Artificial intelligence approaches to the volumetric quantification of glycogen granules in EM images of human tissue.

Skeletal muscle, the major processor of dietary glucose, stores it in myriad glycogen granules. Their numbers vary with cellular location and physiological and pathophysiological states. AI models were developed to derive granular glycogen content from electron-microscopic images of human muscle.

Distinct pathophysiological characteristics in developing muscle from patients susceptible to malignant hyperthermia.

Background: Most patients with malignant hyperthermia susceptibility diagnosed by the in vitro caffeine-halothane contracture test (CHCT) develop excessive force in response to halothane but not caffeine (halothane-hypersensitive). Hallmarks of halothane-hypersensitive patients include high incidence of musculoskeletal symptoms at rest and abnormal calcium events in muscle. By measuring sensitivity to halothane of myotubes and extending clinical observations and cell-level studies to a large group of patients, we reach new insights into the pathological mechanism of malignant hyperthermia susceptibility.

Muscle calcium stress cleaves junctophilin1, unleashing a gene regulatory program predicted to correct glucose dysregulation.

Calcium ion movements between cellular stores and the cytosol govern muscle contraction, the most energy-consuming function in mammals, which confers skeletal myofibers a pivotal role in glycemia regulation. Chronic myoplasmic calcium elevation ("calcium stress"), found in malignant hyperthermia-susceptible (MHS) patients and multiple myopathies, has been suggested to underlie the progression from hyperglycemia to insulin resistance. What drives such progression remains elusive.

A novel method for determining murine skeletal muscle fiber type using autofluorescence lifetimes.

This work describes a simple way to identify fiber types in living muscles by fluorescence lifetime imaging microscopy (FLIM). We quantified the mean values of lifetimes τ1 and τ2 derived from a two-exponential fit in freshly dissected mouse flexor digitorum brevis (FDB) and soleus muscles. While τ1 values changed following a bimodal behavior between muscles, the distribution of τ2 is shifted to higher values in FDB.

Quantification of the calcium signaling deficit in muscles devoid of triadin.

Triadin, a protein of the sarcoplasmic reticulum (SR) of striated muscles, anchors the calcium-storing protein calsequestrin to calcium release RyR channels at the junction with t-tubules, and modulates these channels by conformational effects. Triadin ablation induces structural SR changes and alters the expression of other proteins. Here we quantify alterations of calcium signaling in single skeletal myofibers of constitutive triadin-null mice.

A multi-dimensional analysis of genotype-phenotype discordance in malignant hyperthermia susceptibility.

Background: Malignant hyperthermia (MH) susceptibility is an inherited condition, diagnosed either by the presence of a pathogenic genetic variant or by in vitro caffeine-halothane contracture testing. Through a multi-dimensional approach, we describe the implications of discordance between genetic and in vitro test results in a patient with a family history of possible MH.

Methods: The patient, whose brother had a possible MH reaction, underwent the caffeine-halothane contracture test (CHCT) according to the North American MH Group protocol.

Intracellular calcium leak lowers glucose storage in human muscle, promoting hyperglycemia and diabetes.

Most glucose is processed in muscle, for energy or glycogen stores. Malignant Hyperthermia Susceptibility (MHS) exemplifies muscle conditions that increase [Ca]. 42% of MHS patients have hyperglycemia.

Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle.

Calsequestrin, the only known protein with cyclical storage and supply of calcium as main role, is proposed to have other functions, which remain unproven. Voluntary movement and the heart beat require this calcium flow to be massive and fast. How does calsequestrin do it? To bind large amounts of calcium in vitro, calsequestrin must polymerize and then depolymerize to release it.

The couplonopathies: A comparative approach to a class of diseases of skeletal and cardiac muscle.

A novel category of diseases of striated muscle is proposed, the couplonopathies, as those that affect components of the couplon and thereby alter its operation. Couplons are the functional units of intracellular calcium release in excitation-contraction coupling. They comprise dihydropyridine receptors, ryanodine receptors (Ca2+ release channels), and a growing list of ancillary proteins whose alteration may lead to disease.

Altered Ca2+ concentration, permeability and buffering in the myofibre Ca2+ store of a mouse model of malignant hyperthermia.

  Malignant hyperthermia (MH) is linked to mutations in the type 1 ryanodine receptor, RyR1, the Ca2+ channel of the sarcoplasmic reticulum (SR) of skeletal muscle. The Y522S MH mutation was studied for its complex presentation, which includes structurally and functionally altered cell 'cores'. Imaging cytosolic and intra-SR [Ca2+] in muscle cells of heterozygous YS mice we determined Ca2+ release flux activated by clamp depolarization, permeability (P) of the SR membrane (ratio of flux and [Ca2+] gradient) and SR Ca2+ buffering power (B).

Confocal imaging of transmembrane voltage by SEER of di-8-ANEPPS.

Imaging, optical mapping, and optical multisite recording of transmembrane potential (V(m)) are essential for studying excitable cells and systems. The naphthylstyryl voltage-sensitive dyes, including di-8-ANEPPS, shift both their fluorescence excitation and emission spectra upon changes in V(m). Accordingly, they have been used for monitoring V(m) in nonratioing and both emission and excitation ratioing modes.

Using two dyes with the same fluorophore to monitor cellular calcium concentration in an extended range.

We extend the sensitivity of quantitative concentration imaging to an approximately 1000-fold range of concentrations by a method that uses two fluorescent dyes with the same fluorophore, having different affinity for the monitored species. While the formulation and illustration refer to a monitor of calcium concentration, the method is applicable to any species that binds to multiple indicators with the same spectral properties. The use of a common fluorophore has the virtue of leaving vast regions of the electromagnetic spectrum available for other applications.

Dynamic measurement of the calcium buffering properties of the sarcoplasmic reticulum in mouse skeletal muscle.

The buffering power, B, of the sarcoplasmic reticulum (SR), ratio of the changes in total and free [Ca(2+)], was determined in fast-twitch mouse muscle cells subjected to depleting membrane depolarization. Changes in total SR [Ca(2+)] were measured integrating Ca(2+) release flux, determined with a cytosolic [Ca(2+)] monitor. Free [Ca(2+)](SR) was measured using the cameleon D4cpv-Casq1.

Synthetic localized calcium transients directly probe signalling mechanisms in skeletal muscle.

The contribution of Ca2+-induced Ca2+ release (CICR) to trigger muscle contraction is controversial. It was studied on isolated muscle fibres using synthetic localized increases in Ca2+ concentration, SLICs, generated by two-photon photorelease from nitrodibenzofuran (NDBF)-EGTA just outside the permeabilized plasma membrane. SLICs provided a way to increase cytosolic [Ca2+] rapidly and reversibly, up to 8 μM, levels similar to those reached during physiological activity.

Measurement of RyR permeability reveals a role of calsequestrin in termination of SR Ca(2+) release in skeletal muscle.

The mechanisms that terminate Ca(2+) release from the sarcoplasmic reticulum are not fully understood. D4cpv-Casq1 (Sztretye et al. 2011.

D4cpv-calsequestrin: a sensitive ratiometric biosensor accurately targeted to the calcium store of skeletal muscle.

Current fluorescent monitors of free [Ca(2+)] in the sarcoplasmic reticulum (SR) of skeletal muscle cells are of limited quantitative value. They provide either a nonratio signal that is difficult to calibrate and is not specific or, in the case of Forster resonant energy transfer (FRET) biosensors, a signal of small dynamic range, which may be degraded further by imperfect targeting and interference from endogenous ligands of calsequestrin. We describe a novel tool that uses the cameleon D4cpv, which has a greater dynamic range and lower susceptibility to endogenous ligands than earlier cameleons.