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Action potential-evoked calcium release is impaired in single skeletal muscle fibers from heart failure patients.

ABSTRACT: Exercise intolerance in chronic heart failure (HF) has been attributed to abnormalities of the skeletal muscles. Muscle function depends on intact excitation-contraction coupling (ECC), but ECC studies in HF models have been inconclusive, due to deficiencies in the animal models and tools used to measure calcium (Ca2+) release, mandating investigations in skeletal muscle from HF patients. The purpose of this study was to test the hypothesis that Ca2+ release is significantly impaired in the skeletal muscle of HF patients in whom exercise capacity is severely diminished compared to age-matched healthy volunteers.Using state-of-the-art electrophysiological and optical techniques in single muscle fibers from biopsies of the locomotive vastus lateralis muscle, we measured the action potential (AP)-evoked Ca2+ release in 4 HF patients and 4 age-matched healthy controls. The mean peak Ca2+ release flux in fibers obtained from HF patients (10±1.2 µM/ms) was markedly (2.6-fold) and significantly (p<0.05) smaller than in fibers from healthy volunteers (28±3.3 µM/ms). This impairment in AP-evoked Ca2+ release was ubiquitous and was not explained by differences in the excitability mechanisms since single APs were indistinguishable between HF patients and healthy volunteers.These findings prove the feasibility of performing electrophysiological experiments in single fibers from human skeletal muscle, and offer a new approach for investigations of myopathies due to HF and other diseases. Importantly, we have demonstrated that one step in the ECC process, AP-evoked Ca2+ release, is impaired in single muscle fibers in HF patients.

SUBMITTER: DiFranco M

PROVIDER: S-EPMC4195605 | biostudies-other | 2014

REPOSITORIES: biostudies-other

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Action potential-evoked calcium release is impaired in single skeletal muscle fibers from heart failure patients.

DiFranco Marino M Quiñonez Marbella M Shieh Perry P Fonarow Gregg C GC Cruz Daniel D Deng Mario C MC Vergara Julio L JL Middlekauff Holly R HR

PloS one 20141013 10

<h4>Background</h4>Exercise intolerance in chronic heart failure (HF) has been attributed to abnormalities of the skeletal muscles. Muscle function depends on intact excitation-contraction coupling (ECC), but ECC studies in HF models have been inconclusive, due to deficiencies in the animal models and tools used to measure calcium (Ca2+) release, mandating investigations in skeletal muscle from HF patients. The purpose of this study was to test the hypothesis that Ca2+ release is significantly i ...[more]

PMID: 25310188

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Project description:Skeletal muscle fibers have been used to examine a variety of cellular functions and pathologies. Among other parameters, skeletal muscle action potential (AP) propagation has been measured to assess the integrity and function of skeletal muscle. In this work, we utilize 1-(3-sulfonatopropyl)-4[?[2-(Di-n-octylamino)-6-naphtyl]vinyl]pyridinium betaine, a potentiometric dye, and mag-fluo-4, a low-affinity intracellular Ca2+indicator, to noninvasively and reliably measure AP conduction velocity in skeletal muscle. We used remote extracellular bipolar electrodes to generate an alternating polarity electric field that initiates an AP at either end of the fiber. Using enzymatically dissociated flexor digitorum brevis (FDB) fibers and high-speed line scans, we determine the conduction velocity to be ?0.4 m/s. We applied these methodologies to FDB fibers under elevated extracellular potassium conditions and confirmed that the conduction velocity is significantly reduced in elevated [K+]o. Because our recorded velocities for FDB fibers were much slower than previously reported for other muscle groups, we compared the conduction velocity in FDB fibers to that of extensor digitorum longus (EDL) fibers and measured a significantly faster velocity in EDL fibers than FDB fibers. As a basis for this difference in conduction velocity, we found a similarly higher level of expression of Na+ channels in EDL than in FDB fibers. In addition to measuring the conduction velocity, we can also measure the passive electrotonic potentials elicited by pulses by applying tetrodotoxin and have constructed a circuit model of a skeletal muscle fiber to predict passive polarization of the fiber by the field stimuli. Our predictions from the model fiber closely resemble the recordings acquired from in vitro assays. With these techniques, we can examine how various pathologies and mutations affect skeletal muscle AP propagation. Our work demonstrates the utility of using 1-(3-sulfonatopropyl)-4[?[2-(Di-n-octylamino)-6-naphtyl]vinyl]pyridinium betaine or mag-fluo-4 to noninvasively measure AP initiation and conduction.

Project description:In the mature auditory system, inner hair cells (IHCs) convert sound-induced vibrations into electrical signals that are relayed to the central nervous system via auditory afferents. Before the cochlea can respond to normal sound levels, developing IHCs fire calcium-based action potentials that disappear close to the onset of hearing. Action potential firing triggers transmitter release from the immature IHC that in turn generates experience-independent firing in auditory neurons. These early signaling events are thought to be essential for the organization and development of the auditory system and hair cells. A critical component of the action potential is the rise in intracellular calcium that activates both small conductance potassium channels essential during membrane repolarization, and triggers transmitter release from the cell. Whether this calcium signal is generated by calcium influx or requires calcium-induced calcium release (CICR) is not yet known. IHCs can generate CICR, but to date its physiological role has remained unclear. Here, we used high and low concentrations of ryanodine to block or enhance CICR to determine whether calcium release from intracellular stores affected action potential waveform, interspike interval, or changes in membrane capacitance during development of mouse IHCs. Blocking CICR resulted in mixed action potential waveforms with both brief and prolonged oscillations in membrane potential and intracellular calcium. This mixed behavior is captured well by our mathematical model of IHC electrical activity. We perform two-parameter bifurcation analysis of the model that predicts the dependence of IHCs firing patterns on the level of activation of two parameters, the SK2 channels activation and CICR rate. Our data show that CICR forms an important component of the calcium signal that shapes action potentials and regulates firing patterns, but is not involved directly in triggering exocytosis. These data provide important insights into the calcium signaling mechanisms involved in early developmental processes.

Project description:Experiments were performed to characterize the properties of the intrinsic Ca(2+) buffers in the sarcoplasmic reticulum (SR) of cut fibers from frog twitch muscle. The concentrations of total and free calcium ions within the SR ([Ca(T)](SR) and [Ca(2+)](SR)) were measured, respectively, with the EGTA/phenol red method and tetramethylmurexide (a low affinity Ca(2+) indicator). Results indicate SR Ca(2+) buffering was consistent with a single cooperative-binding component or a combination of a cooperative-binding component and a linear binding component accounting for 20% or less of the bound Ca(2+). Under the assumption of a single cooperative-binding component, the most likely resting values of [Ca(2+)](SR) and [Ca(T)](SR) are 0.67 and 17.1 mM, respectively, and the dissociation constant, Hill coefficient, and concentration of the Ca-binding sites are 0.78 mM, 3.0, and 44 mM, respectively. This information can be used to calculate a variable proportional to the Ca(2+) permeability of the SR, namely d[Ca(T)](SR)/dt ÷ [Ca(2+)](SR) (denoted release permeability), in experiments in which only [Ca(T)](SR) or [Ca(2+)](SR) is measured. In response to a voltage-clamp step to -20 mV at 15°C, the release permeability reaches an early peak followed by a rapid decline to a quasi-steady level that lasts ~50 ms, followed by a slower decline during which the release permeability decreases by at least threefold. During the quasi-steady level of release, the release amplitude is 3.3-fold greater than expected from voltage activation alone, a result consistent with the recruitment by Ca-induced Ca(2+) release of 2.3 SR Ca(2+) release channels neighboring each channel activated by its associated voltage sensor. Release permeability at -60 mV increases as [Ca(T)](SR) decreases from its resting physiological level to ~0.1 of this level. This result argues against a release termination mechanism proposed in mammalian muscle fibers in which a luminal sensor of [Ca(2+)](SR) inhibits release when [Ca(T)](SR) declines to a low level.

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Action potential-evoked calcium release is impaired in single skeletal muscle fibers from heart failure patients.

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Action potential-evoked calcium release is impaired in single skeletal muscle fibers from heart failure patients.

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