Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability.

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, including voltage-gated ion channels. This approach has been used primarily in heterologous expression systems to simultaneously measure membrane currents, the electrical manifestation of the channels' activity, and fluorescence measurements, reporting local domain rearrangements. Functional site-directed fluorometry combines electrophysiology, molecular biology, chemistry, and fluorescence into a single wide-ranging technique that permits the study of real-time structural rearrangements and function through fluorescence and electrophysiology, respectively. Typically, this approach requires an engineered voltage-gated membrane channel that contains a cysteine that can be tested by a thiol-reactive fluorescent dye. Until recently, the thiol-reactive chemistry used for the site-directed fluorescent labeling of proteins was carried out exclusively in Xenopus oocytes and cell lines, restricting the scope of the approach to primary non-excitable cells. This report describes the applicability of functional site-directed fluorometry in adult skeletal muscle cells to study the early steps of excitation-contraction coupling, the process by which muscle fiber electrical depolarization is linked to the activation of muscle contraction. The present protocol describes the methodologies to design and transfect cysteine-engineered voltage-gated Ca2+ channels (CaV1.1) into muscle fibers of the flexor digitorum brevis of adult mice using in vivo electroporation and the subsequent steps required for functional site-directed fluorometry measurements. This approach can be adapted to study other ion channels and proteins. The use of functional site-directed fluorometry of mammalian muscle is particularly relevant to studying basic mechanisms of excitability.

Voltage sensor current, SR Ca2+ release, and Ca2+ channel current during trains of action potential-like depolarizations of skeletal muscle fibers.

In skeletal muscle, CaV 1.1 serves as the voltage sensor for both excitation-contraction coupling (ECC) and L-type Ca2+ channel activation. We have recently adapted the technique of action potential (AP) voltage clamp (APVC) to monitor the current generated by the movement of intramembrane voltage sensors (IQ ) during single imposed transverse tubular AP-like depolarization waveforms (IQAP ). We now extend this procedure to monitoring IQAP , and Ca2+ currents during trains of tubular AP-like waveforms in adult murine skeletal muscle fibers, and compare them with the trajectories of APs and AP-induced Ca2+ release measured in other fibers using field stimulation and optical probes. The AP waveform remains relatively constant during brief trains (<1 sec) for propagating APs in non-V clamped fibers. Trains of 10 AP-like depolarizations at 10 Hz (900 ms), 50 Hz (180 ms), or 100 Hz (90 ms) did not alter IQAP amplitude or kinetics, consistent with previous findings in isolated muscle fibers where negligible charge immobilization occurred during 100 ms step depolarizations. Using field stimulation, Ca2+ release did exhibit a considerable decline from pulse to pulse during the train, also consistent with previous findings, indicating that the decline of Ca2+ release during a short train of APs is not correlated to modification of charge movement. Ca2+ currents during single or 10 Hz trains of AP-like depolarizations were hardly detectable, were minimal during 50 Hz trains, and became more evident during 100 Hz trains in some fibers. Our results verify predictions on the behavior of the ECC machinery in response to AP-like depolarizations and provide a direct demonstration that Ca2+ currents elicited by single AP-like waveforms are negligible, but can become more prominent in some fibers during short high-frequency train stimulation that elicits maximal isometric force.

Contribution of skeletal muscle-specific microRNA-133b to insulin resistance in heart failure.

Insulin resistance (IR) is one of the hallmarks of heart failure (HF). Abnormalities in skeletal muscle (SM) metabolism have been identified in patients with HF. However, the underlying mechanisms of IR development in SM in HF are poorly understood. Herein, we hypothesize that HF upregulates miR-133b in SM and in turn alters glucose metabolism and the propensity toward IR. Mitochondria isolated from SM of mice with HF induced by transverse aortic constriction (TAC) showed lower respiration and downregulation of muscle-specific components of the tricarboxylic acid (TCA) cycle, AMP deaminase 1 (AMPD1), and fumarate compared with those from control animals. RNA-Seq and subsequent qPCR validation confirmed upregulation of SM-specific microRNA (miRNA), miR-133b, in TAC versus sham animals. miR-133b overexpression alone resulted in significantly lower mitochondrial respiration, cellular glucose uptake, and glycolysis along with lower ATP production and cellular energy reserve compared with the scramble (Scr) in C2C12 cells. miR-133b binds to the 3'-untranslated region (UTR) of KLF15, the transcription factor for the insulin-sensitive glucose transporter, GLUT4. Overexpression of miR-133b lowers GLUT4 and lowers pAkt in presence of insulin in C2C12 cells. Finally, lowering miR-133b in primary skeletal myocytes isolated from TAC mice using antagomir-133b reversed the changes in KLF15, GLUT4, and AMPD1 compared with the scramble-transfected myocytes. Taken together, these data demonstrate a role for SM miR-133b in altered glucose metabolism in HF and suggest the therapeutic potential in HF to improve glucose uptake and glycolysis by restoring GLUT4 abundance. The data uncover a novel mechanism for IR and ultimately SM metabolic abnormalities in patients with HF.NEW & NOTEWORTHY Heart failure is associated with systemic insulin resistance and abnormalities in glucose metabolism but the underlying mechanisms are poorly understood. In the skeletal muscle, the major peripheral site of glucose utilization, we observe an increase in miR-133b in heart failure mice, which reduces the insulin-sensitive glucose transporter (GLUT4), glucose uptake, and metabolism in C2C12 and in myocytes. The antagomir for miR-133b restores GLUT4 protein and markers of metabolism in skeletal myocytes from heart failure mice demonstrating that miR-133b is an exciting target for systemic insulin resistance in heart failure and an important player in the cross talk between the heart and the periphery in the heart failure syndrome.

Selective posttranslational inhibition of CaVß1-associated voltage-dependent calcium channels with a functionalized nanobody.

Ca2+ influx through high-voltage-activated calcium channels (HVACCs) controls diverse cellular functions. A critical feature enabling a singular signal, Ca2+ influx, to mediate disparate functions is diversity of HVACC pore-forming α1 and auxiliary CaVß1-CaVß4 subunits. Selective CaVα1 blockers have enabled deciphering their unique physiological roles. By contrast, the capacity to post-translationally inhibit HVACCs based on CaVß isoform is non-existent. Conventional gene knockout/shRNA approaches do not adequately address this deficit owing to subunit reshuffling and partially overlapping functions of CaVß isoforms. Here, we identify a nanobody (nb.E8) that selectively binds CaVß1 SH3 domain and inhibits CaVß1-associated HVACCs by reducing channel surface density, decreasing open probability, and speeding inactivation. Functionalizing nb.E8 with Nedd4L HECT domain yielded Chisel-1 which eliminated current through CaVß1-reconstituted CaV1/CaV2 and native CaV1.1 channels in skeletal muscle, strongly suppressed depolarization-evoked Ca2+ influx and excitation-transcription coupling in hippocampal neurons, but was inert against CaVß2-associated CaV1.2 in cardiomyocytes. The results introduce an original method for probing distinctive functions of ion channel auxiliary subunit isoforms, reveal additional dimensions of CaVß1 signaling in neurons, and describe a genetically-encoded HVACC inhibitor with unique properties.

Voltage sensor movements of CaV1.1 during an action potential in skeletal muscle fibers.

The skeletal muscle L-type Ca2+ channel (CaV1.1) works primarily as a voltage sensor for skeletal muscle action potential (AP)-evoked Ca2+ release. CaV1.1 contains four distinct voltage-sensing domains (VSDs), yet the contribution of each VSD to AP-evoked Ca2+ release remains unknown. To investigate the role of VSDs in excitation-contraction coupling (ECC), we encoded cysteine substitutions on each S4 voltage-sensing segment of CaV1.1, expressed each construct via in vivo gene transfer electroporation, and used in cellulo AP fluorometry to track the movement of each CaV1.1 VSD in skeletal muscle fibers. We first provide electrical measurements of CaV1.1 voltage sensor charge movement in response to an AP waveform. Then we characterize the fluorescently labeled channels' VSD fluorescence signal responses to an AP and compare them with the waveforms of the electrically measured charge movement, the optically measured free myoplasmic Ca2+, and the calculated rate of Ca2+ release from the sarcoplasmic reticulum for an AP, the physiological signal for skeletal muscle fiber activation. A considerable fraction of the fluorescence signal for each VSD occurred after the time of peak Ca2+ release, and even more occurred after the earlier peak of electrically measured charge movement during an AP, and thus could not directly reflect activation of Ca2+ release or charge movement, respectively. However, a sizable fraction of the fluorometric signals for VSDs I, II, and IV, but not VSDIII, overlap the rising phase of charge moved, and even more for Ca2+ release, and thus could be involved in voltage sensor rearrangements or Ca2+ release activation.

CaMKII oxidation is a critical performance/disease trade-off acquired at the dawn of vertebrate evolution.

Antagonistic pleiotropy is a foundational theory that predicts aging-related diseases are the result of evolved genetic traits conferring advantages early in life. Here we examine CaMKII, a pluripotent signaling molecule that contributes to common aging-related diseases, and find that its activation by reactive oxygen species (ROS) was acquired more than half-a-billion years ago along the vertebrate stem lineage. Functional experiments using genetically engineered mice and flies reveal ancestral vertebrates were poised to benefit from the union of ROS and CaMKII, which conferred physiological advantage by allowing ROS to increase intracellular Ca2+ and activate transcriptional programs important for exercise and immunity. Enhanced sensitivity to the adverse effects of ROS in diseases and aging is thus a trade-off for positive traits that facilitated the early and continued evolutionary success of vertebrates.

Mechanoactivation of NOX2-generated ROS elicits persistent TRPM8 Ca2+ signals that are inhibited by oncogenic KRas.

Changes in the mechanical microenvironment and mechanical signals are observed during tumor progression, malignant transformation, and metastasis. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. Here, we report that normal breast epithelial cells are mechanically sensitive, responding to transient mechanical stimuli through a two-part calcium signaling mechanism. We observed an immediate, robust rise in intracellular calcium (within seconds) followed by a persistent extracellular calcium influx (up to 30 min). This persistent calcium was sustained via microtubule-dependent mechanoactivation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on transient receptor potential cation channel subfamily M member 8 (TRPM8) channels to prolong calcium signaling. In contrast, the introduction of a constitutively active oncogenic KRas mutation inhibited the magnitude of initial calcium signaling and severely blunted persistent calcium influx. The identification that oncogenic KRas suppresses mechanically-induced calcium at the level of ROS provides a mechanism for how KRas could alter cell responses to tumor microenvironment mechanics and may reveal chemotherapeutic targets for cancer. Moreover, we find that expression changes in both NOX2 and TRPM8 mRNA predict poor clinical outcome in estrogen receptor (ER)-negative breast cancer patients, a population with limited available treatment options. The clinical and mechanistic data demonstrating disruption of this mechanically-activated calcium pathway in breast cancer patients and by KRas activation reveal signaling alterations that could influence cancer cell responses to the tumor mechanical microenvironment and impact patient survival.

Alternative signaling pathways from IGF1 or insulin to AKT activation and FOXO1 nuclear efflux in adult skeletal muscle fibers.

Muscle atrophy is regulated by the balance between protein degradation and synthesis. FOXO1, a transcription factor, helps to determine this balance by activating pro-atrophic gene transcription when present in muscle fiber nuclei. Foxo1 nuclear efflux is promoted by AKT-mediated Foxo1 phosphorylation, eliminating FOXO1's atrophy-promoting effect. AKT activation can be promoted by insulin-like growth factor 1 (IGF1) or insulin via a pathway including IGF1 or insulin, phosphatidylinositol 3-kinase, and AKT. We used confocal fluorescence time-lapse imaging of FOXO1-GFP in adult isolated living muscle fibers maintained in culture to explore the effects of IGF1 and insulin on FOXO1-GFP nuclear efflux with and without pharmacological inhibitors. We observed that although AKT inhibitor blocks the IGF1- or insulin-induced effect on FOXO1 nuclear efflux, phosphatidylinositol 3-kinase inhibitors, which we show to be effective in these fibers, do not. We also found that inhibition of the protein kinase ACK1 or ATM contributes to the suppression of FOXO1 nuclear efflux after IGF1. These results indicate a novel pathway that has been unexplored in the IGF1- or insulin-induced regulation of FOXO1 and present information useful both for therapeutic interventions for muscle atrophy and for further investigative areas into insulin insensitivity and type 2 diabetes.

High Time Resolution Analysis of Voltage-Dependent and Voltage-Independent Calcium Sparks in Frog Skeletal Muscle Fibers.

In amphibian skeletal muscle calcium (Ca2+) sparks occur both as voltage-dependent and voltage-independent ligand-activated release events. However, whether their properties and their origin show similarities are still in debate. Elevated K+, constant Cl- content solutions were used to initiate small depolarizations of the resting membrane potential to activate dihydropyridine receptors (DHPR) and caffeine to open ryanodine receptors (RyR) on intact fibers. The properties of Ca2+ sparks observed under control conditions were compared to those measured on depolarized cells and those after caffeine treatment. Calcium sparks were recorded on intact frog skeletal muscle fibers using high time resolution confocal microscopy (x-y scan: 30 Hz). Sparks were elicited by 1 mmol/l caffeine or subthreshold depolarization to different membrane potentials. Both treatments increased the frequency of sparks and altered their morphology. Images were analyzed by custom-made computer programs. Both the amplitude (in ΔF/F0; 0.259 ± 0.001 vs. 0.164 ± 0.001; n = 24942 and 43326, respectively; mean ± SE, p < 0.001) and the full width at half maximum (FWHM, in µm; parallel with fiber axis: 2.34 ± 0.01 vs. 1.92 ± 0.01, p < 0.001; perpendicular to fiber axis: 2.08 ± 0.01 vs. 1.68 ± 0.01, p < 0.001) of sparks was significantly greater after caffeine treatment than on depolarized cells. 9.8% of the sparks detected on depolarized fibers and about one third of the caffeine activated sparks (29.7%) overlapped with another one on the previous frame on x-y scans. Centre of overlapping sparks travelled significantly longer distances between consecutive frames after caffeine treatment then after depolarization (in µm; 1.66 ± 0.01 vs. 0.95 ± 0.01, p < 0.001). Our results suggest that the two types of ryanodine receptors, the junctional RyRs controlled by DHPRs and the parajunctional RyRs are activated independently, using alternate ways, with the possibility of cooperation between neighboring release channels.

Disturbed intracellular calcium homeostasis in neural tube defects in diabetic embryopathy.

Pregnancies complicated by preexisting maternal diabetes mellitus are associated with a higher risk of birth defects in infants, known as diabetic embryopathy. The common defects seen in the central nervous system result from failure of neural tube closure. The formation of neural tube defects (NTDs) is associated with excessive programmed cell death (apoptosis) in the neuroepithelium under hyperglycemia-induced intracellular stress conditions. The early cellular response to hyperglycemia remains to be identified. We hypothesize that hyperglycemia may disturb intracellular calcium (Ca2+) homeostasis, which perturbs organelle function and apoptotic regulation, resulting in increased apoptosis and embryonic NTDs. In an animal model of diabetic embryopathy, we performed Ca2+ imaging and observed significant increases in intracellular Ca2+ ([Ca2+]i) in the embryonic neural epithelium. Blocking T-type Ca2+ channels with mibefradil, but not L-type with verapamil, significantly blunted the increases in [Ca2+]i, implicating an involvement of channel type-dependent Ca2+ influx in hyperglycemia-perturbed Ca2+ homeostasis. Treatment of diabetic pregnant mice with mibefradil during neurulation significantly reduced NTD rates in the embryos. This effect was associated with decreases in apoptosis, alleviation of endoplasmic reticulum stress, and increases of anti-apoptotic factors. Taken together, our data suggest an important role of Ca2+ influx in hyperglycemia-induced NTDs and of T-type Ca2+ channels as a potential target to prevent birth defects in diabetic pregnancies.

Mathematical Modeling of Nuclear Trafficking of FOXO Transcription Factors.

Nuclear cytoplasmic flux of Foxo transcription factors is paramount in cellular gene regulation. For example, excluding Foxo from skeletal muscle nuclei is necessary to avoid muscle wasting through elevated protein breakdown. Constructing a mathematical model of the signaling process leading to alteration of Foxo nuclear cytoplasm ratio is useful in predicting and interpreting such ratio changes. In this chapter we derive a general mathematical model for nuclear cytoplasmic flux. We apply this model to Foxo flux and take advantage of rapid phosphorylation approximation and conservation conditions to reduce the Foxo flux model. We constrain our model with data from mouse skeletal muscle with applied IGF. This procedure provides an example of what might be called the central approach of mathematical modeling: The cycling of a biological question through mathematical formulation and back to biological interpretation.

LRP1 (Low-Density Lipoprotein Receptor-Related Protein 1) Regulates Smooth Muscle Contractility by Modulating Ca2+ Signaling and Expression of Cytoskeleton-Related Proteins.

Objective- Mutations affecting contractile-related proteins in the ECM (extracellular matrix), microfibrils, or vascular smooth muscle cells can predispose the aorta to aneurysms. We reported previously that the LRP1 (low-density lipoprotein receptor-related protein 1) maintains vessel wall integrity, and smLRP1-/- mice exhibited aortic dilatation. The current study focused on defining the mechanisms by which LRP1 regulates vessel wall function and integrity. Approach and Results- Isometric contraction assays demonstrated that vasoreactivity of LRP1-deficient aortic rings was significantly attenuated when stimulated with vasoconstrictors, including phenylephrine, thromboxane receptor agonist U-46619, increased potassium, and L-type Ca2+ channel ligand FPL-64176. Quantitative proteomics revealed proteins involved in actin polymerization and contraction were significantly downregulated in aortas of smLRP1-/- mice. However, studies with calyculin A indicated that although aortic muscle from smLRP1-/- mice can contract in response to calyculin A, a role for LRP1 in regulating the contractile machinery is not revealed. Furthermore, intracellular calcium imaging experiments identified defects in calcium release in response to a RyR (ryanodine receptor) agonist in smLRP1-/- aortic rings and cultured vascular smooth muscle cells. Conclusions- These results identify a critical role for LRP1 in modulating vascular smooth muscle cell contraction by regulating calcium signaling events that potentially protect against aneurysm development.

Loss of S100A1 expression leads to Ca2+ release potentiation in mutant mice with disrupted CaM and S100A1 binding to CaMBD2 of RyR1.

Calmodulin (CaM) and S100A1 fine-tune skeletal muscle Ca2+ release via opposite modulation of the ryanodine receptor type 1 (RyR1). Binding to and modulation of RyR1 by CaM and S100A1 occurs predominantly at the region ranging from amino acid residue 3614-3640 of RyR1 (here referred to as CaMBD2). Using synthetic peptides, it has been shown that CaM binds to two additional regions within the RyR1, specifically residues 1975-1999 and 4295-4325 (CaMBD1 and CaMBD3, respectively). Because S100A1 typically binds to similar motifs as CaM, we hypothesized that S100A1 could also bind to CaMBD1 and CaMBD3. Our goals were: (1) to establish whether S100A1 binds to synthetic peptides containing CaMBD1 and CaMBD3 using isothermal calorimetry (ITC), and (2) to identify whether S100A1 and CaM modulate RyR1 Ca2+ release activation via sites other than CaMBD2 in RyR1 in its native cellular context. We developed the mouse model (RyR1D-S100A1KO), which expresses point mutation RyR1-L3625D (RyR1D) that disrupts the modulation of RyR1 by CaM and S100A1 at CaMBD2 and also lacks S100A1 (S100A1KO). ITC assays revealed that S100A1 binds with different affinities to CaMBD1 and CaMBD3. Using high-speed Ca2+ imaging and a model for Ca2+ binding and transport, we show that the RyR1D-S100A1KO muscle fibers exhibit a modest but significant increase in myoplasmic Ca2+ transients and enhanced Ca2+ release flux following field stimulation when compared to fibers from RyR1D mice, which were used as controls to eliminate any effect of binding at CaMBD2, but with preserved S100A1 expression. Our results suggest that S100A1, similar to CaM, binds to CaMBD1 and CaMBD3 within the RyR1, but that CaMBD2 appears to be the primary site of RyR1 regulation by CaM and S100A1.

Voltage sensing mechanism in skeletal muscle excitation-contraction coupling: coming of age or midlife crisis?

The process by which muscle fiber electrical depolarization is linked to activation of muscle contraction is known as excitation-contraction coupling (ECC). Our understanding of ECC has increased enormously since the early scientific descriptions of the phenomenon of electrical activation of muscle contraction by Galvani that date back to the end of the eighteenth century. Major advances in electrical and optical measurements, including muscle fiber voltage clamp to reveal membrane electrical properties, in conjunction with the development of electron microscopy to unveil structural details provided an elegant view of ECC in skeletal muscle during the last century. This surge of knowledge on structural and biophysical aspects of the skeletal muscle was followed by breakthroughs in biochemistry and molecular biology, which allowed for the isolation, purification, and DNA sequencing of the muscle fiber membrane calcium channel/transverse tubule (TT) membrane voltage sensor (Cav1.1) for ECC and of the muscle ryanodine receptor/sarcoplasmic reticulum Ca2+ release channel (RyR1), two essential players of ECC in skeletal muscle. In regard to the process of voltage sensing for controlling calcium release, numerous studies support the concept that the TT Cav1.1 channel is the voltage sensor for ECC, as well as also being a Ca2+ channel in the TT membrane. In this review, we present early and recent findings that support and define the role of Cav1.1 as a voltage sensor for ECC.

Real-time scratch assay reveals mechanisms of early calcium signaling in breast cancer cells in response to wounding.

Aggressive cellular phenotypes such as uncontrolled proliferation and increased migration capacity engender cellular transformation, malignancy and metastasis. While genetic mutations are undisputed drivers of cancer initiation and progression, it is increasingly accepted that external factors are also playing a major role. Two recently studied modulators of breast cancer are changes in the cellular mechanical microenvironment and alterations in calcium homeostasis. While many studies investigate these factors separately in breast cancer cells, very few do so in combination. This current work sets a foundation to explore mechano-calcium relationships driving malignant progression in breast cancer. Utilizing real-time imaging of an in vitro scratch assay, we were able to resolve mechanically-sensitive calcium signaling in human breast cancer cells. We observed rapid initiation of intracellular calcium elevations within seconds in cells at the immediate wound edge, followed by a time-dependent increase in calcium in cells at distances up to 500µm from the scratch wound. Calcium signaling to neighboring cells away from the wound edge returned to baseline within seconds. Calcium elevations at the wound edge however, persisted for up to 50 minutes. Rigorous quantification showed that extracellular calcium was necessary for persistent calcium elevation at the wound edge, but intercellular signal propagation was dependent on internal calcium stores. In addition, intercellular signaling required extracellular ATP and activation of P2Y2 receptors. Through comparison of scratch-induced signaling from multiple cell lines, we report drastic reductions in response from aggressively tumorigenic and metastatic cells. The real-time scratch assay established here provides quantitative data on the molecular mechanisms that support rapid scratch-induced calcium signaling in breast cancer cells. These mechanisms now provide a clear framework for investigating which short-term calcium signals promote long-term changes in cancer cell biology.

Foxo1 nucleo-cytoplasmic distribution and unidirectional nuclear influx are the same in nuclei in a single skeletal muscle fiber but vary between fibers.

Foxo transcription factors promote protein breakdown and atrophy of skeletal muscle fibers. Foxo transcriptional effectiveness is largely determined by phosphorylation-dependent nucleo-cytoplasmic shuttling. Imaging Foxo1-green fluorescent protein (GFP) over time in 124 nuclei in 68 multinucleated adult skeletal muscle fibers under control culture conditions reveals large variability between fibers in Foxo1-GFP nucleo-cytoplasmic concentration ratio (N/C) and in the apparent rate coefficient ( kI') for Foxo1-GFP unidirectional nuclear influx (measured with efflux blocked by leptomycin B). Pairs of values of N/C or of kI' from different nuclei in the same fiber were essentially the same, but only weakly correlated in nuclei from different fibers in the same culture well. Thus, fiber to fiber variability of cellular factors, but not extracellular factors, determines Foxo1 distribution. Over all nuclei, N/C and kI' were closely proportional, indicating that kI' is the major determinant of Foxo1 distribution. IGF-I activation of Foxo kinase Akt reduces variability by decreasing kI' and N/C in all fibers. However, inhibiting Akt did not drive kI' uniformly high, indicating other pathways in Foxo1 regulation.

Acute Elevated Glucose Promotes Abnormal Action Potential-Induced Ca2+ Transients in Cultured Skeletal Muscle Fibers.

A common comorbidity of diabetes is skeletal muscle dysfunction, which leads to compromised physical function. Previous studies of diabetes in skeletal muscle have shown alterations in excitation-contraction coupling (ECC)-the sequential link between action potentials (AP), intracellular Ca2+ release, and the contractile machinery. Yet, little is known about the impact of acute elevated glucose on the temporal properties of AP-induced Ca2+ transients and ionic underlying mechanisms that lead to muscle dysfunction. Here, we used high-speed confocal Ca2+ imaging to investigate the temporal properties of AP-induced Ca2+ transients, an intermediate step of ECC, using an acute in cellulo model of uncontrolled hyperglycemia (25 mM, 48 h.). Control and elevated glucose-exposed muscle fibers cultured for five days displayed four distinct patterns of AP-induced Ca2+ transients (phasic, biphasic, phasic-delayed, and phasic-slow decay); most control muscle fibers show phasic AP-induced Ca2+ transients, while most fibers exposed to elevated D-glucose displayed biphasic Ca2+ transients upon single field stimulation. We hypothesize that these changes in the temporal profile of the AP-induced Ca2+ transients are due to changes in the intrinsic excitable properties of the muscle fibers. We propose that these changes accompany early stages of diabetic myopathy.

The Activation of Protein Kinase A by the Calcium-Binding Protein S100A1 Is Independent of Cyclic AMP.

Biochemical and structural studies demonstrate that S100A1 is involved in a Ca2+-dependent interaction with the type 2α and type 2ß regulatory subunits of protein kinase A (PKA) (RIIα and RIIß) to activate holo-PKA. The interaction was specific for S100A1 because other calcium-binding proteins (i.e., S100B and calmodulin) had no effect. Likewise, a role for S100A1 in PKA-dependent signaling was established because the PKA-dependent subcellular redistribution of HDAC4 was abolished in cells derived from S100A1 knockout mice. Thus, the Ca2+-dependent interaction between S100A1 and the type 2 regulatory subunits represents a novel mechanism that provides a link between Ca2+ and PKA signaling, which is important for the regulation of gene expression in skeletal muscle via HDAC4 cytosolic-nuclear trafficking.

Impaired calcium signaling in muscle fibers from intercostal and foot skeletal muscle in a cigarette smoke-induced mouse model of COPD.

INTRODUCTION: Respiratory and locomotor skeletal muscle dysfunction are common findings in chronic obstructive pulmonary disease (COPD); however, the mechanisms that cause muscle impairment in COPD are unclear. Because Ca2+ signaling in excitation-contraction (E-C) coupling is important for muscle activity, we hypothesized that Ca2+ dysregulation could contribute to muscle dysfunction in COPD. METHODS: Intercostal and flexor digitorum brevis muscles from control and cigarette smoke-exposed mice were investigated. We used single cell Ca2+ imaging and Western blot assays to assess Ca2+ signals and E-C coupling proteins. RESULTS: We found impaired Ca2+ signals in muscle fibers from both muscle types, without significant changes in releasable Ca2+ or in the expression levels of E-C coupling proteins. CONCLUSIONS: Ca2+ dysregulation may contribute or accompany respiratory and locomotor muscle dysfunction in COPD. These findings are of significance to the understanding of the pathophysiological course of COPD in respiratory and locomotor muscles. Muscle Nerve 56: 282-291, 2017.

Altered nuclear dynamics in MDX myofibers.

Duchenne muscular dystrophy (DMD) is a genetic disorder in which the absence of dystrophin leads to progressive muscle degeneration and weakness. Although the genetic basis is known, the pathophysiology of dystrophic skeletal muscle remains unclear. We examined nuclear movement in wild-type (WT) and muscular dystrophy mouse model for DMD (MDX) (dystrophin-null) mouse myofibers. We also examined expression of proteins in the linkers of nucleoskeleton and cytoskeleton (LINC) complex, as well as nuclear transcriptional activity via histone H3 acetylation and polyadenylate-binding nuclear protein-1. Because movement of nuclei is not only LINC dependent but also microtubule dependent, we analyzed microtubule density and organization in WT and MDX myofibers, including the application of a unique 3D tool to assess microtubule core structure. Nuclei in MDX myofibers were more mobile than in WT myofibers for both distance traveled and velocity. MDX muscle shows reduced expression and labeling intensity of nesprin-1, a LINC protein that attaches the nucleus to the microtubule and actin cytoskeleton. MDX nuclei also showed altered transcriptional activity. Previous studies established that microtubule structure at the cortex is disrupted in MDX myofibers; our analyses extend these findings by showing that microtubule structure in the core is also disrupted. In addition, we studied malformed MDX myofibers to better understand the role of altered myofiber morphology vs. microtubule architecture in the underlying susceptibility to injury seen in dystrophic muscles. We incorporated morphological and microtubule architectural concepts into a simplified finite element mathematical model of myofiber mechanics, which suggests a greater contribution of myofiber morphology than microtubule structure to muscle biomechanical performance.NEW & NOTEWORTHY Microtubules provide the means for nuclear movement but show altered organization in the muscular dystrophy mouse model (MDX) (dystrophin-null) muscle. Here, MDX myofibers show increased nuclear movement, altered transcriptional activity, and altered linkers of nucleoskeleton and cytoskeleton complex expression compared with healthy myofibers. Microtubule architecture was incorporated in finite element modeling of passive stretch, revealing a role of fiber malformation, commonly found in MDX muscle. The results suggest that alterations in microtubule architecture in MDX muscle affect nuclear movement, which is essential for muscle function.