Short-Term Very High Carbohydrate Diet and Gut-Training Have Minor Effects on Gastrointestinal Status and Performance in Highly Trained Endurance Athletes.

We implemented a multi-pronged strategy (MAX) involving chronic (2 weeks high carbohydrate [CHO] diet + gut-training) and acute (CHO loading + 90 g·h−1 CHO during exercise) strategies to promote endogenous and exogenous CHO availability, compared with strategies reflecting lower ranges of current guidelines (CON) in two groups of athletes. Nineteen elite male race walkers (MAX: 9; CON:10) undertook a 26 km race-walking session before and after the respective interventions to investigate gastrointestinal function (absorption capacity), integrity (epithelial injury), and symptoms (GIS). We observed considerable individual variability in responses, resulting in a statistically significant (p < 0.

Dietary Intake and Gastrointestinal Integrity in Runners Undertaking High-Intensity Exercise in the Heat.

Gastrointestinal disturbances are one of the most common issues for endurance athletes during training and competition in the heat. The relationship between typical dietary intake or nutritional interventions and perturbations in or maintenance of gut integrity is unclear. Twelve well-trained male endurance athletes (peak oxygen consumption = 61.

Running at Increasing Intensities in the Heat Induces Transient Gut Perturbations.

Purpose: The risk of exercise-induced endotoxemia is increased in the heat and is primarily attributable to changes in gut permeability resulting in the translocation of lipopolysaccharides (LPS) into the circulation. The purpose of this study was to quantify the acute changes in gut permeability and LPS translocation during submaximal continuous and high-intensity interval exercise under heat stress.

Methods: A total of 12 well-trained male runners (age 37 [7] y, maximal oxygen uptake [VO2max] 61.

Sarcoplasmic reticulum calcium mishandling: central tenet in heart failure?

Excitation-contraction coupling links excitation of the sarcolemmal surface membrane to mechanical contraction. In the heart this link is established via a Ca-induced Ca release process, which, following sarcolemmal depolarisation, prompts Ca release from the sarcoplasmic reticulum (SR) though the ryanodine receptor (RyR2). This substantially raises the cytoplasmic Ca concentration to trigger systole.

Ion channel gating in cardiac ryanodine receptors from the arrhythmic RyR2-P2328S mouse.

Mutations in the cardiac ryanodine receptor Ca release channel (RyR2) can cause deadly ventricular arrhythmias and atrial fibrillation (AF). The RyR2-P2328S mutation produces catecholaminergic polymorphic ventricular tachycardia (CPVT) and AF in hearts from homozygous RyR2 (denoted RyR2) mice. We have now examined P2328S RyR2 channels from RyR2 hearts.

Calmodulin inhibition of human RyR2 channels requires phosphorylation of RyR2-S2808 or RyR2-S2814.

Calmodulin (CaM) is a Ca-binding protein that binds to, and can directly inhibit cardiac ryanodine receptor calcium release channels (RyR2). Animal studies have shown that RyR2 hyperphosphorylation reduces CaM binding to RyR2 in failing hearts, but data are lacking on how CaM regulates human RyR2 and how this regulation is affected by RyR2 phosphorylation. Physiological concentrations of CaM (100 nM) inhibited the diastolic activity of RyR2 isolated from failing human hearts by ~50% but had no effect on RyR2 from healthy human hearts.

Recent advances in understanding the ryanodine receptor calcium release channels and their role in calcium signalling.

The ryanodine receptor calcium release channel is central to cytoplasmic Ca signalling in skeletal muscle, the heart, and many other tissues, including the central nervous system, lymphocytes, stomach, kidney, adrenal glands, ovaries, testes, thymus, and lungs. The ion channel protein is massive (more than 2.2 MDa) and has a structure that has defied detailed determination until recent developments in cryo-electron microscopy revealed much of its structure at near-atomic resolution.

Ryanodine receptor Ca release channel post-translational modification: Central player in cardiac and skeletal muscle disease.

Calcium release from internal stores is a quintessential event in excitation-contraction coupling in cardiac and skeletal muscle. The ryanodine receptor Ca release channel is embedded in the internal sarcoplasmic reticulum Ca store, which releases Ca into the cytoplasm, enabling contraction. Ryanodine receptors form the hub of a macromolecular complex extending from the extracellular space to the sarcoplasmic reticulum lumen.

The Anthracycline Metabolite Doxorubicinol Abolishes RyR2 Sensitivity to Physiological Changes in Luminal Ca through an Interaction with Calsequestrin.

The chemotherapeutic anthracycline metabolite doxorubicinol (doxOL) has been shown to interact with and disrupt the function of the cardiac ryanodine receptor Ca release channel (RyR2) in the sarcoplasmic reticulum (SR) membrane and the SR Ca binding protein calsequestrin 2 (CSQ2). Normal increases in RyR2 activity in response to increasing diastolic SR [Ca] are influenced by CSQ2 and are disrupted in arrhythmic conditions. Therefore, we explored the action of doxOL on RyR2's response to changes in luminal [Ca] seen during diastole.

Association of FK506 binding proteins with RyR channels - effect of CLIC2 binding on sub-conductance opening and FKBP binding.

Ryanodine receptor (RyR) Ca channels are central to striated muscle function and influence signalling in neurons and other cell types. Beneficially low RyR activity and maximum conductance opening may be stabilised when RyRs bind to FK506 binding proteins (FKBPs) and destabilised by FKBP dissociation, with submaximal opening during RyR hyperactivity associated with myopathies and neurological disorders. However, the correlation with submaximal opening is debated and quantitative evidence is lacking.

Physiology and Pharmacology of Ryanodine Receptor Calcium Release Channels.

Ryanodine receptor (RyR) ion channels are essential for skeletal and cardiac muscle function. Their knockout leads to perinatal death from respiratory and cardiac failure. Acquired changes or mutations in the protein cause debilitating skeletal myopathy and cardiac arrhythmia which can be deadly.

Core skeletal muscle ryanodine receptor calcium release complex.

The core skeletal muscle ryanodine receptor (RyR1) calcium release complex extends through three compartments of the muscle fibre, linking the extracellular environment through the cytoplasmic junctional gap to the lumen of the internal sarcoplasmic reticulum (SR) calcium store. The protein complex is essential for skeletal excitation-contraction (EC)-coupling and skeletal muscle function. Its importance is highlighted by perinatal death if any one of the EC-coupling components are missing and by myopathies associated with mutation of any of the proteins.

Regions of ryanodine receptors that influence activation by the dihydropyridine receptor β1a subunit.

Background: Although excitation-contraction (EC) coupling in skeletal muscle relies on physical activation of the skeletal ryanodine receptor (RyR1) Ca(2+) release channel by dihydropyridine receptors (DHPRs), the activation pathway between the DHPR and RyR1 remains unknown. However, the pathway includes the DHPR β1a subunit which is integral to EC coupling and activates RyR1. In this manuscript, we explore the isoform specificity of β1a activation of RyRs and the β1a binding site on RyR1.

C-terminal residues of skeletal muscle calsequestrin are essential for calcium binding and for skeletal ryanodine receptor inhibition.

Background: Skeletal muscle function depends on calcium signaling proteins in the sarcoplasmic reticulum (SR), including the calcium-binding protein calsequestrin (CSQ), the ryanodine receptor (RyR) calcium release channel, and skeletal triadin 95 kDa (trisk95) and junctin, proteins that bind to calsequestrin type 1 (CSQ1) and ryanodine receptor type 1 (RyR1). CSQ1 inhibits RyR1 and communicates store calcium load to RyR1 channels via trisk95 and/or junctin.

Methods: In this manuscript, we test predictions that CSQ1's acidic C-terminus contains binding sites for trisk95 and junctin, the major calcium binding domain, and that it determines CSQ1's ability to regulate RyR1 activity.

A new cytoplasmic interaction between junctin and ryanodine receptor Ca2+ release channels.

Junctin, a non-catalytic splice variant encoded by the aspartate-β-hydroxylase (Asph) gene, is inserted into the membrane of the sarcoplasmic reticulum (SR) Ca(2+) store where it modifies Ca(2+) signalling in the heart and skeletal muscle through its regulation of ryanodine receptor (RyR) Ca(2+) release channels. Junctin is required for normal muscle function as its knockout leads to abnormal Ca(2+) signalling, muscle dysfunction and cardiac arrhythmia. However, the nature of the molecular interaction between junctin and RyRs is largely unknown and was assumed to occur only in the SR lumen.

Differences in the regulation of RyR2 from human, sheep, and rat by Ca²⁺ and Mg²⁺ in the cytoplasm and in the lumen of the sarcoplasmic reticulum.

Regulation of the cardiac ryanodine receptor (RyR2) by intracellular Ca(2+) and Mg(2+) plays a key role in determining cardiac contraction and rhythmicity, but their role in regulating the human RyR2 remains poorly defined. The Ca(2+)- and Mg(2+)-dependent regulation of human RyR2 was recorded in artificial lipid bilayers in the presence of 2 mM ATP and compared with that in two commonly used animal models for RyR2 function (rat and sheep). Human RyR2 displayed cytoplasmic Ca(2+) activation (K(a) = 4 µM) and inhibition by cytoplasmic Mg(2+) (K(i) = 10 µM at 100 nM Ca(2+)) that was similar to RyR2 from rat and sheep obtained under the same experimental conditions.

Cardiac ryanodine receptor activation by a high Ca²⁺ store load is reversed in a reducing cytoplasmic redox environment.

Here, we report the impact of redox potential on isolated cardiac ryanodine receptor (RyR2) channel activity and its response to physiological changes in luminal [Ca(2+)]. Basal leak from the sarcoplasmic reticulum is required for normal Ca(2+) handling, but excess diastolic Ca(2+) leak attributed to oxidative stress is thought to lower the threshold of RyR2 for spontaneous sarcoplasmic reticulum Ca(2+) release, thus inducing arrhythmia in pathological situations. Therefore, we examined the RyR2 response to luminal [Ca(2+)] under reducing or oxidising cytoplasmic redox conditions.

Adverse effects of doxorubicin and its metabolic product on cardiac RyR2 and SERCA2A.

The use of anthracycline chemotherapeutic drugs is restricted owing to potentially fatal cardiotoxic side effects. It has been hypothesized that anthracycline metabolites have a primary role in this cardiac dysfunction; however, information on the molecular interactions of these compounds in the heart is scarce. Here we provide novel evidence that doxorubicin and its metabolite, doxorubicinol, bind to the cardiac ryanodine receptor (RyR2) and to the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA2A) and deleteriously alter their activity.

Skeletal muscle excitation-contraction coupling: who are the dancing partners?

There is an overwhelming body of work supporting the idea that excitation-contraction coupling in skeletal muscle depends on a physical interaction between the skeletal muscle isoform of the dihydropyridine receptor L-type Ca(2+) channel and the skeletal isoform of the ryanodine receptor Ca(2+) release channel. A general assumption is that this physical interaction is between "critical" residues that have been identified in the II-III loop of the dihydropyridine receptor alpha subunit and the ryanodine receptor. However, despite extensive searches, the complementary "critical" residues in the ryanodine receptor have not been identified.

Multiple actions of phi-LITX-Lw1a on ryanodine receptors reveal a functional link between scorpion DDH and ICK toxins.

We recently reported the isolation of a scorpion toxin named U1-liotoxin-Lw1a (U1-LITX-Lw1a) that adopts an unusual 3D fold termed the disulfide-directed hairpin (DDH) motif, which is the proposed evolutionary structural precursor of the three-disulfide-containing inhibitor cystine knot (ICK) motif found widely in animals and plants. Here we reveal that U1-LITX-Lw1a targets and activates the mammalian ryanodine receptor intracellular calcium release channel (RyR) with high (fM) potency and provides a functional link between DDH and ICK scorpion toxins. Moreover, U1-LITX-Lw1a, now described as ϕ-liotoxin-Lw1a (ϕ-LITX-Lw1a), has a similar mode of action on RyRs as scorpion calcines, although with significantly greater potency, inducing full channel openings at lower (fM) toxin concentrations whereas at higher pM concentrations increasing the frequency and duration of channel openings to a submaximal state.

ß-Adrenergic stimulation increases RyR2 activity via intracellular Ca2+ and Mg2+ regulation.

Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca(2+) and Mg(2+) and the role of these changes in SR Ca(2+) release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N2 to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling.

A skeletal muscle ryanodine receptor interaction domain in triadin.

Excitation-contraction coupling in skeletal muscle depends, in part, on a functional interaction between the ligand-gated ryanodine receptor (RyR1) and integral membrane protein Trisk 95, localized to the sarcoplasmic reticulum membrane. Various domains on Trisk 95 can associate with RyR1, yet the domain responsible for regulating RyR1 activity has remained elusive. We explored the hypothesis that a luminal Trisk 95 KEKE motif (residues 200-232), known to promote RyR1 binding, may also form the RyR1 activation domain.