In the human heart, cytosolic Ca2+ signals are well characterized but the contribution of nuclear Ca2+ is essentially unexplored. The present study investigated nuclear Ca2+ signaling in hPSC-VCMs. Calcium transient or sparks in hPSC-VCMs were measured by line scanning using a spinning disc confocal microscope. We observed that nuclear Ca2+, which stems from unitary sparks due to the diffusion of cytosolic Ca2+ that are mediated by RyRs on the nuclear reticulum, is functional. Parvalbumin- (PV-) mediated Ca2+ buffering successfully manipulated Ca2+ transient and stimuli-induced apoptosis in hPSC-VCMs. We also investigated the effect of Ca2+ on gene transcription in hPSC-VCMs, and the involvement of nuclear factor of activated T-cell (NFAT) pathway was identified. The overexpression of Ca2+-sensitive, nuclear localized Ca2+/calmodulin-dependent protein kinase II δB (CaMKIIδB) induced cardiac hypertrophy through nuclear Ca2+/CaMKIIδB/HDAC4/MEF2 pathway. These findings provide insights into nuclear Ca2+ signal in hPSC-VCMs, which may lead to novel strategies for maturation as well as improved systems for disease modeling, drug discovery, and cell-based therapies.The loss of nonregenerative, terminally differentiated cardiomyocytes (CMs) due to aging or diseases is generally considered irreversible. Human pluripotent stem cells (hPSCs) can self-renew while maintaining their pluripotency to differentiate into all cell types, including ventricular (V) cardiomyocytes (CMs), to provide a potential unlimited  These were completely abolished by ryanodine  were clearly separated into the cytoplasmic and nuclear groups, with a clear delay for the latter during electrical stimulation-induced Ca2+ transients, resembling those of extCa2+-induced. Although the onsets of cytosolic and nuclear Ca2+ signals upon electrical stimulation were virtually indistinguishable, both the rise and decay times of nuclear Ca2+ were significantly prolonged. Of note, no autonomous nuclear Ca2+ transients or waves could be observed in hESC-VCMs during pacing, distinctive from neonatal rat cardiomyocytes [2+ inhibitor CCCP and oligomycin did not prevent the delayed kinetics of nuclear Ca2+ signals    implied 2+ level . Therefoecreased . Interes Figures . This ob2+ sparks in hESC-VCMs as the elementary units. 2+ sparks in a Hoechst-labeled nucleus. Pharmacologically, both cytosolic and nuclear Ca2+ sparks could be abolished by pretreatment with the RyR blocker ryanodine or tetracaine but were unaffected by the IP3R blocker Xec or 2-APB  but not IP3  induced a significant increase in nucleoplasmic Ca2+. Consistently, Ca2+ measurement in nuclear envelop by Fluo-5N indicated that caffeine , but not IP3 , led to this Ca2+ release , from the nuclear envelope (NE) into the center of the nucleus . Consist cytosol . Western nucleus . This is nucleus . To seekM nuclei  loaded w release . Collect2+ binding protein, has been used as an endogenous Ca2+ buffer [2+ in the desired cellular compartment. We confirmed the expected subcellular localization patterns in GFP+ LV-MLC2v-GFP-transduced hESC-VCMs by confocal imaging and western blot analyses  exclusively reduced that of nuclear but not cytosolic Ca2+ peaks. Transduction of a mutated form of PV with inactivated calcium binding sites targeted to the nucleus (PV-NLS-CD) had no effect. Interestingly, PV-NLS but not PV-NES altered the ratio of nuclear Ca2+ transient amplitude to that of the cytosol   [2+ buffering protected hESC-VCMs from stimuli (H2O2 and hypoxia)-induced apoptosis , a 9-11 kDa Ca+ buffer , 26, 27.+ buffer , we atte cytosol , consisthv-CMTs) . In rat de (ANP) . However Figures .2+ in pluripotency and differentiation, we generated stably LV-PV-NLS- and LV-PV-NES-transduced hESC lines with persistent PV expression in the nucleus and cytosol, respectively  pretreatment, indicating that it was Ca2+, rather than ionomycin itself, that caused the elevated expression of ANP. Moreover, this Ca2+-induced ANP expression was dramatically inhibited by CsA, the calcineurin (CN) inhibitor, but not altered by KN62, the CaMKII inhibitor. To further illustrate the functional CN/NFAT pathway in hESC-VCMs, western blotting using NFATc3 antibody was performed. The results indicated that ionomycin addition led to the shift of NFATc3 band to a lower molecular weight, suggesting the dephosphorylation of NFATc3, which was also blocked by CsA but not KN62  CaMKIIδB were successfully expressed in hESC-VCMs as examined by western blotting ; the nuclear envelope (NE) is composed of an outer membrane contiguous with the ER and an inner membrane separated by a luminal space that serves as a Ca2+ store (2+ is regulated by (1) Ca2+ entry into the nucleoplasm by passive diffusion from cytosol through nuclear membrane pores [2+ release from the inner membrane of NE into the nucleoplasm [2+ entry and exit. NR has been visualized by two-photon microscopy in a variety of mammalian cells including CMs [3R as well as phospholamban (PLB) expressed on the NR membrane [2+ to regulate cellular functions independently of cytosolic Ca2+ change [2+ signaling is functional in hESC and hESC-VCMs.The basis of how Cacificity , 36, 37.cription , 9 to cecription  as well cription , 39. Cyt2+ store . Dependine pores ; (2) dirleoplasm ; and (3)leoplasm , 43, theding CMs , 44, witmembrane –47, allo+ change . Our pre2+ could diffuse to hESC-VCM nucleoplasm from cytosol, resulting in a delayed kinetics of nuclear signals which is not dye-specific and not due to different Ca2+ dye characteristics in the nucleus and the cytosol  was able to differentially regulate nuclear Ca2+: compared to cytosolic Ca2+ transient, nuclear Ca2+ transients turned into a more sustained (higher diastolic Ca2+) Ca2+ signal with lower fluctuation (lower transient amplitude) by high stimulation frequency in hESC-VCMs. The functions of this frequency-dependent modulation of nuclear Ca2+ signal remain to be determined [We found that Ca cytosol . This iso the NE . More imtermined .2+ transients are triggered by IP3-mediated Ca2+ release, which is then amplified and modulated by RyR-mediated Ca2+ release [3R is highly expressed in conductive CMs in either embryonic or adult hearts [3 has been theorized to increase intracellular Ca2+ for directing developmental pathways rather than for EC coupling. This is supported by the preferential intra and perinuclear location of IP3R for nuclear signaling and IP3R downregulation after birth [2+ of hESC-VCMs is regulated only by RyRs, like in the SR, but not IP3R. It is reported that IP3Rs in ventricular cardiomyocytes are around 5-6 times less abundant than those in atrial cells [3R in nuclear Ca2+ signal since only ventricular cardiomyocytes derived from stem cells are used in this study.In murine and human ESC-CMs, it has been suggested that spontaneous Ca release –51. Indet hearts . Developer birth , 53. Oural cells –56, whic2+ reduces the global Ca2+ transient amplitude (by ~50%) [2+ buffering prolongs APD, suggesting that nuclear Ca2+ is specifically responsible for electrophysiological remodeling. To correlate cytosolic or nuclear Ca2+ changes early on during pluripotency or late after ventricular specification to phenotypic properties of hESC-VCM, we attempted to regulate the Ca2+ levels in different cell compartments by targeted expression of calcium buffer protein PV. PV targeted to the cytosol (PV-NES) diminished both cytosolic and nuclear Ca2+ rise elicited electrically because any reduced cytosolic Ca2+ increase would decrease the contribution of passive diffusion to nuclear Ca2+ increase (cf. isolation of active release and diffusion components in our experiments with intact cells and isolated nuclei provides a basis for interpretation here). At the physiological level, cytosolic Ca2+, but not nuclear Ca2+, blockade prevented stimuli-induced apoptosis -dependent relationship was observed  and causes NFAT translocation from cytosol to nucleus to modulate gene transcription. Numerous publications indicate the connection between CN/NFAT pathway and cardiac hypertrophy. For instance, in mice, CN expression level and activity are increased during early pregnancy, which may be caused by the pregnancy-associated sex hormone progesterone. This enhanced expression and activity of CN increase NFAT activity and lead to cardiac hypertrophy during pregnancy. Moreover, CsA treatment blocks pregnancy-induced hypertrophy in mice [2+, indicating a functional CN/NFAT pathway in hESC-VCMs. On the other hand, CaMKII is a serine/threonine-specific protein kinase that can be regulated by Ca2+/calmodulin complex. CaMKII is well known to be involved in cardiac hypertrophy and heart failure [δB induces hypertrophy [2+ in hESC-VCMs, CaMKII was mainly expressed in cytosol, which explains its limited role in gene transcription in hESC-VCMs. However, when we overexpressed the nucleus-targeted isoform CaMKIIδB in hESC-VCMs, we found that CaMKIIδB could indeed induce cardiac hypertrophy through nuclear Ca2+/CaMKIIδB/HDAC4/MEF2 pathway  nuclear Ca2+ signal has delayed kinetics compared to cytosolic Ca2+ signal during global Ca2+ changes like waves or transients, indicating that nuclear Ca2+ is initiated by Ca2+ diffusion from cytosol and (2) local nuclear Ca2+ sparks in hPSC-VCMs are mediated by RyRs, and NR serves as structural basis. In addition, PV-mediated Ca2+ buffering can successfully modulate Ca2+ transient and stimuli-induced apoptosis in hPSC-VCMs. Moreover, the endogenous cytosolic Ca2+/CN/NFAT pathway is functional in hESC-VCMs, and the overexpression of nucleus-localized CaMKIIδB led to cardiac hypertrophy through nuclear Ca2+/CaMKIIδB/HDAC4/MEF2 pathway. Collectively, these findings provide the insights of nuclear Ca2+ signal in hPSC-VCMs, which may lead to novel strategies for maturation as well as improved systems for disease modeling, drug discovery, and cell-based therapies.Overall, we sought to develop a cause-and-effect relationship between any changes in nuclear or cytosolic Ca


Podocytes are specialized cells that form interdigitating foot processes as an essential component of the glomerular filter. These cells, which participate in generation of the primary urine, are often affected during primary glomerular diseases, such as glomerulonephritis and secondary hypertensive or diabetic nephropathy, which always leads to proteinuria. Because the function of podocytes is known to be controlled by intracellular Ca2+ signaling, it is important to know about the role of Ca2+-activated TMEM16F in these cells. To that end, we generated an inducible TMEM16F knockdown in the podocyte cell line AB8, and produced a conditional mouse model with knockout of TMEM16F in podocytes and renal epithelial cells of the nephron. We found that knockdown of TMEM16F did not produce proteinuria or any obvious phenotypic changes. Knockdown of TMEM16F affected cell death of tubular epithelial cells but not of glomerular podocytes when analyzed in TUNEL assays. Surprisingly, and in contrast to other cell types, TMEM16F did not control intracellular Ca2+ signaling and was not responsible for Ca2+-activated whole cell currents in podocytes. TMEM16F levels in podocytes were enhanced after inhibition of the endolysosomal pathway and after treatment with angiotensin II. Renal knockout of TMEM16F did not compromise renal morphology and serum electrolytes. Taken together, in contrast to other cell types, such as platelets, bone cells, and immune cells, TMEM16F shows little effect on basal properties of podocytes and does not appear to be essential for renal function.The Ca The formula used to calculate [Ca2+]i was [Ca2+]i = Kd × (R − Rmin)/(Rmax − R) × (Sf2/Sb2), where R is the observed fluorescence ratio. The values Rmax and Rmin (maximum and minimum ratios) and the constant Sf2/Sb2 (fluorescence of free and Ca2 ± bound Fura-2 at 380 nm) were calculated using 1 μM ionomycin , 5 μM nigericin, 10 μM monensin (Sigma), and 5 mM EGTA to equilibrate intracellular and extracellular Ca2+ in intact Fura-2-loaded cells. The dissociation constant for the Fura-2·Ca2+ complex was taken as 224 nmoL/L. Control of experiments, imaging acquisition, and data analysis were done with the software package Meta-Fluor  and Origin .Intracellular Ca2PO4 1.2, Na2HPO4 4.8, EGTA 1, Ca-gluconate 0.758, MgCl2 1.03, d-glucose 5, ATP 3, pH 7.2. TMEM16A was activated by high-pipette Ca2+ (1 µM). Extracellular Cl− (145 mM) was replaced by gluconate, HCO3−, or I−. Shifts in the reversal potential of current voltage relationships were used to calculate permeability ratios. (Pipette solution 145 mM). Coverslips were mounted in a perfused bath chamber on the stage of an inverted microscope  and kept at 37 °C. The bath was perfused continuously with Ringer solution at a rate of 8 mL/min. Patch clamp experiments were performed in the fast whole-cell configuration. Patch pipettes had an input resistance of 2–4 MΩ when filled with cytosolic-like solution. Currents were corrected for serial resistance. Access conductances were monitored continuously and were 60–140 nS. Currents (voltage clamp) and voltages (current clamp) were recorded using a patch clamp amplifier , the LIH1600 interface and PULSE software , as well as Chart software . Data were stored continuously on a computer hard disc and analyzed using PULSE software. In regular intervals, membrane voltages (Vc) were clamped from −100 to +100 mV in steps of 20 mV (holding voltage of −100 mV). Current densities were calculated by dividing whole-cell currents by cell capacitance.Patch clamp experiments were performed as described earlier . In brie


Drosophila to investigate the role of impaired Ca2+ handling in memory deficits caused by expression of human 0N4R isoform of tau which is associated with AD. Expression of tau in mushroom body neuropils, or a subset of mushroom body output neurons, led to impaired memory. By using the Ca2+ reporter GCaMP6f, we observed changes in Ca2+ signaling when tau was expressed in these neurons, an effect that could be blocked by the L-type Ca2+ channel antagonist nimodipine or reversed by RNAi knock-down of the L-type channel gene. The L-type Ca2+ channel itself is required for memory formation, however, RNAi knock-down of the L-type Ca2+ channel in neurons overexpressing human tau resulted in flies whose memory is restored to levels equivalent to wild-type. Expression data suggest that Drosophila L-type Ca2+ channel mRNA levels are increased upon tau expression in neurons, thus contributing to the effects observed on memory and intracellular Ca2+ homeostasis. Together, our Ca2+ imaging and memory experiments suggest that expression of the 0N4R isoform of human tau increases the number of L-type Ca2+ channels in the membrane resulting in changes in neuronal excitability that can be ameliorated by RNAi knockdown or pharmacological blockade of L-type Ca2+ channels. This highlights a role for L-type Ca2+ channels in tauopathy and their potential as a therapeutic target for AD.The cellular underpinnings of memory deficits in Alzheimer’s disease (AD) are poorly understood. We utilized the tractable neural circuits sub-serving memory in  The accumulation of the microtubule-associated protein tau (MAPT) within central neurons is a key histopathological feature of Alzheimer’s disease (AD) . Post-mo2+) homeostasis including increased Ca2+ levels causing excitotoxicity has been posited as a key pathophysiology in AD, which may underpin the early stages of disease and precipitate neurodegeneration , have been shown to be upregulated in rodent models of AD, with blockers, such as nifedipine, being effective in trails to prevent the cognitive decline that occurs in AD  and dorsal paired medial (DPM) neurons (amn(c316)-Gal4, pGal4 = 0.002, pUAS = 0.03), which consolidate and stabilize labile memory  concentration resulted in a robust and transient elevation in fluorescence which could be ameliorated by removing Ca2+ from the external solution  or by adding cadmium , a general blocker of Drosophila voltage-gated Ca2+ channels . The elevated Ca2+ influx seen in these neurons may, therefore, be due to augmented L-type Ca2+ channels. To further investigate which type of voltage-gated Ca2+ channel mediated the Ca2+ influx, we also tested the effect of addition of the L-type and T-type Ca2+ channel blocker, amiloride (1 mM), to nimodipine on the KCl-induced Ca2+ transient, as this compound greatly reduced Ca2+ currents in cultured embryonic Drosophila giant neurons , that has been shown to reduce L-type Ca2+ channel currents and protein levels by over 75% in Drosophila neurons . The KCl-evoked Ca2+ response in this large population of neurons was reduced by the expression of tau  to ensure changes in gene expression were detectable (Ca-α1D-RNAi knock-down reduced expression of the channel by 58% (p < 0.05), while expression of tau lead to a 68% (p = 0.04) increase in Ca-α1D. Interestingly, co-expression of tau and Ca-α1D-RNAi restored Ca-α1D to 72% (p = 0.3) of control fly levels. This strongly suggests that the effects observed in behavior and Ca2+ imaging are due to a change in Ca-α1D expression in the brain.The mechanism underlying tau-mediated augmentation of Catectable . Ca-α1D-In vivo Ca2+ imaging of different MB neuronal subsets has revealed the importance of Ca2+ transient plasticity in olfactory associative memory , which are a period of prolonged hyperpolarized membrane potential following action potential firing, in hippocampal neurons is dependent on Ca2+ entry through L-type channels  the elevated Ca2+ influx through L-type channels is likely small relative to the reduced Ca2+ levels due to suppressed neuronal activity, therefore a reduction in the Ca2+ transient was observed. The reduced excitability of MB neurons was rescued by co-expression of Ca-α1D-RNAi as this manipulation opposes the suppression of excitability caused by tau  was observed in a neuroblastoma cell line following transgenic Aβ42 expression; this resulted in reduced cell viability and six-fold increase in Ca2+ influx, that could be ameliorated by the L-type Ca2+ channel dihydropyridine blockers, nimodipine and isradipine (Drosophila overexpressing human amyloid precursor protein (APP695), as well as decreasing the accumulation of Aβ and phosphorylated tau in the triple transgenic AD mice (3 × TgAD) which express human Presenilin 1M146V, APPSwedish and tauP30L. This indicates that correcting defective Ca2+ handling in AD may be of therapeutic benefit, particularly as L-type Ca2+ channels appear to be relevant in the human AD brain, too.Raised L-type Ca Tg mice , as were Tg mice . Our datradipine . This st2+ channel expression has been documented in the brains of AD patients  at 25°C. Wild-type control was Canton S w- (CSw-) and R21D02-GAL4, UAS-GCaMP6f were kind gifts from Prof. Scott Waddell (University of Oxford). UAS-human MAPT (TAU 0N4R) wild-type (UAS-GFP was a gift from Prof. Mark Wu (John Hopkins University). The following flies were obtained from the Bloomington and Vienna fly stock centers: OK107-Gal4 (Bloomington Drosophila stock center number BDSC:854), c305a-Gal4 (BDSC:30829), amn(c316)-Gal4 (BDSC:30830), MB247-Gal4 (BDSC:50742), elav-Gal4 (BDSC:8760), UAS-GCaMP6f (BDSC:42747) and UAS-Ca-α1D-RNAi flies [Vienna Drosophila resource center GD51491 (Flies were raised at a standard density with a 12 h:12 h light dark (LD) cycle on standard ild-type  was a ki GD51491 ].n = 1. Moreover, the order of delivery of CS+ and CS− was alternately reversed.All memory experiments were carried out at 25°C and 70% relative humidity under dim red light. Flies were used for experiments after 2–5 days of aging at 25°C and 70% relative humidity in a 12-hour light: 12-hour dark environment. Using a previously published protocol , groups To test memory, flies were placed at the choice point of a T-maze with one pathway exposed to CS+ and the other to CS−. After 120 s, the number of flies choosing each pathway was counted. Memory was quantified using the performance index (PI):CS– and NCS+ is the number of flies choosing CS− and CS+, respectively. A PI = 1 indicates perfect learning where all flies chose CS−, and PI = 0 indicates a 50:50 split between CS− and CS+ and, therefore, no learning.where N2, decapitated and their brains dissected out of the head in extracellular saline containing (in mM): 101 NaCl, 1 CaCl2, 4 MgCl2, 3 KCl, 5 D-glucose, 1.25 NaH2PO4, and 20.7 NaHCO3 adjusted to a final pH of pH 7.2 with an osmolality of 247–253 mmol/kg. Brains were held ventral side up in a recording chamber using a custom-made anchor and visualized with a 40× water-immersion lens on an upright microscope (Zeiss Examiner Z1).GCaMP imaging was performed using previously published protocols  with fli2+-lacking solutions were superfused over the brain for 60 s prior to imaging. The Ca2+-lacking external solution contained 8 mM MgCl2.Brains were superfused with extracellular saline (5 mL/min) as above and cells were depolarized by bath application of 100 mM KCl in extracellular solution (362 mmol/kg) for 15 s at 5 mL/min. Drug-containing or Ca0) was taken as the mean fluorescence during the 10 s (80 images) prior to the start of KCl perfusion. The change in fluorescence relative to baseline  following KCl addition was recorded, and the peak change [(Fmax-F0)/F0] was used as a metric of transient [Ca2+] increase. Example traces were plotted using Origin 9 (Origin Lab).Images were acquired at 8 Hz with 50 ms exposure using a CCD camera (ZEISS Axiocam) and a 470 nm LED light source  and recorded with ZEN . Baseline fluorescence , except for nimodipine which was purchased from Tocris  and amiloride which was donated by Prof. David Sheppard (University of Bristol).2 and decapitated, obtaining six biological replicates with 23 heads each. Total RNA was extracted from head lysates by organic phenol/chloroform method using TRIzol reagent (Invitrogen). RNA quantification was carried out in Nanodrop spectrophotometer (Thermo Scientific) and RNA integrity was checked by electrophoresis in 1% agarose gel. Samples were treated with TURBO DNA-free kit (Invitrogen) in order to remove genomic DNA contamination. Reverse transcription was carried out using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) following manufacturer’s instructions, with 500 ng of RNA as template and Oligo (dT) as primer to amplify total mRNA. cDNA samples were stored at −20°C or used immediately for qPCR reactions.Relative measure of Ca-α1D expression levels was assessed by two-step qPCR. 2–5 days old male flies were anesthetized with COmRNA were as follows: Ca1DF 5′-CCTTGAGGGCTGGACTGATG-3′ and Ca1DR 5′-ATCACGAAGAAGGCACCCAG-3′ with a PCR product expected size of 108 bp and 104% primer efficiency  method and data was expressed as a percentage of change.Quantitative PCR reactions were carried out in QuantStudio 3 Real-Time PCR system (Applied Biosystems) using HOT FIREPol EvaGreen qPCR Mix Plus (Solis BioDyne). The primers used to amplify Ca-α1D ficiency . As a hosoftware . QuantifAll data were analyzed using Prism 7 (GraphPad Inc.). Data were scrutinized to check they met the assumptions of parametric statistical tests, and non-parametric, rank-based alternatives were used where appropriate. Details of statistical tests used are in figure legends. Data are presented as mean ± standard error of mean.The raw data supporting the conclusion of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.All authors devised and performed the experiments, and wrote and edited the manuscript. JH secured the funding.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


What is the central question of this study?in vivo?What is the cellular basis of the protection conferred on the heart by overexpression of caveolin‐3 (Cav‐3 OE) against many of the features of heart failure normally observed What is the main finding and its importance?ICa, but not local t‐tubular Ca2+ release, in heart failure induced by pressure overload in mice. Thus Cav‐3 overexpression provides specific but limited protection following induction of heart failure, although other factors disrupt Ca2+ release.Cav‐3 overexpression has little effect in normal ventricular myocytes but reduces cellular hypertrophy and preserves t‐tubular 2+ release at the t‐tubules. However, the extent of cardiac and cellular hypertrophy was reduced in Cav‐3 OE compared to WT mice, and t‐tubular Ca2+ current (ICa) density was maintained. These data suggest that Cav‐3 OE helps prevent hypertrophy and loss of t‐tubular ICa following TAC, but that other factors disrupt local Ca2+ release.Caveolin‐3 (Cav‐3) is an 18 kDa protein that has been implicated in t‐tubule formation and function in cardiac ventricular myocytes. During cardiac hypertrophy and failure, Cav‐3 expression decreases, t‐tubule structure is disrupted and excitation–contraction coupling (ECC) is impaired. Previous work has suggested that Cav‐3 overexpression (OE) is cardio‐protective, but the effect of Cav‐3 OE on these cellular changes is unknown. We therefore investigated whether Cav‐3 OE in mice is protective against the cellular effects of pressure overload induced by 8 weeks’ transverse aortic constriction (TAC). Cav‐3 OE mice developed cardiac dilatation, decreased stroke volume and ejection fraction, and hypertrophy and pulmonary congestion in response to TAC. These changes were accompanied by cellular hypertrophy, a decrease in t‐tubule regularity and density, and impaired local Ca The consequent Ca2+ influx (ICa) triggers Ca2+ release from adjacent sarcoplasmic reticulum (SR) via Ca2+ release channels . This Ca2+‐induced Ca2+ release  in cardiac myocytes is initiated by the action potential, which activates sarcolemmal L‐type CaFabiato,  producesICa to the t‐tubules  is a cholesterol‐binding protein that has been implicated in the genesis of t‐tubules  and conducted in accordance with UK legislation  Act 1986 Amendment Regulations 2012 incorporating European Directive 2010/63/EU); the study also complies with the ethical principles under which 2.2ad libitum access to food and water.Adult (12 week) male homozygous cardiac‐specific Cav‐3 OE mice, produced as described previously  and medetomidine  and given buprenorphine  Ltd, Hull, UK) for pain relief; the surgical plane of anaesthesia was monitored using the limb withdrawal reflex. The aortic arch was exposed via a medial sternal thoracotomy and a silk ligature (6‐0) placed between the innominate and left carotid arteries and tied round a 27G needle (0.4 mm OD). Sham animals underwent the same operation but without placement of the banding suture. Animals were maintained post‐operatively for 8 weeks before use.Eight weeks of TAC was used to produce pressure overload, since this has previously been shown to result in cardiac hypertrophy and failure , heart rate was monitored, and measurements of contractile performance made from M‐mode images acquired from the parasternal short axis view using a Vevo 3100  and MX550D transducer.2.4Animals were killed by cervical dislocation and ventricular myocytes isolated using standard enzymatic digestion via Langendorff perfusion as described previously  in Airyscan ‘super‐resolution’ mode, with a 1.2 NA, ×40 water immersion objective, sampled at 40 nm in‐plane and 180 nm along the optical axis. Airyscan uses a 32‐channel photomultiplier tube detector that collects a pinhole‐plane image at every scan position, thus improving spatial resolution. In super‐resolution mode, linear deconvolution provides further improvement to achieve spatial resolution that is 1.7× that of a conventional confocal microscope. The regularity of t‐tubule staining was quantified by applying a two‐dimensional (2D) fast Fourier transform (FFT) to an offset‐subtracted square region of the cell interior, and the power of the first harmonic normalized to that of the average image intensity (P1/P0). T‐tubule density was calculated from an intracellular volume marked by hand. The 3D skeleton of the t‐tubules was obtained by processing the volumetric data with a tubule‐enhancing 3D filter, segmenting using an Otsu threshold in MATLAB R2015a , and converting to a skeleton using Skeletonize (2D/3D) in ImageJ . The skeleton was used to calculate t‐tubule density  and local Eigenvectors for t‐tubule angles. Tubule orientation is expressed relative to the transverse plane, so that 0° corresponds to a transverse tubule, while 90° corresponds to a tubule that extends along the cell .Surface and t‐tubular cell membranes were labelled by incubating cells with 5 μmol l2.6m Tris–HCl pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% (v/v) Triton X‐100, 1% (w/v) sodium deoxycholate, 0.1% (v/v) SDS, 1 mm phenylmethylsulfonyl fluoride, complete protease inhibitors, 8 μg ml−1 calpain inhibitor I, 8 μg ml−1 calpain inhibitor II, 50 mm sodium fluoride, 1 mm sodium orthovanadate, and 16 mm sodium pyrophosphate, homogenized by pipetting and incubated on ice for 15 min. After centrifugation at 13,000 g 4°C for 15 min the supernatants were collected, protein concentrations estimated using the Pierce BCA protein assay  and adjustments made to allow for equal protein loading on SDS‐PAGE. Ten‐microgram samples of the myocyte lysates were run on 4–15% gradient SDS‐PAGE gels and transferred onto Immobilon‐P membrane. Blots were probed with antibodies against Cav‐3 , junctophilin‐2  and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) . Protein bands were visualized and images captured using horseradish peroxidase‐conjugated secondary antibodies , chemiluminescence and a G:BOX Chemi XT4 imaging system . Gels were first probed with the antibody to Cav‐3 or JPH‐2, then stripped using a commercial stripping solution  and re‐probed with the loading control antibody to GAPDH, before being stripped and re‐probed for JPH‐2 or Cav‐3. The density of the bands was measured using ImageJ and normalized to GAPDH.Following myocyte isolation, aliquots were pelleted by centrifugation, the supernatant removed and the cell pellet snap frozen in liquid nitrogen and stored at −80°C. Once all samples had been acquired, the pellets were processed simultaneously by thawing directly into lysis buffer containing 50 m2.7ICa amplitude (pA) in intact myocytes was measured as the difference between peak inward current and current at the end of the depolarizing pulse; absolute ICa in the t‐tubular and surface membranes was calculated from measurements of ICa and membrane capacitance in intact and detubulated myocytes with correction for incomplete detubulation as described previously  to calculate ICa density (pA pF−1). ICa density in the t‐tubule membrane was calculated from the loss of membrane current and capacitance following DT; ICa density in the surface membrane was calculated from currents measured in DT myocytes with correction for incomplete detubulation as described previously . Pipette resistance was typically 1.5–3 MΩ when filled with pipette solution (see below), and pipette capacitance and series resistance were compensated by ∼70% to optimize the measurement of membrane current. Currents were activated from a holding potential of −80 mV by step depolarization to −40 mV for 200 ms (to inactivate the sodium current) followed by steps to potentials between −50 and +80 mV for 500 ms, before repolarization to the holding potential, at a frequency of 0.2 Hz. Absolute 2.82+ and membrane potential were recorded simultaneously along single t‐tubules as described previously  and the voltage sensitive dye di‐4‐AN(F)EPPTEA EPPTEA fluorescence was used to determine the time of the AP upstroke, and the latency from the AP upstroke to the initial  and maximum rate of rise of Ca2+ was determined at each point along the Fluo‐4 line‐scan image. The SD of latencies for each cell was used as a measure of the heterogeneity of release. Whole‐cell Ca2+ transients were obtained using line‐scans along the long axis of cells loaded with Fluo‐4/AM only. Cells were field‐stimulated at 0.2 Hz at 1.5 × threshold using parallel Pt electrodes.Intracellular Ca2.9m): 133 NaCl, 5 KCl, 1 MgSO4, 1 CaCl2, 1 Na2HPO4, 10 d‐glucose, 10 HEPES, pH 7.4 (NaOH). During electrophysiological recordings, KCl was replaced with CsCl to inhibit K+ currents and the pipette solution contained (in mm): 110 CsCl, 20 TEACl, 0.5 MgCl2, 5 MgATP, 5 BAPTA, 10 HEPES, 0.4 GTP‐Tris, pH 7.2 (CsOH). For some experiments, cells were incubated in C3SD peptide  for 1 h at room temperature before use. C3SD was designed to mimic the ‘scaffolding domain’ of Cav‐3 and thus to interfere with Cav‐3 binding to its endogenous partners and, as used in the present study, has previously been shown to inhibit Cav‐3‐dependent signalling in cardiac myocytes  for cellular measurements). Data normality was assessed using the Shapiro–Wilk test and subsequent testing was performed using Student's t test or the Mann–Whitney U test, one‐way ANOVA, or the Kruskal–Wallis test, as appropriate. ICa density–voltage relationship curves were analysed using repeated measures (RM) ANOVA with voltage and intervention as factors. Single myocyte properties including those elicited by a step depolarization to a single voltage were analysed with two‐way ANOVA; post hoc tests used the Bonferroni correction. The errors in derived variables , and the subsequent statistical analysis (unpaired Student's t test), were calculated using propagation of errors from the source measurements  for a given phenotype and by † between phenotypes (WT vs. Cav‐3 OE).Data are expressed as mean ± SD  or left ventricular mass . Following TAC, diastolic and systolic left ventricular volume increased, and ejection fraction decreased, in both WT and Cav‐3 OE mice  decreased significantly in Cav‐3 OE, but not WT mice. However, none of the measurements of cardiac function were significantly different between WT and Cav‐3 OE mice following TAC. Thus, it appears that Cav‐3 OE has little effect on in vivo cardiac function either in sham animals or following TAC. However, although TAC increased left ventricular mass in both types of mouse , left ventricular mass was significantly smaller (P < 0.01) in Cav‐3 OE than in WT mice following TAC. Heart rate was not significantly different between groups .Cardiac structure and function were assessed in vivo, following TAC both WT and Cav‐3 OE mice showed a significant increase in wet heart weight  and wet lung weight , but no change in body weight  or tibia length . Thus, in both WT and Cav‐3 OE mice, TAC resulted in cardiac hypertrophy, indicated by a significant increase in heart weight:tibia length ratio  and width , and thus calculated cell volume  and TAC (P < 0.001) Cav‐3 OE myocytes  phase of the whole cell Ca2+ transient, which was not significantly different in WT and Cav‐3 OE myocytes and showed no change in either time to peak or amplitude  decrease in cardiac function in Cav‐3 OE mice, but a significant decrease in WT mice  two mechanisms. The first is by altering cell growth. It has previously been suggested that Cav‐3 is anti‐hypertrophic  compared with the mouse's normal heart rate, to enable comparison with ICa, which was recorded at this frequency to allow recovery from inactivation between voltage clamp pulses. It remains possible, therefore, that changes in local Ca2+ release may decrease Ca2+ transient amplitude, and thus impair cardiac performance, at physiological frequencies. However, the impairment occurred in the presence of increased heart size, so that reduced stroke volume and ejection fraction do not necessarily imply reduced contractility because myocyte contraction will have to overcome the higher wall tension that will result from the increase in heart size (law of Laplace).Regardless of the mechanism underlying the maintenance of t‐tubular 2+ release despite the recovery of t‐tubular ICa suggests that the decrease in ICa is not the primary cause of disruption of Ca2+ release following TAC. This may be because of redundancy in the Ca2+‐induced Ca2+ release process (Cannell, Berlin, & Lederer, ICa, which are regulated by Cav‐3 are predominantly extra‐dyadic (Glukhov et al., ICa amplitude produced by Ca2+ channel blockers, which will affect all Ca2+ channels, inhibit release (Bryant et al., 2+ release may, therefore, be due predominantly to disruption of the dyad (Louch et al., ICa may have summative effects that impair Ca2+ release along t‐tubules. Interestingly, however, Cav‐3 OE decreased the heterogeneity of Ca2+ release in both sham and TAC myocytes, suggesting that Cav‐3 may increase the uniformity of t‐tubular Ca2+ release by altering the distribution of ICa or the response to ICa along the t‐tubule.The lack of effect of Cav‐3 OE on the latency of Ca5ICa; this is not secondary to the smaller increase in t‐tubular membrane area in OE myocytes, because absolute ICa increases. This attenuation of the TAC phenotype by Cav‐3 OE is consistent with previous work suggesting that Cav‐3 plays a role in the local regulation of ICa and is anti‐hypertrophic, but it remains unclear whether these effects are direct or secondary.These data show that Cav‐3 OE alone has little effect on the structure or function of either the whole heart or ventricular myocytes. However, following TAC, Cav‐3 OE is anti‐hypertrophic and helps to maintain t‐tubular All laboratory experiments were performed at the University of Bristol. S.M.B., C.H.T.K., D.M.R., H.H.P., A.F.J., M.B.C. and C.H.O. conceived the study and designed the project methods. S.M.B., C.H.T.K. and J.J.W. contributed to acquisition, analysis or interpretation of data and S.M.B., C.H.T.K., J.J.W., D.M.R., H.H.P., A.F.J., M.B.C. and C.H.O. contributed to the drafting of the manuscript and its revision. All authors approved the final version of the manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.


Recently, we developed a non-invasive method to analyze receipt of motor commands in terms of movement kinematics for the wrist joint (r/KrB ratio). In the present study, we have identified two components (F1 and F2) of the smooth pursuit movement. We found that the two components were in different control modes with different r/KrB ratios. The major F1 component in a lower frequency range encodes both velocity and position of the moving target (r/Krhigher B ratio) to synchronize movement of the wrist joint with motion of the target in a predictive manner. The minor F2 component in a higher frequency range is biased to position control in order to generate intermittent small step-wise movements. In cerebellar patients, the F1 component shows a selective decrease in the r/KrB ratio, which is correlated with decrease in accuracy of the pursuit movement. We conclude that the r/KrB ratio of the F1 component provides a unique parameter to evaluate accuracy of the predictive control. We also discuss the pathophysiological and clinical implications for clinical ataxiology.Goal-directed movements are predictive and multimodal in nature, especially for moving targets. For instance, during a reaching movement for a moving target, humans need to predict both motion of the target and movement of the limb. Recent computational studies show that the cerebellum predicts current and future states of the body and its environment using internal forward models. Sensory feedback signals from the periphery have delays in reaching the central nervous system, ranging between tens to hundreds of milliseconds. It is well known in engineering that feedback control based on time-delayed inputs can result in oscillatory and often unstable movements. In contrast, the brain predicts a current state from a previous state using forward models. This predictive mechanism most likely underpins stable and dexterous control of reaching movements. Although the  Goal-directed movements are predictive in nature, especially for moving targets in the environment of daily life. The prediction is in essence multimodal. For instance, during a reaching task for a moving target, humans need to predict both motion of the target and movement of the limb to match them optimally. Making predictions and validating the predictions against actual sensory information is a fundamental function of the nervous system. Prediction errors and assessment of the discrepancy between predicted and actual information are critical parameters .Recent computational studies posit a mechanism that predicts current and future states of the body and its environments by integrating an estimate of previous state and efference copies of motor signals, the computation known as an internal forward model . SensoryThe predictive control  and corrective control (known as sensory feedback)  togetherreanalyzed the same data to find that the smooth pursuit movement actually contained two distinct components, corresponding to separate frequency bands. We further identified that the two components were in different control modes that corresponded to predictive and corrective control reviewed above, respectively. The major component in a lower frequency range (referred to as F1) encodes velocity and position of the moving target in a predictive manner, whereas the minor component in a higher frequency range (F2) generates intermittent small step-wise movements to correct positional errors. In cerebellar patients, however, the predictive component is associated with a selective decrease in the velocity component, which results in poorer accuracy of the pursuit movement. The impairment in cerebellar patients was succinctly characterized by a ratio of viscosity to elasticity coefficients (r/KrB ratio defined below) in the F1 component, thereby providing a reliable metric to assess the performance of forward-model prediction. We propose that our new method provides a unique tool to evaluate accuracy of the predictive control in patients with cerebellar ataxia.Although the cerebellum, especially its hemispheric part, has long been suggested as containing loci of various forward models , there iThirteen healthy control subjects with no history of neurological disorders  of the monitor. After a fixed hold period (4 s), the target started moving along the path of the The subjects were asked to perform the smooth pursuit task of the wrist joint  employedflexor carpi radialis (FCR), flexor carpi ulnaris (FCU), extensor carpi ulnaris (ECU), and extensor carpi radialis (ECR)]  and muscle activities [electromyography (EMG) signals] from four wrist prime movers [s (ECR)] .We recorded the EMG signals with Ag-AgCl electrode pairs spaced 10 mm apart. EMG signals were amplified  and band-pass filtered  using an amplifier , and sampled at 2 kHz. The typical locations of the recording electrodes are shown in The EMG signals were rectified and filtered with a second-order low-pass filter ].t) represents the wrist joint torque. iT represents muscle tension processed as explained above  of each muscle (t), t), and t) represent the angle, angular velocity, and angular acceleration of the wrist joint, respectively. M, B, and K are the inertia parameter (kgm2), the viscous coefficient (Nms/rad) and the elastic coefficient (Nm/rad).where τ(h muscle . The varkinematic torque: right-hand side of equation (1)] and the wrist joint torque that is calculated from the muscle activities [muscle torque: middle of equation (1)]. To identify the relationships between the muscle activities and the movement kinematics for the pursuit task, it is necessary to find the two sets of parameters 1a, 2a, 3a, 4a, and M, B, K that optimize the match between the kinematic torque and the muscle torque. We used canonical correlation analysis (CCA) (1T(t), 2T(t), 3T(t), 4T(t)], and the movement kinematics, i.e.,  in each subject with SAS . The program yielded two parameter vectors , and  such that the pair of canonical variates  TM, B, K) T [= Mt) + Bθ(t) + Kθ(t)] maximize their correlation   is justified if there is a high correlation between the wrist joint torque that is calculated from the movement kinematics [is (CCA)  for the CC)] see . In the Furthermore, in our previous study , we demorB) to the elastic (rK) coefficient: r/KrB. To obtain rB and rK, we used the equation (2) and CCA as mentioned above, and calculated r/Kr ratioB. See section “Different B/K Ratios for the Two Components of the Pursuit Movements” in the results for more detail.In the pursuit task in which the subjects tracked a smooth motion of the target, the joint torque was characterized by the velocity-dependent term and the position-dependent term in Eq. (2). Therefore, we introduce a metric to characterize the contributions of velocity and position as a ratio of the viscous coefficient (B/K Ratios for the Two Components of the Pursuit Movements” in the results for more detail), we performed a frequency analysis  for the velocity of the movement. The wrist movement was decomposed into a low-frequency (≤0.5 Hz) component and a high-frequency (>0.5 Hz) component, referred to as the F1 and F2 components, respectively. The r/KrB ratio, defined above, was computed for the F1 and F2 components separately.To analyze components of the wrist movement  as a sum of instantaneous difference  between target position and cursor position of the F1 component throughout the trial. We name it an F1 error.t-test  or Mann–Whitney U test .Statistical tests were made using two-sample a1, a2, a3, a4, and r, KrB that maximize the CC between the two canonical variates  (middle). muscle torque {i.e.,  T}  and kinematic torque {i.e.,  T}  with high values of canonical correlation (CCs) (CC = 0.94). For all control subjects, the average CC was 0.93 ± 0.01  for the pursuit task.We used CCA to analyze the causality relationship between the muscle activities and the movement kinematics of the wrist joint in 13 control subjects using the wrist joint model (2). With CCA , we obta torque) middle. per se (F1) . Thus, the major muscle activity in the F1 domain encoded both velocity and position of the wrist to reproduce the motion of the target. In contrast, muscle activity of the F2 domain (0.5–3 Hz) demonstrated low r/KrB ratios  (F2), like muscle activity for the step-tracking movement . In other words, the minor muscle activity of the F2 domain appeared to be concerned with frequent small adjustments of wrist position.Next, we separated the movement kinematics and activity of each of the four muscles into F1 and F2 components . We thenn = 130) , F2, likr/KrB ratios, and the two components appeared to play distinct roles in the pursuit movement. The F1 component appeared to play the primary role to synchronize the movements of the wrist and the motion of the target. To test this hypothesis, we calculated the cross-correlation of the target motion and the F1 component of the wrist movement. As demonstrated by one control subject (n = 13) (Controls). This short lead time means that the F1 component of the wrist movement cannot be generated with a visuomotor feedback control of the target motion, because the conduction time of the peripheral motor nerve (∼10 ms) and electromechanical delay (∼50 ms) alone would take that long. Thus, the delay was too short to be a visuomotor feedback delay. Rather, generation of the motor command in the CNS must have preceded the corresponding motion of the target, if we take the average lead time of neuron activity in the motor cortex of the monkey for the wrist movement (∼100 ms) into account . Nevertheless, the dissociation of r/KrB ratios for the two components observed in the control subject (r/KrB ratios for the F1 component (r/KrB ratio for F1 (r/KrB = 1.75), while the r/KrB ratio for F2 (r/KrB = 0.15) was the same as that of the control subject (r/KrB = 0.15).Next, we determined the  subject  was signomponent . The Br/o for F1  was no mr/KrB ratios for the F1 component and relative preservation of low r/KrB ratios for the F2 component were shared by all cerebellar patients  (F1) were significantly lower than those of the control subjects  (F1) (p < 0.001). In contrast, r/KrB ratios of the F2 component were comparable for both groups (compare F2 and F2). To summarize, the poor performance of target tracking in the cerebellar patients was attributed to the selective decrease in Br/Kr ratios for the F1 component (The marked decrease in patients . Br/Kr r ± 0.42) , F1 were ± 0.28) , F1 , we found that the F1 component of the patients was delayed on average by about 100 ms  than that of the controls  (p < 0.0001).It should be noted that the decrease in r/KrB ratios of the F1 component and performance of pursuit movement in the cerebellar patients and the control subjects . As shown in r/KrB ratio of the F1 component and the F1 error demonstrated a clear negative correlation, although the F1 error showed little decrease for higher r/KrB ratio (>1.5). In other words, relative decrease of muscle activity proportional to velocity resulted in poorer accuracy of tracking. However, there remains a possibility that an increase in F1 error  may be compensated by a feedback control and does not affect the overall performance of the pursuit movement. In order to test this possibility, we examined the relationship between the r/KrB ratio of the F1 component and the tracking score. The tracking score is defined as a percentage of time when the cursor was kept within the target in a single trial . Furthermore, the F1 error and the tracking score demonstrated a strikingly linear (negative) correlation , suggesting that the F2 component is recruited to compensate for increase in F1 error.Finally, we have examined a possibility that the F2 component could be related to an error-correction mechanism. The power  of F2 component see , F2 and r/KrB ratios in control subjects. The major F1 component with higher r/KrB ratio appears to play the primary role to reproduce both velocity and position of the target motion in a predictive manner. In contrast, the minor F2 component with lower r/KrB ratio encodes mostly position of small step-wise movements. Therefore, the two control modes, predictive control based on the forward-model prediction and corrective control based on sensory feedback, were identified as the F1 and F2 components, respectively. In cerebellar patients, the predictive F1 component demonstrates a selective decrease in the r/KrB ratio. Notably, the r/KrB ratios of the F1 component has a strong correlation with accuracy of the pursuit movement. In contrast, there was no significant difference between the r/KrB ratios of the F2 component for control and patient groups. Taken together, our results support the hypothesis that cerebellar patients have an impairment in the forward-model prediction while maintaining corrective control in response to sensory feedback. In the following sections, we will focus on five points: (1) dissociation of two components of pursuit movement; (2) functional interpretation of the r/KrB ratio; (3) the r/KrB ratios for F1 and F2 components in patients with cerebellar ataxia and the role of the cerebellum in predictive control; (4) the F1 (predictive) component of the pursuit movement and precision of motor control; (5) quantitative evaluation of motor function of patients with cerebellar ataxia based on the r/KrB ratio.We demonstrate that the smooth pursuit movement of the wrist joint consists of two components with distinct The basic design of this study owes to r/KrB ratio). In contrast, for the smooth pursuit task in which the target moves with known velocity and position, the muscle activities were correlated comparably with the velocity and position of the target motion (higher r/KrB ratio). In contrast, the ability of cerebellar patients to select a proper r/KrB ratio depending on the task requirement was markedly deteriorated  of the smooth target motion (predictive manner (r/KrB ratio) of small step-wise movements  during the pursuit movement. Indeed, the F2 component appeared to provide quick corrective  mechanism , cerebellar patients must rely on position-dominant step-wise movements (top inset), which are probably manageable only with position control. The step-wise position-dominant movement appears to be a default mode of motor control that utilized by patients with cerebellar ataxia as a compensation method. Indeed, the low r/KrB ratio for the F2 component in cerebellar patients was similar to that in control subjects (F2). On the other hand, velocity control is continuous and predictive in nature. Therefore, the impaired velocity control and decrease in tracking accuracy  could be generated with muscle activities with even lower r/KrB ratios compared to those observed in cerebellar patients. Nevertheless, the r/KrB ratio of the F1 (predictive) component demonstrated a strong negative correlation with the error of the predictive movement (r/KrB ratio of the predictive (F1) component provides a unique parameter that represents accuracy of the predictive control for the pursuit movement in patients with ataxia.The movement  and a stmovement . Therefor/KrB ratio for the F1 component strongly reflected the pathophysiological changes in these patients (r/KrB ratios for the F1 component of the pursuit task in ataxic patients for a long period.Precise evaluations of motor functions of patients with neurological disorders are essential for both monitoring the progress of disease and evaluation of effects of treatment. Although several groups have tried to perform quantitative evaluations of cerebellar ataxia with arm movements , their epatients , 6B, 8. r/KrB ratio of the F1 component provides a unique parameter to characterize the accuracy in terms of predictive control of voluntary goal-directed motion. This method can be applied in the numerous forms of cerebellar ataxias encountered in daily practice.In conclusion, the This study was carried out in accordance with the recommendations of the ethics committees of the Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Neurological Hospital, and Tokyo Medical University with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the ethics committees of the Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Neurological Hospital, and Tokyo Medical University.SK and JL conceived and designed the experiments. JL, SK, and HM conducted the experiments. JL, SK, HM, and HT analyzed the data. SK, JL, HM, HT, MM, and CH wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Type 1 diabetes mellitus (T1DM) has been linked to many autoimmune problems. The association between T1DM and urticaria warrants investigation. Data were extracted from the National Health Insurance Research Database (NHIRD) of Taiwan. Participants with T1DM were recruited as the case group, and that group was matched by sex and age at a ratio of 1:4 to the control group comprising those without T1DM. The study period was 1998–2011. All participants were followed up to the diagnosis of urticaria, withdrawal from the insurance program, death, or the end of the study. A multivariable Cox proportional hazard model was used to calculate the adjusted and crude hazard ratios for urticaria. A total of 5895 participants (1179 in the case group and 4716 in the control group) were followed up in the study. The total incidence rate of urticaria in patients with type 1 DM was 26.6 per 1000 person-years, and that in controls was 6.85 per 1000 person-years. Compared with the control group, the hazard ratio of urticaria in the case group was 2.84 (95% CI = 2.27–3.56). Compared with age-matched participants without T1DM, patients with type 1 DM aged <18 years had a 3.62-fold higher risk of urticaria (95% CI = 2.85–4.59). The hazard ratio in patients with an adjusted Diabetes Complications Severity Index (aDCSI) score of 1.01–2.00 per year was 2.57 (95% CI = 1.18–5.57), and that in patients with an aDCSI score of >2.00 per year was 4.47 (95% CI = 2.68–7.47). T1DM patients aged <18 years had an increased risk of urticaria, but a similar phenomenon was not observed among T1DM patients older than 18 years. Urticaria, either acute or chronic, is a troublesome skin disorder with the presentation of hives and an itching sensation . Acute uPrevious studies have indicated that chronic urticaria is less prevalent in children than in adults, and most urticaria studies have focused on adults ,11. HoweWhether urticaria in children is also associated with autoimmune disease, similar to the phenomenon observed in adults remains unknown. Type 1 diabetes mellitus is a well-recognized autoimmune disease associated with autoantibodies in pancreatic islet cells. Therefore, we determined a type 1 diabetes mellitus disease model. We conducted a nationwide cohort study aiming to investigate the incidence of urticaria in type 1 diabetes mellitus children and adults.The National Health Insurance Administration of Taiwan established a national health insurance program in 1995. The National Health Insurance Research Database (NHIRD) of Taiwan was subsequently constructed to improve medical research resources in Taiwan. Specifically, 99% of the residents of Taiwan are enrolled into the NHI program, and their medical information is recorded in the NHIRD. Catastrophic illness data in the NHIRD were utilized to conduct the present study. The identification data for all patients were encrypted to ensure their privacy. The disease codes were recorded by physician specialists according to the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM). This study was approved by the Research Ethics Committee of China Medical University and Hospital in Taiwan (CMUH104-REC2-115-CR4).The study cohort consisted of case and control groups. Participants with T1DM (ICD-9-CM codes 250.x1 and 250.x3) were recruited as the case group. To ensure the correct diagnosis of urticaria, we defined patients with newly diagnosed urticaria as those with at least three claims for outpatient and/or hospitalization visits. The control group included individuals without T1DM. This study excluded patients with type 2 DM (ICD-9-CM code 250), gestational diabetes mellitus (GDM) (ICD-9-CM code 648.83), and atopic diseases . The age criterion was younger than 30 years of age. The study period was 1998–2011. One T1DM patient was matched by sex and age to four people in the control group. The study cohort was followed up to the point of diagnosis of urticaria, withdrawal from the insurance program, death, or the end of the study.2), proportion of residents with higher education, elderly and agricultural population, and the number of physicians per 100,000 people in each area. Level 1 represents areas with a higher population density and socioeconomic status, and level 7 represents the lowest. Because few people lived in more rural areas of levels 4–7, our study included these areas in the level 4 group. [The primary outcome of this study was the diagnosis of urticaria (ICD-9-CM code 708). The cohort was divided into two groups by using 18 years of age as the cutoff. The aim of this study was to investigate whether T1DM “children” had a lower or higher incidence of urticaria. Therefore, we compared T1DM patients less than and older than 18 years. However, it has been reported that chronic urticaria is associated with metabolic syndrome , which i4 group.  The cate4 group. ,18 was at test. Incidence densities of urticaria according to each variable were calculated for the two groups. The crude hazard ratio was estimated using a univariable Cox proportional hazard model. Sex, age, urbanization level, parental occupation, and inflammatory bowel disease were included in a multivariable Cox proportional hazard model for calculating the adjusted crude hazard ratio. The effect of the adjusted Diabetes Complications Severity Index (aDCSI) score change on urticaria in T1DM patients was also analyzed. The Kaplan-Meier method was utilized to determine the cumulative incidence of urticaria for the two groups. The level of statistical significance was set at a p value of <0.05.The chi-square test was performed to compare the difference in categorical variables between the case and control groups. Continuous variables were compared between the two groups using the Student’s A total of 5895 participants (1179 in the case group and 4716 in the control group) were followed up in the study. p < 0.001). As shown in p for trend < 0.001).The association between aDCSI score and urticaria in the case group is shown in Urticaria has been reported to have a connection with autoimmune disease, such as Grave’s disease, type 1 diabetes, and autoimmune thyroiditis . HoweverA similar association between T1DM and urticaria was not observed in adults in this study. This may be because we excluded those diagnosed with urticaria before enrollment into the study. It has been estimated that 15–23% of adults have experienced at least one episode of urticaria ; thus, cThis study has several limitations. First, information on the results of autoantibodies, total and specific IgE, differentiated blood count, erythrocyte sedimentation rate, C-reactive protein, autologous serum skin test, thyroid hormones and thyroid autoantibodies, antinuclear autoantibodies, surveys for infection and intolerance of specific food, urine analysis, skin prick tests, and investigation of autoimmune diseases is not available in the NHIRD. However, in our preliminary analysis, we considered and adjusted for many autoimmune diseases, including autoimmune thyroid disease, Addison disease, vitiligo, ankylosing spondylitis, inflammatory bowel disease, pernicious anemia, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, Sjögren syndrome, myasthenia gravis, and others. We excluded the aforementioned autoimmune diseases in our formal study because the case numbers were too few and nonsignificant. Second, we did not obtain detailed data through patient questionnaires, diaries, and calendars. Third, we used ICD-9 codes for ensuring the diagnosis of urticaria, which made it difficult to differentiate between acute or chronic urticaria and identify urticaria subtypes. Therefore, we could not perform further analysis based on the subtype of urticaria, such as physical urticaria, cold urticaria, or food urticaria, which is most associated with type 1 diabetes. Fourth, we matched gender and index year between study and control cohort. Therefore, the mean ages of these two cohorts were significantly different. Finally, we did not adjust for medical visits in our analysis. Because T1DM patients have a higher likelihood of utilizing medical care, surveillance bias may exist.Our study showed that T1DM patients aged <18 years had an increased risk of urticaria, but a similar phenomenon could not be observed among T1DM patients older than 18 years. Our results indicate the possibility of urticaria development in children with T1DM.


Background and Purpose: Store-operated calcium entry (SOCE) is an important physiological phenomenon that extensively mediates intracellular calcium ion (Ca2+) load. It has been previously found in myocytes isolated from neonatal or diseased hearts. We aimed to determine its existence, molecular nature in undiseased hearts and its potential arrhythmogenic implications under hyperactive conditions.Experimental Approach: Ventricular myocytes isolated from adult FVB mice were studied by using Ca2+ imaging and whole-cell perforated patch-clamp recording. In addition, lead II ECGs were recorded in isolated Langendorff-perfused mice hearts. Functional TRPC channel antibodies and inhibitors, and TRPC6 activator hyperforin were used.Key Results: In this study, we demonstrate the existence and contribution of SOCE in normal adult mouse cardiac myocytes. For an apparent SOCE activation, complete depletion of sarcoplasmic reticulum (SR) Ca2+ by employing both caffeine (10 mM) and thapsigargin (1 μM) or cyclopiazonic acid (10 μM) was required. Consistent with the notion that SOCE may be mediated by heteromultimeric TRPC channels, SOCEs observed from those myocytes were significantly reduced by the pretreatment with anti-TRPC1, 3, and 6 antibodies as well as by gadolinium, a non-selective TRPC channel blocker. In addition, we showed that SOCE may regulate spontaneous SR Ca2+ release, Ca2+ waves, and triggered activities which may manifest cardiac arrhythmias. Since the spontaneous depolarization in membrane potential preceded the elevation of intracellular Ca2+, an inward membrane current presumably via TRPC channels was considered as the predominant cause of cellular arrhythmias. The selective TRPC6 activator hyperforin (0.1–10 μM) significantly facilitated the SOCE, SOCE-mediated inward current, and calcium load in the ventricular myocytes. ECG recording further demonstrated the proarrhythmic effects of hyperforin in ex vivo mouse hearts.Conclusion and Implications: We suggest that SOCE, which is at least partially mediated by TRPC channels, exists in adult mouse ventricular myocytes. TRPC channels and SOCE mechanism may be involved in cardiac arrhythmogenesis via promotion of spontaneous Ca2+ waves and triggered activities under hyperactivated conditions. TRPC cheostasis . The parproaches . Recent proaches , 2009. F2+ entry . Recent 2+ entry , Ca2+ pax injury , as wellx injury . Hyperfolear yet . It has lear yet . TherefoIn the present study, we demonstrate the presence of SOCE in adult mouse ventricular myocytes, and elucidate the possible roles of SOCE/TRPCs in promoting cardiac arrhythmias via facilitating the generation of inward current and calcium load. Our findings support a missing link between upregulated SOCE/TRPCs activities, especially TRPC6, to lethal arrhythmias in the heart.2+ concentration ([Ca2+]) upon restoration of external calcium load (1.8 mM)  antibodies for TRPC1, 3 and 6 were a generous gift from Dr. Robert M. Graham . These antibodies recognize the putative pore-forming region of mouse TRPC channels specifically, and pre-incubation with anti-TRPC6 antibody could effectively block the rise of intracellular Ca(1.8 mM) . The SER2+-free Tyrode’s solution containing 1.0 mg/mL collagenase  and 0.1 mg/mL thermolysin (Sigma) for 15 min. The hearts were removed from the perfusion apparatus after washing out the enzyme solution, the left ventricle were gently teased apart with forceps in a petri dish and the myocytes were filtered through a nylon mesh. The Ca2+ concentration was gradually increased to 1.0 mM, and the cells were stored at room temperature and used within 8 h . Briefly, hearts were removed from mice anesthetized with overdosed isoflurane, and were perfused retrogradely at 37°C in Langendorff fashion with nominally Cathin 8 h .2+ indicator Fluo-4 AM by incubating them for ∼30 min in bath solution containing 4 μM Fluo-4 AM (Molecular Probes), after which the cells were washed and placed in a heated chamber on an inverted microscope. Fluo-4 fluorescence was excited at ∼485 nm and the emission was measured at ∼530 nm using a Nikon Eclipse TE200 inverted microscope with a Fluor x40 oil objective lens . The fluorescence signals were recorded using an Andor Ixon charge-coupled device (CCD) camera (Andor Technology) operated with Imaging Workbench software (INDEC BioSystems) at 50 frames per second with a spatial resolution of 500 × 400 pixels. Fluorescence intensity was measured as the ratio of the fluorescence (F) over the basal diastolic fluorescence (0F). The spontaneous waves were quantified by counting the wave frequency (per minute) appeared after a 3-min perfusion of HF 0.1 μM, compared to the control state.Myocytes were loaded with the Ca2-phosphocreatine, 0.05 cAMP, pH 7.2 with KOH. The cells were superfused with Tyrode’s solution containing (in mM): 136 NaCl, 4.0 KCl, 0.33 Na2PO4, 1.0 CaCl2, 1 MgCl2, 10 glucose and 10 HEPES, pH 7.4 adjusted with NaOH. APs were elicited with 2-ms, 2- to 4-nA square pulses at a PCL of 6 s.Myocytes were patch-clamped using the whole-cell configuration of the perforated patch-clamp technique (240 μg/ml amphotericin B). Voltage or current signals were measured with a MultiClamp 700A patch-clamp amplifier controlled by a personal computer using a Digidata 1322 acquisition board driven by pCLAMP 10 software . For AP recordings, patch pipettes (resistance 2–5 MΩ) were filled with internal solution containing (in mM): 110 K-aspartate, 30 KCl, 5 NaCl, 10 HEPES, 0.1 EGTA, 5 MgATP, 5 Na+, L-type Ca2+, Na+-Ca2+ exchange currents were previously blocked with a K+-free extracellular solution with (in mM) 140 NaCl, 1.0 CaCl2, 0.5 MgCl2, 0.33 NaH2PO4, 5.5 glucose, 0.01 Nifedipine, 0.002 SEA400 and 5 HEPES ( pH 7.4 adjusted with NaOH), and a pipette solution containing (in mM) 90 Cs-aspartate, 30 CsCl, 20 traethylammonium chloride (TEA-Cl), 2 MgCl2, 5 Tris-ATP, 0.1 Li2-GTP, 5 EGTA, 2 CaCl2 and 5 HEPES (pH adjusted to 7.2 with CsOH)[17]. All patch clamp and Ca2+ imaging experiments were performed at 37°C.The whole-cell I–V relationship was measured by applying ramp pulses from -110 to +50 mV and the holding potential was -80 mV. K1-S2 arrhythmia induction protocol at twice the pacing threshold intensity was adapted from 1), 3 extra stimuli (S2) with a coupling cycle length of 50, 40, or 30 ms, respectively, were introduced. Each of these 3 sets of stimulation were repeated 3 times. Arrhythmias will be categorized into 5 groups and assigned the following points: 0 points, no arrhythmia; 1 point, 1–3 premature ventricular complexes (PVCs); 2 points, non-sustained VT ; 3 points, sustained VT (>10 consecutive PVCs); and 4 points, VF/SCD ((Pseudo-)Lead II ECGs were recorded in isolated Langendorff-perfused hearts. A pair of Ag-AgCl electrodes were placed close to the apex of the left ventricle as well as at the right atrial appendage to obtain ECG signals at a sampling rate of 1 kHz. Two additional platinum electrodes were placed on the free wall of the right ventricle for stimulation using a stimulator (Grass) triggered by a custom-designed computer program. To induce ventricular arrhythmias, a standard S, VF/SCD .t-tests or ANOVA analysis, with P < 0.05 considered significant.Data are presented as mean ± SEM, and at least 3 animals were used for each group. Statistical significance was assessed using paired, unpaired Student’s 2+. The changes of Ca2+ level (reflected by Fluo-4 fluorescence intensity) were measured by increasing extracellular Ca2+ concentration ([Ca2+]) from 0 to 1 mM  was observed when [Ca2+] was changed from 0 to 1 mM. In order to maximally/completely deplete SR Ca2+, in addition to CPA, we also employed 10 mM caffeine to fully open RyR. As a result, a much larger elevation of Ca2+ level was induced when [Ca2+] was changed from 0 to 1 mM . As shown in Figure 0 = 2.7 ± 0.7) was markedly higher than that after Tha/CPA only , suggesting the existence of SOCE in adult cardiac myocytes, and a maximal SOCE activation requires the complete depletion of SR Ca2+. This SOCE was effectively blocked by SOCE/TRPC blockers gadolinium  and ML-9 , but not by Na+/Ca2+ exchanger (NCX) inhibitor SEA0400  + caffeine (10 mM). Control myocytes without pretreatment with antibodies exhibited a peak SOCE amplitude of F/F0 = 2.4 ± 0.2, n = 39, whereas all three TRPC1, 3 or 6 antibodies significantly inhibited the peak of SOCE to F/F0 = 1.4 ± 0.2 (n = 10), 1.2 ± 0.2 (n = 8), 1.1 ± 0.1 (n = 8), respectively . Similar inhibitory effects to 1.2 ± 0.1 (n = 5) by Gd3+ or to 1.2 ± 0.1 (n = 6) by the TRPC3 blocker Pyr3 pre-perfused in the bath for 10–15 min were also observed   antibodies for TRPC1, 3 and 6 were added, respectively, to pretreat the myocytes for 30 min before SOCE was evaluated following the standard protocol with complete depletion of SR Ca0 = 1.5 ± 0.1, n = 26). Hyperforin further potentiated the SOCE level in a concentration-dependent manner in adult mouse ventricular myocytes. The amplitudes (F/F0) of SOCE in the presence of hyperforin at 0.1, 1, and 10 μM were, 1.9 ± 0.1 (n = 15), 2.4 ± 0.2 (n = 8) and 3.7 ± 0.4 (n = 7), respectively , which were all significantly higher than the control SOCE value before hyperforin application.Furthermore, the participation of TRPC6 in SOCE was confirmed by using various concentrations of hyperforin, a potent activator of TRPC6 . As show2+ transients and Ca2+ waves. Ca2+ transients were measured by pacing myocytes continuously at a PCL of 2 s. As shown in a representative trace  by 0.1 μM hyperforin, and from 2.0 ± 0.2 to 2.4 ± 0.1  by 1 μM hyperforin. To exclude the possibility that the enhancement of Ca2+ transients might be due to the potentiation any other voltage-gated Ca influx mechanisms, we then used a different protocol in which the cells were kept quiescent when they were perfused with hyperforin (0.1 or 1 μM) for 1 min  as indicated by the arrow in each panel). We found that Ca2+ transient amplitude was enhanced by hyperforin treatment even the cells remained in resting condition. Ca2+ transient amplitudes increased from 1.8 ± 0.2 to 2.1 ± 0.3  by 0.1 μM hyperforin and from 2.4 ± 0.5 to 3.0 ± 0.4  by 1 μM hyperforin . These data suggest that hyperforin promoted Ca2+ entry through sarcolemmal membrane and increase of cellular/SR Ca2+ load, unlikely via voltage-gated Ca2+ channels.Next, we evaluated how hyperforin-activated TRPC may affect Cae Figure , the ampM Figure  and 1 μMM Figure . Summarin Figure   without being stimulated. As shown in Figure 2+ waves were observed under baseline in normal cells (2+ waves were significantly facilitated by hyperforin (0.1 μM) with a frequency increase from 22 ± 3/min under control condition to 38 ± 4/min after hyperforin treatment . Taken together, the results shown in Figures 2+ depletion condition and TRPC6 channels independent of SR Ca2+ depletion.Next, we further assessed the effect of hyperforin on spontaneous Caal cells . These sr [Ca2+]  mM withoOCE) after SR Ca2+ depletion was subsequently measured. As described in the Method section, K+, Na+, L-type Ca, and Na+-Ca2+ exchange currents were pre-blocked. Whole-cell currents were recorded under the voltage clamp condition using a ramp protocol from -110 to +50 mV, and the inward current amplitudes at -110 mV were evaluated. As shown in time course and representative traces . As summarized in Figure n = 12 each, ∗p < 0.01) in the inward current, which was attenuated by the application of 1 mM Gd3+ . The I/V relationship revealed a reversal potential around 0 mV, indicating its non-selective property, which is consistent with TRPC channels as reported by n = 8, ∗p < 0.05 compared to control), which was effectively blocked by 1 mM Gd3+ .The inward current when SOCE occurred  of membrane potential and corresponding elevation of intracellular Ca2+. As shown in the expanded regions  had been removed in this condition when arrhythmias occurred, it seemed that Ca2+ release and NCX current did not play a causal role in spontaneous membrane depolarization, in which case the initiation of intracellular Ca2+ elevation should occur earlier than membrane potential changes. This relation has been well discussed in our previous publication  remained longer compared to control  were used to assess the propensity to develop arrhythmias in each group. As shown in Figures n = 6), which was significantly higher than hearts without treatment with hyperforin .To examine the arrhythmogenic effect of activation of TRPC6 channel by hyperforin at the whole-heart level, hyperforin (0.1 μM) was perfused in 2+ signaling. In addition to replenish the SR stores, its long-term and immediate physiological significances have been profoundly explored in previous studies, includes cell proliferation, apoptosis, muscle contraction and intracellular Ca2+ oscillations   , the rolomyocyte , and theomyocyte .OCE exist in normal adult ventricular myocytes subsequent to a complete depletion of SR Ca2+ storage. SR Ca2+ level can be physiologically reduced following the activation of calcium-releasing signals mediated by IP3R or RyR. Whereas under our experimental condition, specific inhibitors of SR Ca2+ pump  are usually employed to induce SOCE, previous studies seemed to produce only moderate increase in normal adult cardiac myocytes when exposed to CPA or Tha separately (0: over 2.5) compared to that induced only by SERCA inhibitor. This Ca2+ entry can be effectively inhibited by acute application of SOCE blockers Gd3+ and ML-9, but not by the NCX inhibitor SEA0400. We have noticed that neither Gd3+  nor ML-9  is completely selective to TRPC channels. However, our experiments using selective antibodies of TRPC channels provide more convincing evidence for the involvement of TRPC channels.Our present study demonstrated that SOCE and Iparately . Similarparately . Alternaparately . In the 2+ load-independent pathway, e.g., PKA-dependent activation. This issue needs to be further elucidated.It should be pointed out that caffeine also functions as a PDE inhibitor , which i2+ selectivity, the involvement of TRPCs in SOCE is still under debate. For example, the expression of TRPC3 has been examined in DT40 B lympohocytes by two groups individually. One group demonstrated the TRPC3 activation is triggered in response to PLC-γ or PLC-β enzymes, but independent of SR depletion , and it was reported to selectively activate TRPC6 channels. Our data clearly demonstrated that hyperforin at concentrations of 0.1 and 1 μM predominantly potentiated the SOCE and IOCE in adult mouse ventricular myocytes. It also facilitated the spontaneous Ca2+ waves and promoted the calcium transient amplitudes. A previous study has shown that hyperforin exerts complex actions on cortical neurons. In addition to promoting TRPC6 channels in plasma membrane, hyperforin may exert protonophore-like effect, triggering the release of Ca2+ and Zn2+ from mitochondria  in the setting of our experiments, but imply that hyperforin gives rise to Ca2+ most likely via activation of TRPC6 in cardiac myocytes. Our results have demonstrated that although the activation of SOCE requires SR Ca2+ depletion, its potential composing entity, TRPC channels, are able to be activated by HF independent of SR Ca2+ depletion (Figure ex vivo hearts, implying a possible arrhythmogenic role of SOCE/TRPC6 in the whole heart setting. TRPC6 channel was found to be important in the pathologic cardiac remodeling as a key component of a calcium-dependent regulatory loop (Hyperforin is known as an antidepressant compound extracted from St. John’s wort (chondria . Howevern Figure . This isn Figure . We obseory loop . Herein,HW, ZZ, NF, and L-HX performed the experiments and analyzed the data. HW and L-HX conceived and designed the research, interpreted results of experiments, prepared figures, and wrote manuscript. HW, ZZ, NF, and L-HX approved final version of manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.