Quantitative Analysis of Acetyl-CoA, Malonyl-CoA, and Succinyl-CoA in Myocytes.

Several analytical challenges make it difficult to accurately measure coenzyme A (CoA) metaboforms, including insufficient stability and a lack of available metabolite standards. Consequently, our understanding of CoA biology and the modulation of human diseases may be nascent. CoA's serve as lipid precursors, energy intermediates, and mediators of post-translational modifications of proteins. Here, we present a liquid chromatography-mass spectrometry (LC-MS) approach to measure malonyl-CoA, acetyl-CoA, and succinyl-CoA in complex biological samples. Additionally, we evaluated workflows to increase sample stability. We used reference standards to optimize CoA assay sensitivity and test CoA metabolite stability as a function of the reconstitution solvent. We show that using glass instead of plastic sample vials decreases CoA signal loss and improves the sample stability. We identify additives that improve CoA stability and facilitate accurate analysis of CoA species across large sample sets. We apply our optimized workflow to biological samples of skeletal muscle cells cultured under hypoxic and normoxia conditions. Together, our workflow improves the detection and identification of CoA species through targeted analysis in complex biological samples.

Effects of omecamtiv mecarbil on calcium-transients and contractility in a translational canine myocyte model.

Omecamtiv mecarbil (OM) is a selective cardiac myosin activator (myotrope), currently in Phase 3 clinical investigation as a novel treatment for heart failure with reduced ejection fraction. OM increases cardiac contractility by enhancing interaction between myosin and actin in a calcium-independent fashion. This study aims to characterize the mechanism of action by evaluating its simultaneous effect on myocyte contractility and calcium-transients (CTs) in healthy canine ventricular myocytes. Left ventricular myocytes were isolated from canines and loaded with Fura-2 AM. With an IonOptix system, contractility parameters including amplitude and duration of sarcomere shortening, contraction and relaxation velocity, and resting sarcomere length were measured. CT parameters including amplitude at systole and diastole, velocity at systole and diastole, and duration at 50% from peak were simultaneously measured. OM was tested at 0.03, 0.1, 0.3, 1, and 3 µmol\L concentrations to simulate therapeutic human plasma exposure levels. OM and isoproterenol (ISO) demonstrated differential effects on CTs and myocyte contractility. OM increased contractility mainly by prolonging duration of contraction while ISO increased contractility mainly by augmenting the amplitude of contraction. ISO increased the amplitude and velocity of CT, shortened duration of CT concurrent with increasing myocyte contraction, while OM did not change the amplitude, velocity, and duration of CT up to 1 µmol\L. Decreases in relaxation velocity and increases in duration were present only at 3 µmol\L. In this translational myocyte model study, therapeutically relevant concentrations of OM increased contractility but did not alter intracellular CTs, a mechanism of action distinct from traditional calcitropes.

Mitochondrial transplantation ameliorates ischemia/reperfusion-induced kidney injury in rat.

No real therapeutic modality is currently available for Acute kidney injury (AKI) and if any, they are mainly supportive in nature. Therefore, developing a new therapeutic strategy is crucial. Mitochondrial dysfunction proved to be a key contributor to renal tubular cell death during AKI. Thus, replacement or augmentation of damaged mitochondria could be a proper target in AKI treatment. Here, in an animal model of AKI, we auto-transplanted normal mitochondria isolated from healthy muscle cells to injured kidney cells through injection to renal artery. The mitochondria transplantation prevented renal tubular cell death, restored renal function, ameliorated kidney damage, improved regenerative potential of renal tubules, and decreased ischemia/reperfusion-induced apoptosis. Although further studies including clinical trials are required in this regard, our findings suggest a novel therapeutic strategy for treatment of AKI. Improved quality of life of patients suffering from renal failure and decreased morbidity and mortality rates would be the potential advantages of this therapeutic strategy.

Simultaneous speculation of 401 monomeric or homo-oligomeric subunit structures of human cellular proteins, mining the information in 1901 native 2D protein maps reconstructed from one nondenaturing 2DE gel.

Subunit structures of proteins are essential for their properties and functions, but there is a lack of method for global detection of the status of proteins being monomers or homo-oligomers. In this work, we report on a new method to simultaneously speculate hundreds of monomeric or homo-oligomeric subunit structures of cellular proteins, based on in-depth analysis of native 2D protein maps. Previously we have reported on the analysis of soluble proteins of human bronchial muscle cells (HBSMC) by combining nondenaturing 2DE, grid gel-cutting and quantitative LC-MS/MS. Totally 4323 proteins were detected and for each protein the quantity distribution on the gel was reconstructed as a native 2D map. In this work, this large dataset of maps were further mined with bioinformatic analysis. The native 2D maps of 1901 HBSMC proteins that were detected in at least five out of the grid-cut 972 gel squares were examined and 658 proteins that showed one major quantity-peak distribution were subjected to further analysis. After excluding those that mainly formed hetero-oligomeric structures, the monomeric or homo-oligomeric subunit structures of 505 proteins were speculated. The quotient of the apparent molecular mass of the quantity-peak position on the native 2D map divided by the theoretical molecular mass was calculated for each protein, to speculate the number of monomers which constituted its subunit structure. The suggested composition was then compared with the "Subunit structure" record of the protein in UniProtKB. When the database record included possible interactions with other proteins, their native 2D maps were extracted from the native map dataset, presented together and compared to confirm the prominent subunit structure. With this new approach, the monomeric or homo-oligomeric subunit structures of 401 proteins were speculated. Among them, 162 proteins had the speculated subunit structures coinciding with their database records, and 91 proteins with matched database records as being monomers or homo-oligomers but mismatched at the numbers of the composing monomers. For 148 proteins that did not have database record, their subunit structures were newly speculated. We expect this method, combining nondenaturing 2DE separation with in-depth proteomic and bioinformatic analysis, would suggest a means to achieve large-scale information on monomeric and homo-oligomeric subunit structures of cellular proteins.

Phosphoproteome analysis of sarcoplasmic and myofibrillar proteins in stress-induced dysfunctional broiler pectoralis major muscle.

Postmortem changes of sarcoplasmic and myofibrillar protein phosphorylation in pectoralis major (PM) muscle of broilers under pre-slaughter stress were investigated. Broiler chickens were randomly distributed to unstressed control and transport under high environmental temperature groups. PM muscle samples of transport-stressed broilers were classified into normal or pale, soft and exudative (PSE)-like. Sarcoplasmic fraction in PSE-like meat had a higher global phosphorylation level than that in normal meat at the early postmortem stage. The myofibrillar proteins showed diverse phosphorylation patterns at different postmortem times. The stress-induced highly phosphorylated sarcoplasmic proteins were glycometabolic enzymes, which partially contributed to accelerated glycolysis rate. The phosphorylation levels of most sarcomeric proteins identified in the myofibrillar fraction were affected by postmortem time, implying their roles in regulating muscle rigor mortis development. This work contributes to a deeper understanding of the biochemical processes that may lead to stress-induced changes in meat quality.

Autotaxin and vascular endothelial growth factor receptor-2 and -3 are related to vascular development during the progression of chronic viral hepatitis C.

Vascular endothelial growth factor (VEGF) and autotaxin (ATX) play important roles in embryonic vasculogenesis and cancer progression. This study examines whether these two angiogenic factors cooperate in the mechanism that regulates vascular development during the progression of chronic viral hepatitis C (CVH-C) (Inuyama classification, F1-F4). First, surgical wedge biopsy specimens and needle biopsy specimens were obtained. Immunohistochemical staining for ATX and vascular endothelial growth factor receptor was assessed in serial sections. Immunoelectron microscopy was conducted with a perfusion-fixation method. In normal control liver tissue specimens, ATX was expressed at low levels within the branches of the hepatic artery and hepatic sinusoids. In F1 CVH-C liver tissue specimens, ATX was expressed within the branches of the hepatic artery. Additionally, VEGFR-2 was expressed within the branches of the hepatic artery and capillaries. In F3-F4 CVH-C liver tissue specimens, positive staining for ATX and VEGFR-2 or VEGFR-3 was detected in the branches of the hepatic artery or microlymphatic vessels. ATX-1 reaction products were specifically expressed on the plasma membrane of some microvascular endothelial cells (ECs) in the proliferative capillary artery. VEGFR-2 was expressed on caveolae in ECs and vascular smooth muscle cells. VEGFR-3 immunogold particles were also observed in lymphatic ECs. These results suggest functional interactions among ATX, VEGFR-2, and VEGFR-3 in the modulation of hemovascular and lymphovascular cell activation during vascular development.

Mining the Secretome of C2C12 Muscle Cells: Data Dependent Experimental Approach To Analyze Protein Secretion Using Label-Free Quantification and Peptide Based Analysis.

Secretome analysis faces several challenges including detection of low abundant proteins and the discrimination of bona fide secreted proteins from false-positive identifications stemming from cell leakage or serum. Here, we developed a two-step secretomics approach and applied it to the analysis of secreted proteins of C2C12 skeletal muscle cells since the skeletal muscle has been identified as an important endocrine organ secreting myokines as signaling molecules. First, we compared culture supernatants with corresponding cell lysates by mass spectrometry-based proteomics and label-free quantification. We identified 672 protein groups as candidate secreted proteins due to their higher abundance in the secretome. On the basis of Brefeldin A mediated blocking of classical secretory processes, we estimated a sensitivity of >80% for the detection of classical secreted proteins for our experimental approach. In the second step, the peptide level information was integrated with UniProt based protein information employing the newly developed bioinformatics tool "Lysate and Secretome Peptide Feature Plotter" (LSPFP) to detect proteolytic protein processing events that might occur during secretion. Concerning the proof of concept, we identified truncations of the cytoplasmic part of the protein Plexin-B2. Our workflow provides an efficient combination of experimental workflow and data analysis to identify putative secreted and proteolytic processed proteins.

Low-current & high-frequency electrical stunning increased oxidative stress, lipid peroxidation, and gene transcription of the mitogen-activated protein kinase/nuclear factor-erythroid 2-related factor 2/antioxidant responsive element (MAPK/Nrf2/ARE) signaling pathway in breast muscle of broilers.

Mechanism of electrical stunning (ES) methods on lipid peroxidation and antioxidant protection were studied by determining meat color, serum variables, antioxidant-related enzyme activities, gene expressions of mitogen-activated protein kinases (MAPKs), nuclear factor-erythroid 2-related factor 2 (Nrf2), glutathione S-transferases (GSTs), and superoxide dismutases (SODs). Broilers were sacrificed without stunning, or after ES with 65V, 86mA, 1000Hz (E65V) or 150V, 130mA, 60Hz (E150V). Serum cortisol and uric acid, muscular malondialdehyde and mRNA levels of MAPKs, Nrf2, GSTA3, GSTT1 and SOD2 were increased, whereas, serum free triiodothyronine, free thyroxine, muscular GST1d activity were decreased in E65V compared with E150V. Overall, the serum uric acid and transcription of the MAPK/Nrf2/ARE (antioxidant responsive element) signaling pathway were elevated, but didn't overcome the oxidative stress stimulated by low-current & high-frequency ES, leading to aggravated lipid peroxidation at 1d and 9d postmortem in breast muscle compared with high-current & low-frequency ES.

Premature contractions of the bladder are suppressed by interactions between TRPV4 and SK3 channels in murine detrusor PDGFRα+ cells.

During filling, urinary bladder volume increases dramatically with little change in pressure. This is accomplished by suppressing contractions of the detrusor muscle that lines the bladder wall. Mechanisms responsible for regulating detrusor contraction during filling are poorly understood. Here we describe a novel pathway to stabilize detrusor excitability involving platelet-derived growth factor receptor-α positive (PDGFRα+) interstitial cells. PDGFRα+ cells express small conductance Ca2+-activated K+ (SK) and TRPV4 channels. We found that Ca2+ entry through mechanosensitive TRPV4 channels during bladder filling stabilizes detrusor excitability. GSK1016790A (GSK), a TRPV4 channel agonist, activated a non-selective cation conductance that coupled to activation of SK channels. GSK induced hyperpolarization of PDGFRα+ cells and decreased detrusor contractions. Contractions were also inhibited by activation of SK channels. Blockers of TRPV4 or SK channels inhibited currents activated by GSK and increased detrusor contractions. TRPV4 and SK channel blockers also increased contractions of intact bladders during filling. Similar enhancement of contractions occurred in bladders of Trpv4 -/- mice during filling. An SK channel activator (SKA-31) decreased contractions during filling, and rescued the overactivity of Trpv4 -/- bladders. Our findings demonstrate how Ca2+ influx through TRPV4 channels can activate SK channels in PDGFRα+ cells and prevent bladder overactivity during filling.

SarConfoCal: simultaneous sarcomere length and cytoplasmic calcium measurements for laser scanning confocal microscopy images.

Summary: Simultaneous recordings of myocytes contractility and their cytoplasmic calcium concentration allow powerful studies, particularly on heart failure and other cardiac dysfunctions. Such studies require dedicated and expensive experimental devices that are difficult to use. Thus we propose SarConfoCal, the first and only software to simultaneously analyse both cytoplasmic calcium variations (from fluorescence signal) and myocytes contractility (from sarcomere length measurement) on laser scanning confocal microscopy images. SarConfoCal is easy to set up and use, especially by people without programming skills. Availability and implementation: The software is freely distributed under the GNU General Public License. Download and setup instructions are available at http://pccv.univ-tours.fr/ImageJ/SarConfoCal . It is provided as a toolset for ImageJ (the open-source program for image analysis provided by the National Institutes of Health). SarConfoCal has been tested under Windows, Mac and Linux operating systems. Contact: come.pasqualin@univ-tours.fr. Supplementary information: Supplementary data are available at Bioinformatics online.

Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer.

The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homooligomers that increase the catalytic rate of calcium transport. We investigated SERCA dimerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher-molecular-weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer measurements in cells transiently transfected with fluorescently labeled SERCA2a revealed that SERCA readily forms homodimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation or changes in calcium or ATP. Fluorescence lifetime data are compatible with a model in which PLB interacts with a SERCA homodimer in a stoichiometry of 1:2. Together, these results suggest that SERCA forms constitutive homodimers in live cells and that dimer formation is not modulated by SERCA conformational poise, PLB binding, or PLB phosphorylation.

Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues.

Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol to the plasma membrane is a critical step in the transfer of information from membrane-embedded receptors or transporters to the cell interior. To understand how membrane proteins work, it is important to separate the membrane fraction of cells. This unit provides a protocol for rapidly obtaining plasma membrane fractions for western blot analysis. © 2016 by John Wiley & Sons, Inc.

Innervation of dystrophic muscle after muscle stem cell therapy.

INTRODUCTION: Duchenne muscular dystrophy (DMD) is caused by loss of the structural protein, dystrophin, resulting in muscle fragility. Muscle stem cell (MuSC) transplantation is a potential therapy for DMD. It is unknown whether donor-derived muscle fibers are structurally innervated. METHODS: Green fluorescent protein (GFP)-expressing MuSCs were transplanted into the tibials anterior of adult dystrophic mdx/mTR mice. Three weeks later the neuromuscular junction was labeled by immunohistochemistry. RESULTS: The percent overlap between pre- and postsynaptic immunolabeling was greater in donor-derived GFP(+) myofibers, and fewer GFP(+) myofibers were identified as denervated compared with control GFP(-) fibers (P = 0.001 and 0.03). GFP(+) fibers also demonstrated acetylcholine receptor fragmentation and expanded endplate area, indicators of muscle reinnervation (P = 0.008 and 0.033). CONCLUSION: It is unclear whether GFP(+) fibers are a result of de novo synthesis or fusion with damaged endogenous fibers. Either way, donor-derived fibers demonstrate clear histological innervation. Muscle Nerve 54: 763-768, 2016.

Red alert: labile heme is an alarmin.

Alarmins are a heterogeneous group of endogenous molecules that signal cellular damage when sensed extracellularly. Heme is an endogenous molecule that acts as a prosthetic group of hemoproteins, such as hemoglobin and myoglobin. When released from damaged red blood cells or muscle cells, oxidized hemoglobin and myoglobin release their prosthetic heme groups, respectively. This generates labile heme, which is sensed by pattern recognition receptors (PRR) expressed by innate immune cells and possibly regulatory T cells (TREG). The ensuing adaptive response, which alerts for the occurrence of red blood cell or muscle cell damage, regulates the pathologic outcome of hemolysis or rhabdomyolysis, respectively. In conclusion, we propose that labile heme is an alarmin.

Cannabinoids regulate intestinal motor function and electrophysiological activity of myocytes in rodents.

BACKGROUND AND AIMS: This study aims to investigate the effects of cannabinoid (CB)1 and CB2 receptor ligands on intestinal motor function and muscular electrophysiological activity in rodent gastrointestinal (GI) tract. METHODS: Lipopolysaccharide (LPS) was used to induce intestinal hypomotility. The effect of selective CB1 and CB2 agonists and antagonists on contractility of the muscle strips from rat jejunum was measured using organ bath, and the membrane potential of the jejunal smooth muscle cells was recorded with intracellular microelectrodes. The single cell patch clamp technique was applied to record delayed rectifying potassium currents (IKV) and spontaneous transient outward currents (STOC). RESULTS: LPS significantly reduced contractility of the smooth muscle strips (p <0.010) and caused hyperpolarization of membrane potential of the smooth muscle cells (p <0.010). This LPS-induced effect was reversed by AM251 and AM630, selective CB1 and CB2 antagonists, respectively, which promoted contractions of smooth muscle strips and triggered cell depolarization (p <0.010). LPS-induced changes were further enhanced in the presence of CB agonists, HU210 and WIN55 (p <0.050 or p <0.010). No effect of HU210 or AM251 on IKV and STOC has been observed. This ex vivo study suggests that CB1 and CB2 receptors are involved in intestinal motor function in normal and LPS-induced pathological states and the regulation of the membrane potential of smooth muscle cells is very likely one of the effective mechanisms. CONCLUSIONS: This is one of the first reports on neuronal regulation of intestinal motility through CB-dependent pathways with potential application in the treatment of inflammatory and functional GI disorders.

[Biophysical Model of Contractile Activity of Muscle Cells].

Muscle cells have the specific structure, the advanced cytoskeleton, which takes most of cell volume and forms a contractile apparatus. On the basis of the equations of continuum mechanics we proposed a mathematical model of the biomechanical behavior of cells as a whole, and it was modified to describe the contractile activity in muscle cells as an elastic rod. The model takes into account the result of transduction of external influences, namely the development of internal deformation, and allows for estimation of mobility and/or tension developed in muscle cells in the case of change in external influence.

A pilot validation of multi-echo based echo-planar correlated spectroscopic imaging in human calf muscles.

A current limitation of MR spectroscopic imaging of multiple skeletal muscles is prolonged scan duration. A significant reduction in the total scan duration using the echo-planar correlated spectroscopic imaging (EP-COSI) sequence was accomplished using two bipolar readout trains with different phase-encoded echoes for one of two spatial dimensions within a single repetition time (TR). The second bipolar readout was used for spatially encoding the outer k-space, whereas the first readout was used for the central k-space only. The performance of this novel sequence, called multi-echo based echo-planar correlated spectroscopic imaging (ME-EPCOSI), was demonstrated by localizing specific key features in calf muscles and bone marrow of 11 healthy volunteers and five subjects with type 2 diabetes (T2D). A 3 T MRI-MRS scanner equipped with a transmit-receive extremity coil was used. Localization of the ME-EPCOSI sequence was in good agreement with the earlier single-readout based EP-COSI sequence and the required scan time was reduced by a factor of two. In agreement with an earlier report using single-voxel based 2D MRS, significantly increased unsaturated pools of intramyocellular lipid (IMCL) and extramyocellular lipid (EMCL) and decreased IMCL and EMCL unsaturation indices (UIs) were observed in the soleus and tibialis anterior muscle regions of subjects with T2D compared with healthy controls. In addition, significantly decreased choline content was observed in the soleus of T2D subjects compared with healthy controls. Multi-voxel characterization of IMCL and EMCL ratios and UI in the calf muscle may be useful for the non-invasive assessment of altered lipid metabolism in the pathophysiology of T2D.

Changes in meat quality traits and sarcoplasmic proteins during aging in three different cattle breeds.

The effects of breed and aging time (1, 7, 14, 21d) were evaluated on physical meat properties and on sarcoplasmic protein changes in 24 young bulls from Romagnola×Podolian, Podolian and Friesian breeds. Aging affects lightness showing an increase in all breeds while changes in redness varied according to the breed. Podolian breed showed meat with the darkest and the reddest color and the lowest drip loss compared to the other breeds. Extending aging to 21d reduced drip loss from meat. SDS-PAGE and 2DE showed that many changes in the sarcoplasmic proteins occurred among breeds and during aging. During post-mortem some sarcoplasmic proteins decline in intensity after 21d highlighting that they were susceptible to aging. Protein identification and western blotting showed the presence of myosin light chains, Troponin T and tropomyosin proteins during aging, suggesting a degradation of myofibers and a more intense proteolysis especially in the Podolian breed.

Adenosine A2B and A3 receptor location at the mouse neuromuscular junction.

To date, four subtypes of adenosine receptors have been cloned (A(1)R, A(2A)R, A(2B)R, and A(3)R). In a previous study we used confocal immunocytochemistry to identify A(1)R and A(2A)R receptors at mouse neuromuscular junctions (NMJs). The data shows that these receptors are localized differently in the three cells (muscle, nerve and glia) that configure the NMJs. A(1)R localizes in the terminal teloglial Schwann cell and nerve terminal, whereas A(2A)R localizes in the postsynaptic muscle and in the axon and nerve terminal. Here, we use Western blotting to investigate the presence of A(2B)R and A(3)R receptors in striated muscle and immunohistochemistry to localize them in the three cells of the adult neuromuscular synapse. The data show that A(2B)R and A(3)R receptors are present in the nerve terminal and muscle cells at the NMJs. Neither A(2B)R nor A(3)R receptors are localized in the Schwann cells. Thus, the four subtypes of adenosine receptors are present in the motor endings. The presence of these receptors in the neuromuscular synapse allows the receptors to be involved in the modulation of transmitter release.