Guillermo Avila

Excitation–contraction uncoupling by a human central core disease mutation in the ryanodine receptor

Central core disease (CCD) is a human congenital myopathy characterized by fetal hypotonia and proximal muscle weakness that is linked to mutations in the gene encoding the type-1 ryanodine receptor (RyR1). CCD is thought to arise from Ca2+-induced damage stemming from mutant RyR1 proteins forming “leaky” sarcoplasmic reticulum (SR) Ca2+ release channels. A novel mutation in the C-terminal region of RyR1 (I4898T) accounts for an unusually severe and highly penetrant form of CCD in humans [Lynch, P. J., Tong, J., Lehane, M., Mallet, A., Giblin, L., Heffron, J. J., Vaughan, P., Zafra, G., MacLennan, D. H. & McCarthy, T. V. (1999) Proc. Natl. Acad. Sci. USA 96, 4164–4169]. We expressed in skeletal myotubes derived from RyR1-knockout (dyspedic) mice the analogous mutation engineered into a rabbit RyR1 cDNA (I4897T). Here we show that homozygous expression of I4897T in dyspedic myotubes results in a complete uncoupling of sarcolemmal excitation from voltage-gated SR Ca2+ release without significantly altering resting cytosolic Ca2+ levels, SR Ca2+ content, or RyR1-mediated enhancement of dihydropyridine receptor (DHPR) channel activity. Coexpression of both I4897T and wild-type RyR1 resulted in a 60% reduction in voltage-gated SR Ca2+ release, again without altering resting cytosolic Ca2+ levels, SR Ca2+ content, or DHPR channel activity. These findings indicate that muscle weakness suffered by individuals possessing the I4898T mutation involves a functional uncoupling of sarcolemmal excitation from SR Ca2+ release, rather than the expression of overactive or leaky SR Ca2+ release channels.

The Pore Region of the Skeletal Muscle Ryanodine Receptor Is a Primary Locus for Excitation-Contraction Uncoupling in Central Core Disease

Human central core disease (CCD) is caused by mutations/deletions in the gene that encodes the skeletal muscle ryanodine receptor (RyR1). Previous studies have shown that CCD mutations in the NH2-terminal region of RyR1 lead to the formation of leaky SR Ca2+ release channels when expressed in myotubes derived from RyR1-knockout (dyspedic) mice, whereas a COOH-terminal mutant (I4897T) results in channels that are not leaky to Ca2+ but lack depolarization-induced Ca2+ release (termed excitation-contraction [EC] uncoupling). We show here that store depletion resulting from NH2-terminal (Y523S) and COOH-terminal (Y4795C) leaky CCD mutant release channels is eliminated after incorporation of the I4897T mutation into the channel (Y523S/I4897T and Y4795C/I4897T). In spite of normal SR Ca2+ content, myotubes expressing the double mutants lacked voltage-gated Ca2+ release and thus exhibited an EC uncoupling phenotype similar to that of I4897T-expressing myotubes. We also show that dyspedic myotubes expressing each of seven recently identified CCD mutations located in exon 102 of the RyR1 gene (G4890R, R4892W, I4897T, G4898E, G4898R, A4905V, R4913G) behave as EC-uncoupled release channels. Interestingly, voltage-gated Ca2+ release was nearly abolished (reduced ∼90%) while caffeine-induced Ca2+ release was only marginally reduced in R4892W-expressing myotubes, indicating that this mutation preferentially disrupts voltage-sensor activation of release. These data demonstrate that CCD mutations in exon 102 disrupt release channel permeation to Ca2+ during EC coupling and that this region represents a primary molecular locus for EC uncoupling in CCD.

Role of TGF-β on cardiac structural and electrical remodeling

The type β transforming growth factors (TGF-βs) are involved in a number of human diseases, including heart failure and myocardial arrhythmias. In fact, during the last 20 years numerous studies have demonstrated that TGF-β affects the architecture of the heart under both normal and pathological conditions. Moreover, TGF-β signaling is currently under investigation, with the aim of discovering potential therapeutic roles in human disease. In contrast, only few studies have investigated whether TGF-β affects electrophysiological properties of the heart. This fact is surprising since electrical remodeling represents an important substrate for cardiac disease. This review discusses the potential role of TGF-β on cardiac excitation-contraction (EC) coupling, action potentials, and ion channels. We also discuss the effects of TGF-β on cardiac development and disease from structural and electrophysiological points of view.

Role of the Sequence Surrounding Predicted Transmembrane Helix M4 in Membrane Association and Function of the Ca2+ Release Channel of Skeletal Muscle Sarcoplasmic Reticulum (Ryanodine Receptor Isoform 1)

The role of the sequence surrounding M4 in ryanodine receptors (RyR) in membrane association and function was investigated. This sequence contains a basic, 19-amino acid M3/M4 loop, a hydrophobic 44–49 amino acid sequence designated M4 (or M4a/M4b), and a hydrophilic M4/M5 loop. Enhanced green fluorescent protein (EGFP) was inserted into RyR1 and truncated just after the basic sequence, just after M4, within the M4/M5 loop, just before M5 and just after M5. The A52 epitope was inserted into RyR2 and truncated just after M4a. Analysis of these constructs ruled out a M3/M4 transmembrane hairpin and narrowed the region of membrane association to M4a/M4b. EGFP inserted between M4a and M4b in full-length RyR2 was altered conformationally, losing fluorescence and gaining trypsin sensitivity. Although it was accessible to an antibody from the cytosolic side, tryptic fragments were membrane-bound. The expressed protein containing EGFP retained caffeine-induced Ca2+ release channel function. These results suggest that M4a/M4b either forms a transmembrane hairpin or associates in an unorthodox fashion with the cytosolic leaflet of the membrane, possibly involving the basic M3/M4 loop. The expression of a mutant RyR1, Δ4274–4535, deleted in the sequence surrounding both M3 and M4, restored robust, voltage-gated L-type Ca2+ currents and Ca2+ transients in dyspedic myotubes, demonstrating that this sequence is not required for either orthograde (DHPR activation of sarcoplasmic reticulum Ca2+ release) or retrograde (RyR1 increase in DHPR Ca2+ channel activity) signals of excitation-contraction coupling. Maximal amplitudes of L-currents and Ca2+ transients with Δ4274–4535 were larger than with wild-type RyR1, and voltage-gated sarcoplasmic reticulum Ca2+ release was more sensitive to activation by sarcolemmal voltage sensors. Thus, this region may act as a negative regulatory module that increases the energy barrier for Ca2+ release channel opening.

Sustained CGRP1 receptor stimulation modulates development of EC coupling by cAMP/PKA signalling pathway in mouse skeletal myotubes

We investigated modulation of excitation–contraction (EC) coupling by calcitonin gene‐related peptide (CGRP), which is released by motorneurons during neuromuscular transmission. Mouse skeletal myotubes were cultured either under control conditions or in the presence of 100 nm CGRP (∼4–72 h). T‐ and L‐type Ca2+ currents, immobilization resistant charge movement, and intracellular Ca2+ transients were characterized in whole‐cell patch‐clamp experiments. CGRP treatment increased the amplitude of voltage‐gated Ca2+ release ((ΔF/F)max) ∼75–350% and moderately increased both maximal L‐current conductance (Gmax) and charge movement (Qmax). In contrast, CGRP treatment did not affect their corresponding voltage dependence of activation (V1/2 and k) or T‐current density. CGRP treatment enhanced voltage‐gated Ca2+ release in ∼4 h, whereas the effect on L‐channel magnitude took longer to develop (∼24 h), suggesting that short‐term potentiation of EC coupling may lead to subsequent long‐term up‐regulation of DHPR expression. CGRP treatment also drastically increased caffeine‐induced Ca2+ release in ∼4 h (∼400%). Thus, short‐term potentiation of EC coupling is due to an increase in sarcoplasmic reticulum Ca2+ content. Both application of a phosphodiesterase inhibitor (papaverine) and a membrane‐permeant cAMP analogue (Db‐cAMP) produced a similar potentiation of EC coupling. Conversely, this potentiation was prevented by pretreatment with either CGRP1 receptor antagonist (CGRP8‐37) or a PKA inhibitor (H‐89). Thus, CGRP acts through CGRP1 receptors and the cAMP/PKA signalling pathway to enhance voltage‐gated Ca2+ release. Effects of CGRP on both EC coupling and L‐channels were attenuated at later times during myotube differentiation. Therefore, we conclude that CGRP accelerates maturation of EC coupling.

Ca2+ channel regulation by transforming growth factor-β1 and bone morphogenetic protein-2 in developing mice myotubes

In skeletal muscle myogenesis, precursor cells or myoblasts fuse to form multinucleated cells (myotubes), which then further develop into functional muscle. We investigated if the inhibition of myogenesis by transforming growth factor‐β1 (TGF‐β1) and bone morphogenetic protein‐2 (BMP‐2) involve regulation of voltage‐dependent Ca2+ channels. Primary cultured myoblasts were kept in fusion medium (0–6 days) in either the absence (control conditions) or the presence of 40 pm TGF‐β1 or 5 nm BMP‐2. Subsequently, the developing myotubes were transferred to a growth factor‐free recording solution, and subjected to whole cell patch‐clamp experiments. At day 0, 14% of non‐fusing myoblasts exhibited T‐current, whereas the L‐current was practically absent. Under control conditions, however, the percentage of T‐ and L‐channel‐expressing myotubes increased sharply, from 25% at day 1 to ∼100% at days 2–6. In addition, parallel increases were determined for Ca2+‐currents density and cell membrane capacitance (Cm), which is proportional to the size of myotubes. Interestingly, at days 1–2 TGF‐β1 and BMP‐2 eliminated the T‐current on initial 14% of T‐channel‐expressing myoblasts. Moreover, at day 6 the growth factors significantly reduced the maximal values of both T‐current density (80%) and Cm (60%). The effect of BMP‐2 was selective on T‐channels, whereas TGF‐β1 decreased also the L‐current density (90%). A similar reduction in maximal conductance of the Ca2+ channels was determined, in the absence of significant alterations in other essential properties of the channels, including the time course and voltage dependence of activation and inactivation. The results suggest these growth factors markedly reduce the number of functional T‐ (both TGF‐β1 and BMP‐2) and L‐channels (only TGF‐β1) in the surface of the plasma membrane, and contribute to explaining the associated effects on myogenesis.

Nerve growth factor affects Ca2+ currents via the p75 receptor to enhance prolactin mRNA levels in GH3 rat pituitary cells

In clonal pituitary GH3 cells, spontaneous action potentials drive the opening of Cav1 (L‐type) channels, leading to Ca2+ transients that are coupled to prolactin gene transcription. Nerve growth factor (NGF) has been shown to stimulate prolactin synthesis by GH3 cells, but the underlying mechanisms are unknown. Here we studied whether NGF influences prolactin gene expression and Ca2+ currents. By using RT‐PCR, NGF (50 ng ml−1) was found to augment prolactin mRNA levels by ∼80% when applied to GH3 cells for 3 days. A parallel change in the prolactin content was detected by Western blotting. Both NGF‐induced responses were mimicked by an agonist (Bay K 8644) and prevented by a blocker (nimodipine) of L‐type channels. In whole‐cell patch‐clamp experiments, NGF enhanced the L‐type Ca2+ current by ∼2‐fold within 60 min. This effect reversed quickly upon growth factor withdrawal, but was maintained for days in the continued presence of NGF. In addition, chronic treatment (≥ 24 h) with NGF amplified the T‐type current, which flows through Cav3 channels and is thought to support pacemaking activity. Thus, NGF probably increases the amount of Ca2+ that enters per action potential and may also induce a late increase in spike frequency. MC192, a specific antibody for the p75 neurotrophin receptor, but not tyrosine kinase inhibitors (K252a and lavendustin A), blocked the effects of NGF on Ca2+ currents. Overall, the results indicate that NGF activates the p75 receptor to cause a prolonged increase in Ca2+ influx through L‐type channels, which in turn up‐regulates the prolactin mRNA.

CGRP, a vasodilator neuropeptide that stimulates neuromuscular transmission and EC coupling.

Calcitonin gene related peptide (CGRP) is a vasodilator; its plasma levels are altered in several human diseases, including migraine, hypertension and diabetes. CGRP is locally released by motor neurons, and is overexpressed in response to surgical or pharmacological blockage of neuromuscular transmission. Additionally to a brief discussion with regard to the clinical relevance of CGRP, this review focuses on the effects of CGRP on skeletal muscle excitation-contraction (EC) coupling, as well as the corresponding pathophysiological consequences. EC coupling involves activation of 2 different types of calcium channels: dihydropyridine receptors (DHPRs) located at the sarcolemma, and ryanodine receptors (RyR1s) located at the sarcoplasmic reticulum (SR). In response to electrical depolarization, DHPRs activate nearby and physically bound RyR1s, allowing Ca(2+) from the SR to move into the cytosol (termed voltage-gated Ca(2+) release, or VGCR). We recently found that CGRP stimulates VGCR by 350 % in as short as 1h. This effect, which lasts for at least 48 h, is due to activation of the CGRP receptor, and requires activation of the cAMP/PKA signaling pathway. CGRP also increases the amplitude of caffeine-induced Ca(2+) release (400 %); suggesting increased SR Ca(2+) content underlies stimulation of VGCR. Interestingly, in the long-term CGRP also increases the density of sarcolemmal DHPRs (up to 30%, within 24-48 h). We propose that these CGRP effects may contribute to prevent and/or restore symptoms in central core disease (CCD); a congenital myopathy that is linked to mutations in the gene encoding RyR1.

Ryanodine receptors as leak channels

Ryanodine receptors are Ca(2+) release channels of internal stores. This review focuses on those situations and conditions that transform RyRs from a finely regulated ion channel to an unregulated Ca(2+) leak channel and the pathological consequences of this alteration. In skeletal muscle, mutations in either CaV1.1 channel or RyR1 results in a leaky behavior of the latter. In heart cells, RyR2 functions normally as a Ca(2+) leak channel during diastole within certain limits, the enhancement of this activity leads to arrhythmogenic situations that are tackled with different pharmacological strategies. In smooth muscle, RyRs are involved more in reducing excitability than in stimulating contraction so the leak activity of RyRs in the form of Ca(2+) sparks, locally activates Ca(2+)-dependent potassium channels to reduce excitability. In neurons the enhanced activity of RyRs is associated with the development of different neurodegenerative disorders such as Alzheimer and Huntington diseases. It appears then that the activity of RyRs as leak channels can have both physiological and pathological consequences depending on the cell type and the metabolic condition.

Calcitonin gene-related peptide restores disrupted excitation-contraction coupling in myotubes expressing central core disease mutations in RyR1.

Non‐Technical Summary  Central core disease (CCD) is linked to mutations in the skeletal muscle ryanodine receptor (RyR1) gene that are believed to disrupt excitation–contraction (EC) coupling. EC coupling requires activation of RyR1s to release Ca2+ from the sarcoplasmic reticulum (SR). Subsequently, the SR Ca2+‐ATPase (SERCA) returns cytoplasmic Ca2+ to intracellular stores. Here, we have investigated the effects of two CCD RyR1 mutants (I4897T and Y523S) in C2C12 myotubes. Both mutations significantly altered myogenesis and SERCA gene expression – inhibition by I4897T and stimulation by Y523S. They are thought to behave differently (‘Ca2+‐impermeable’ and ‘leaky’, respectively), but disrupted EC coupling through the same mechanism (store depletion). It is conceivable that reduced SERCA expression by I4897T could explain this paradox. In both cases, the neuropeptide CGRP restored EC coupling by increasing SR Ca2+ content. We propose that CGRP and its corresponding signalling pathway exert beneficial effects in myotubes expressing CCD mutants.