal muscle. Data fitting with a stochastic model provides a self–consistent estimate of all the mechanokinetic properties of the motor ensemble including the motor force, the fraction of actin–attached motors and the rate of transition through the attachment–detachment cycle. The achievements in this paper set the stage for any future study on the emergent mechanokinetic properties of an ensemble of myosin molecules either engineered or purified from mutant animal models or human biopsies.

Sequence Alignment-Based Prediction of Myosin 7A: Structural Implications and Protein Interactions.

Myosin, a superfamily of motor proteins, obtain the energy they require for movement from ATP hydrolysis to perform various functions by binding to actin filaments. Extensive studies have clarified the diverse functions performed by the different isoforms of myosin. However, the unavailability of resolved structures has made it difficult to understand the way in which their mechanochemical cycle and structural diversity give rise to distinct functional properties. With this study, we seek to further our understanding of the structural organization of the myosin 7A motor domain by modeling the tertiary structure of myosin 7A based on its primary sequence. Multiple sequence alignment and a comparison of the models of different myosin isoforms and myosin 7A not only enabled us to identify highly conserved nucleotide binding sites but also to predict actin binding sites. In addition, the actomyosin-7A complex was predicted from the protein-protein interaction model, from which the core interface sites of actin and the myosin 7A motor domain were defined. Finally, sequence alignment and the comparison of models were used to suggest the possibility of a pliant region existing between the converter domain and lever arm of myosin 7A. The results of this study provide insights into the structure of myosin 7A that could serve as a framework for higher resolution studies in future.

Motor neurons and endothelial cells additively promote development and fusion of human iPSC-derived skeletal myocytes

BackgroundNeurovascular cells have wide-ranging implications on skeletal muscle biology regulating myogenesis, maturation, and regeneration. Although several in vitro studies have investigated how motor neurons and endothelial cells interact with skeletal myocytes independently, there is limited knowledge about the combined effect of neural and vascular cells on muscle maturation and development.MethodsHere, we report a triculture system comprising human-induced pluripotent stem cell (iPSC)-derived skeletal myocytes, human iPSC-derived motor neurons, and primary human endothelial cells maintained under controlled media conditions. Briefly, iPSCs were differentiated to generate skeletal muscle progenitor cells (SMPCs). These SMPCs were seeded at a density of 5 × 104 cells/well in 12-well plates and allowed to differentiate for 7 days before adding iPSC-derived motor neurons at a concentration of 0.5 × 104 cells/well. The neuromuscular coculture was maintained for another 7 days in coculture media before addition of primary human umbilical vein endothelial cells (HUVEC) also at 0.5 × 104 cells/well. The triculture was maintained for another 7 days in triculture media comprising equal portions of muscle differentiation media, coculture media, and vascular media. Extensive morphological, genetic, and molecular characterization was performed to understand the combined and individual effects of neural and vascular cells on skeletal muscle maturation.ResultsWe observed that motor neurons independently promoted myofiber fusion, upregulated neuromuscular junction genes, and maintained a molecular niche supportive of muscle maturation. Endothelial cells independently did not support myofiber fusion and downregulated expression of LRP4 but did promote expression of type II specific myosin isoforms. However, neurovascular cells in combination exhibited additive increases in myofiber fusion and length, enhanced production of Agrin, along with upregulation of several key genes like MUSK, RAPSYN, DOK-7, and SLC2A4. Interestingly, more divergent effects were observed in expression of genes like MYH8, MYH1, MYH2, MYH4, and LRP4 and secretion of key molecular factors like amphiregulin and IGFBP-4.ConclusionsNeurovascular cells when cultured in combination with skeletal myocytes promoted myocyte fusion with concomitant increase in expression of various neuromuscular genes. This triculture system may be used to gain a deeper understanding of the effects of the neurovascular niche on skeletal muscle biology and pathophysiology.

Homologous mutations in human β, embryonic, and perinatal muscle myosins have divergent effects on molecular power generation

Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in β-cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman-Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus-pseudocamptodactyly syndrome. It is not known whether their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human β, embryonic, and perinatal myosin subfragment-1. We found large effects in the developmental myosins but minimal effects in β myosin, and magnitude of changes correlated partially with clinical severity. The mutations in the developmental myosins dramatically decreased the step size and load-sensitive actin-detachment rate of single molecules measured by optical tweezers, in addition to decreasing overall enzymatic (ATPase) cycle rate. In contrast, the only measured effect of R671C in β myosin was a larger step size. Our measurements of step size and bound times predicted velocities consistent with those measured in an in vitro motility assay. Finally, molecular dynamics simulations predicted that the arginine to cysteine mutation in embryonic, but not β, myosin may reduce pre-powerstroke lever arm priming and ADP pocket opening, providing a possible structural mechanism consistent with the experimental observations. This paper presents direct comparisons of homologous mutations in several different myosin isoforms, whose divergent functional effects are a testament to myosin's highly allosteric nature.

Mitochondrial Quantity and Quality in Age-Related Sarcopenia.

Sarcopenia, the age-associated decline in skeletal muscle mass and strength, is a condition with a complex pathophysiology. Among the factors underlying the development of sarcopenia are the progressive demise of motor neurons, the transition from fast to slow myosin isoform (type II to type I fiber switch), and the decrease in satellite cell number and function. Mitochondrial dysfunction has been indicated as a key contributor to skeletal myocyte decline and loss of physical performance with aging. Several systems have been implicated in the regulation of muscle plasticity and trophism such as the fine-tuned and complex regulation between the stimulator of protein synthesis, mechanistic target of rapamycin (mTOR), and the inhibitor of mTOR, AMP-activated protein kinase (AMPK), that promotes muscle catabolism. Here, we provide an overview of the molecular mechanisms linking mitochondrial signaling and quality with muscle homeostasis and performance and discuss the main pathways elicited by their imbalance during age-related muscle wasting. We also discuss lifestyle interventions (i.e., physical exercise and nutrition) that may be exploited to preserve mitochondrial function in the aged muscle. Finally, we illustrate the emerging possibility of rescuing muscle tissue homeostasis through mitochondrial transplantation.

MYH3 mutations associated with distal arthrogryposis alter the contractility and myosin isoform switching of hiPSC-derived skeletal muscle

MYH3 mutations associated with distal arthrogryposis alter the contractility and myosin isoform switching of hiPSC-derived skeletal muscle

NLRP3 Contributes to Sarcopenia Associated to Dependency Recapitulating Inflammatory-Associated Muscle Degeneration.

Sarcopenia, a complex and debilitating condition characterized by progressive deterioration of skeletal muscle, is the primary cause of age-associated disability and significantly impacts healthspan in elderly patients. Despite its prevalence among the aging population, the underlying molecular mechanisms are still under investigation. The NLRP3 inflammasome is crucial in the innate immune response and has a significant impact on diseases related to inflammation and aging. Here, we investigated the expression of the NLRP3 inflammasome pathway and pro-inflammatory cytokines in skeletal muscle and peripheral blood of dependent and independent patients who underwent hip surgery. Patients were categorized into independent and dependent individuals based on their Barthel Index. The expression of NLRP3 inflammasome components was significantly upregulated in sarcopenic muscle from dependent patients, accompanied by higher levels of Caspase-1, IL-1β and IL-6. Among older dependent individuals with sarcopenia, there was a significant increase in the MYH3/MYH2 ratio, indicating a transcriptional shift in expression from mature to developmental myosin isoforms. Creatine kinase levels and senescence markers were also higher in dependent patients, altogether resembling dystrophic diseases and indicating muscle degeneration. In summary, we present evidence for the involvement of the NLRP3/ASC/NEK7/Caspase-1 inflammasome pathway with activation of pro-inflammatory SASP in the outcome of sarcopenia in the elderly.

N-Terminal Fragment of Cardiac Myosin Binding Protein C Modulates Cooperative Mechanisms of Thin Filament Activation in Atria and Ventricles.

Cardiac myosin binding protein C (cMyBP-C) is one of the essential control components of the myosin cross-bridge cycle. The C-terminal part of cMyBP-C is located on the surface of the thick filament, and its N-terminal part interacts with actin, myosin, and tropomyosin, affecting both kinetics of the ATP hydrolysis cycle and lifetime of the cross-bridge, as well as calcium regulation of the actin-myosin interaction, thereby modulating contractile function of myocardium. The role of cMyBP-C in atrial contraction has not been practically studied. We examined effect of the N-terminal C0-C1-m-C2 (C0-C2) fragment of cMyBP-C on actin-myosin interaction using ventricular and atrial myosin in an in vitro motility assay. The C0-C2 fragment of cMyBP-C significantly reduced the maximum sliding velocity of thin filaments on both myosin isoforms and increased the calcium sensitivity of the actin-myosin interaction. The C0-C2 fragment had different effects on the kinetics of ATP and ADP exchange, increasing the affinity of ventricular myosin for ADP and decreasing the affinity of atrial myosin. The effect of the C0-C2 fragment on the activation of the thin filament depended on the myosin isoforms. Atrial myosin activates the thin filament less than ventricular myosin, and the C0-C2 fragment makes these differences in the myosin isoforms more pronounced.

Fiber-type traps: revisiting common misconceptions about skeletal muscle fiber types with application to motor control, biomechanics, physiology, and biology.

Skeletal muscle is a highly complex tissue that is studied by scientists from a wide spectrum of disciplines, including motor control, biomechanics, exercise science, physiology, cell biology, genetics, regenerative medicine, orthopedics, and engineering. Although this diversity in perspectives has led to many important discoveries, historically, there has been limited overlap in discussions across fields. This has led to misconceptions and oversimplifications about muscle biology that can create confusion and potentially slow scientific progress across fields. The purpose of this synthesis paper is to bring together research perspectives across multiple muscle fields to identify common assumptions related to muscle fiber type that are points of concern to clarify. These assumptions include 1) classification by myosin isoform and fiber oxidative capacity is equivalent, 2) fiber cross-sectional area (CSA) is a surrogate marker for myosin isoform or oxidative capacity, and 3) muscle force-generating capacity can be inferred from myosin isoform. We address these three fiber-type traps and provide some context for how these misunderstandings can and do impact experimental design, computational modeling, and interpretations of findings, from the perspective of a range of fields. We stress the dangers of generalizing findings about "muscle fiber types" among muscles or across species or sex, and we note the importance for precise use of common terminology across the muscle fields.

Selective fast fiber damage after leg press exercise leading to failure: a pole vaulter case report

The aim of this study was to investigate, in a trained pole vaulter (PV) and in an endurance-trained physical education student (PE), the effect of a leg press exercise leading to failure (LPF) on changes in serum activity of muscle enzymes and serum concentration of fast (FM) and slow (SM) myosin isoforms, while simultaneously examining mechanical output components as indicators of performance and fatigue developed throughout exercise. A case report study design based on an observational comparison of response between two dichotomous participants, PV and PE, was used. Differences between the participants’ exercise outputs were examined by unpaired t-test or Mann-Whitney test and serum levels of muscle enzymes and myosin isoforms were analyzed at baseline and 24 and 48 hours after LPF. Exercise output analyses showed that the PV’s average fatigue index was significantly higher (P = 0.004). Moreover, during the first six sets, the concentric average power exerted by the PV was significantly (P < 0.01) higher (range: 14% to 35%) than that of the PE. The PV only showed acute mild increases of serum creatine kinase (CK) and FM 24 hours after exercise. In contrast, the PE presented persistent serum rises of several muscle enzymes and SM until 48 h after exercise. The PV’s exercise output revealed an explosive (power-oriented) profile leading to selective mild damage of fast fibers. In contrast, the PE exercise output showed a fatigue-resistant profile, which induced greater muscle enzyme activity and SM serum concentration, suggesting a higher extent of slow fiber damage. Keywords: muscle damage, creatine kinase, myosin isoforms, power output.

Protocols for Myosin and Actin-Myosin Assays Using Rapid, Stopped-Flow Kinetics.

Fast transient kinetics using stopped-flow fluorimetry is now a powerful method for defining the ATPase cycle of myosin and its subfragments and has foundwide use in defining the difference between myosin isoforms, myosins carrying disease linked mutations, and the effect of small molecules on the ATPase cycle. Here the protocols for completing the classical assays of myosin and actin.myosin using the stopped-flow are described. The assays include ATP and ADP binding to myosin and actin.myosin, displacement of ADP from myosin and actin.myosin, and the cleavage of ATP to ADP and phosphate on myosin. Single and multiple turnover assays are also described.

Pharmacological and mechanical properties of isolated pig coronary veins.

Recent successful cardiac transplantation from pig to non-human primates and the first pig-to-human transplantation has put the focus on the properties of the pig heart. In contrast to the coronary arteries, the coronary veins are less well characterized and the aim was to examine the mechanical and pharmacological properties of coronary veins in comparison to the arteries. Vessel segments from the left anterior descending coronary artery (LAD) and the concomitant vein were isolated from pig hearts in cardioplegia and examined in vitro. The wall thickness, active tension and active stress at optimal circumference were lower in coronary veins, reflecting the lower intravascular pressure in vivo. Reverse transcription polymerase chain reaction (RT-PCR) analysis of myosin isoforms showed that the vein could be characterized as having a slower smooth muscle phenotype compared to the artery. Both vessel types contracted in response to the thromboxane agonist U46619 with EC50 values of about 20nM. The artery contracted in response to acetylcholine. Precontracted arteries relaxed in noradrenaline and substance P. In contrast, the veins relaxed in acetylcholine, contracted in noradrenaline and were unresponsive to substance P. In conclusion, these results demonstrate significant differences between the coronary artery and vein in the smooth muscle properties and in the responses to sympathetic and parasympathetic stimuli.