Mammalian Muscle Cells Research Articles

Fat chance: the effect of lipid supplementation on adapting to hypoxia in skeletal muscle cells

Diving mammals are a unique type of endurance athlete that undergo apnea during exercise, yet rely on an aerobic lipid based metabolism to fuel their primary swimming muscles. A hallmark adaptation that allows diving mammals to endure aerobic exercise in a seemingly hypoxic environment is the high abundance of myoglobin in their working skeletal muscle. Our previous research found in response to hypoxic conditions alone the expression of myoglobin significantly decreased in mouse cell lines. In contrast, recent results showed that unlike terrestrial mammal muscle cells the concentration of myoglobin increased under hypoxic conditions in seal primary muscle cells. The purpose of this study was to determine the effects of lipid supplementation on myoglobin expression in mouse C2C12 and seal primary muscle cells. Myoglobin assays were used to quantify differences in myoglobin expression. As previously seen in our seal muscle cells, myoglobin expression was significantly increased in C2C12 cell lines with lipid supplementation under hypoxic but not normoxic conditions, thus implying that lipids play a role in driving the expression of myoglobin under hypoxic conditions in mammalian muscle cells.

Influence of reaction and diffusion on spatial organization of mitochondria and effectiveness factors in skeletal muscle cell design

A mathematical model is developed to analyze the influence of chemical reaction and diffusion processes on the intracellular organization of mitochondria in skeletal muscle cells. The mathematical modeling approach uses a reaction-diffusion analysis of oxygen, ATP, and ADP involved in energy metabolism and mitochondrial function as governed by oxygen supply, volume fraction of mitochondria, and rates of reaction. Superimposed upon and coupled to the continuum species material balances is a cellular automata (CA) approach governing mitochondrial life cycles in response to the metabolic state of the cell. The effectiveness factor (η), defined as the ratio of reaction rate in the system with finite rates of diffusion to those in the absence of any diffusion limitation is used to assess diffusional constraints in muscle cells. The model shows the dramatic effects that the governing parameters have on the mitochondrial cycle of life and death and how these effects lead to changes in the distribution patterns of mitochondria observed experimentally. The model results showed good agreement with experimental results on mitochondrial distributions in mammalian muscle fibers. The η increases as the mitochondrial population is redistributed toward the fiber periphery in response to a decreased availability of oxygen. Modification of the CA parameters so that the mitochondrial lifecycle is more sensitive to the oxygen concentration caused larger mitochondrial shifts to the edge of the cell with smaller changes in oxygen concentration, and thus also lead to increased values of η. The present study shows that variation in oxygen supply, muscle activity and mitochondrial ATP supply influence the η and are the important parameters that can cause diffusion limitations. In order to prevent diffusion constraints, the cell resorts to shifts in their mitochondrial population towards the cell periphery, thus increasing η.

Patterns of tropomyosin and troponin-T isoform expression in jaw-closing muscles of mammals and reptiles that express masticatory myosin

We recently reported that masticatory ('superfast') myosin is expressed in jaw-closing muscles of some rodent species. Most mammalian limb muscle fibers express tropomyosin-β (Tm-β), along with fast-type or slow-type tropomyosin-β (Tm-β), but jaw-closing muscle fibers in members of Carnivora express a unique isoform of Tm [Tm-masticatory (Tm-M)] and little or no Tm-β. The goal of this study was to determine patterns of Tm and troponin-T (TnT) isoform expression in the jaw-closing muscles of rodents and other vertebrate species that express masticatory myosin, and compare the results to those from members of Carnivora. Comparisons of electrophoretic mobility, immunoblotting and mass spectrometry were used to probe the Tm and fast-type TnT isoform composition of jaw-closing and limb muscles of six species of Carnivora, eight species of Rodentia, five species of Marsupialia, big brown bat, long-tailed macaque and six species of Reptilia. Extensive heterogeneity exists in Tm and TnT isoform expression in jaw-closing muscles between phylogenetic groups, but there are fairly consistent patterns within each group. We propose that the differences in Tm and TnT isoform expression patterns between phylogenetic groups, which share the expression of masticatory myosin, may impart fundamental differences in thin-filament-mediated muscle activation to accommodate markedly different feeding styles that may require high force generation in some species (e.g. many members of Carnivora) and high speed in others (e.g. Rodentia).

Characterization of the ClC-1 Conductance at the Surface and Transverse Tubular System Membranes of Mammalian Skeletal Muscle Fibers

ClC-1 is believed to be primarily responsible for the resting conductance in mammalian skeletal muscle fibers. However, the actual distribution of ClC-1 channels between the surface and transverse tubular system (TTS) membranes has not been assessed in intact muscle fibers. To investigate this issue, we voltage-clamped enzymatically dissociated short fibers using a 2-microelectrode configuration and simultaneously recorded chloride currents (ICl), and di-8-ANEPPS fluorescence signals to assess membrane potential changes in the TTS. Experiments were conducted in conditions that blocked all, but the chloride conductance. Fibers were equilibrated with 40 or 70 mM intracellular chloride in order to enhance the magnitude of inward ICl, and the specific ClC-1 blocker 9-ACA was used to eliminate these currents whenever necessary. Voltage-dependent di-8-ANEPPS signals and ICl acquired before (control) and after the addition of 9-ACA were comparatively assessed. Early after the onset of stimulus pulses, di-8-ANEPPS signals under control conditions were smaller than those recorded in the presence of 9-ACA. We defined as attenuation the normalized time-dependent difference between these signals. Attenuation was discovered to be ICl-dependent since its magnitude varied in close correlation with the amplitude and time course of ICl. While the properties of ICl, and those of the attenuation seen in optical records, could be simultaneously predicted by model simulations when the chloride permeability (PCl) at the surface and TTS membranes were approximately equal, the model failed to explain the optical data if PCl was precluded from the TTS membranes. Since the ratio between the areas of TTS membranes and the sarcolemma is large in mammalian muscle fibers, our results demonstrate that a significant fraction of the experimentally recorded ICl arises from the TTS. Supported by NIH grants AR047664, AR041802, and AR054816.

Chloride currents from the transverse tubular system in adult mammalian skeletal muscle fibers

Chloride fluxes are the main contributors to the resting conductance of mammalian skeletal muscle fibers. ClC-1, the most abundant chloride channel isoform in this preparation, is believed to be responsible for this conductance. However, the actual distribution of ClC-1 channels between the surface and transverse tubular system (TTS) membranes has not been assessed in intact muscle fibers. To investigate this issue, we voltageclamped enzymatically dissociated short fibers using a two-microelectrode configuration and simultaneously recorded chloride currents (ICl) and di-8-ANEPPS fluorescence signals to assess membrane potential changes in the TTS. Experiments were conducted in conditions that blocked all but the chloride conductance. Fibers were equilibrated with 40 or 70 mM intracellular chloride to enhance the magnitude of inward ICl, and the specific ClC-1 blocker 9-ACA was used to eliminate these currents whenever necessary. Voltage-dependent di-8-ANEPPS signals and ICl acquired before (control) and after the addition of 9-ACA were comparatively assessed. Early after the onset of stimulus pulses, di-8-ANEPPS signals under control conditions were smaller than those recorded in the presence of 9-ACA. We defined as attenuation the normalized time-dependent difference between these signals. Attenuation was discovered to be ICl dependent since its magnitude varied in close correlation with the amplitude and time course of ICl. While the properties of ICl, and those of the attenuation seen in optical records, could be simultaneously predicted by model simulations when the chloride permeability (PCl) at the surface and TTS membranes were approximately equal, the model failed to explain the optical data if PCl was precluded from the TTS membranes. Since the ratio between the areas of TTS membranes and the sarcolemma is large in mammalian muscle fibers, our results demonstrate that a significant fraction of the experimentally recorded ICl arises from TTS contributions.

Stimulation Of Glucose Uptake In Rainbow Trout Myotubes By Ampk Activating Compounds

AMP-activated protein kinase (AMPK), a master metabolic switch, mediates the observed increase of glucose uptake in locomotory muscle of mammals during exercise. AICAR and metformin, two synthetic compounds, have been shown to activate AMPK, producing an increase in glucose transport in mammalian muscle cells. Despite the physiological importance of exercise as a modulator of glucose disposal by locomotory muscle, the effect of both drugs has not been investigated in fish cells. Our results show that AICAR and metformin significantly (p= 0.010) stimulate glucose uptake (1.6 and 1.8 fold, respectively) in rainbow trout myotubes. These results are consistent with the AMPK-mediated response demonstrated in mammals. Future studies will investigate further the activity of AMPK in fish muscle in addition to incorporating in vitro experimental models to study how glucose uptake and gene expression are regulated during conditions that mimic exercise. Study supported by a MICINN grant (AGL2009-07006, Spain) to J. V. Planas, and a FP7-PEOPLE-IIF-2008 fellowship (Marie Curie Action, EU) to L. J. Magnoni.

Force and power generating mechanism(s) in active muscle as revealed from temperature perturbation studies.

The basic characteristics of the process of force and power generation in active muscle that have emerged from temperature studies are examined. This is done by reviewing complementary findings from temperature-dependence studies and rapid temperature-jump (T-jump) experiments and from intact and skinned fast mammalian muscle fibres. In isometric muscle, a small T-jump leads to a characteristic rise in force showing that crossbridge force generation is endothermic (heat absorbed) and associated with increased entropy (disorder). The sensitivity of the T-jump force generation to added inorganic phosphate (Pi) indicates that a T-jump enhances an early step in the actomyosin (crossbridge) ATPase cycle before Pi-release. During muscle lengthening when steady force is increased, the T-jump force generation is inhibited. Conversely, during shortening when steady force is decreased, the T-jump force generation is enhanced in a velocity-dependent manner, showing that T-jump force generation is strain sensitive. Within the temperature range of ∼5–35◦C, the temperature dependence of steady active force is sigmoidal both in isometric and in shortening muscle. However, in shortening muscle, the endothermic character of force generation becomes more pronounced with increased velocity and this can, at least partly, account for the marked increase with warming of the mechanical power output of active muscle.

S100A1: a physiological modulator of RYR1, Ca2+ release, and contractility in skeletal muscle. Focus on “S100A1 promotes action potential-initiated calcium release flux and force production in skeletal muscle”

SKELETAL MUSCLES adjust their contractile output over an exceptionally wide dynamic range, as needed to produce movements ranging from sitting and standing, to athletics and dance. At the single fiber level, the force generated by skeletal muscles is determined, ultimately, by the formation of cycling cross bridges between actin and myosin. This interaction requires Ca 2 and is governed by the simple relationship: force is proportional to intracellular ionized Ca 2 . Cross-bridge formation is inhibited in resting muscle when Ca 2 is sequestered within the sarcoplasmic reticulum (SR); it is activated when stored Ca 2 is released into the cytosol, allowing actin-myosin interactions to proceed. Contraction stops as Ca 2 is actively resequestered by the Ca 2 -ATPase of the SR (SERCA1). Thus Ca 2 is the central messenger in muscle activation, and the amplitude and time course of the intracellular Ca 2 change is the major determinant of contractile output. The key mechanisms that activate Ca 2 release from the SR are now well defined. This sequence, termed excitation-contraction coupling, begins when an excitatory input from the motor nerve triggers a propagating action potential on the muscle membrane. The action potential rapidly depolarizes the entire sarcolemma, including transverse tubules, which invaginate from the surface membrane and form specialized junctions (triads) with the SR along much of their length. The triad junction serves as a platform for assembling sarcolemmal calcium

Transient Inactivation of Rb and ARF Yields Regenerative Cells from Postmitotic Mammalian Muscle

An outstanding biological question is why tissue regeneration in mammals is limited, whereas urodele amphibians and teleost fish regenerate major structures, largely by cell cycle reentry. Upon inactivation of Rb, proliferation of postmitotic urodele skeletal muscle is induced, whereas in mammalian muscle this mechanism does not exist. We postulated that a tumor suppressor present in mammals but absent in regenerative vertebrates, the Ink4a product ARF (alternative reading frame), is a regeneration suppressor. Concomitant inactivation of Arf and Rb led to mammalian muscle cell cycle reentry, loss of differentiation properties, and upregulation of cytokinetic machinery. Single postmitotic myocytes were isolated by laser micro-dissection-catapulting, and transient suppression of Arf and Rb yielded myoblast colonies that retained the ability to differentiate and fuse into myofibers upon transplantation in vivo. These results show that differentiation of mammalian cells is reversed by inactivation of Arf and Rb and support the hypothesis that Arf evolved at the expense of regeneration.

Dihydrotestosterone activates the MAPK pathway and modulates maximum isometric force through the EGF receptor in isolated intact mouse skeletal muscle fibres

It is generally believed that steroid hormones have both genomic and non-genomic (rapid) actions. Although the latter form an important component of the physiological response of these hormones, little is known about the cellular signalling pathway(s) mediating these effects and their physiological functions in adult mammalian skeletal muscle fibres. Therefore, the primary aim of this study was to investigate the non-genomic actions of dihydrotestosterone (DHT) and their physiological role in isolated intact mammalian skeletal muscle fibre bundles. Our results show that treating the fibre bundles with physiological concentrations of DHT increases both twitch and tetanic contractions in fast twitch fibres. However, it decreases them in slow twitch fibres. These changes in force are accompanied by an increase in the phosphorylation of MAPK/ERK1/2 in both fibre types and that of regulatory myosin light chains in fast twitch fibres. Both effects were insensitive to inhibitors of Src kinase, androgen receptor, insulin-like growth factor 1 receptor and platelet-derived growth factor receptor. However, they were abolished by the MAPK/ERK1/2 kinase inhibitor PD98059 and the epidermal growth factor (EGF) receptor inhibitor tyrphostin AG 1478. In contrast, testosterone had no effect on force and increased the phosphorylation of ERK1/2 in slow twitch fibres only. From these results we conclude that sex steroids have non-genomic actions in isolated intact mammalian skeletal muscle fibres. These are mediated through the EGF receptor and one of their main physiological functions is the enhancement of force production in fast twitch skeletal muscle fibres.

Tropomyosin and Troponin-T Isoform Differences in Jaw-Closing Muscles of Rodentia and Carnivora that Express Masticatory Myosin

We recently reported that, contrary to prevailing dogma, masticatory (“superfast”) myosin is expressed in jaw-closing muscles of some members of Rodentia (Reiser et al., J. Exp. Biol. 212:2511-2519, 2009). Whereas virtually all mammalian limb muscle fibers express tropomyosin-β (Tm-β), along with fast-type or slow-type Tm-α, jaw-closing muscle fibers that express masticatory myosin in members of Carnivora express a unique isoform of Tm-α and do not express Tm-β (Rowlerson et al., Biochem. Biophys. Res. Commun. 113:519-525, 1983; Hoh et al., Proc. Aust. Physiol. Pharmacol. Soc. 20:192P, 1989). The goal of this study was to examine thin filament protein isoform composition in jaw-closing muscles of rodents that express masticatory myosin and compare the results to those from members of Carnivora. Tm or troponin-T (TnT) specific antibodies and immunoblotting were used to probe homogenates of limb and jaw-closing muscles of six species of Rodentia and three species of Carnivora. The results verify the almost exclusive expression of Tm-α, presumably the unique isoform reported earlier, in the jaw-closing muscles of Carnivora and reveal that members of Rodentia express both Tm-α and Tm-β in jaw-closing muscles, similar to limb muscles in the same species. The results also indicate that the same complement of TnT isoforms are expressed in jaw-closing and limb muscles of Carnivora, but differ markedly between the two muscle groups in Rodentia. Fast-type TnC and TnI appear to be expressed in jaw-closing muscles of both orders. It is postulated that the differences in Tm and TnT isoform expression patterns between Carnivora and Rodentia may impart fundamental differences in calcium-sensitivity of force generation to accommodate markedly different feeding styles, with shared expression of masticatory myosin, between these two animal orders. Supported by the National Science Foundation.

Effects of Domain Peptide DP4 on Depolarization-Induced Calcium Transients and Calcium Currents in Voltage-Clamped Fibers from Mouse FDB Skeletal Muscle

Skeletal muscle excitation-contraction coupling involves sequential activation of dihydropyridine receptors (DHPRs) and type-1 ryanodine receptors (RyR1) to produce depolarization-dependent sarcoplasmic reticulum Ca2+ release via “orthograde” signaling. Another form of DHPRs-RyR1 communication is “retrograde” signaling, in which RyRs modulate the gating of DHPR. Domain peptide 4 (DP4), a peptide corresponding to residues Leu2442-Pro2477 of RyR1, interferes with inter-domain interactions within RyR1 that normally stabilize the closed state of the RyR1 channel. DP4 has been shown to potentiate force in response to submaximal depolarization by ionic substitution and caffeine-induced Ca2+ release in peeled muscle fibers. In sarcoplasmic reticulum vesicles, DP4 directly potentiates RyR1 opening as shown by increased ryanodine binding and sensitization of Ca2+ release. Here we explore possible effects of DP4 on excitation-contraction coupling in whole-cell voltage clamped adult FDB skeletal muscle fibers. Depolarization-induced fluo-4 fluorescence transients (F/F0) became detectable at smaller depolarizations, and were increased at larger depolarizations in fibers dialyzed with DP4 compared to those without DP4 in the patch pipette, as were the Ca2+ transients and Ca2+ release calculated from F/F0. The amplitude of peak Ca2+ release (R) depended on voltage according to a two-state Boltzmann function. DP4 increased the amplitude of maximum release rate (Rmax) by ca 54% when compared to control fibers. DP4 also induced a negative 5 mV shift in the potential at which R = 0.5 of Rmax and an augmentation of macroscopic DHPR Ca2+ current density at all voltages tested. Thus, DP4 potentiates both depolarization-dependent Ca2+ release via RyR1 and Ca2+ influx via DHPR, the later possibly mediated by retrograde signaling between the RyR1 and DHPR Ca2+ channels in adult mammalian muscle fibers. Supported by NIH-NIAMS Grants R01-AR055099 and T32-AR007592.

Interactions Between Connected Half-Sarcomeres Produce Emergent Mechanical Behavior in a Mathematical Model of Muscle

Most reductionist theories of muscle attribute a fiber's mechanical properties to the scaled behavior of a single half-sarcomere. Mathematical models of this type can explain many of the known mechanical properties of muscle but have to incorporate a passive mechanical component that becomes ∼300% stiffer in activating conditions to reproduce the force response elicited by stretching a fast mammalian muscle fiber. The available experimental data suggests that titin filaments, which are the mostly likely source of the passive component, become at most ∼30% stiffer in saturating Ca2+ solutions. The work described in this manuscript used computer modeling to test an alternative systems theory that attributes the stretch response of a mammalian fiber to the composite behavior of a collection of half-sarcomeres. The principal finding was that the stretch response of a chemically permeabilized rabbit psoas fiber could be reproduced with a framework consisting of 300 half-sarcomeres arranged in 6 parallel myofibrils without requiring titin filaments to stiffen in activating solutions. Ablation of inter-myofibrillar links in the computer simulations lowered isometric force values and lowered energy absorption during a stretch. This computed behavior mimics effects previously observed in experiments using muscles from desmin-deficient mice in which the connections between Z-disks in adjacent myofibrils are presumably compromised. The current simulations suggest that muscle fibers exhibit emergent properties that reflect interactions between half-sarcomeres and are not properties of a single half-sarcomere in isolation. It is therefore likely that full quantitative understanding of a fiber's mechanical properties requires detailed analysis of a complete fiber system and cannot be achieved by focusing solely on the properties of a single half-sarcomere.

Functional diversity among a family of human skeletal muscle myosin motors

Human skeletal muscle fibers express five highly conserved type-II myosin heavy chain (MyHC) genes in distinct spatial and temporal patterns. In addition, the human genome contains an intact sixth gene, MyHC-IIb, which is thought under most circumstances not to be expressed. The physiological and biochemical properties of individual muscle fibers correlate with the predominantly expressed MyHC isoform, but a functional analysis of homogenous skeletal muscle myosin isoforms has not been possible. This is due to the difficulties of separating the multiple isoforms usually coexpressed in muscle fibers, as well as the lack of an expression system that produces active recombinant type II skeletal muscle myosin. In this study we describe a mammalian muscle cell expression system and the functional analysis of all six recombinant human type II skeletal muscle myosin isoforms. The diverse biochemical activities and actin-filament velocities of these myosins indicate that they likely have distinct functions in muscle. Our data also show that ATPase activity and motility are generally correlated for human skeletal muscle myosins. The exception, MyHC-IIb, encodes a protein that is high in ATPase activity but slow in motility; this is the first functional analysis of the protein from this gene. In addition, the developmental isoforms, hypothesized to have low ATPase activity, were indistinguishable from adult-fast MyHC-IIa and the specialized MyHC-Extraocular isoform, that was predicted to be the fastest of all six isoforms but was functionally similar to the slower isoforms.

Interactions between Connected Half-Sarcomeres Produce Emergent Mechanical Behavior in a Mathematical Model of Muscle

Most reductionist theories of muscle attribute a fiber's mechanical properties to the scaled behavior of a single half-sarcomere. Mathematical models of this type can explain many of the known mechanical properties of muscle but have to incorporate a passive mechanical component that becomes ∼300% stiffer in activating conditions to reproduce the force response elicited by stretching a fast mammalian muscle fiber. The available experimental data suggests that titin filaments, which are the mostly likely source of the passive component, become at most ∼30% stiffer in saturating Ca2+ solutions. The work described in this manuscript used computer modeling to test an alternative systems theory that attributes the stretch response of a mammalian fiber to the composite behavior of a collection of half-sarcomeres. The principal finding was that the stretch response of a chemically permeabilized rabbit psoas fiber could be reproduced with a framework consisting of 300 half-sarcomeres arranged in 6 parallel myofibrils without requiring titin filaments to stiffen in activating solutions. Ablation of inter-myofibrillar links in the computer simulations lowered isometric force values and lowered energy absorption during a stretch. This computed behavior mimics effects previously observed in experiments using muscles from desmin-deficient mice in which the connections between Z-disks in adjacent myofibrils are presumably compromised. The current simulations suggest that muscle fibers exhibit emergent properties that reflect interactions between half-sarcomeres and are not properties of a single half-sarcomere in isolation. It is therefore likely that full quantitative understanding of a fiber's mechanical properties requires detailed analysis of a complete fiber system and cannot be achieved by focusing solely on the properties of a single half-sarcomere.

The Qγ component of intra‐membrane charge movement is present in mammalian muscle fibres, but suppressed in the absence of S100A1

S100A1 is a Ca(2+) binding protein that modulates excitation-contraction (EC) coupling in skeletal and cardiac muscle. S100A1 competes with calmodulin for binding to the skeletal muscle SR Ca(2+) release channel (the ryanodine receptor type 1, RyR1) at a site that also interacts with the C-terminal tail of the voltage sensor of EC coupling, the dihydropyridine receptor. Ablation of S100A1 leads to delayed and decreased action potential evoked Ca(2+) transients, possibly linked to altered voltage sensor activation. Here we investigate the effects of S100A1 on voltage sensor activation in skeletal muscle utilizing whole-cell patch clamp electrophysiology to record intra-membrane charge movement currents in isolated flexor digitorum brevis (FDB) muscle fibres from wild-type and S100A1 knock-out (KO) mice. In contrast to recent reports, we found that FDB fibres exhibit two distinct components of intra-membrane charge movement, an initial rapid component (Q(beta)), and a delayed, steeply voltage dependent 'hump' component (Q(gamma)) previously recorded primarily in amphibian but not mammalian fibres. Surprisingly, we found that Q(gamma) was selectively suppressed in S100A1 KO fibres, while the Q(beta) component of charge movement was unaffected. This result was specific to S100A1 and not a compensatory result of genetic manipulation, as transient intracellular application of S100A1 restored Q(gamma). Furthermore, we found that exposure to the RyR1 inhibitor dantrolene suppressed a similar component of charge movement in FDB fibres. These results shed light on voltage sensor activation in mammalian muscle, and support S100A1 as a positive regulator of the voltage sensor and Ca(2+) release channel in skeletal muscle EC coupling.

Enrichment and terminal differentiation of striated muscle progenitors in vitro

Enrichment and terminal differentiation of mammalian striated muscle cells is severely hampered by fibroblast overgrowth, de-differentiation and/or lack of functional differentiation. Herein we report a new, reproducible and simple method to enrich and terminally differentiate muscle stem cells and progenitors from mice and humans. We show that a single gamma irradiation of muscle cells induces their massive differentiation into structurally and functionally intact myotubes and cardiomyocytes and that these cells can be kept in culture for many weeks. Similar results are also obtained when treating skeletal muscle-derived stem cells and progenitors with Mitomycin C.

A multisegmental cross-bridge kinetics model of the myofibril

Striated muscle is a mechanical system that develops force and generates power in serving vital activities in the body. Striated muscle is a complex biological system; a single mammalian muscle fibre contains up to hundred or even more myofibrils in parallel connected via an inter-myofibril filament network. In one single myofibril thousands of sarcomeres are lined up as a series of linear motors. We recently demonstrated that half-sarcomeres (hS) in a single myofibril operate non-uniformly. We outline a mathematical framework based on cross-bridge kinetics for the simulation of the force response and length change of individual hS in a myofibril. The model describes the muscle myofibril in contraction experiments under various conditions. The myofibril is modeled as a multisegmental mechanical system of hS models, which have active and viscoelastic properties. In the first approach, a two-state cross-bridge formalism relates the hS force to the chemical kinetics of ATP hydrolysis, as first described by Huxley [1957. Muscle structure and theories of contraction. Prog. Biophys. Mol. Biol. 7, 255–318]. Two possible types of biological variability are introduced and modeled. Numerical simulations of a myofibril composed of four to eight hS show a non-uniform hS length distribution and complex internal dynamics upon activation. We demonstrate that the steady-state approximation holds only in restricted time zones during activation. Simulations of myofibril contraction experiments that reproduce the classic steady-state force–length and force–velocity relationships, strictly constrained or “clamped” in either end-held isometric or isotonic contraction conditions, reveal a small but conspicuous effect of hS dynamics on force.

Promoter cloning and characterization of the rabbit BK channel β1 subunit gene

The β1 subunit of the voltage-dependent and Ca 2+-activated large-conductance K + channel (BK) in mammalian smooth muscle cells (SMCs) plays an important role in regulating smooth muscle tone and is closely linked with a series of smooth muscle tone associated diseases. However, knowledge of the transcriptional regulation of the BK β1 is still largely unclear. For the first time, we cloned and characterized the full-length genomic sequence of the rabbit BK β1 containing a 5′-flanking region of 2021 bp. The full-reading frame of the BK β1 spans ~ 7.7 kb and is organized into 4 exons and 3 introns. All of the exon/intron junction sequences contain the GT/AG consensus junction sequence. The transcription initiation site (+ 1G) is located at 447 bp upstream of the translation initiation codon. Bioinformatics analysis indicated that, without any canonical TATA-box, the 5′-flanking region possesses a high GC content and contains a number of putative transcription factor binding sites. 5′-deletion analysis demonstrated that the region of − 93/+ 30 potentially functions as a core promoter region. A gel mobility shift assay and chromatin immunoprecipitation assay revealed that Sp1 specifically interacts with a putative Sp1-binding site (− 91/− 85) in vitro and in vivo. Mutation of this site significantly diminished the promoter activities. Over-expression of Sp1 in smooth muscle cells of rabbit sphincter of Oddi enhanced the promoter activities of the BK β1 in a dose-dependent manner. Thus, we suggest that the Sp1-binding site (− 91/− 85) is essential to the basal transcription of the rabbit BK β1. Our studies provide a basic knowledge of the transcription regulation of the rabbit BK β1.

Atrophin Proteins Interact with the Fat1 Cadherin and Regulate Migration and Orientation in Vascular Smooth Muscle Cells

Fat1, an atypical cadherin induced robustly after arterial injury, has significant effects on mammalian vascular smooth muscle cell (VSMC) growth and migration. The related Drosophila protein Fat interacts genetically and physically with Atrophin, a protein essential for development and control of cell polarity. We hypothesized that interactions between Fat1 and mammalian Atrophin (Atr) proteins might contribute to Fat1 effects on VSMCs. Like Fat1, mammalian Atr expression increased after arterial injury and in VSMCs stimulated with growth and chemotactic factors including angiotensin II, basic fibroblast growth factor, and platelet-derived growth factor BB. Two distinct Atr2 transcripts, atr2L and atr2S, were identified by Northern analysis; in VSMCs, atr2S mRNA expression was more responsive to stimuli. By immunocytochemistry, Fat1 and Atrs colocalized at cell-cell junctions, in the perinuclear area, and in the nucleus. In coimmunoprecipitation studies, Fat1 interacted with both Atr1 and Atr2; these interactions required Fat1 amino acids 4300-4400 and an intact Atro-box in the Atrs. Knock-down of Atrs by small interfering RNA did not affect VSMC growth but had complex effects on migration, which was impaired by Atr1 knockdown, enhanced by Atr2L knockdown, and unchanged when both Atr2S and Atr2L were depleted. Enhanced migration caused by Atr2L knockdown required Fat1 expression. Similarly, orientation of cells after monolayer denudation was impaired in cells with Atr1 knockdown but enhanced in cells selectively depleted of Atr2L. Together these findings suggest that Fat1 and Atrs act in concert after vascular injury but show further that distinct Atr isoforms have disparate effects on VSMC directional migration.