Journal of General Physiology

Published by Rockefeller University Press
Online ISSN: 1540-7748
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Article
The epithelial Na+ channel (ENaC) is a heterotrimeric protein whose assembly, trafficking, and function are highly regulated. To better understand the biogenesis and activation of the channel, we quantified the expression of individual subunits of ENaC in rat kidneys and colon using calibrated Western blots. The estimated abundance for the three subunits differed by an order of magnitude with the order γENaC ∼ βENaC ≫ αENaC in both organs. Transcript abundance in the kidney, measured with digital-drop PCR and RNAseq, was similar for the three subunits. In both organs, the calculated protein expression of all subunits was much larger than that required to account for maximal Na+ currents measured in these cells, implying a large excess of subunit protein. Whole-kidney biotinylation indicated that at least 5% of β and γ subunits in the kidney and 3% in the colon were expressed on the surface under conditions of salt restriction, which maximizes ENaC-dependent Na+ transport. This indicates a 10- to 100-fold excess of βENaC and γENaC subunits at the surface relative to the requirement for channel activity. We conclude that these epithelia make much more ENaC protein than is required for the physiological function of the channel. This could facilitate rapid regulation of the channels at the cell surface by insuring a large population of inactive, recruitable subunits.
 
Article
Piezo2 is a mechanosensitive ion channel that plays critical roles in sensing touch and pain, proprioception, and regulation of heart rate. Global knockout of Piezo2 leads to perinatal lethality in mice, and Piezo2 gain-of-function mutations are associated with distal arthrogryposis, a disease characterized by congenital joint contractures. Emerging evidence suggests that Piezo channels (Piezo1 and Piezo2) can be regulated by their local membrane environment and particularly by cholesterol and phosphoinositides. To characterize the local Piezo2 lipid environment and investigate key lipid-protein interactions, we carried out coarse-grained molecular dynamics simulations of Piezo2 embedded in a complex mammalian membrane containing >60 distinct lipid species. We show that Piezo2 alters its local membrane composition such that it becomes enriched with specific lipids, such as phosphoinositides, and forms specific, long-term interactions with a variety of lipids at functionally relevant sites.
 
Article
Ca2+ signals regulate the function of many immune cells and promote immune responses to infection, cancer, and autoantigens. Ca2+ influx in immune cells is mediated by store-operated Ca2+ entry (SOCE) that results from the opening of Ca2+ release-activated Ca2+ (CRAC) channels. The CRAC channel is formed by three plasma membrane proteins, ORAI1, ORAI2, and ORAI3. Of these, ORAI1 is the best studied and plays important roles in immune function. By contrast, the physiological role of ORAI3 in immune cells remains elusive. We show here that ORAI3 is expressed in many immune cells including macrophages, B cells, and T cells. To investigate ORAI3 function in immune cells, we generated Orai3-/- mice. The development of lymphoid and myeloid cells in the thymus and bone marrow was normal in Orai3-/- mice, as was the composition of immune cells in secondary lymphoid organs. Deletion of Orai3 did not affect SOCE in B cells and T cells but moderately enhanced SOCE in macrophages. Orai3-deficient macrophages, B cells, and T cells had normal effector functions in vitro. Immune responses in vivo, including humoral immunity (T cell dependent or independent) and antitumor immunity, were normal in Orai3-/- mice. Moreover, Orai3-/- mice showed no differences in susceptibility to septic shock, experimental autoimmune encephalomyelitis, or collagen-induced arthritis. We conclude that despite its expression in myeloid and lymphoid cells, ORAI3 appears to be dispensable or redundant for physiological and pathological immune responses mediated by these cells.
 
Article
Excitation-contraction coupling kinetics is dictated by the action potential rate of sinoatrial-nodal cells. These cells generate local Ca releases (LCRs) that activate Na/Ca exchanger current, which accelerates diastolic depolarization and determines the pace. LCRs are generated by clusters of ryanodine receptors, Ca release units (CRUs), residing in the sarcoplasmic reticulum. While CRU distribution exhibits substantial heterogeneity, its functional importance remains unknown. Using numerical modeling, here we show that with a square lattice distribution of CRUs, Ca-induced-Ca-release propagation during diastolic depolarization is insufficient for pacemaking within a broad range of realistic ICaL densities. Allowing each CRU to deviate randomly from its lattice position allows sparks to propagate, as observed experimentally. As disorder increases, the CRU distribution exhibits larger empty spaces and simultaneously CRU clusters, as in Poisson clumping. Propagating within the clusters, Ca release becomes synchronized, increasing action potential rate and reviving pacemaker function of dormant/nonfiring cells. However, cells with fully disordered CRU positions could not reach low firing rates and their β-adrenergic-receptor stimulation effect was substantially decreased. Inclusion of Cav1.3, a low-voltage activation L-type Ca channel isoform into ICaL, strongly increases recruitment of CRUs to fire during diastolic depolarization, increasing robustness of pacemaking and complementing effects of CRU distribution. Thus, order/disorder in CRU locations along with Cav1.3 expression regulates pacemaker function via synchronization of CRU firing. Excessive CRU disorder and/or overexpression of Cav1.3 boosts pacemaker function in the basal state, but limits the rate range, which may contribute to heart rate range decline with age and disease.
 
Article
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. To understand the origin of this difference, we obtained maps of autofluorescence lifetimes of flavin mononucleotide and dinucleotide (FMN/FAD) in cryosections, where excitation was set at 440 nm and emission at a bandwidth of between 500 and 570 nm, and paired them with immunofluorescence images of myosin heavy chain isoforms, which allowed identification of fiber types. In soleus, τ2 was 3.16 ns for type I (SD 0.11, 97 fibers), 3.45 ns for IIA (0.10, 69), and 3.46 ns for IIX (0.12, 65). In FDB muscle, τ2 was 3.17 ns for type I (0.08, 22), 3.46 ns for IIA (0.16, 48), and 3.66 ns for IIX (0.15, 43). From τ2 distributions, it follows that an FDB fiber with τ2 > 3.3 ns is expected to be of type II, and of type I otherwise. This simple classification method has first and second kind errors estimated at 0.02 and 0.10, which can be lowered by reducing the threshold for identification of type I and increasing it for type II. Lifetime maps of autofluorescence, therefore, constitute a tool to identify fiber types that, for being practical, fast, and noninvasive, can be applied in living tissue without compromising other experimental interventions.
 
Article
The zebrafish has emerged as a very relevant animal model for probing the pathophysiology of human skeletal muscle disorders. This vertebrate animal model displays a startle response characterized by high-frequency swimming activity powered by contraction of fast skeletal muscle fibers excited at extremely high frequencies, critical for escaping predators and capturing prey. Such intense muscle performance requires extremely fast properties of the contractile machinery but also of excitation-contraction coupling, the process by which an action potential spreading along the sarcolemma induces a change in configuration of the dihydropyridine receptors, resulting in intramembrane charge movements, which in turn triggers the release of Ca2+ from the sarcoplasmic reticulum. However, thus far, the fastest Ca2+ transients evoked by vertebrate muscle fibers has been described in muscles used to produce sounds, such as those in the toadfish swim bladder, but not in muscles used for locomotion. By performing intracellular Ca2+ measurements under voltage control in isolated fast skeletal muscle fibers from adult zebrafish and mouse, we demonstrate that fish fast muscle fibers display superfast kinetics of action potentials, intramembrane charge movements, and action potential-evoked Ca2+ transient, allowing fusion and fused sustained Ca2+ transients at frequencies of excitation much higher than in mouse fast skeletal muscle fibers and comparable to those recorded in muscles producing sounds. The present study is the first demonstration of superfast kinetics of excitation-contraction coupling in skeletal muscle allowing superfast locomotor behaviors in a vertebrate.
 
Article
It is controversial whether the cardiac type-2 ryanodine receptor harboring a catecholaminergic polymorphic ventricular tachycardia-associated point mutation is regulated by luminal or cytosolic Ca2+. This commentary discusses new findings supporting the cytosolic Ca2+-dependent regulation.
 
Article
Dysfunction of the sinoatrial node (SAN), the natural heart pacemaker, is common in heart failure (HF) patients. SAN spontaneous activity relies on various ion currents in the plasma membrane (voltage clock), but intracellular Ca2+ ([Ca2+]i) release via ryanodine receptor 2 (RYR2; Ca2+ clock) plays an important synergetic role. Whereas remodeling of voltage-clock components has been revealed in HF, less is known about possible alterations to the Ca2+ clock. Here, we analyzed [Ca2+]i handling in SAN from a mouse HF model after transverse aortic constriction (TAC) and compared it with sham-operated animals. ECG data from awake animals showed slower heart rate in HF mice upon autonomic nervous system blockade, indicating intrinsic sinus node dysfunction. Confocal microscopy analyses of SAN cells within whole tissue showed slower and less frequent [Ca2+]i transients in HF. This correlated with fewer and smaller spontaneous Ca2+ sparks in HF SAN cells, which associated with lower RYR2 protein expression level and reduced phosphorylation at the CaMKII site. Moreover, PLB phosphorylation at the CaMKII site was also decreased in HF, which could lead to reduced sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) function and lower sarcoplasmic reticulum Ca2+ content, further depressing the Ca2+ clock. The inhibition of CaMKII with KN93 slowed [Ca2+]i transient rate in both groups, but this effect was smaller in HF SAN, consistent with less CaMKII activation. In conclusion, our data uncover that the mechanism of intrinsic pacemaker dysfunction in HF involves reduced CaMKII activation.
 
Article
Type 2 ryanodine receptor (RYR2) is a cardiac Ca2+ release channel in the ER. Mutations in RYR2 are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT is associated with enhanced spontaneous Ca2+ release, which tends to occur when [Ca2+]ER reaches a threshold. Mutations lower the threshold [Ca2+]ER by increasing luminal Ca2+ sensitivity or enhancing cytosolic [Ca2+] ([Ca2+]cyt)-dependent activity. Here, to establish the mechanism relating the change in [Ca2+]cyt-dependent activity of RYR2 and the threshold [Ca2+]ER, we carried out cell-based experiments and in silico simulations. We expressed WT and CPVT-linked mutant RYR2s in HEK293 cells and measured [Ca2+]cyt and [Ca2+]ER using fluorescent Ca2+ indicators. CPVT RYR2 cells showed higher oscillation frequency and lower threshold [Ca2+]ER than WT cells. The [Ca2+]cyt-dependent activity at resting [Ca2+]cyt, Arest, was greater in CPVT mutants than in WT, and we found an inverse correlation between threshold [Ca2+]ER and Arest. In addition, lowering RYR2 expression increased the threshold [Ca2+]ER and a product of Arest, and the relative expression level for each mutant correlated with threshold [Ca2+]ER, suggesting that the threshold [Ca2+]ER depends on the net Ca2+ release rate via RYR2. Modeling reproduced Ca2+ oscillations with [Ca2+]cyt and [Ca2+]ER changes in WT and CPVT cells. Interestingly, the [Ca2+]cyt-dependent activity of specific mutations correlated with the age of disease onset in patients carrying them. Our data suggest that the reduction in threshold [Ca2+]ER for spontaneous Ca2+ release by CPVT mutation is explained by enhanced [Ca2+]cyt-dependent activity without requiring modulation of the [Ca2+]ER sensitivity of RYR2.
 
Article
The skeletal muscle voltage-gated calcium channel (CaV1.1) primarily functions as a voltage sensor for excitation–contraction coupling. Conversely, its ion-conducting function is modulated by multiple mechanisms within the pore-forming α1S subunit and the auxiliary α2δ-1 and γ1 subunits. In particular, developmentally regulated alternative splicing of exon 29, which inserts 19 amino acids in the extracellular IVS3-S4 loop of CaV1.1a, greatly reduces the current density and shifts the voltage dependence of activation to positive potentials outside the physiological range. We generated new HEK293 cell lines stably expressing α2δ-1, β3, and STAC3. When the adult (CaV1.1a) and embryonic (CaV1.1e) splice variants were expressed in these cells, the difference in the voltage dependence of activation observed in muscle cells was reproduced, but not the reduced current density of CaV1.1a. Only when we further coexpressed the γ1 subunit was the current density of CaV1.1a, but not that of CaV1.1e, reduced by >50%. In addition, γ1 caused a shift of the voltage dependence of inactivation to negative voltages in both variants. Thus, the current-reducing effect of γ1, unlike its effect on inactivation, is specifically dependent on the inclusion of exon 29 in CaV1.1a. Molecular structure modeling revealed several direct ionic interactions between residues in the IVS3-S4 loop and the γ1 subunit. However, substitution of these residues by alanine, individually or in combination, did not abolish the γ1-dependent reduction of current density, suggesting that structural rearrangements in CaV1.1a induced by inclusion of exon 29 may allosterically empower the γ1 subunit to exert its inhibitory action on CaV1.1 calcium currents.
 
Article
Cycling of Ca2+ between the sarcoplasmic reticulum (SR) and myoplasm is an important component of skeletal muscle resting metabolism. As part of this cycle, Ca2+ leaks from the SR into the myoplasm and is pumped back into the SR using ATP, which leads to the consumption of O2 and generation of heat. Ca2+ may leak through release channels or ryanodine receptors (RYRs). RYR Ca2+ leak can be monitored in a skinned fiber preparation in which leaked Ca2+ is pumped into the t-system and measured with a fluorescent dye. However, accurate quantification faces a number of hurdles. To overcome them, we developed a mathematical model of Ca2+ movement in these preparations. The model incorporated Ca2+ pumps that move Ca2+ from the myoplasm to the SR and from the junctional space (JS) to the t-system, Ca2+ buffering by EGTA in the JS and myoplasm and by buffers in the SR, and Ca2+ leaks from the SR into the JS and myoplasm and from the t-system into the myoplasm. The model accurately simulated Ca2+ uptake into the t-system, the relationship between myoplasmic [Ca2+] and steady-state t-system [Ca2+], and the effect of blocking RYR Ca2+ leak on t-system Ca2+ uptake. The magnitude of the leak through the RYRs would contribute ∼5% of the resting heat production of human muscle. In normal resting fibers, RYR Ca2+ leak makes a small contribution to resting metabolism. RYR-focused pathologies have the potential to increase RYR Ca2+ leak and the RYR leak component of resting metabolism.
 
Article
In skeletal muscle, depolarization of the plasma membrane (PM) causes conformational changes of the calcium channel CaV1.1 that then activate RYR1 to release calcium from the SR. Being independent of extracellular calcium entry, this process is termed voltage-induced calcium release. In skeletal muscle, junctophilins (JPHs) 1 and 2 form the SR–PM junctions at which voltage-induced calcium release occurs. Previous work demonstrated that JPH2 is able to recapitulate voltage-induced calcium release when expressed in HEK293 cells together with CaV1.1, β1a, Stac3, and RYR1. However, it is unknown whether JPH1 and the more distantly related neuronal JPH3 and JPH4 might also function in this manner, a question of interest because different JPH isoforms diverge in their interactions with RYR1. Here, we show that, like JPH2, JPH1 and JPH3, coexpressed with CaV1.1, β1a, Stac3, and RYR1 in HEK293 cells, cause colocalization of CaV1.1 and RYR1 at ER–PM junctions. Furthermore, potassium depolarization elicited cytoplasmic calcium transients in cells in which WT CaV1.1 was replaced with the calcium impermeant mutant CaV1.1(N617D), indicating that JPH1, JPH2, and JPH3 can all support voltage-induced calcium release, despite sequence divergence and differences in interaction with RYR1. Conversely, JPH4-induced ER–PM junctions contain CaV1.1 but not RYR1, and cells expressing JPH4 are unable to produce depolarization-induced calcium transients. Thus, JPHs seem to act primarily to form ER–PM junctions and to recruit the necessary signaling proteins to these junctions but appear not to be directly involved in the functional interactions between these proteins.
 
Article
The inhibitor of store-operated Ca2+ entry (SOCE) BTP2 was reported to inhibit ryanodine receptor Ca2+ leak and electrically evoked Ca2+ release from the sarcoplasmic reticulum when introduced into mechanically skinned muscle fibers. However, it is unclear how effects of intracellular application of a highly lipophilic drug like BTP2 on Ca2+ release during excitation–contraction (EC) coupling compare with extracellular exposure in intact muscle fibers. Here, we address this question by quantifying the effect of short- and long-term exposure to 10 and 20 µM BTP2 on the magnitude and kinetics of electrically evoked Ca2+ release in intact mouse flexor digitorum brevis muscle fibers. Our results demonstrate that neither the magnitude nor the kinetics of electrically evoked Ca2+ release evoked during repetitive electrical stimulation were altered by brief exposure (2 min) to either BTP2 concentration. However, BTP2 did reduce the magnitude of electrically evoked Ca2+ release in intact fibers when applied extracellularly for a prolonged period of time (30 min at 10 µM or 10 min at 20 µM), consistent with slow diffusion of the lipophilic drug across the plasma membrane. Together, these results indicate that the time course and impact of BTP2 on Ca2+ release during EC coupling in skeletal muscle depends strongly on whether the drug is applied intracellularly or extracellularly. Further, these results demonstrate that electrically evoked Ca2+ release in intact muscle fibers is unaltered by extracellular application of 10 µM BTP2 for <25 min, validating this use to assess the role of SOCE in the absence of an effect on EC coupling.
 
Article
Atrial fibrillation (AF) has been linked to the remodeling of membrane receptors and alterations in downstream cAMP-dependent regulation. However, to date, no study has elucidated how the increase on cAMP upon different G-protein-coupled receptors (GPCRs) can lead to different physiological compartmentalized responses. The aim of this study was to investigate the compartmentally specific effects of GPCRs on cAMP levels in human atrial myocytes (HAMs) from patients with AF and control patients without AF (Ctl), and how these compartmentalized effects are altered in AF. HAMs were isolated from 60 AF and 76 Ctl patient tissues. Cells were transduced with adenoviruses (Epac1-camps, pm-Epac1-camps and Epac1-JNC) and cultured for 48 hours to express the FRET-based cAMP sensor in the cytosolic, membrane, and RYR2 nanodomains. Förster-resonance energy transfer (FRET) was used to measure cAMP levels in 525 HAMs stimulated with isoprenaline (100 µM), serotonin (100 µM), or the A2AR agonist CGS (200 nM). A desensitization to β-adrenergic receptor stimulation was exclusively found in the cytosol of AF myocytes, while no difference was seen in the RYR2 or LTCC compartment. Similar effects were observed upon serotonin stimulation with a significant desensitization in the cytosol, and no difference in the RYR2 compartment. In response to A2ARs stimulation AF myocytes displayed a significantly higher cytosolic increase in cAMP levels. However, no response was seen in the LTCC compartment in response to serotonin or A2AR stimulation. Collectively, our data show that cAMP levels are highly compartmentalized and differentially regulated by GPCRs. Furthermore, these results provide a mechanistic insight for the previously reported functional effects seen upon stimulation of these three receptors.
 
Article
We previously showed that RYR2 tetramers are distributed nonuniformly within ventricular dyads, and that physiological and pathological factors can alter their relative positions. Agents that decreased Ca2+ spark frequency, high Mg2+, and saturating concentrations of the immunophilins FKBP12 and FKBP12.6 drew the receptors together, minimizing their nearest-neighbor distance and reducing the size of the clusters. Activating kinases with a phosphorylation cocktail did the opposite. The purpose of this study is to test the hypothesis that phosphorylation of RYR2 is required for the structural changes we have observed. We measured junctional sarcoplasmic reticulum (jSR) lengths using 2-D transmission electron microscopy (TEM) and directly visualized RYR2 distribution using dual-tilt electron tomography in phosphomutant mice S2808A, S2814A, S2814D, and S2030A. Mouse hearts were hung on a Langendorff and treated with either saline or 300 nmol/liter isoproterenol (ISO) for 2 min before being fixed and sectioned for analysis. We found that (1) RYR2 distribution in mouse ventricles is comparable to that reported for rats and humans, (2) the response to ISO applied to an intact, beating heart is identical to a phosphorylation cocktail applied to isolated permeabilized myocytes, and (3) all of the mutations produced significant changes in the tetramer arrangements and/or NND relative to wild-type (WT) mice. Our 2-D TEM measurements showed that (1) in WT mice, ISO significantly increased the length of the jSR, (2) ISO significantly increased the jSR lengths of WT, S2814A, and S2808A mice, but not the S2030A mouse, and (3) the jSR length of the S2814D mouse was significantly greater than WT, but not WT + ISO or S2814D + ISO, indicating that a mutation of the RYR2 alone caused a significant change in the jSR length. These results indicate that the tetramers and the jSR form a structural syncytium.
 
Article
Lead is a heavy metal pollutant that constitutes frequent exposomes. It is nonbiodegradable and has a nonsafe limit of exposure. It has multisystemic effects, and most of the cardiac effects have been discovered to be indirect. There are strong similarities between Ca2+ and Pb2+ in their chemistry. Because cardiac function is dramatically dependent in extracellular Ca2+, as well as in precise control of intracellular Ca2+, we tested if Pb2+ could antagonize Ca2+-dependent effects in a short amount of time. Acute exposure of isolated hearts showed a negative inotropic effect. In guinea pig isolated cardiomyocytes loaded with a Pb2+-specific dye (Leadmium green), our results showed that there was an associated increment in fluorescence related to extracellular stimulation blocked by 1–5 µM DHP. Calcium currents were partially blocked by extracellular Pb2+, though currents seemed to last longer after a fast inactivation. Charge movement from gating currents was slightly hastened over time, giving an appearance of a slight reduction in the Cav1.2 gating currents. Action potentials were prolonged in Pb2+ compared with Ca2+. In isolated cardiomyocytes loaded with Ca2+-sensitive dyes, Ca2+ variations promoted by extracellular stimuli were affected in space/time. As Pb2+ could interfere with Ca2+-sensitive dyes, we measured contraction of isolated cardiomyocytes under extracellular stimuli in Pb2+. In both Ca2+ dye fluorescence and contractions, Pb2+ disorganizes the pattern of contraction and intracellular Ca2+ homeostasis. Our results suggest that (1) Pb2+ enters to cardiomyocytes through Cav1.2 channels, and (2) once it enters the cell, Pb2+ may substitute Ca2+ in Ca2+-binding proteins. In addition to these direct mechanisms related to Pb2+ competition with Ca2+-binding sites, we cannot discard a direct contribution of Pb2+ redox properties.
 
Article
Septins are considered as the fourth component of the cytoskeleton, with septin-7 isoform playing a critical role in myogenic cell division and fusion. Skeletal muscle regeneration is a highly orchestrated process that requires many steps, including proper cell division to achieve functional recovery. Here, the role of septin-7 was investigated in this complex process. To this end, muscle injury was induced in wild type BL6/C57 and septin-7–conditional (mer-Cre-mer) knock-down mice by in vivo BaCl2 injection to the left m. tibialis anterior muscle (TA) of the mice (the right m. tibialis anterior muscle was nontreated control). Mice were sacrificed 4 and 14 d later to reflect the early (monitored by PAX7 level) and late (monitored by myogenin level) phases of muscle regeneration. Western blotting was used to follow the changes of septin-7, PAX7, and myogenin expression at the protein level, while changes of mRNA were detected by qPCR. Morphological differences were visualized by HE staining. Levels of septin-7 protein increased 4 and 14 d after injury in BL6/C57 mice and mRNA expression of SEPT7 showed significant elevation both 4 and 14 d after injection in Cre+ mice only, considered to be a compensatory increase of mRNA expression of SEPT7 in order to ensure the appropriate regeneration process. Furthermore, up-regulation of septin-7 protein was more pronounced on day 14 in both Cre− and Cre+ mice, which may indicate its importance in the later phase of regeneration. Level of PAX7 and myogenin were also increased 4 and 14 d after injury in BL6/C57, Cre−, and Cre+ mice, respectively. Taken together, our data suggest the importance of septin-7 in skeletal muscle regeneration.
 
Article
Twitch force potentiation of fast-twitch skeletal muscle is produced by repetitive stimulation that can be achieved from either (1) the staircase effect (continual low frequency stimulation) or (2) post-tetanic potentiation (a 1–2 s high-frequency tetanic stimulation). Previous studies examining twitch force potentiation have been conducted in vitro and shown that it is related to phosphorylation of myosin regulatory light chain (pRLC). We previously found, in vitro, reduced potentiation of twitch force and decreased pRLC in ovariectomized (Ovx, estrogen-deficient) compared with sham-operated (estrogen-replete) mice. Thus, we questioned whether this phenomenon occurred in vivo and whether age and sex would affect the potentiation of twitch force. Using an in vivo post-tetanic potentiation method (one twitch contraction followed by a tetanic contraction—100 Hz for 1,000 ms with 0.01 ms pulses, and two post-tetanic twitch contractions), we investigated twitch torque potentiation in C57BL/6 young and old, male and female mice. There were significant main effects of sex (P < 0.001) and age (P < 0.001) on body mass and significant main effects of sex (P < 0.001) on tibialis anterior and extensor digitorum longus muscle masses, with males and aged being relatively greater. Analysis of twitch torque using a three-way ANOVA across time, age, and sex showed a significant main effect of time (pre < post; P < 0.001), time × age (P = 0.038), and time × sex (P = 0.028), indicating potentiation occurred in young and old, males and females. Analysis of twitch torque potentiation (percent increase) using a two-way ANOVA revealed a significant main effect of age (young = 45.16 ± 2.04 versus old = 27.88 ± 9.96; P < 0.001) with no effect of sex (P = 0.215). In summary, enhanced generation of twitch force of skeletal muscle using a post-tetanic potentiation method does occur in vivo and is affected by age but not sex, as there is greater twitch torque potentiation in young than old mice.
 
Article
The recurrent attacks of weakness in hypokalemic periodic paralysis (HypoPP) are caused by failure to maintain the resting potential, with paradoxical depolarization in low K+. Remarkably, 24 out of 25 HypoPP mutations are R/X substitutions in S4 segments of voltage-sensing domains of CaV1.1 (70% of cases) or NaV1.4 (10% of cases). Expression studies in oocytes and murine muscle show anomalous gating pore leakage currents (ω-pore) for six of eight CaV1.1-HypoPP mutations, with one exception being the charge-conserving R897K. The proposed consensus pathomechanism, whereby a gating pore leak predisposes to paradoxical depolarization in low K+, is now verified by continuous recording of Vm. Selective measurement of voltage-dependent Ca2+ release, in “healthy appearing” HypoPP fibers, shows only a modest decrease in the Ca2+-dependent peak fluorescence (Oregon green 488/EGTA), and supports the notion that stabilizing Vrest will be sufficient to prevent low-K+–induced loss of force. In our knockin mouse models of HypoPP (CaV1.1-R528H and NaV1.4-R669H), pretreatment with K+-channel openers protects against the loss of force with a 2 mM K+ challenge. Alternatively, gene editing offers the possibility of sustained protection from attacks of weakness, and may prevent the late-onset permanent myopathy. In a proof-of-principle study of cultured myoblasts and in vivo electroporation, we show selective editing of the mutant HypoPP allele, without compromise of the WT allele, using CRISPR/Cas-mediated indel formation to destroy the HypoPP allele or a CRISPR/Cas base editor to correct the missense mutation.
 
Article
Pancreatic β cells express several high voltage-gated Ca2+ channel (HVCC) isoforms critical for insulin release, cell differentiation, and survival. RNaseq and qPCR analyses demonstrated that CACNA1D gene encoding for CaV1.3-α1D isoform is highly expressed in pancreatic islets of both mice and men. Additionally, CACNA1D genetic polymorphisms were associated with increased susceptibility for diabetes while CaV1.3 gain-of-function mutations cause hyperinsulinemia in humans. Nevertheless, functional evidence for the role of CaV1.3 on β-cell electrical activity, insulin release, and β-cell mass is contradictory and largely unknown. Here, we show that CaV1.3 deletion led to a sixfold increase in DNA damage and a threefold decrease in proliferation markers in pancreatic β cells of 14-d-old mice, while adult mice were largely unaffected. However, β-cell mass was reduced by ∼20% in both young and old mice, resulting in a diminished sustained insulin release. Voltage-clamp recordings in β-cells of 14-d-old mice showed an ∼20% reduction in total Ca2+ influx (WT Ipeak = −19.76 ± 1.04 pA/pF; CaV1.3−/− Ipeak = −14.84 ± 0.61 pA/pF, P = 0.001) accompanied by slower inactivation and an ∼5 mV rightwards shift in the voltage dependence of activation (WT V1/2 = −7.71 ± 0.82 mV; CaV1.3−/− V1/2 = −2.32 ± 1.09 mV, P = 0.0003). Although to a lower extent, Ca2+ influx in adult CaV1.3−/− β cells was similarly affected. Moreover, current-clamp recordings showed that CaV1.3 deletion delayed the glucose-induced action potential (AP) onset, reduced AP firing frequency (e.g., at 7.5 mM glucose, WT = 4.3 Hz; CaV1.3−/− = 2.1 Hz, P = 0.001) and AP-train frequency (e.g., at 7.5 mM glucose intertrain interval, WT = 49.3 ± 9.6 s; CaV1.3−/− = 120.3 ± 25.5 s, P = 0.04) in both young and adult β cells. Therefore, our data demonstrate that the CaV1.3 channel is required for the initiation of glucose-induced β-cell electrical activity and modulates β-cell mass and insulin release in both young and old mice.
 
Article
Heart failure (HF) is a complex syndrome in which death rates are over 50%. The main cause of death among HF patients is pump failure and ventricular arrhythmias, but severe bradycardia is also a common cause of sudden cardiac death, pointing to sinoatrial node (SAN) dysfunction. SAN pacemaker activity is regulated by voltage-clock and Ca2+-clock mechanisms and, although voltage-clock dysfunction in SAN has been largely proved in HF, Ca2+-clock dysfunction mechanisms in SAN remains undiscovered. Here, we used a HF model in mice with transverse aortic constriction (TAC) and using telemetry saw slower heart rhythm under autonomic nervous system blockade. Then, using confocal microscopy we analyzed Ca2+ handling in HF SAN tissue and found that intracellular Ca2+ transient rates were slower in addition to less frequency of Ca2+ sparks than in SHAM SAN tissue. Next, we studied protein expression of key excitation–contraction coupling proteins and found reduced expression of the Na+/Ca2+ exchanger and reduced phosphorylated status of ryanodine receptor and phospholamban in the CaMKII sites for the SAN in TAC mice. Finally, the application of the CaMKII inhibitor KN93 caused less effect in slowing the Ca2+ transient rates in HF SAN tissue, confirming the reduced CaMKII activation. In conclusion, our data demonstrate a reduction in CaMKII activation in the Ca2+-clock function of the SAN tissue in a mouse model of HF.
 
Article
Duchenne muscular dystrophy (DMD) is a fatal X-linked genetic disease characterized by progressive loss of skeletal muscle. The mechanisms underlying the DMD pathology likely involve the complex interaction between reactive oxygen species (ROS) impaired Ca2+ handling and chronic inflammation, characterized by the presence of immune cells such as neutrophils. Hypochlorous acid (HOCl) is a highly reactive form of ROS produced endogenously via the actions of myeloperoxidase, an enzyme secreted by neutrophils. Myeloperoxidase activity is significantly elevated in dystrophic muscle. This study aimed to determine the effect of HOCl exposure on excitation–contraction coupling and its potential contribution to the dystrophic pathology. Isolated extensor digitorum longus (EDL) muscles and single fibers from C57 (wild type) and mdx (dystrophic) mice were used to investigate the effects of HOCl on whole muscle function, intracellular Ca2+ handling, and myofilament force production. HOCl exposure significantly decreased maximum specific force in isolated EDL muscles by 26% and 49%, respectively, in C57 and mdx mice (P < 0.0001). In single interosseous fibers, HOCl exposure significantly increased resting intracellular Ca2+ concentration by ∼17–19% (P < 0.05) and decreased the amplitude of electrically induced Ca2+ transients by ∼45% and 50%, respectively, in C57 and mdx fibers (C57, P < 0.05; mdx, P < 0.01). These effects of HOCl on resting Ca2+ could be blocked via application of tetracaine (ryanodine receptor blocker) or Gd3+ (stretch-activated channel blocker; C57, P < 0.01; mdx, P < 0.01 for both). The effect of HOCl on Ca2+ transient amplitude was significantly reduced by Gd3+ (C57, P < 0.05; mdx, P < 0.01). In chemically skinned EDL fibers, HOCl exposure decreased maximum Ca2+-activated force by ∼40% in both C57 and mdx fibers (P < 0.001). These results indicate that HOCl potently affects excitation–contraction coupling via impaired Ca2+ handling and myofilament force production. Hence, HOCl potentially links the chronic inflammation, oxidative stress, and impaired Ca2+ handling that underlies the dystrophic pathology.
 
Article
Exertional/environmental heat strokes (EHSs) are hyperthermic crises triggered by strenuous physical exercise and/or exposure to environmental heat, and are caused by an altered intracellular Ca2+ homeostasis in muscle. We recently demonstrated that a single bout of exercise on treadmill leads to formation of calcium entry units (CEUs), intracellular junctions that promote interaction between STIM1 and Orai1, the two proteins that mediate store-operated Ca2+ entry (SOCE). SOCE is a mechanism that is activated during muscle fatigue and that allows for recovery of extracellular Ca2+ during prolonged activity. The hypothesis underlying this work is that assembly of CEUs during prolonged exercise may predispose to EHSs when exercise is performed in challenging environmental conditions. To test this hypothesis, 4-mo-old mice were (1) divided into three experimental groups: control, trained-1m (1 mo of voluntary running in wheel cages), and exercised-1h (1 h of incremental treadmill run); and (2) subjected to an exertional stress (ES) protocol consisting of an incremental 45-min treadmill run at 34°C and 40% humidity. We then (a) measured the internal temperature of mice, which was higher in the two pre-exercised groups (trained-1m: 38.9°C ± 0.33; exercised-1h: 38.7°C ± 0.40) compared with control animals (37.9°C ± 0.17); (b) applied an ex vivo ES protocol to isolated EDL muscles (tetanic stimulation performed at 30°C) and verified that samples from trained-1m and exercised-1h mice generated a tension significantly greater than control samples; and (c) analyzed CEUs by electron microscopy (EM) and verified that EDL muscles of trained-1m and exercised-1h mice contained a greater number of membranes elements forming CEUs. The data collected indicates that the presence of CEUs correlates with a greater increase in body temperature and could, in principle, predispose to EHS when exercise is performed in challenging environmental conditions.
 
Article
Cerebral blood flow (CBF) is exquisitely controlled to meet the ever-changing demands of active neurons in the brain. Brain capillaries are equipped with sensors of neurovascular coupling agents released from neurons/astrocytes onto the outer wall of a capillary. While capillaries can translate external signals into electrical and Ca2+ changes, control mechanisms from the lumen are less clear. The continuous flux of red blood cells and plasma through narrow-diameter capillaries imposes mechanical forces on the luminal (inner) capillary wall. Whether—and, if so, how—the ever-changing CBF could be mechanically sensed in capillaries is not known. Here, we propose and provide evidence that the mechanosensitive Piezo1 channels operate as mechanosensors in CNS capillaries to ultimately regulate CBF. Patch clamp electrophysiology confirmed the expression and function of Piezo1 channels in brain cortical and retinal capillary endothelial cells. Mechanical or pharmacological activation of Piezo1 channels evoked currents that were sensitive to Piezo1 channel blockers. Using genetically encoded Ca2+ indicator (Cdh5-GCaMP8) mice, we observed that Piezo1 channel activation triggered Ca2+ signals in endothelial cells. An ex vivo pressurized retina preparation was employed to further explore the mechanosensitivity of capillary Piezo1-mediated Ca2+ signals. Genetic and pharmacologic manipulation of Piezo1 in endothelial cells had significant impacts on CBF, reemphasizing the crucial role of mechanosensation in blood flow control. In conclusion, this study shows that Piezo1 channels act as mechanosensors in capillaries, and that these channels initiate crucial Ca2+ signals. We further show that Piezo1 modulates CBF, an observation of profound significance for the control of brain blood flow in health and in disorders where hemodynamic forces are disrupted, such as hypertension.
 
Article
Although it is well known that ion channels conduct ions across biomembranes, whether ions are conducted by some non-membrane proteins is not known because of the lack of a detection method. Calsequestrin-2 (CSQ2) is a sarcoplasmic reticulum (SR) Ca2+-binding protein suppling Ca2+ for the ryanodine receptor Ca2+ release during the excitation–contraction coupling in cardiomyocytes. CSQ2 mutations, even in some heterozygous occasions, causes catecholaminergic polymorphic ventricular tachycardia (CPVT2), suggesting that CSQ2 may function beyond a Ca2+ buffer. Here, we identify a non-transmembrane channel in Ca2+-enriched CSQ2 dimers, which facilitates fast Ca2+ mobilization. Using crystallography, we solved the high-resolution structure of Ca2+-bound CSQ2 and discovered that the negatively charged residues at the dimer interface encompassed a tubular channel-like structure, dubbed “tunnel,” in which ∼15 Ca2+ ions aligned across the ∼5 nm tunnel path. To determine the potential tunnel conductance, we developed a graphene-based nanoelectronic technology to connect a CSQ2 dimer into a nanocircuit. In the Tyrode solution containing 1 mM Ca2+, a CSQ2 dimer exhibited a conductance one order of magnitude higher than the background level. This conductance was Ca2+ dependent, and was largely suppressed by the single-residue mutation D309N at the bottleneck region of the tunnel path, indicating that the tunnel conducted Ca2+ fluxes. When the D309N mutant CSQ2 was expressed in wild-type rat cardiomyocytes by adenoviral vectors, isoproterenol treatment induced chaotic Ca2+ waves, delayed after-depolarizations and trigged activities resembling those occurring in CPVT2 models. This dominant negative effect of CSQ2 mutation agreed well with our structural observation that CSQ2 tunnels were interconnected to form a tunnel network. Taken together, these results revealed that CSQ2 builds a nano-highway network for energy-efficient Ca2+ mobilization in the SR. Factors that block the Ca2+ highway may lead to arrhythmogenesis.
 
Article
The zebrafish has emerged as a very relevant animal model to decipher the pathophysiology of human muscle disorders. However, the vast majority of studies on zebrafish skeletal muscle have investigated genetic, histological, and molecular aspects, but functional approaches at the cellular level, especially in the field of excitation–contraction (EC) coupling, are scarcer and generally limited to cultured myotubes or fibers from embryonic zebrafish. Considering that zebrafish undergoes profound metamorphosis during transition from larval to adult stage and that number of muscle pathologies come up at ages far beyond embryonic stages, there is an actual need to investigate EC coupling in fully differentiated zebrafish skeletal muscle. In the present study, we were able to implement current and voltage clamp combined with intracellular Ca2+ measurements using the intracellularly loaded Ca2+ dye indo-1 in enzymatically isolated fast skeletal muscle fibers from 1-yr old zebrafish. Recording of action potentials (AP) in current-clamp conditions revealed very fast kinetics of the repolarization phase of AP. Measurements of intramembrane charge movements in voltage-clamp conditions showed that charge movement density was half that measured in mammalian fibers, but they displayed much faster kinetics. Ca2+ transients elicited by depolarization displayed a voltage-dependent phase of activation and voltage- and time-dependent phase of inactivation. Recording of Ca2+ signals elicited by trains of AP at different rates in current-clamp conditions indicated that Ca2+ signals fused at very high stimulation frequencies with no sign of Ca2+ signal decay for the entire 0.5 s duration of the stimulation, giving evidence that fibers were still able to generate AP and the sarcoplasmic reticulum to release Ca2+ with stimulation rates as high as 200 Hz. These data indicate that adult zebrafish fast skeletal muscle fibers exhibit strikingly fast kinetics of EC coupling from AP firing to charge movements and sarcoplasmic reticulum Ca2+ release.
 
Article
The mechanisms that link the primary increase in SR Ca2+ leak of MH susceptibility and related conditions to their disease phenotypes are not well understood. We found that abnormal Ca2+ homeostasis in MHS individuals induces proteolysis of junctophilin1 (JPh1), an essential structural protein of EC coupling (Perni, in 2017). Guo (in 2018) and Lahiri (in 2020) reported similar fragmentation of JPh2 in stressed hearts. Western blot of patients’ muscle with domain-specific antibodies showed a deficit of full-length JPh1 and excess of a 44-kD C-terminal fragment (JPh44) in MHS subjects. While JPh1 was located in T-SR junctions, JPh44 was found anywhere within the I band, and at high densities within nuclei—a location forbidden for JPh1. Expression and cleavage in mice of a JPh1 plasmid tagged at both ends showed that its N-terminal fragment remained in triads, and the C-terminal fragment, orthologue to JPh44, entered nuclei, which indicates that JPh44 is the C-terminal cleavage product. Endogenous calpain1 appeared in T-SR junctions, colocalized with JPh1. On muscle extracts and primary cultures, Ca2+-activated calpain1 cleaved a 44-kD JPh1 piece, consistent with the C-terminal fragment that starts at Ser241, the highest probability cleavage site found by calpain1 algorithms. Completing the identification of Ser241 as the likely start of JPh44, the tagged deletion plasmid GFP-JPh1_Δ1-240, expressed in mice, copied the location and migration of JPh44. Expression of GFP-JPh1_Δ1-240 in C2C12 myoblasts reduced by more than twofold the transcription of PI3K-Akt genes that inhibit muscle uptake and storage of glucose, including GSK3β, an inhibitor of glycogen synthase that is activated in MHS patients. In agreement with the genetic profile, GSK3β protein content decreased upon expression of GFP-JPh1_Δ1-240. In sum, the identified gene control roles of JPh44 oppose the deleterious effects of chronically elevated cytosolic [Ca2+], including late-onset hyperglycemia and type-2 diabetes (Tammineni, in 2020).
 
Article
Diamide insecticides target insect ryanodine receptors (RYRs) and cause dysregulation of calcium signaling in insect muscles and neurons, generating worldwide sales over 2 billion US dollars annually. Several resistance mutations have been reported to reduce the efficacy of the diamides, but the exact binding sites and mechanism of resistance mutations were not clear. Recently, we solved the cryo-electron microscopy (cryo-EM) structure of RYR in complex with the anthranilic diamide chlorantraniliprole (CHL). CHL binds to the pseudo–voltage-sensor domain (pVSD) of RYR, a site in proximity to the previously identified resistance mutations. Mutagenesis studies in silico, in mutant cell lines, and in transgenic Drosophila strains revealed the key residues involved in diamide coordination and the molecular mechanism under species-selectivity and resistance mutations. We also proposed that CHL may alleviate the loss-of-function effects of some central core disease (CCD) mutations by increasing the opening probability (Po) of RYR1. In addition, we solved the crystal structures of several RYR domains from the diamondback moth and the bee, revealing insect-specific structural features which could be potentially targeted by novel insecticides. Interestingly, we found that the phosphorylation of insect RYR is temperature dependent, facilitated by the low thermal stability and dynamic structure of the insect RYR. Our structures provide a foundation for developing novel pesticides to overcome the resistance crisis.
 
Article
An important question in neuromuscular biology is how skeletal muscle cells decipher the stimulation pattern coming from motoneurons to define their phenotype-activating transcriptional changes in a process named excitation–transcription coupling. We have shown in adult muscle fibers that 20 Hz electrical stimulation (ES) activates a signaling cascade that starts with Cav1.1 activation, ATP release trough pannexin-1 channel, activation of purinergic receptors, and IP3-dependent Ca2+ signals inducing transcriptional changes related to muscle plasticity from fast to slow phenotype. Extracellular addition of 30 µM ATP mimics transcriptional changes induced by ES at 20 Hz. ATP release occurs in two peaks, the first around 15 s after ES and a second around 300 s after ES. In the present work, we used apyrase to hydrolyze ATP 60 s after ES, maintaining the first peak and eliminating the second peak. In this condition, transcriptional changes were abolished, indicating that the second peak is the one crucial to activate transcription. Additionally, we observed a small depolarization of fibers after ES. The addition of 30 to 100 µM external ATP also induced depolarization of muscle fibers. This depolarization was unable to activate contraction but was able to induce transcriptional changes induced by 20 Hz ES. These changes were completely inhibited by the IP3R blocker xestospongin B, suggesting that IP3-dependent events are triggered at these membrane depolarization values. Moreover, transcriptional changes induced by addition of 30 µM extracellular ATP was blocked by incubation of fibers with 25 µM Nifedipine. These results suggest that the second ATP peak observed after 20 Hz ES is responsible for transcriptional activation by inducing small depolarizations of fiber membranes that are also sensed by Cav1.1. Finally, we show evidence that downstream of purinergic receptors, PKC is activated, likely causing phosphorylation of ClC-1 chloride channels, possibly responsible for depolarization after 20 Hz.
 
Article
Calmodulin (CaM) prevents proarrhythmic late sodium current (INa) by facilitating normal inactivation of sodium channels (NaV). Since dysfunction of NaV1.6 has been implicated in late INa-mediated arrhythmias, we investigated its role in arrhythmias promoted by CaM mutant D96V. Super-resolution STED microscopy revealed enlarged NaV1.6 clusters in NaV1.6-expressing Chinese hamster ovary cells transfected with D96V-CaM relative to those transfected with WT-CaM. Therefore, we examined NaV1.6 clustering in transgenic mice with cardiac-specific expression of D96V-CaM (cD96V) with a C-terminal FLAG tag. Confocal microscopy confirmed expression of NaV1.6 and FLAG-tagged D96V-CaM in a striated pattern along with RYR2 in cD96V hearts, consistent with T-tubular localization. In both WT and cD96V hearts, STORM single molecule localization microscopy revealed that ∼50% of NaV1.6 clusters localized <100 nm from RYR2. However, NaV1.6 density within these regions was 67% greater in cD96V relative to WT. Consistent with this result, SICM-guided “smart” patch clamp recording of NaV activity from T-tubule openings revealed more frequent late-burst openings involving larger NaV clusters in cD96V myocytes relative to WT. Previous work identifies the sodium-calcium exchanger (NCX) as a key link between aberrant late NaV1.6 activity and proarrhythmic Ca2+ mishandling. Therefore, we explored the spatial organization of NaV1.6 and NCX using STORM. Consistent with their close association, 89% of NaV1.6 clusters localized <100 nm from NCX in cD96V hearts, compared with 77% in WT. Notably, density of both NaV1.6 and NCX was increased at these sites by 48% and 31%, respectively, in cD96V relative to WT. Consistent with these data, cD96V myocytes displayed larger, more frequent Ca2+ sparks relative to WT. These proarrhythmic functional effects were abrogated by cardiac-specific knockout of NaV1.6. To our knowledge, this is the first demonstration of proarrhythmic cardiac structural remodeling secondary to a defect in calmodulin, offering novel mechanistic insight into calmodulinopathy.
 
Article
Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. While ∼50% of patients with HCM carry a sarcomere gene mutation (sarcomere mutation-positive, SMP), the genetic background is unknown in the other half of the patients (sarcomere mutation-negative, SMN). Gene mutations are most often present in genes encoding the sarcomere proteins myosin heavy chain, myosin-binding protein C, and troponin T. Studies in cardiac tissue samples from patients with obstructive HCM that were obtained during myectomy surgery showed increased myofilament calcium sensitivity, increased kinetics and tension cost, and a reduction of the super-relaxed state of myosin, which is associated with an energy-conserving status of the crossbridges. The increase in myofilament calcium sensitivity is observed at a low dose of mutant protein, while the magnitude of the increase in calcium sensitivity depends on the specific mutation location. These mutation-mediated myofilament changes may underlie inefficient in vivo cardiac performance in mutation carriers. Reduced cardiac efficiency has been observed before onset of cardiac hypertrophy and at advanced disease stages. In addition, impaired diastolic function is an early disease characteristic of HCM. Our recent proteomics studies revealed increased detyrosination of microtubules, which may be a cause of diastolic dysfunction. Recent treatments that target inefficient cardiac performance, such as myosin inhibitors and metabolic drug therapies, may have the potential to prevent, delay, or even reverse disease in HCM-mutation carriers. Treatment response may depend on the specific gene mutation in SMP individuals and may explain diverse response of HCM patients to therapy. While mutation-mediated cardiomyocyte defects have become clear in past years, more research is warranted to define the cellular pathomechanisms of cardiac dysfunction in SMN patients.
 
Article
In 2017, Boncompagni, Michelucci et al. demonstrated that during exercise the sarcotubular system of extensor digitorum longus (EDL) fibers undergoes a profound remodeling that leads to the assembly of new junctions between T-tubule extensions at the I band and sarcoplasmic reticulum (SR) stacks. As these junctions contain colocalized STIM1 and Orai1 and enhance store-operated Ca2+ entry (SOCE), they have been named Ca2+ entry units (CEUs). In addition, it has been more recently shown that (1) CEUs disassemble following recovery, with T-tubules retraction from the I band faster than SR stacks disassembly, and (2) lack of calsequestrin-1 (CASQ1) induces a constitutive assembly of CEUs, resulting in enhanced SOCE that counteracts the SR Ca2+ depletion. We have now analyzed (1) CEUs during postnatal maturation (at 2 wk of age) and (2) whether CEUs form in slow-twitch fibers (soleus). (a) Compared with adult (4 mo) EDL fibers of resting WT mice, at 2 wk of age we found a greater longitudinal disposition of T-tubules associated to SR membranes forming junctions virtually identical to CEUs in adult EDLs of exercised WT mice, which promote increased STIM1/Orai1-mediated SOCE. (b) We also compared structure and function of soleus (which also express the cardiac isoform CASQ2) from WT mice and from mice lacking either CASQ1 (CASQ1-null) or CASQ1/2 (dCASQ-null). In soleus from both genotypes, CEUs are constitutively assembled although they appear structurally smaller than those described previously in exercised WT or CASQ1-null EDLs. A detailed EM quantitative analysis revealed that CEUs were more abundant in dCASQ-null than CASQ1-null mice. The amount of CEUs strictly correlated with the ability of soleus fibers to recover extracellular Ca2+ via SOCE to support contractility during high-frequency stimulation. These data were supported by molecular analysis of Western blots, showing that Orai1 expression was enhanced following ablation of CASQ.
 
Article
It is well established that abnormalities in [Ca2+] regulation occur in heart diseases. Actually, independent studies demonstrated that Orai1/2/3 and TRPC protein related with store-operated calcium channels (SOCC) have a role in cardiac pathologies. Ischemia/reperfusion (I/R) stimulates transcription factor activation that modifies the expression of genes implicated in the pathogenesis of this process. Previous results described an increase in the expression of Orai1 and TRPC5 in cardiomyocytes after I/R, although the molecular mechanisms that mediate this regulation are still unknown. The aim of this study is to examine the molecular mechanisms implicated in the regulation of SOCC in cardiomyocytes after I/R focusing on the handling of intracellular [Ca2+]. Experiments were performed in a rat model of myocardial I/R, in adult (ARVM) and neonatal rat ventricular myocytes (NRVM), and in ventricular samples of heart-failure patients. Immunofluorescence was used to investigate CREB activation, and the protein expression was analyzed by Western blot. Calcium diastolic studies were realized using microfluorimetric technic with FURA-2AM. To evoke intracellular Ca2+ transients, ARVMs were field stimulated at 0.5 Hz and NRVMs at 1 Hz. An activation of CREB after I/R was observed in adult and neonatal rat cardiomyocytes. Furthermore, it was demonstrated that this activation was mediated by PKA, but not for EPAC2 or ERK. I/R induced an CREB-dependent ORAI protein expression increase and also an increase in the diastolic calcium in NRVM and ARVM from I/R animal models. Additionally, it was observed that ORAI1 inhibition with SYNTA-66 or GSK reduced the calcium diastolic increase induced by I/R. We demonstrated, for the first time, the activation of the transcription factor CREB in cardiomyocytes after I/R. This activation induces an up-regulation of ORAI1, suggesting that this channel plays a role in the I/R induced calcium diastolic increase.
 
Article
Subcellular calcium variations are involved in physiological and pathological mechanisms. Whereas elementary calcium release events (CREs) have been known for almost three decades in intact muscle cells isolated from vertebrates, they remained not characterized in invertebrates until recently. Dynamic confocal imaging was used on intact skeletal muscle cells isolated enzymatically from the adult honeybee legs to characterize spatio-temporal features of subcellular CREs. The frequency of these insect CREs, measured in x–y time lapse series, was higher than frequencies usually described in vertebrates. Spatial spread at half maximum was larger than in vertebrates and had a slightly ellipsoidal shape, two characteristics that may be related to ultrastructural features specific to invertebrate cells. In line-scan experiments, the histogram of CREs’ duration followed a bimodal distribution, supporting the existence of both sparks and embers. Unlike in vertebrates, embers and sparks had similar amplitudes, a difference that could be related to genomic differences and/or excitation–contraction coupling specificities in honeybee skeletal muscle fibers. Arthropods muscle cells show strong genomic, ultrastructural and physiological differences with vertebrates and a comparative analysis may help to better understanding the roles and regulations of CREs. From a toxicological point of view, such a comparison will lead to better anticipating the myotoxicity of new insecticides targeting ryanodine receptors. Recent studies described the effects of these insecticides on macroscopic calcium homeostasis in bee neurons and muscle cells. Here, cyantraniliprole, the most recently approved anthranilic diamide in Europe, triggers calcium transients in bee muscle cell as well. Cyantraniliprole effects on Ca2+ sparks are currently under study.
 
Article
Cancer and cardiovascular diseases are the main causes of death in Uruguay and developed countries. In clinical practice, there is often the need to administrate chemotherapy with cisplatin (CTP) to patients with cardiovascular comorbidities. The aim of this work is to characterize the possible detrimental effects in cardiac function by the acute exposition to CPT using isolated heart and cardiomyocytes from guinea pigs (Cavia porcellus). All the procedures regarding animal experimentation were performed following approved protocols by the university ethics committee. Isolated hearts were placed in a Langendorff system and perfused with Tyrode 1.8 mM Ca2+ as control medium, or with extracellularly added CPT (0–100 µM). Tension was recorded with a gauge force transducer attached to the papillary muscle and electrical responses were measured with Ag-AgCl electrodes placed in surface extremes near the papillary muscle. Cardiomyocytes were isolated by enzymatic methods. Data were obtained by patch clamp and confocal microscopy with Rhodamine and Fluo dyes sensitive to Ca2+ binding. Non-parametric t tests were used for data comparison. The best fit of Hill’s equation to dose–response curves was done using nonlinear regression methods. In isolated hearts, CPT showed a biphasic effect over the development of tension, increasing up to 5–10 µM to decrease at higher concentrations. In isolated cardiomyocytes, Ca2+ currents were stimulated and inhibited by CPT in a similar dose. Confocal microscopy showed an increment and a reduction of relative fluorescence of the calcium-sensitive dyes with CPT as well. Our results suggest that CPT may affect cardiac contraction and automatism upon acute exposure of the heart, presumably by blocking L-type (Cav1.2) calcium channels and interference with molecules involved in maintaining the homeostasis of intracellular Ca2+.
 
Article
The P2328S mutation in mice is associated with arrhythmia and spontaneous diastolic calcium release in atrial and ventricular myocytes and there is a corresponding leftward shift in the Ca2+-activation curve for mutant RYR2 channels from homozygous mouse hearts (Salvage et al. 2019. J Cell Sci. https://doi.org/10.1242/jcs.229039). P2328 is located in helical domain 1 (HD1) of RYR2. Local structural changes likely result when structurally active proline residues are replaced by structurally inert serine residues. We speculate that local structural changes in HD1 lead to sequential intradomain and interdomain stearic changes through the protein to the distant channel gate, which favor the open pore conformation. The drug flecainide prevents arrhythmia in humans and mouse models of CPVT by blocking NaV1.5 and RYR2 channels. Conventionally, flecainide blocks RYR2 channels in a voltage-dependent manner. We did not observe voltage-dependent pore block. This was possibly because, in contrast to previous studies, the only channel modulators that we used to produce end-diastolic control channel activity were 1 µM cytoplasmic Ca2+ and 1 mM luminal Ca2+. We observed previously unreported, voltage-independent increases in WT and P2328S channel activity at low flecainide concentrations, followed by a decline in activity at higher concentrations. The increase in activity dominated the effect of flecainide on P2328S channels. These effects suggested high-affinity flecainide binding to an activation site and lower-affinity binding to an inhibition site, both distant from the channel pore (Salvage et al. 2021. Cells. https://doi.org/10.3390/cells10082101). Unlike channel block by flecainide, the drug under our conditions stabilized intrinsic sub-conductance activity at +40 mV and −40 mV. Since flecainide effectively reduces CPVT arrythmia clinically and in animal models, we conclude that voltage-independent inhibition and voltage-dependent channel block prevail under cellular conditions. However, channel activation is important to note as it may be unmasked in other circumstances such as acquired cardiac disorders, mutations, or additional drug applications.
 
Correspondence of lifetime images with immunostaining in whole muscles. (A a and c) Autofluorescence lifetime images (see Materials and methods). (A b and d) Immunostaining of MHC type I (red), IIA (green), or IIX (blue) in soleus or FDB as indicated. (B) Box representations of fitted τ 2 values for ROIs in the fiber types identified with immunostaining in soleus (a) and FDB (b). (C a) Histograms of the fast time constant τ 1 . (C b) Slow time constant τ 2 in soleus (N = 4 mice; m = 179 fibers) and FDB (N = 5; m = 351).
Correlation of τ 2 maps with immunostaining images in muscle fiber cross-sections. (A a and c) Autofluorescence lifetime images of soleus and FDB as indicated. (A b and d) Consecutive cryosections immunostained for fiber types I (red), IIA (green), and IIX (blue). (B) Statistics of τ 2 values in fibers identified by immunostaining of cross-sections (N = 3, m = 231) for soleus; (N = 3, m = 113) for FDB.
Identification of fiber types in longitudinal sections of live muscles. (A a and b) Lifetime images of autofluorescence in FDB and soleus. (B a and b) Phasor maps, which display the approximate regions within fibers corresponding to a particular τ value. (B c and d) Phasor plots, resulting from application of the phasor transform (described in the Appendix) to the lifetime data in A; higher values of the abscissa correspond to lower arrival times, hence lower τ 2 . The maps in B a and b are created by selecting the regions of high signal in the phasor plots.
Fluorescence properties of FAD in solution compared with muscle autofluorescence. (A) Emission spectrum of FAD; excitation was set at 440 nm. Measurements were performed in vivo in soleus and FDB muscles, and in vitro with two solutions of different free FAD concentrations. (B) In vitro measurements of τ at increasing [FAD]. The arrival time histograms or fluorescence responses I(t) obtained at the extremes of the FAD concentration range are shown in the inset. They are well fitted by single exponentials of time constant τ.
Article
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 derived from a two-exponential fit (τ1 and τ2) in freshly dissected mouse FDB and soleus muscles. While τ1 values did not change between muscles, the distribution of τ2 shifted to higher values in FDB. To understand the origin of this difference, we obtained maps of autofluorescence lifetimes in cryosections of both muscles and paired them with immunofluorescence images of myosin heavy chain isoforms (MHC), which allow identification of fiber types. In soleus, τ2 was 3.1 ns for type I (SEM = 0.009, n = 49), 3.4 ns for type IIA (SEM = 0.01, n = 30), and 3.3 ns for type IIX (SEM = 0.01, n = 21). In FDB muscle, τ2 was 3.17 ns for type I (SEM = 0.04, n = 18), 3.5 ns for type IIA (SEM = 0.03, n = 27), and 3.62 ns for type IIX (SEM = 0.03, n = 22). From the distribution of measures, it follows that an FDB fiber with τ2 >3.3 ns is expected to be of type II, and of type I otherwise. This simple classification method has first- and second-class errors estimated at 0.06 and 0.27, respectively. Studies in progress aim at further elucidating the reasons for the different lifetimes, not just among fiber types but between fibers of the same type in the two muscles. Preliminary results point at differences in both the oxidation-reduction and protein-bound versus free states of flavins as causes for the observed divergence of fluorescence lifetimes. Lifetime maps of autofluorescence therefore constitute a tool to identify fiber type that, being practical, fast, and noninvasive, can be applied in living tissue without compromising other experimental interventions.
 
Article
Ryanodine receptor type-1 (RYR1) and Calsequestrin-1 (CASQ1) proteins, located in the sarcoplasmic reticulum (SR), are two of the main players in skeletal excitation–contraction (EC) coupling. Mutations in the human RYR1 gene (encoding for the SR Ca2+ release channel) and ablation in mice of CASQ1 (a SR Ca2+ binding protein) cause hypersensitivity to halogenated anesthetics (malignant hyperthermia [MH] susceptibility) and to heat (heat stroke; HS). As both MH and HS are characterized by excessive cytosolic Ca2+ levels and hypermetabolic responses, we studied the metabolism of 4-mo-old mice from two different lines that are MH/HS susceptible: knock-in mice carrying a human MH mutation (RYR1YS) and CASQ1-knockout (ko) mice. RYR1YS and, to a lesser degree, CASQ1-null mice show an increased volume of oxygen consumption (VO2) and a lower respiratory quotient (RQ) compared with WT mice (indicative of a metabolism that relies more on lipids). This finding is accompanied by a reduction in total body fat mass in both Y522S and CASQ1-null mice (again, compared with WT). In addition, we found that RYR1YS and CASQ1-null mice have an increased food consumption (+26.04% and +25.58% grams/day, respectively) and higher basal core temperature (+0.57°C and +0.54°C, respectively) compared with WT mice. Finally, Western blots and electron microscopy indicated that, in hyperthermic mice, (1) SERCA (used to remove myoplasmic Ca2+) and UCP3 (responsible for a thermogenic process that dissipates mitochondrial H+ gradient) are overexpressed, and (2) mitochondrial volume and percentage of damaged mitochondria are both increased. In conclusion, the MH/HS phenotype in RYR1YS and CASQ1-null mice is associated with an intrinsically increased basal metabolism.
 
Article
The purpose of this study is to investigate the mechanism underlying sarcoplasmic reticulum (SR) Ca2+ leakage at recovery phase after in vivo contractions. Rat gastrocnemius muscles were electrically stimulated in vivo, and then mechanically skinned fibers were prepared from the muscles excised 30 min after repeated high-intensity contractions. SR Ca2+ leakage was increased in the skinned fibers from stimulated muscles. Thereafter, SR Ca2+ leakage in skinned fibers was measured (1) under a continuously depolarized condition and (2) in the presence of nifedipine in the sealed transverse tubular system. In either of the two conditions, SR Ca2+ leakage in the rested fibers reached a level similar to that in the stimulated fibers. Furthermore, 1 mM tetracaine (Tet) treatment, but not 3 mM Mg2+ (3 Mg) treatment, lessened SR Ca2+ leakage in stimulated fibers. Depolarization-induced force in skinned fibers was more greatly decreased by Tet treatment than by 3 Mg treatment (92% reduction in Tet versus 31% reduction in 3 Mg), whereas caffeine-induced force in skinned fibers was similarly decreased by either treatment (73% reduction in Tet versus 75% reduction in 3 Mg). This difference indicates that Tet exerts a greater inhibitory effect on the dihydropyridine receptor (DHPR) signal to ryanodine receptor (RYR) than 3 Mg, although their inhibitory effects on RYR are almost similar. These results suggest that the increased Ca2+ leakage after muscle contractions is mainly caused by the orthograde signal of DHPRs to RYRs.
 
Article
Phospholamban (PLN) is the natural inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2a). Heterozygous PLN-R14del mutation is associated with an arrhythmogenic dilated cardiomyopathy (DCM), whose pathogenesis has been attributed to SERCA2a “superinhibition.” The aim of the project is to test in human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CM) harvested from a PLN-R14del carrier whether (1) Ca2+ dynamics and protein localization were compatible with SERCA2a superinhibition and (2) functional abnormalities could be reverted by pharmacological SERCA2a activation with PST3093. Ca2+ transients (CaT) were recorded at 36°C in hiPSC-CMs clusters during field stimulation. SERCA2a and PLN were immunolabeled in single hiPSC-CMs. Mutant (MUT) preparations were compared with isogenic WT ones obtained by mutation reversal. WT and MUT differed for the following properties: (1) CaT time to peak (tpeak) and half-time of CaT decay were shorter in MUT, (2) several CaT profiles were identified in WT, whereas “hyperdynamic” ones largely prevailed in MUT, (3) whereas tpeak rate-dependently declined in WT, it was shorter and rate independent in MUT, and (4) diastolic Ca2+ rate-dependently accumulated in WT, but not in MUT. When applied to WT, PST3093 changed all of the above properties to resemble those of MUT; when applied to MUT, PST3093 had no effect. Preferential perinuclear SERCA2a-PLN localization was lost in MUT hiPSC-CMs. In conclusion, functional data converge to argue for PLN-R14del incompetence in inhibiting SERCA2a in the tested case, thus weakening the rationale for therapeutic SERCA2a activation. Mechanisms alternative to SERCA2a superinhibition should be considered in the pathogenesis of DCM, including dysregulation of Ca2+-dependent transcription.
 
Article
Trimeric intracellular cation channels (TRIC-A and TRIC-B), found in the sarco/endoplasmic reticulum (SR/ER) and nuclear membranes, are thought to provide countercurrents to balance Ca2+-movements across the SR, but there is also evidence that they physically interact with ryanodine receptors (RYR). We therefore investigated if TRIC channels could modulate the single-channel function of RYR2 after incorporation of vesicles isolated from HEK293 cells expressing TRIC-A or TRIC-B with RYR2 into artificial membranes under voltage clamp. We also examined the gating and conductance properties of TRIC channels. Co-expression of RYR2 with either TRIC-A or TRIC-B significantly altered the gating behavior of RYR2; however, co-expression with TRIC-A was particularly effective at potentiating the activating effects of cytosolic Ca2+. Fusing membrane vesicles containing TRIC-A or TRIC-B together with RYR2 into bilayers produced large currents of rapidly gating current fluctuations of multiple amplitudes. In 740 cytosolic/210 luminal mM KCl gradient, current-voltage relationships of macroscopic currents revealed average reversal potentials (Erev) of −13.67 ± 9.02 (n = 7), −2.11 ± 3.84 (n = 11), and 13.19 ± 3.23 (n = 13, **, P = 0.0025) from vesicles from RYR2 only, RyR2 + TRIC-A, or RyR2 + TRIC-B cells, respectively. Thus, with the incorporation of TRIC channels, the Erevs depart further from the calculated Erev for ideally selective cation channels than occurs when vesicles from RYR2-only cells are incorporated, suggesting that TRIC channels are permeable to both K+ and Cl−. In conclusion, our results indicate that both TRIC-A and TRIC-B regulate the gating of RYR2, but that TRIC-A has greater capacity to stimulate the RYR2 opening. The results also suggest that TRIC channels may be relatively nonselective ion channels being permeable to both cations and anions. This property would enable TRIC channels to be versatile providers of counter-ion current throughout the SR of many cell types.
 
Article
Orai1 and STIM1, molecular components of store-operated calcium entry (SOCE), have been associated with vascular smooth muscle cell (VSMC) proliferation in vascular remodeling. Nevertheless, the role of SARAF (SOCE-associated regulatory factor), a regulatory protein involved in STIM1 inhibition, in vascular remodeling has not been examined. The aim of this study is to examine the role of SARAF and Orai1 in VSMC proliferation and neointima formation after balloon injury of rat carotid arteries. Experiments were conducted in an animal model of rat carotid angioplasty to characterize neointima formation. VSMC isolated from rat coronary arteries was also used to examine cell proliferation. The formation of neointima after balloon injury of rat carotid arteries was confirmed by hematoxylin and eosin staining of tissue sections up to 3 wk after surgery. Injured arteries showed significantly higher expression of SARAF, STIM1, and Orai1 compared with control tissues, corroborating the presence of these regulatory proteins in the neointima layer. Proximity ligation and coimmunoprecipitation assays revealed that SARAF interacts with Orai1 in the neointima. Furthermore, selective silencing of SARAF and Orai1 by small interfering RNA (siRNA) inhibited IGF-1–induced VSMC proliferation. Our data suggest that SARAF interacts with Orai1 to modulate SOCE and VSMC proliferation after vascular injury.
 
Article
Cardiac RYR2-mediated sarcoplasmic Ca2+ (SR) release is essential for matching increased energy demand during fight-or-flight response with mitochondrial metabolic output by delivering Ca2+ into the mitochondrial matrix to activate Ca2+-dependent Krebs cycle dehydrogenases. RYR2 complex gain-of-function mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) have been linked to mitochondrial structural damage and enhanced production of reactive oxygen species (ROS). Despite being critical for arrhythmogenesis in CPVT, the exact causes of these phenomena remain undetermined. Taking advantage of a new rat model of CPVT induced by heterozygous RYR2 gain-of-function mutation S2222L, we tested how RYR2 overactivity alters mitochondrial Ca2+ and ROS handling, and how these changes cause mitochondrial structural defects. Injection of epinephrine (1 mg/kg) and caffeine (120 mg/kg) induced bigamy and bidirectional VT in vivo in 100% of CPVT rats. Simultaneous whole-cell patch clamp and confocal Ca2+-imaging demonstrated that under β-adrenergic stimulation with isoproterenol (50 nM), CPVT ventricular myocytes (VMs) exhibited severe Ca2+ mishandling and high propensity for generation of spontaneous Ca2+ waves (SCWs) that cause arrhythmogenic afterdepolarizations. Diminished Ca2+ transient amplitude in CPVT VMs resulted in a significant reduction in mitochondrial matrix–[Ca2+], and thereby a mito-ROS surge, visualized using matrix-targeted biosensors mtRCaMP1h and MLS-HyPer, respectively. Importantly, using novel Ca2+-biosensors targeted to intermembrane space (IMS-GECO), we uncovered that [Ca2+] in this compartment reaches 1 µM, sufficient for activation of Ca2+-dependent protease μ-calpain. Adenoviral overexpression of IMS-targeted calpastatin, an endogenous calpain inhibitor, reduced mito-ROS, restored cytosolic Ca2+ transient amplitude and SR Ca2+ content, and reduced RYR2-mediated SCWs in CPVT VMs. These changes were paralleled by restored expression levels of OPA1, a mitochondrial structural protein responsible for tight cristae organization. Our data suggest that enhanced mito-ROS due to matrix-[Ca2+] reduction in CPVT VMs and unexpectedly high IMS-[Ca2+] promotes IMS-calpain–mediated degradation of OPA1, resulting in mitochondrial structural damage that contributes to proarrhythmic remodeling.
 
Article
Skeletal muscle function is regulated by intracellular Ca2+ levels. Two main mechanisms control movements of Ca2+ ions from intracellular stores (i.e., the sarcoplasmic reticulum; SR) and from extracellular space: (1) excitation–contraction (EC) coupling and (2) store-operated Ca2+ entry (SOCE). SOCE allows recovery of extracellular Ca2+ during prolonged muscle activity, when the SR undergoes depletion. We recently discovered that prolonged exercise leads to formation of calcium entry units (CEUs), intracellular junctions located at the I band that are formed by two distinct elements: SR stacks and transverse tubules (TTs). Assembly of CEUs during exercise promotes the interaction between STIM1 and Orai1, the two main proteins that mediate SOCE, and increases muscle resistance to fatigue in the presence of extracellular Ca2+. The molecular mechanisms underlying the exercise-dependent remodeling of SR and TT leading to CEU assembly remain to be fully elucidated. Here, we first verified whether CEUs can assemble ex vivo (in the absence of blood supply and innervation), subjecting excised EDL muscles from mice to an ex vivo incremental fatigue protocol (80 Hz tetanus stimulation lasting 45 min): the data collected demonstrate that CEUs can assemble ex vivo in isolated EDL muscles. We then evaluated if intracellular parameters that are affected by exercise, such as temperature and pH, may influence the assembly of CEUs. We found that higher temperature (36°C versus 25°C) and lower pH (7.2 versus 7.4) promotes formation of CEUs increasing the percentage of fibers containing SR stacks, the number of SR stacks/area, and the elongation of TTs at the I band. Importantly, increased assembly of CEUs at higher temperature (36°C) or at lower pH (7.2) correlated with increased fatigue resistance of EDL muscles in the presence of extracellular Ca2+, suggesting that CEUs assembled ex vivo are functional.
 
Article
Rodents are commonly used as models in electrophysiology. However, distinct differences exist between large animals and rodents in terms of their ion channel expression and action potential shapes, possibly limiting the translational value of findings obtained in rodents. We aimed for a direct comparison of the possible impact of selective inhibition of ion channels on the cardiac repolarization in preparations from human hearts and from model species. We applied the standard microelectrode technique at 37°C on cardiac ventricular preparations (papillary muscles and trabecules) from human (n = 63), dog (n = 47), guinea pig (n = 53), rat (n = 43), and rabbit (n = 16) hearts, paced at 1 Hz. To selectively block the IKur current, 1 µM XEN-D101; IK1 current, 10 µM barium chloride; IKr current, 50 nM dofetilide; IKs current, 500 nM HMR-1556; and Ito current, 100 µM chromanol-293B were applied directly to the tissue bath. The block of IKur and IK1 elicited significantly more prominent prolongation of APD in rats (35.6% and 67.9%, respectively) when compared with the other species, including that of human (1.0% and 2.6%, respectively). On the other hand, IKr block did not affect APD in rat preparations (1.6%), whereas it elicited marked prolongation in other species (9.0–47.7%), especially being pronounced in human preparations (60.3%). IKs inhibition elicited similar but minor APD prolongation (0.3–11.4%) in all species. Inhibition of Ito moderately lengthened APD in dog (22.3%) and rabbit (17.5%) preparations but elicited no change of APD in human preparations. In contrast, block of Ito caused marked APD prolongation in rat preparations (33.2%). Our findings suggest that the specific inhibition of various ion channels elicits fundamentally different effects in rodent ventricular action potential when compared with those of other species, including human. Therefore, from a translational standpoint, rodent models in cardiac electrophysiological and arrhythmia research should be used with great caution.
 
Article
In humans, type 2 diabetes mellitus (T2DM) has a higher incidence in males compared to females, a phenotype recapitulated by many rodent models. While the sex difference in insulin sensitivity partially accounts for this phenomenon, hitherto uncharacterized differences in pancreatic β-cell insulin release strongly contribute. Here, we show that stepwise increase in extracellular glucose concentration (2, 5, 7.5, 10, 15, 20 mM) induced electrical activity in β cells of both sexes with similar glucose sensitivity (female, EC50 = 9.45 ± 0.15 mM; male, EC50 = 9.42 ± 0.16 mM). However, female β cells’ resting membrane potential (RMP) and inter-spike potential (IP) were significantly higher compared to males (e.g., at 15 mM glucose: male RMP = −82.7 ± 6.3, IP = −74.3 ± 6.8 mV; female RMP = −50.0 ± 7.1, IP = −41.2 ± 7.3 mV). Females also showed higher frequency of trains of action potential (AP; at 10 mM glucose: male F = 1.13 ± 0.15 trains/min; female F = 1.78 ± 0.25 trains/min) and longer AP-burst duration (e.g., at 10 mM glucose: male, 241 ± 30.8 ms; female, 419 ± 60.2 ms). The higher RMP in females reduced the voltage-gated calcium channel (CaV) availability by ∼60%. This explains the paradoxical observation that, despite identical CaV expression levels and higher electrical activity, the islet Ca2+ transients were smaller in females compared to males. Interestingly, the different RMPs are not caused by altered KATP, TASK, or TALK K+ currents. However, stromatoxin-1–sensitive KV2.1 K+ current amplitude was almost double in males (IK = 130.93 ± 7.05 pA/pF) compared to females (IK = 75.85 ± 11.3 pA/pF) when measured at +80 mV. Our results are in agreement with previous findings showing that KV2.1 genetic deletion or pharmacological block leads to higher insulin release and β-cell survival. Therefore, we propose the sex-specific expression of KV2.1 to be the mechanism underlying the observed sexual dimorphism in insulin release and the incidence of T2DM.
 
Article
Single-point mutations in ryanodine receptors (RYRs), large intracellular Ca2+ channels that play a critical role in EC coupling, are linked to debilitating and lethal disorders such as central core disease, malignant hyperthermia (for the skeletal isoform, RYR1), catecholaminergic polymorphic ventricular tachycardia, and ARVD2 (for the cardiac isoform, RYR2). Mutant RYRs result in elevated [Ca2+]cyto due to steady leak from the sarcoplasmic reticulum. To explore the nature of long-range allosteric mechanisms of malfunction, we determined the structure of two N-terminal domain mutants of RYR1, situated far away from the pore. Cryo-electron microscopy of the N-terminal subdomain A (NTDA) and subdomain C (NTDC) full-length mutants, RYR1 R163C (determined to 3.5 Å resolution), and RYR1 Y522S (determined to 4.0 Å resolution), respectively, reveal large-scale conformational changes in the cytoplasmic assembly under closed-state conditions (i.e., absence of activating Ca2+). The multidomain changes suggest that the mutations induce a preactivated state of the channel in R164C by altering the NTDA+/CD interface, and in Y522S by rearrangement of the α-helical bundle in NTDC. However, the extent of preactivation is considerably higher in Y522S as compared with R163C, which agrees with the increased severity of the Y522S mutation as established by various functional studies. The Y522S mutation represents loss of a spacer residue that is crucial for maintaining optimal orientation of α helices in NTDC, alteration of which has long-range effects felt as far away as ∼100 Å. Additionally, the structure of the Y522S mutant channel under open-state conditions also differs from RYR1 WT open channels. Our developing work with RYR mutants exhibits the diverse mechanisms by which these single-point mutations exert an effect on the channel’s function and highlight the complexity of the multidomain channel, as well as the need for targeted therapies.
 
Article
In excitation–contraction coupling (ECC), when the skeletal muscle action potential (AP) propagates into the transverse tubules, it modifies the conformational state of the voltage-gated calcium channels (CaV1.1). CaV1.1 serves as the voltage sensor for activation of calcium release from the sarcoplasmic reticulum (SR); however, many questions about this function persist. CaV1.1 α1 subunits contain four distinct homologous domains (I–IV). Each repeat includes six transmembranal helical segments; the voltage-sensing domain (VSD) is formed by S1–S4 segments, and the pore domain is formed by helices S5–S6. Because, in other voltage-gated channels, individual VSDs appear to be differentially involved in specific aspects of channel gating, here we thus hypothesized that not all the VSDs in CaV1.1 contribute equally to calcium-release activation. Yet, the voltage-sensor movements during an AP (the physiological stimulus for the muscle fiber) have not been previously measured in muscle. Reorientation of VSDs I–IV in CaV1.1 during an AP should generate a small but measurable electrical current. Still, neither the voltage-sensor charge movement during the AP nor the contribution of the individual VSDs to voltage-gated calcium release have been previously monitored. Here, we electrically monitor VSD movements using an AP voltage-clamp technique applied to muscle fibers. We introduce AP-fluorometry, a variant of the functional site-directed fluorescence, to track the movement of each VSD via a cysteine substitution on the extracellular region of S4 of each VSD and its labeling with a cysteine-reacting fluorescent probe, which served as an optical reporter of local rearrangements. Independent optical recordings of AP and calcium transients were performed to establish the temporal correlation between AP, AP-elicited charge movement, VSDs conformational changes, and calcium release flux. Our results support the hypothesis that not all VSDs in CaV1.1 contribute to ECC.
 
Top-cited authors
Francisco Bezanilla
  • University of Chicago
Bertil Hille
  • University of Washington Seattle
Benoît Roux
  • The University of Chicago Medical Center
Baron Chanda
  • University of Wisconsin–Madison
Björn H Falkenburger
  • Technische Universität Dresden