In this study, we explored the contractile and electrophysiological properties of nerol and demonstrated its antiarrhythmic effects in guinea pig heart preparation. Nerol effects were evaluated on atrial and ventricular tissue contractility, electrocardiogram (ECG), voltage-dependent L-type Ca2+ current , and ouabain-triggered arrhythmias. Overall our results revealed that by increasing concentrations of nerol (from 0.001 to 30 mM) there was a significant decrease in left atrium contractile force. This effect was completely and rapidly reversible after washing out (~ 2 min). Nerol (at 3 mM concentration) decreased the left atrium positive inotropic response evoked by adding up CaCl2 in the extracellular medium. Interestingly, when using a lower concentration of nerol (30 μM), it was not possible to clearly observe any significant ECG signal alterations but a small reduction of ventricular contractility was observed. In addition, 300 μM nerol promoted a significant decrease on the cardiac rate and contractility. Important to note is the fact that in isolated cardiomyocytes, peak ICa,L was reduced by 58.9 ± 6.31% after perfusing 300 μM nerol . Nerol, at 30 and 300 μM, delayed the time of onset of ouabain-triggered arrhythmias and provoked a decrease in the diastolic tension induced by the presence of ouabain (50 μM). Furthermore, nerol preincubation significantly attenuated arrhythmia severity index without changes in the positive inotropism elicited by ouabain exposure. Taken all together, we may be able to conclude that nerol primarily by reducing Ca2+ influx through L-type Ca2+ channel blockade lessened the severity of ouabain-triggered arrhythmias in mammalian heart.Nerol (C Cardiac myocytes exhibit an exquisite dynamic control of intracellular Ca2+ homeostasis. Perturbations in this control process have been recognized as a major contributor to life-threatening ventricular arrhythmias i, which enhances cell contractility by increasing intracellular [Ca2+] due to NCX [2+ efflux by the NCX as [Na+]i rises. In other words, less Ca2+ efflux for a given Ca2+ influx would increase intracellular Ca2+. The increased [Na+]i could also increase Ca2+ influx via reverse-mode of NCX i increases as a result of Na+/K+ ATPase inhibition. Nerol at 30 and 300 μM delayed the onset time for cardiac arrhythmias but the ouabain-triggered inotropy remained largely intact (see 2+ dynamics. Furthermore, nerol decreased the occurrence of more severe arrhythmias such as tachycardia and ventricular fibrillation.Several groups have previously demonstrated that the positive inotropism observed during the exposure to ouabain is mostly related to Catact see . These rIn our study, nerol corrected key adverse effects of ouabain-triggered arrhythmias: it abolished heart diastolic contracture, it recovered electrical activity, and it reduced the propensity to generate arrhythmias. This is the first study to actually demonstrate that, mechanistically, nerol at low concentration can preserve inotropy and the electrical properties of cardiac muscle while at the same time acting as antiarrhythmic agent. Taken all together, nerol may be useful adjunct to digitalis treatment and justifies further investigation.Small animal models are commonly used in cardiovascular research showing advantages and disadvantages . The pre


We used (i) the hiPSC-CM Paci2013 model as starting point, (ii) a new dataset of Ca2+ transient measurements to tune the parameters of the inward and outward Ca2+ fluxes of sarcoplasmic reticulum, and (iii) an automatic parameter optimization to fit action potentials and Ca2+ transients. The Paci2018 model simulates, together with the typical hiPSC-CM spontaneous action potentials, more refined Ca2+ transients and delayed afterdepolarizations-like abnormalities, which the old Paci2013 was not able to predict due to its mathematical formulation. The Paci2018 model was validated against (i) the same current blocking experiments used to validate the Paci2013 model, and (ii) recently published data about effects of different extracellular ionic concentrations. In conclusion, we present a new and more versatile in silico model, which will provide a platform for modeling the effects of drugs or mutations that affect Ca2+ handling in hiPSC-CMs.The growing importance of human induced pluripotent stem cell-derived cardiomyoyctes (hiPSC-CMs), as patient-specific and disease-specific models for studying cellular cardiac electrophysiology or for preliminary cardiotoxicity tests, generated better understanding of hiPSC-CM biophysical mechanisms and great amount of action potential and calcium transient data. In this paper, we propose a new hiPSC-CM  Especially hiPSC-CMs' patient- and disease-specificity is fundamental to assess the effects of genetic mutations, such as Long QT (LQT)  (http://cipaproject.org/) in 2013, hiPSC-CMs have had a dramatic impact on pharmacology, serving as a powerful in vitro model to test the in silico model predictions regarding cardiac safety or drug toxicity at cellular level.Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are cardiac cells derived from stem cells, which have been produced by donor's differentiated cells by means of reprogramming  measurements. Ca2+ is fundamental in the heart excitation-contraction (EC) coupling, i.e., how the electrical and the mechanical properties of the heart are linked together and how the AP leads to the cardiomyocyte contraction. The elements involved in this phenomenon are the L-type Ca2+ channels, the sarcoplasmic reticulum (SR) and the sarcomeres, i.e., the contractile unit of the cardiomyocyte. During the AP upstroke, the L-type Ca2+ channels open and Ca2+ flows into the cardiomyocyte. This Ca2+ influx is sufficient to trigger the Ca2+ release from SR through the ryanodine-sensitive Ca2+ channels, which increases the cytosolic Ca2+. Such amount of Ca2+ allows starting the crossbridge cycle, which is at the basis of the cardiomyocite contraction and continues until Ca2+ is restored to its basal cytosolic concentration. This is mainly done by the SERCA-2 pump, which reabsorbs Ca2+ from the cytosol into the SR. Moreover, cytosolic Ca2+ is extruded into the extracellular space by the Na+/Ca2+ exchanger (INaCa) and by the sarcolemmal Ca2+ pump (IpCa). All these mechanisms make the intracellular Ca2+ concentration change, thus producing Ca2+ transients associated to the APs  is formulated with the functional but quite elementary Ca2+ release from the TenTusscher2004 model  voltage-dependent activation gate, and g is the Irel specific inactivation gate. Therefore, Irel is not activated by the cytosolic Ca2+ concentration Cai sensed by the Ryanodine-sensitive receptor located on the SR membrane, but it is triggered the same way as ICaL. Due to this mechanism, the model was not able to produce proarrhythmic triggers, such as delayed afterdepolarization (DADs)  gating variable, RyRc the closed (inactivation) gating variable. A third gating variable, RyRa, was used to modulate the working point (adaptation) of the RyRo and RyRc gates according to the cytosolic Ca2+ concentration as in Koivumäki et al.  as described in section “Parameter Optimization.” We report the full set of equations in section 1 of the Supplementary Material.where the subscript ss indicates the steady state value of the gating variable and the subscripts half and k indicate the half Caup) and the leakage current (Ileak) were not changed with respect to the Paci2013 model , maximum diastolic potential (MDP), cycle length (CL), maximum upstroke velocity (Vmax), AP duration at 10, 30, and 90% of repolarization  and AP shape factor (Triangulation). Triangulation is a shape factor used by Ma et al. , time to peak (TPEAK), rise time from 10 to 50% (RT1050), rise time from 10 to 90% (RT1090), decay time from 90 to 10% (DT9010) and the Ca2+ transient rate (FREQ). The full list of the parameters optimized by this method and their original values are reported in Table 2+ handling and additional parameters of INaCa and the Na+/K+ pump (INaK), i.e., those currents for which the Ma et al. dataset  only.The Cai et al.  before ti et al.  for the 2+ transient recordings performed at the BioMediTech Institute .To constrain the model parameters, two different experimental datasets were used. AP data were taken from Ma et al. . This is2+ transient dataset were recorded at the BioMediTech Institute from healthy control hiPSC-CMs at 35–37°C. The generation and characterization of the control hiPSC line and cardiac differentiation were done as described earlier . pH of the perfusate medium was adjusted to 7.4 with NaOH (Sigma Aldrich). Perfusate was heated with an inline heater SH-27B controlled with a TC-324B controller unit and input into a RC-25 imaging chamber . Calcium kinetics of spontaneously beating cardiomyocytes were imaged with an inverted Olympus IX70 microscope using UApo/340 0,75NA 20x air objective  and recorded with ANDOR iXon 885 EM-CCD camera  using 2 × 2 binning and synchronized with a Polychrome V light source by a real time DPS control unit. LiveAcquisition software  was used to control light source and camera during recording. Fluo-4 was excited at 490 nm wavelength and the emission was recorded through Olympus U-MF2 Alexa 488 band-pass filter cube .This study was carried out in accordance with the recommendations of Guidelines of the Ethics Committee of Pirkanmaa Hospital District . The protocol was approved by the Ethics Committee of Pirkanmaa Hospital District . All subjects gave written informed consent in accordance with the Declaration of Helsinki. New CaData were recorded from 15 cells for a total of 218 transients. For each biomarker mean value and standard deviation (SD) were computed for model calibration. The experimental values for the biomarkers and their weights are reported in Table rel formulation and the parameters optimization by means of our Ca2+ transient data, we chose to replicate the current blocker simulations performed in Paci et al. (Na blocker), nifedipine , E4031 (IKr blocker) and 3R4S-Chromanol 293B . For TTX, NIFED and E4031 we considered the following IC50 values: 0.64 μM , as reported in the last column of Table 2+ transients and traces from four illustrative cells: the comparison highlights that the simulated Ca2+ transient contour is fully in agreement with our experiments. In Figure 2+ traces in steady state are reported, together with the main ionic currents and concentrations. Irel gating variables, together with the Ca2+ transients, are detailed in Figure 2+ release from SR is not directly dependent on the ICaL activation, but on the cytosolic Ca2+ concentration Cai, which rules the behavior of the RyRo, RyRc, and RyRa gating variables.The automatic optimization process provided a new set of parameters, which is reported in Table rel formulation and parameter optimization, the behavior of the Paci2018 model is still consistent with the Paci2013 simulations  and no external stimulation, i.e., both models produced spontaneous APs. As shown in Figure t =  s Irel underwent a small reactivation, slowing down the decay of the Ca2+ transients , characterized with premature Ca2+ releases. These traces were obtained with a normal extracellular Ca2+ concentration (Cao = 1.8 mM) but simulating more “immature” RyR machinery . In these conditions of more “immature” RyR machinery, we tested also the effects of INaCa block on DADs generation. In Supplementary Figure NaCa block levels  while pacing the Paci2018 model at 1 Hz. With no INaCa block (solid blue trace), APs show small DADs comparable to those reported in Figure t =  s. By blocking 50% of INaCa (dashed orange trace), we observed that the amplitude of DADs increases. Finally, for the higher block levels, we did not observe any DAD.Figure f block by ivabradine, (ii) hyperkalemia, (iii) hypocalcemia.In order to compare the Paci2018 model with the experimental data, we challenged our model in the following conditions: (i) If block by 3 μM ivabradine as a 41% block of If maximum conductance , in agreement with : for Ko = 8 mM and Ko = 16 mM the Ca2+ transient spontaneous rate dropped by 11% and 15% respectively. An extracellular K+ concentration equal to 20 mM stopped the hiPSC-CM spontaneous activity as in Kim et al.  as in Kim et al. . This Irel formulation was then chosen for its simplicity instead of more complex formulations, however it presumes Irel to be directly dependent on the intracellular Ca2+ concentration and it prevents the model to be able to reproduce Ca2+-related anomalies such as DADs. As reported in Fink et al.  we do not observe DADs. It is interesting to note that the 50% block is not enough to cancel DADs: in fact, the inward INaCa component is still strong enough and further enhanced by the cytosolic Ca2+ accumulation, due to the very same INaCa block . This results in even larger DADs than in the no block case. Similar DAD-like abnormalities in the electrical properties of hiPSC-CMs were also observed in mutant hiPSC lines, such as those derived from patients with HCM , where the spontaneous Ca2+ releases, which trigger DADs, finally cause a transition to a higher rate of Ca2+ transients and APs.The result is a new model where spontaneous APs match the Ma et al. dataset and where CaCa block 0 and 90%K1 and a much depolarized MDP (−59.1 ± 3.3 mV), which facilitate the spontaneous electrical activity, as shown also by the high frequency of the spontaneous Ca2+ transients (1 Hz or higher) in control conditions. On the contrary, the experimental datasets we used in this paper showed slower spontaneous activity and more hyperpolarized MDP, as simulated by our model and reported in Table To further validate our model, we challenged it to simulate specific conditions such as hyperkalemia and hypocalcemia, to compare its behavior to published experimental data. The model showed qualitative agreement with such experiments, in spite of the fact that the spontaneous rate reduction is stronger in the experiments. However, such differences can emerge from (i) the multicellular embryoid body culturing and measurements, i.e., on multicellular ensembles, and especially (ii) as consequence of the high inter-cell line and inter-laboratory variability showed by hiPSC-CMs ; (ii) greater INaCa , and (iii) smaller IK1 . It is also interesting to notice that the three parameter sets showed greater IKr compared to baseline: this suggests that in models where IKr is highly expressed, a 90% block can have more dramatic effects than in cells expressing smaller IKr. This small test suggests, as we reported among the limitations, that the use of more refined modeling approaches, e.g., populations of in silico models, is advisable to simulate specific phenomena (in this case EADs as consequence of IKr block) in cell types characterized by high electrophysiological variability. In the perspective of offering researchers a tool to investigate the mechanisms of ventricular arrhythmia development in hiPSC-CMs, we ran also two preliminary tests about the occurrence of alternans or alternans-like patterns in the Paci2018 model . When pacing the Paci2018 model at 120 bpm (2 Hz), the membrane potential exhibited an alternans-like pattern, characterized by a full AP followed by a smaller one  in conditions of Ia et al. , and thaa et al. , we demoi et al.  triggerein silico model, also the Paci2018 is an approximated description of a real system: consequently, new mechanisms can be explored and improved. This is especially true for such a complex cellular system as hiPSC-CM, which is not fully characterized yet. The main limitation that affects the Paci2018 model, as well the other single cell in silico models available in literature, is the fact that the simulated behavior belongs to a theoretical cell, built on averaged experimental data from many cells. In spite of our efforts to build the Paci2018 in a rigorous way, we do not have the presumption to consider it representative of all the hiPSC-CMs available nowadays, both from commercial lines and laboratory-specific lines. This is particularly true in case of an in vitro model like hiPSC-CMs, since the same experiments that we need to build an in silico model are extremely variable, as reported by multiple studies clinical applications, more refined modeling techniques exist, e.g., the population of in silico models  only for two critical biomarkers which characterize hiPSC-CMs, i.e., MDP (depolarized compared to adult cardiomyocytes) and the rate of the spontaneous APs. For all the other biomarkers, the weights were set to one. The tradeoff obtained by the parameter optimization produced a model that simulates Ca2+ transients whose DURATION is 617.8 ms. Despite this value lies within the experimental range 804.5 ± 188.0 ms, it is very close to its lower bound. Conversely, the DT9010 biomarker properly lies within its experimental range, although slightly smaller than the experimental mean DT9010 (409.8 ± 100.1 vs. 367.6 ms). The inset included in Figure 9010 of the simulated transient is included in the experimental interval. Of note, the experimental traces reported in Figure 2+ transient experimental datasets, chosen to develop the Paci2018 model, were recorded at the same temperature (35–37°C) further mitigates the differences between the two datasets. Another potential limitation of our model is related to its simple structure, consisting of two compartments only (cytosol and SR). Such compartmentalization does not allow to simulate a spatial inhomogeneous Ca2+ distribution, as conversely recently done by others , which affect the Ca2+ handling in hiPSC-CMs. Due to its relatively light formulation , our model is suitable also for very large studies on in silico populations, e.g., to support screening of different drug/compounds at various concentrations.In conclusion, in this work we present an updated and more versatile version of our hiPSC-CM in vitro measurements and analyzed the in vitro data. MP and DC performed the in silico model development and validation. MP and SS analyzed the in silico data, prepared the figures and drafted the manuscript. All the authors interpreted the results and revised the manuscript.All the authors conceived and designed the study. R-PP and KP performed the 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. The reviewer JB and handling Editor declared their shared affiliation.


Numerous experimental studies about the Ca2+/cardiolipin (CL) interaction are reported in the literature, but there are few studies in conjunction with theoretical approaches based on ab initio calculations. In the present study, the lipid fraction of the inner mitochondrial membrane was modeled as POPC/CL large unilamellar vesicles (LUVs). POPC/CL and, comparatively, POPC, and CL LUVs were challenged by singlet molecular oxygen using the anionic porphyrin TPPS4 as a photosensitizer and by free radicals produced by Fe2+-citrate. Calcium ion favored both types of lipid oxidation in a lipid composition-dependent manner. In membranes containing predominantly or exclusively POPC, Ca2+ increased the oxidation at later reaction times while the oxidation of CL membranes was exacerbated at the early times of reaction. Considering that Ca2+ interaction affects the lipid structure and packing, density functional theory (DFT) calculations were applied to the Ca2+ association with totally and partially protonated and deprotonated CL, in the presence of water. The interaction of totally and partially protonated CL head groups with Ca2+ decreased the intramolecular P-P distance and increased the hydrophobic volume of the acyl chains. Consistently with the theoretically predicted effect of Ca2+ on CL, in the absence of pro-oxidants, giant unilamellar vesicles (GUVs) challenged by Ca2+ formed buds and many internal vesicles. Therefore, Ca2+ induces changes in CL packing and increases the susceptibility of CL to the oxidation promoted by free radicals and excited species.Ca The authors estimated that protons could exchange between phosphate groups with an energy barrier of ~4–5 kcal/mol. Lemmin et al. stated that in-depth models and characterization of cardiolipins are to date rare and proposed an ab initio parametrization of cardiolipin species for molecular simulation and concluded that the cardiolipin protonation influences the lipid packing . Considering this premise, we modeled the lipid fraction of the inner mitochondrial membrane arranged as POPC /TOCL (tetraoleoyl cardiolipin) large and giant unilamellar vesicles . The PC and CL fractions of the lipid bilayers were also analyzed individually by using POPC and TOCL LUVs. TOCL LUVs were replaced by BBCL (bovine brain cardiolipin) LUVs when Fe2+/citrate was the pro-oxidant agent. LUVs were challenged by the oxidative attack of excited species and free radicals simulating the events occurring in photodynamic therapy (PDT) and chemical oxidative stress promoted by the Fenton reaction. A PDT model was constructed using the anionic porphyrin TPPS4 (meso-tetrakis(4-sulfonatophenyl) porphyrin) as the photosensitizer for the generation of singlet molecular oxygen via energy transfer to ground state molecular oxygen. TPPS4 irradiated by a LED array was the singlet oxygen source for POPC/TOCL (80/20 mol%), POPC (100%) and TOCL (100%). TPPS4 was chosen because this molecule does not have an affinity to negatively charged interfaces . In aerated media, the triplet sensitizer is deactivated by energy transfer to molecular oxygen, which, in turn, is converted to singlet molecular oxygen (1Δg). Singlet molecular oxygen adds to a double bond of the acyl chain and produces the peroxide derivatives. The lipid oxidation promoted by Fe2+/citrate is initiated by the formation of oxyferryl species (Fe(IV) = O) in the presence of Fe3+ . The pH 6.5 corresponds to the expected value at the interface of the outer side of the inner mitochondrial membrane in the energized state of the organelle, i.e., with the transmembrane potential (ΔΨ) generated by the proton gradient in a coupled mitochondrion  of POPC/TOCL at pH around 6.5. The POPC/TOCL GUVs labeled with fluorescent CL (TopFluorCL) were challenged by Ca2+ and analyzed by fluorescence microscopy , although they required higher Ca2+ concentration than normal mitochondria. In a subsequent study, Giorgio et al.  and liposomes. The authors proposed that the Ca2+-induced in-plane rearrangements of lipids respond for the increased production of carbon-centered free radicals and membrane permeability in antimycin A-poisoned SMPs. Therefore, the studies above postulated that Ca2+-induced changes in lipid packing reverberate on the structure and aggregation of proteins involved in the membrane permeabilization. In the present study, it was demonstrated theoretically and experimentally that Ca2+ changes lipid packing and, even in the absence of proteins, the binding of Ca2+ favored lipid oxidation. Interestingly, oxidative effects of chemically generated free radicals and photochemically-generated singlet oxygen were similarly favored by Ca2+, at least concerning the formation of LOOH (lipid peroxide). The factors behind the different LOOH production kinetics determined in this paper are a challenging issue. Some aspects should be considered in trying to answer this question. For singlet oxygen generation, porphyrin TPPS4 was chosen precisely to prevent that the binding affinity of the photosensitizer to the membrane could interfer with oxidation kinetics. Therefore, as already shown in a previous study, with the use of TPPS4, most singlet oxygen is generated in bulk water, and membrane attack depends on the diffusion of the reactive species to the membrane. As Fe2+-citrate is also water-soluble under our experimental conditions, the oxidation by both singlet oxygen and Fe2+-citrate depend on the access of the reactive species to the lipid bilayer. Thus, the similar effects of Ca2+ on the CL oxidation kinetics by singlet oxygen and free radicals are consistent with changes in lipid packing  was purchased from Cambridge Isotope Laboratories, Inc. . The TPPS4 stock solution concentration was checked using the absorption coefficient ε at 412 nm = 5.30 × 105 M−1 cm−1 , tetraoleoyl cardiolipin (TOCL), and bovine brain cardiolipin (BBCL) were obtained from Avanti Polar Lipids, Inc. , TPPS4 (meso-tetrakis(4-sulfonatophenyl) porphyrin) was purchased from Sigma-Aldrich Corp. , Xylenol orange was purchased from Fluka Chemical Corp. . Glucose, sucrose, ammoniacal ferrous sulfate ((NH4)2Fe(SO4)2), chloroform, n-butanol, and ethanol were obtained from Synth , Deuterium oxide  were grown using the electroformation method . Multilamellar liposomes were subjected to 11 passes through two polycarbonate filters  installed in tandem. Liposome solutions were diluted with the buffer to the final lipid concentration of 250 μM.Lipids, POPC/CL (80/20 mol%), POPC (100%), and CL (100%) were initially dissolved in chloroform and the solvent evaporated under a flow of N2+/citrate polyunsaturated BBCL was used and incubated in the presence of 50 μM Fe2+/citrate . Lipid photo-oxidation was promoted by 10 μM of TPPS4. For the oxidation promoted by free radical generated by Fe® water and 100 μL of hydroperoxide reagent that contained 2.5 mM xylenol orange, 2.5 mM (NH4)2Fe(SO4)2, and 1.15 M HClO4 in Milli-Q® water. The sample was rested for 30 min at room temperature. The oxidation of Fe(II) by LOOH generates Fe(III), which reacts with xylenol orange and is converted to the colored product that absorbs at 560 nm. LOOH concentration was calculated by using ε = 560 nm = 6.45 × 104 M−1cm−1. TBARS was determined at 535 nm after reaction with thiobarbituric acid (TBA), using the molar extinction coefficient 1.56 × 105 M−1cm−1.Lipid oxidation was evaluated with lipid peroxide (LOOH) production. Lipid hydroperoxide concentration was determined by the oxidation of Fe(II) in the presence of xylenol orange: aliquots of the sample (10 μL) were extracted from incubation at room temperature and mixed with 890 μL of Milli-QThe electronic absorption spectra of porphyrins were measured by using a Varian Cary 50, Varian Inc.  spectrometer. The spectral resolution for wavelength scan was 0.5 nm. The optical path length was 1 cm for the LOOH dosages, and 0.1 cm for the porphyrin spectra measurements and quantification.in situ attenuated total reflectance-Fourier transform infrared (ATR-FTIR) technique , using Troullier-Martins pseudopotentials with the inclusion of London dispersion forces (DCACPs) (Kohn and Sham, The The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.IN-C conceived experiments. ÉM and JA-C conducted the experiments and analyzed part of the results. ID and JA performed theoretical calculations. IN-C and JA analyzed the results and wrote the manuscript. CK performed GUV production and images. AB performed FTIR experiments. All authors reviewed 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.


RYR1), whereas multi-minicore disease is linked to pathogenic variants of several genes, including selenoprotein N (SELENON), RYR1 and titin (TTN). Understanding the mechanisms that drive core development and muscle weakness remains challenging due to the diversity of the excitation-contraction coupling (ECC) proteins involved and the differential effects of mutations across proteins. Because of this, the use of representative models expressing a mature ECC apparatus is crucial. Animal models have facilitated the identification of disease progression mechanisms for some mutations and have provided evidence to help explain genotype-phenotype correlations. However, many unanswered questions remain about the common and divergent pathological mechanisms that drive disease progression, and these mechanisms need to be understood in order to identify therapeutic targets. Several new transgenic animals have been described recently, expanding the spectrum of core myopathy models, including mice with patient-specific mutations. Furthermore, recent developments in 3D tissue engineering are expected to enable the study of core myopathy disease progression and the effects of potential therapeutic interventions in the context of human cells. In this Review, we summarize the current landscape of core myopathy models, and assess the hurdles and opportunities of future modeling strategies.The core myopathies are a group of congenital myopathies with variable clinical expression – ranging from early-onset skeletal-muscle weakness to later-onset disease of variable severity – that are identified by characteristic ‘core-like’ lesions in myofibers and the presence of hypothonia and slowly or rather non-progressive muscle weakness. The genetic causes are diverse; central core disease is most often caused by mutations in ryanodine receptor 1 ( Summary: The core myopathies are neuromuscular disorders with no cure. In this Review, we outline our current understanding of pathomechanisms, the advantages and limitations of available models, and discuss emerging modeling technologies. First described by Shy and Magee in 1956, CCD is the most common congenital myopathy . CCD is First described by Engel and colleagues , MmD is SELENON  transits between the sarcoplasmic reticulum (SR) and sarcoplasm -actin gene; it encodes a key component of skeletal muscle sarcomeres, the thin filaments.ACTN2: alpha (α)-actinin 2 gene; in sarcomeres, α-actinins are located in the Z-disc, where they anchor actin filaments.Bio-printing: a technique that uses 3D printing technologies to combine cells, growth factors and various matrices  to maximally mimic tissues.CACNA1S: calcium voltage-gated channel subunit α1 gene; encodes the voltage-dependent L-type calcium channel α1S subunit [also known as dihydropyridine receptor (DHPR) and Cav1.1].CASQ1: calsequestrin 1; this skeletal-muscle-specific protein binds Ca2+ with high affinity and acts as a Ca2+ sensor within the sarcoplasmic reticulum.CCDC78: coiled-coil domain-containing protein 78 gene. CCDC78 is localized in the perinuclear region, sarcolemma membrane and T-tubule regions of the sarcoplasm. Mutations are associated with congenital myopathy, but the exact protein function(s) are currently unknown.CDK4: cyclin-dependent kinase 4; a kinase involved in cell cycle regulation.CNFs: centrally nucleated fibers; myofiber nuclei that form in a central, rather than peripheral, location; often a hallmark of regenerating fibers.CRU: calcium release unit; this comprises junctional domains of the sarcoplasmic reticulum and the exterior membranes of the T-tubules, containing RYR1 and DHPRs, respectively. Release of Ca2+ from the CRU causes excitation-contraction coupling (ECC).DHPR: dihydropyridine receptor ; the protein, encoded by CACNA1S, is located on the T-tubule membrane and interacts with RYR1. An action potential alters the T-tubule membrane potential, changing the confirmation of DHPR, which opens RYR1, triggering intracellular Ca2+ release and muscle contraction.ECC: excitation-contraction coupling. The process by which an action potential propagates along the sarcolemma and T-tubule invaginations, triggering Ca2+ release and causing the synchronous shortening of aligned sarcomeres and thus leading to muscle contraction.ER: endoplasmic reticulum; a continuous membrane network within cells involved (amongst other roles) in protein and lipid synthesis and folding.ERO1: endoplasmic reticulum oxidoreductase 1 gene; catalyzes protein disulphide bond formation within the endoplasmic reticulum.Fluo-4: one of several Ca2+ indicator dyes whose fluorescence increases upon binding to intracellular Ca2+. It is used to measure Ca2+ concentrations in living cells.FRET: fluorescence resonance energy transfer; this technique measures whether two molecules interact with each other via the highly distance-dependent energy transfer between two light-sensitive molecules at the nanometer scale. It allows visualization at a subcellular and provides higher resolution compared to immunofluorescence.Fura-2: the first Ca2+ indicator dye; increases in fluorescence upon binding to intracellular Ca2+ and is used to measure Ca2+ concentrations in living cells.FXR1: fragile X-related 1 gene; encodes an RNA-binding protein required for embryonic and postnatal development of muscle.GCaMP6: a genetically encoded fluorescent Ca2+ indicator, consisting of green fluorescent protein (‘G’), calmodulin (‘Ca’) and calmodulin-interacting M13 peptide (‘MP’).HDAC: histone deacetylase; a group of enzymes that, among other functions, regulate gene expression by removing acetyl groups from histones.HEK-293 cells: an immortalized human embryonic kidney cell line widely used in cell and molecular biology research because of their reliable growth and propensity for transfection.hTERT: human telomerase reverse transcriptase; an enzyme that lengthens telomeres. Forced expressed of hTERT prevents primary cells from undergoing replicative senescence, thereby immortalizing them.I-band: an area of the sarcomere in which thin filaments (actin) are not superimposed by thick filaments (myosin) ((myosin) .Kyphoscoliosis: a spinal deformity characterized by abnormal curvature of the vertebral column along the coronal and sagittal planes. Combines kyphosis (convex curvature) and scoliosis (sideways curvature).MEGF10: multiple epidermal growth factor-like domains protein 10 gene; in skeletal muscle, MEGF10 regulates satellite cell proliferation and mutations are associated with MmD.MRI: magnetic resonance imaging; a technique that uses magnetic fields and radio waves to image the structure of organs in 3D in a non-invasive manner.MYH7: myosin heavy chain 7 gene; it encodes for β-myosin heavy chain protein expressed in type 1 myofibers and cardiac cells. β-myosin heavy chain forms part of the type 2 myosin hexamer of sarcomeric thick filaments.Myofibril: an aligned sarcomeric unit that extends along the length of each myofiber.Nemaline rods: thread-like (from Greek ‘nema’) or rod-like protein aggregates within myofibers of patients with nemaline myopathies. These electron-dense structures are visible in muscle biopsies and stain red with the Gomori's trichrome staining.RYR1: ryanodine receptor 1 gene; the RYR1 protein is located on the sarcoplasmic reticulum and releases Ca2+ in response to an action potential.Sarcolemma: myofiber plasma membrane  35-614], the central domain  and the C-terminus   to assess the threshold of muscle contraction  .Box 4. 2+ homeostasis via two proposed pathological mechanisms: the ‘leaky channel’ and ‘EC uncoupling’. Mutations in hotspot 1 or 2 cause RYR1 to be ‘leaky’, decreasing the threshold for channel activation and leading to channel hyperactivity and precocious Ca2+ release from the SR ..MYH7  and non-muscle  was dominant  compared to other Ca2+ storage regions in the cell , which activates proteolysis and leads to sarcomere degeneration (unstructured cores)  .Ryr1 mutations develop phenotypes that do not all align with patient symptoms. The T4826I mutation in the transmembrane domain of RYR1 is associated with MHS without core myopathy in humans ,,RYR1 mutSELENON ..2+-bindiuscle SR . In humagregates  and MHS kinetics . Casq1−/weakness , 2015. Tweakness . Althougructures . Interesngly see , includingly see . Of noteT/+ mice , furtherFXR1 were identified in two families with congenital myopathies of varying severity, exhibiting type 1 fiber predominance, Z-line streaming, CNFs and minicores  channel mutation  is yet to be determined.Notably, the range of mutations affecting different functional domains enables researchers to assess novel therapeutic strategies in a mutation-specific manner. Mitochondrial dysfunction – a hallmark of core myopathies – also occurs in a mutation-specific manner. While mitochondrial swelling, uncoupling from triads, dysfunction and eventual loss occur in many mouse models , studiesS/+ mice , Ryr1AG/ content . Furtherutations , Ca2+-inmutation . Here, mneration . Whether+) supplementation or K+-channel inhibition rescued abnormal K+ homeostasis in AG/+Ryr1 mice, thus improving the phenotype (2+ leakage from the SR in Y522S/+Ryr1 mice with the antioxidant N-acetylcysteine (NAC). In the first study, 8 weeks of 1% w/v NAC in drinking water protected against mitochondrial damage and force degeneration (relatively relaxed zebrafish MmD model and in human myotubes (RYR1-related myopathy (NCT02362425).In another therapeutic strategy, Hanson and colleagues showed that potassium , or due to heterogeneity in relative mitochondrial oxidative capacity  due to the 3D structure of RYR1 and its interaction with other proteins in vitro,Generally, 3D skeletal muscle cell culture involves the encapsulation of muscle progenitor cells within a biomaterial and then deposition of these into a culture well containing two attachment points that mimic the tendons to establish uniaxial tension and drive the self-organization of aligned, multinucleated myofibers in the 3D tissue construct. Unlike 2D culture, in which cells bind a substrate only on one side and maturation is affected by aberrant signaling from the cell surface to the nucleus , a 3D main vivo-like Ca2+ handling , measurements generally cannot be repeated over time. By contrast, transducing 3D myoblasts with a Ca2+ reporter, such as GCaMP6  myofibers . The prein vitro , as wellsistance . These ssistance . Anothersistance . Indeed,sistance . Neverthsistance , and resA further advantage of 3D cell culture models is the possibility to decrease the intrinsic variability often associated with animal models; replicate number is limited to the expansion potential of the cell type used, and variability can be further decreased through the implementation of robotics and their integration with miniaturization strategies . Computein vitro models are a valuable tool to discover physiological and pathological mechanisms in skeletal muscle. They may be successfully utilized to study core myopathies, providing an amenable platform to evaluate Ca2+ fluxes, muscle strength and better fiber maturation compared to traditional 2D culture (Therefore, 3D  culture .RYR1-associated core myopathies. Dowling and colleagues recently noted that the time for research into congenital myopathies ‘is now’ (in vitro or in vivo models, researchers should take care to holistically model the various aspects of mutation-specific disease mechanisms and progress with those models that best recapitulate the tissues and structures affected in the human disorder. In turn, these models will allow the search for effective treatment in these disorders and pave the way to the ‘therapeutic era’ of core myopathies, allowing treatment before clinical signs or muscle damage become apparent.In the past decades, research into core myopathies has provided great insight into the cause and progression of this subgroup of congenital myopathies. However, major gaps in our understanding of pathogenic mechanisms remain, due in part to a lack of recapitulative models, especially for non-‘is now’ . When ge


Soft robotics is a branch of robotics that deals with mechatronics and electromechanical systems primarily made of soft materials. This paper presents a summary of a chronicle study of various soft robotic hand exoskeletons, with different electroencephalography (EEG)- and electromyography (EMG)-based instrumentations and controls, for rehabilitation and assistance in activities of daily living. A total of 45 soft robotic hand exoskeletons are reviewed. The study follows two methodological frameworks: a systematic review and a chronological review of the exoskeletons. The first approach summarizes the designs of different soft robotic hand exoskeletons based on their mechanical, electrical and functional attributes, including the degree of freedom, number of fingers, force transmission, actuation mode and control strategy. The second approach discusses the technological trend of soft robotic hand exoskeletons in the past decade. The timeline analysis demonstrates the transformation of the exoskeletons from rigid ferrous materials to soft elastomeric materials. It uncovers recent research, development and integration of their mechanical and electrical components. It also approximates the future of the soft robotic hand exoskeletons and some of their crucial design attributes. The emerging trend of soft robotics has stimulated the interest of engineers and researchers around the world to look into various applications, ranging from biomedical and rehabilitation to grasping and manipulation ,2. BiomiOne of the major lessons learned from these biostructures was the ability to form and adapt to complexly shaped bodies. This led to various developments such as: (1) an octopus-like robot for flexible manipulation ; (2) a wAnother important development in the emerging field of soft robotics is the use of pneumatic soft grippers for handling fragile objects such as an uncooked egg or an anesthetized mouse . These dThis new trend is especially interesting for biomedical and rehabilitation engineering applications as well, with the hand exoskeleton as one of the examples. A major shift from the use of hard to soft materials can be observed in some of the latest designs of hand exoskeletons, such as the Wyss Institute glove ,7,8, theUnlike a prosthetic hand, a hand exoskeleton is designed and built around the human hand; thus, it has to conform to the hand anatomy and its range of motion to minimize the wearer’s discomfort. More importantly, it has to be light and able to be put on easily so that the wearer can use it daily to perform basic activities. Hand exoskeletons have endured extensive research, primarily in the field of assistive and rehabilitative robotics, and have also been discussed from various perspectives ,30,31. SA chronological review was conducted of several renowned literature databases, i.e., Science Direct, PubMed, IEEE Xplorer, SciVal and Google Scholar, to identify the trend of soft robotics in hand exoskeletons from the year 2008 onward. The keywords used as the search entries to examine more categorical papers were ‘soft hand exoskeleton’ and ‘soft robotic hand exoskeleton’. The search results were further filtered with keywords like ‘rehabilitation’, ‘ADL’, ‘pneumatic’ and ‘tendon’. A set of inclusion and exclusion criteria was then applied to select the right literature.The following inclusion and exclusion criteria were used to funnel down the discrete knowledge that is vital for the systematic review and chronological review of the soft robotic hand exoskeleton.The study introduced and applied soft robotics technology for the development of a hand exoskeleton that included a wearable glove, actuator or bothThe study presented the mechanical or electrical aspect of the hand exoskeleton in at least three of the following attributes: actuation, active degrees of freedom (DOFs), finger movement, output force, range of motion (ROM), weight and functionalityThe hand exoskeleton had at least one soft robotic finger (3 degrees of freedom)The study presented a prototype or at least a design of the hand exoskeletonThe study was published in any language other than EnglishThe device used rigid or ferrous mechanical components for the glove design and linkagesThe device was used other than that for rehabilitation and assistance for ADLThe device was a prosthetic or anthropomorphic handA total of 65 literature works on soft robotic hand exoskeleton were found. However, only 45 devices were selected for the systematic review, because some designs were reported in several articles and used for different purposes. For example, the SNU research group presented their Exo-Glove Poly in ,21,22 anPrevious reviews revolved around short-term narration  and geneThe systematic review describes the merits and demerits of different exoskeletons based on their design attributes, such as ergonomics, complexity, instrumentation and actuation mode. It also discusses different design techniques that distinguish one soft exoskeleton from another. For this purpose, the attributes selected for comparison between different exoskeletons were based on quantitative results such as year of publication, mode of actuation, number of fingers, active degrees of freedom, output force and many other attributes.The chronological review investigates the adoption of soft robotics technology in the design of hand exoskeletons in the recent decade. Here, we review the evolution of the soft robotic hand exoskeleton systems and how the newer designs use soft robotics as a form of improvement from the previous studies. We examine the exoskeleton mechanical and electrical attributes and their functionalities based on the articles published from 2008 to 2017. It is hoped that by evaluating these elements annually, one can realize the importance of soft robotics in designing and developing better hand exoskeletons.Forty-five soft robotic hand exoskeletons were systematically reviewed based on the criteria described in Unlike prosthetics, hand exoskeletons are designed to actuate or mechanize the paralyzed human hand. In its early years, hand exoskeletons were made of rigid linkages. These linkages connected the exoskeleton with the finger, and the rotational axes of the joints were aligned with the human finger joints b. DespitSoft wearable gloves are usually made with fabric, plastic or silicone-based material. They can be designed as per the patient’s finger measurements. The fabric-based materials can be seen in the earlier designs of the SNU Exo-Glove , the gloA tendon/cable-driven mechanism is one of the most common actuation mechanisms embedded in the soft glove and generally requires more than one actuator. In the earlier designs, this mechanism had actuator units for each flexion and extension of the finger . The tenAnother common actuation mechanism is pneumatic based, in which most of the soft pneumatic actuators have strain limiting layers and/or fibers. The NUS glove , the MRCAn actuator is generally defined as having the ability to convert energy into motion. Actuators in a soft robotic hand exoskeleton mainly are comprised of tendon/cable–pulley and pneumatic/hydraulic systems. These actuators are usually placed on the dorsal side or on the sagittal plane of the finger to maximize the power transmission to the fingertip. In the pneumatic system, the air chamber is placed during the fabrication process. The soft actuator is then placed on the dorsal side of the fingers by either attaching it to the hand through a harness , stitching it to fabric-based material or gluing it to a glove . The mosGenerally, the control strategies of the hand exoskeletons can be divided into two categories: low-level and high-level control strategies. In the low-level control strategy, the controller addresses the physical parameters of the device such as force, torque and position . It usesAnother important aspect in designing a hand exoskeleton is the weight, which is also related to the reason behind choosing the actuating mechanism. As can be seen in Most of the hand exoskeletons designed for clinical research purposes are pneumatically-actuated , such asOne of the limitations of a soft pneumatic actuator is the delivered force or torque. The embedded pressure chamber requires high-pressure air to inflate and transmit the force to the end effector. While a driving force can be initiated, it is hard to control the holding force (gripping force). As the holding position requires continuous force, the pneumatic actuator has to continuously supply a specific amount of pressure to sustain the required gripping force . The powThe second part of this review paper is to investigate the evolution of soft robotic hand exoskeletons in the recent decade. It highlights the areas that have been investigated and the contributing factors in opening new avenues for further research. It also evaluates the trend and predicts how soft robotic hand exoskeletons may look in the future. Another important aspect of this review is to explore different parameters and variables used as the design criteria to develop hand exoskeletons.Sixty-five articles were carefully reviewed and are listed chronologically in One of the earliest works on soft robotic hand exoskeletons was the Power-Assist glove reported in  in 2008.In 2011, three articles were published. One of them was an exoskeleton that uses the tendon/cable–pulley mechanism , while tIn 2012, three studies were found, and all of them were tendon/cable-actuated soft exoskeletons ,40. One A five-finger cable-driven hand exoskeleton was proposed in 2013 . It has Three pneumatically-actuated exoskeletons were reported in 2014 ,44,45. OSince 2015, the development of soft robotic hand exoskeletons escalated. Thirteen studies were published, in which 10 of them were on pneumatic actuators ,53,54,55The research and development of soft robotic hand exoskeletons reached its peak in 2016. Fourteen articles were on pneumatically-actuated exoskeletons ,69,70,71In 2017, eight articles reported the use of pneumatic/hydraulic actuators ,49,76,77In order to understand the technological shift in the hand exoskeleton, a chronological analysis was conducted. This analysis is divided into three main areas of interest: mechanical, electrical and functionality of the soft exoskeleton. The electrical evolution of the soft robotic hand exoskeleton is depicted in Here, we summarize various soft robotic hand exoskeletons based on their merits and demerits and reviews the technological trend of these devices in the last decade, which includes 45 soft wearable exoskeletons along with their design criteria and basic properties. The systematic review describes the general trend and actuation mechanisms of the soft robotic hand exoskeleton. The summary shown in Up until now, rehabilitation purposes appear to have led the design and development of soft robotic hand exoskeletons. However, these soft wearable exoskeletons may be used for other functions as well. For example, the virtual reality control used in PneuGlove  allows e


Mesenteric resistance arteries from wild type (WT) and genetically modified mice with PKG resistance to oxidative activation were studied using wire and pressure myography. Ca2+ sparks and Ca2+ transients within vascular smooth muscle cells of intact arteries were characterized using high‐speed confocal microscopy of intact arteries. Arteries were studied under conditions of varying intraluminal pressure and oxidation. Intraluminal pressure specifically, rather than the generic stretch of the artery, was necessary to activate the oxidative pathway. We demonstrated a graded step activation profile for the generation of Ca2+ sparks and also a functional “ceiling” for this pressure –‐sensitive oxidative pathway. During steady state pressure ‐ induced constriction, any additional Ca2+ sensitive‐K+ channel functional availability was independent of oxidant activated PKG. There was an increase in the amplitude, but not the Area under the Curve (AUC) of the caffeine‐induced Ca2+ transient in pressurized arteries from mice with oxidant‐resistant PKG compared with wild type. Overall, we surmise that intraluminal pressure within resistance arteries controls Ca2+ spark vasoregulation through a tightly controlled pathway with a graded onset switch. The pathway, underpinned by oxidant activation of PKG, cannot be further boosted by additional pressure or oxidation once active. We propose that these restrictive characteristics of pressure‐induced Ca2+ spark vasoregulation confer stability for the artery in order to provide a constant flow independent of additional pressure fluctuations or exogenous oxidants.We investigated the biomechanical relationship between intraluminal pressure within small mesenteric resistance arteries, oxidant activation of PKG, Ca Intraluminal pressure within resistance arteries triggers a binary activation of an oxidant‐driven pathway whereby protein kinase G regulates calcium spark vasoregulation. Once activated, the pathway cannot be boosted by additional pressure or oxidants and we propose this confers stability upon the pressure‐induced constriction of the artery. Subsequently, the PKG[C42S]KI arteries were more constricted and this was associated with hypertension.We recently reported that the mechanism by which intraluminal pressure initiates the vasodilatory Ca2+ sparks–BK vasodilation, raised several questions regarding the biomechanics of Ca2+ spark vasoregulation such as the relationship between pressure‐related oxidants and Ca2+ spark frequency, the inflection point necessary to initiate the pathway, and the capacity of oxidants to further modulate PKG and thus Ca2+ spark vasoregulation. In this study, we addressed these questions by comparing diameter changes and Ca2+ signalling between WT and PKG[C42S]KI arteries in response to a variety of physical and pharmacological stimuli. There was steep, almost digital activation of Ca2+ spark generation between 20 and 50 mmHg of intraluminal pressure and higher pressures had no further effect on Ca2+ spark frequency. Together with our previous results, our study contextualizes the functional contribution of pressure‐induced oxidative activation of PKG in the control of small artery diameter via Ca2+ spark vasoregulation.Detection of this pathway: intraluminal pressure–oxidants–PKG–Caα Cys42Ser (referred to in the manuscript as PKG[C42S]KI) were generated on a pure C57BL/6j background by Taconic Artemis  as described previously  mice to generate heterozygous mice. From these heterozygotes, either WT or PKG[C42S]KI colonies were generated and then maintained by breeding and genotyping. Age‐matched (12‐week‐old) male WT C57BL/6j and PKG[Cys42Ser]KI mice were used in all studies. Mice had ad libitum access to standard chow and water and were kept in specific pathogen‐free conditions, under a 12‐h day/night cycle. Mice were euthanized by cervical dislocation. The mesenteric bed was removed and kept in an ice‐cold HEPES‐buffered physiological saline (HEPES‐PSS) with the following composition: 134 mmol/L NaCl, 6 mmol/L KCl, 1 mmol/L MgCl2, 2 mmol/L CaCl2, 7 mmol/L glucose, and 10 mmol/L HEPES, with pH adjusted to 7.4 with 1 mol/L NaOH. All chemicals were from Sigma , aside from the antibodies, which were obtained from Badrilla or the Jackson Immunoresearch Laboratories Inc.Procedures were performed in accordance with the UK Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986 in United Kingdom and were approved by an institutional review committee. Mice constitutively expressing PKGIμmol/L.Mice were genotyped using the REDExtract‐N‐Amp Tissue PCR kit from Sigma‐Aldrich , as described by the manufacturer. Briefly, DNA was extracted from ear snip tissue (2–3 mm) by incubating it with Extraction Solution and Tissue Preparation Solution in a 4:1 ratio for 10 min at room temperature. The mixture was then transferred into a heating block and incubated at 95°C for 3 min, before digestion was stopped by adding the Neutralization solution. The extracted DNA was mixed with PCR ReadyMiX (Sigma‐Aldrich) and the PKG[C42S]KI‐specific primers, 5′‐cag ttt agg gac aga gtt gg‐3′ (forward) and 5′‐aac ctg ctt cat gcg caa gg‐3 (reverse), used at a final concentration of 0.4 α  as previously documented were heated to 65°C for 10 min immediately prior to loading the gel. Nonreducing SDS‐polyacrylamide gel electrophoresis was carried out using a Bio‐Rad system . After electrophoresis, samples were transferred to polyvinylidene difluoride membranes using a Bio‐Rad semidry blotter. Membranes were blocked with 3.5% BSA in Tris‐buffered saline (TBS) containing 0.1% Tween (TBS‐T), overnight at 4°C. All proteins were imaged under UV excitation, using the BioRad Stain‐Free™ technology, to assess protein loading. Digitized immunoblots were collected using a Chemidoc system (BioRad) and analyzed with BioRad ImageLab software. For the PKG blot protocols, membranes were blocked with 10% nonfat dry milk in phosphate‐buffered saline containing 0.1% PBS‐T for 1 h at room temperature and then immunostained for PKG1µm internal diameter) were dissected free of surrounding tissue. Some were snap frozen in liquid N2 and others mounted on borosilicate glass pipettes in an arteriograph chamber . The proximal glass pipette was attached to a servo‐controlled pressure‐regulating device and the distal pipette was blocked. After checking for leaks, arteries were pressurized to 80 mmHg for approximately 45 min in physiological saline (PSS) with the following composition: 119 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgCl2, 2 mmol/L CaCl2, 7 mmol/L glucose, 24 mmol/L NaHCO3, and 2.3 mmol/L EDTA. PSS was warmed to 37°C and continuously gassed with a 95% air and 5% CO2 mixture. Arteries exhibiting a leak were discarded and pharmacological protocols were performed only on arteries that exhibited pressure‐induced myogenic tone. Internal diameter was detected using a CCD camera and edge‐detection software. Pressurized arteries were exposed to circulating PSS and allowed to develop myogenic tone, defined as the difference between the vessel diameter in Ca2+‐containing and Ca2+‐free solutions, expressed as a percentage of the diameter in Ca2+‐free conditions. Dilation or constriction to drugs was expressed as the percent change in active tone. All drugs used were purchased from Sigma‐Aldrich.Third‐order branches of mesenteric arteries  consisting of a Nikon upright microscope, 60× water‐dipping objective (NA 1.0) and electron‐multiplying Andor CCD camera. Images were acquired with Andor Revolution acquisition software at 55 frames/sec. Bound Ca2+ was detected by exciting at 488 nm and collecting emitted fluorescence using a 527.5/49 nm band‐pass filter. Video files of Ca2+ sparks were obtained from arteries before and after incubation with drugs. Ca2+ sparks were identified using custom‐written software , which detects temporally delineated increases in fractional fluorescence (F) greater than 0.26 above the baseline fluorescence level (Fo) in a defined, 1.1 × 1.1 µm area of interest (5 × 5 pixels). We used this threshold amplitude because it is reliably away from the noise of about three times the standard deviation of the baseline. Noise differs from record to record and also depends on the selection box size. We chose the value of 1.26 F/Fo in order to avoid false positives from all of our records. We have published in Physiological Reports previously using this threshold  are expressed as F/Fo. At least two areas from each artery were analyzed for each experiment and the average was used as n = 1 for analysis.Mesenteric arteries were placed in HEPES‐PSS containing 10 μmol/L wortmannin, which does not affect caffeine‐induced [Ca2+] transients in myocytes. Arteries were exposed to caffeine (10 mmol/L) for ~5 sec . In our previous study, the caffeine‐induced [Ca2+] transients were imaged at 1 Hz  by the average fluorescence of 10 preceding images from the same ROI without activity. The area under the curve for each of the caffeine‐induced [Ca2+] transients was calculated using a trapezoidal numerical integration of (F−F0)/F0 over time. The caffeine‐induced Ca2+ transient was considered as any change in F/Fo from the precaffeine baseline.Mesenteric arteries were loaded with Fluo‐4‐AM, mounted in a pressure myograph, pressurized to 20 or 110 mmHg and imaged with a spinning‐disc confocal microscope as described above. To prevent contraction and thus loss of focus, the arteries were incubated with 1 KI mice were mounted in a tension myograph  and stretched to the optimal resting tension using the normalization software module. Arteries were equilibrated in oxygenated (95% air:5% CO2) PSS at 37°C, prior to testing vitality with 60 mmol/L K+‐enriched physiological saline. Vessels were then incubated with vehicle or 1 μmol/L paxilline for 15 min to block BK channels, before adding 3 μmol/L U46619 to induce tension. This was followed by cumulatively increasing concentrations of H2O2 (10–300 μmol/L).Isolated second‐order mesenteric arteries from WT C57BL/6j and PKG[C42S]t‐test or multiple points ANOVA. P < 0.05 was considered statistically significant.Statistical analyses were performed with GraphPad software by the Student's 2+ spark frequency in third order mesenteric resistance arteries from WT (C57Bl6/j background) mice. Ca2+ sparks were imaged in arteries at the following incremental intraluminal pressures: 20, 30, 4, 50, 80, and 110 mmHg. Examples of Ca2+ spark recordings at 20 and 80 mmHg are shown in Figure 2+ spark frequency with increasing pressure, but two potentially notable findings were apparent. First, there was a graded “pressure‐step” between 20 and 50 mmHg and second, once Ca2+ spark frequency had risen to 50 mmHg there was no further increase in frequency at higher intraluminal pressures; that is, there was a “ceiling effect” regarding the generation of Ca2+ sparks through pressure  possibly representing hyperpolarization of the artery and subsequently a reduction in Ca2+ entry through voltage‐gated Ca2+ channels. However, this observation also suggests that once the pressure–oxidant–PKG axis is active and stimulating Ca2+ spark formation, it may not be possible to further boost this pathway using exogenously applied oxidants. We attempted to measure Ca2+ sparks in pressurized arteries by depolarizing the VSMC membrane potential through application of high K+ solution, as previously demonstrated in middle cerebral arteries . From a physiological perspective, the difference between these approaches is potentially important as we previously showed that it is specifically the rise in intraluminal pressure which triggers oxidant activation of PKG and thus Ca2O2‐induced vasodilation of arteries at an intraluminal pressure of 80 mmHg studied using pressure myography. Intriguingly, there was no difference in the vasodilation to H2O2 between the arteries from WT or PKG[C42S]KI mice . In PKG[C42S]KI arteries an increase in pressure causes constriction that is not counteracted by activation of BK channels, as evidenced by a lack of effect of paxilline on artery diameter , pEC50 = 4.77 ± 0.03 and maximal response above 30 µmol/L. When tested on arteries from PKG[C42S]KI mice, or WT arteries in the presence of paxilline, H2O2 at 30 µmol/L had reduced effect on force and furthermore the induced responses often failed to reach a clear maximum at 100 µmol/L, the highest concentration tested, and this further impeded analysis. Accurate EC50 values could not, therefore, be determined for each preparation in these conditions. Comparison of the mean concentration–response curves generated in each condition . The H2O2 concentration–relaxation relationship measured in arteries from PKG[C42S]KI mice, previously described by Prysyazhna and Eaton  for paxilline‐treated arteries from WT mice and 37 µmol/L (pEC50 = 4.4) for arteries from KI mice  in order to magnify any differences between the WT and PKG[C42S]KI arteries and also calculated the Area under the curve (AUC) and the time‐to‐ peak. Furthermore, in this study the caffeine‐induced Ca2+ transient was measured in intact arteries at intraluminal pressures of 20 mmHg, at which point the PKG–Ca2+ spark pathway is dormant, and at 110 mmHg which is an intraluminal pressure at which, Ca2+ sparks in WT vessels are present.To elucidate the effects of oxidized PKG on the SR in resistance arteries, we studied the Ca2+ loading of the SR would be associated with an overall reduction in the caffeine‐induced Ca2+ transient. Conversely, if Ca2+ release were to be disabled, then the SR Ca2+ content would not be affected. Examples of Ca2+ transients induced by rapid application of a caffeine bolus at low (20 mmHg) and high (110 mmHg) intraluminal pressures are shown in Figure KI arteries. As summarized in Figure F/F0) or the time ‐to‐peak of the caffeine‐induced Ca2+ transient between the WT and PKG[C42S]KI arteries. At high intraluminal pressure (110 mmHg), although the AUC and time‐to‐peak were equivalent between groups, the amplitude of the caffeine‐induced [Ca2+] transient was significantly higher in arteries from PKG[C42S]KI mice than from WT mice. Raising intraluminal pressure did not affect AUC, amplitude, or the duration of the caffeine‐induced Ca2+ transient within groups .We hypothesized that any interruption to Ca2+ sparks–BK channel vasodilation. We show the importance of intraluminal pressure specifically as the prompt to activate the oxidant‐driven pathway and illustrate the functional independence of BK channel bioavailability from oxidant‐activated PKG in arteries with steady state constriction. Our data suggest that once oxidized and activated, PKG regulates Ca2+ spark release from the SR rather than influencing SR Ca2+ loading. We propose that these distinctive characteristics enable resistance arteries not only to maintain the requisite degree of BK channel vasodilation to regulate pressure‐induced constriction of the artery but also to prevent excessive vasodilation which would disturb local flow.The principal findings from this study illustrate a distinctive activation step and a functional “ceiling” of the vasodilatory pathway within resistance arteries triggered by intraluminal pressure: oxidant generation–PKG–Ca2+ spark vasoregulation of small arteries is one of the most important determinants of contractile tone and thus microvascular function  is weighted heavily in favor of the contractile mechanism. Thus, at steady state pressure‐induced constriction, inhibiting BK channel function further constricts a mesenteric artery by an additional 10% of the overall passive diameter. This is particularly important for conditions such as obesity   mouse  and elevated (110 mmHg) intraluminal pressures, similar to our measurement of Ca2+ sparks. Compared with WT arteries, at the higher intraluminal pressure, the lower Ca2+ spark frequency of the PKG[C42S]KI arteries was associated with a higher caffeine‐induced Ca2+ transient amplitude. Importantly, however, there were no differences in the “Area Under the Curve” (AUC) or “Time to Peak” for the caffeine‐induced Ca2+ transients; and in this context, the AUC is probably a more accurate interpretation of the overall SR Ca2+ load than the peak amplitude. Our interpretation of these divergent differences  is that whilst Ca2+ loading of the SR is maintained in the PKG[C42S]KI arteries, Ca2+ release may be impeded. These observations contrast to those observed in isolated cardiomyoctes, where a failure of of phospholamban phosphorylation in PKG[C42S]KI mice is associated with a smaller caffeine‐induced Ca2+ transient . However, in wire‐mounted arteries, exogenous H2O2 is required to activate PKG and this accounts for the impaired vasodilation to H2O2 seen in the PKG[C42S]KI arteries compared with WT. This interpretation was reinforced by the western blot protocols which indicated that to induce oxidative dimerization of PKG within wire‐mounted arteries, it was necessary to incubate with H2O2. Whether this difference between the techniques reflects a failure of stretch from the transluminal wire to generate oxidants, or perhaps wire‐induced disruption of the myocyte microarchitecture required to anchor PKG adjacent to oxidant producers or the SR, is an avenue for further study. Nevertheless, while this is perhaps a subtle difference between the approaches (wire vs. pressure myography) and will not be relevant for the majority of vascular studies, our observation here has ramifications for the choice of technique used to study Ca2+ sparks or BK channel vasodilatory function in resistance arteries. A caveat is that wire myography can only be applied to arteries around twice the intraluminal diameter (200–400 µm) of those used for pressure myography. As pressure‐induced constriction is generally seen in arteries of smaller diameter (less than 200 µm), the observed difference between wire and pressure myography could reflect heterogeneity due to size.Our data also illustrate the importance of intraluminal pressure as a specific trigger to activate the oxidant–PKG–Ca2+ spark vasodilation. In cardiomyocytes, a microtubule network detects mechanical stretch and facilitates activation of NADPH Oxidase 2 (Nox2) to generate oxidants which regulate Ca2+ spark frequency (Prosser et al. Also left unanswered is the question of how intraluminal pressure generates oxidants within small arteries to regulate Ca2+ spark–BK channel vasodilatory buffer, which balances against inherent pressure‐induced constriction. The initiation of Ca2+ sparks, via pressure‐induced oxidants that activate PKG, is triggered at an intraluminal pressure threshold between 20 and 50 mmHg, but once the system is activated neither pressure nor oxidants can further increase Ca2+ spark frequency or vasodilation through this mechanism. We suggest that this feature of pressure‐induced vasodilation is crucial for normal microvascular diameter regulation. The principal role of the VSMC layer of resistance arteries is to provide a steady basal blood flow to distal beds, which can be both quickly and easily modulated, for example by the endothelium, but then reverts back to the original steady state. The characteristics of the pressure–oxidant–PKG–Ca2+ spark pathway, a steep sensitivity to intraluminal pressure and functional ceiling, suit this role well. Our work also suggests that oxidant‐activated PKG regulates Ca2+ spark release rather than Ca2+ loading of the SR, but further research is required to elucidate the microdomain that is likely to accommodate the pressure‐sensitive oxidant generators, PKG and the SR.In summary, our work clarifies a mechanism that lies at the heart of small artery autoregulation; specifically, the processes underpinning the CaThe authors declare no conflict of interest.Figure S1. Representative Western blots illustrating presence of significant dimerization of PKG in wire‐mounted arteries only following incubation/vasodilation with H2O2.Click here for additional data file.


Cold-induced urticaria is a significant condition, especially among young females. Despite the morbidity of this disease, studies that fully characterize the disease are limited.We analyzed the characteristics of patients diagnosed with cold-induced urticaria at a community-based allergy practice in Vancouver, BC, Canada between 2003 and 2016. Detailed patient history, diagnostic measures and treatment were evaluated.A total of 50 patients were found to have active cold-induced urticaria with a median age of 28.5 (range 2–67) years and 35 patients (70%) were female. 16 patients (32%) had co-occurring physical urticarias while 26 patients (52%) had secondary allergic diagnoses and 3 patients (6%) were thought to have a provoking factor. Of those with a clinical history of suspected cold-induced urticaria that were evaluated with ice cube testing, a positive test was obtained in 84.7% of patients. Treatment was largely with non-sedating antihistamines, with the majority of patients receiving this modality.Cold-induced urticaria is a complex disease with significant overlap with other chronic inducible urticarias and other allergic diseases. Diagnostic testing shows inconsistent results and the mainstay of treatment consists of non-sedating antihistamines, with other options available for those who do not respond. Cold-induced urticaria is the second most common type of chronic inducible urticaria and has been reported as early as 1866 . SymptomConfusion in the classification and nomenclature of the different forms of urticaria is an ongoing issue. In the 2013 international guidelines, the urticarial diseases were divided into acute (duration of symptoms less than 6 weeks) and chronic  . Chronic® measurements, both of which were listed as acceptable in recent guidelines outlined by the 2016 EAACI/GA2LEN/EDFUNEV [Cold-induced urticaria is divided into primary and secondary forms and there have been many associations reported in the literature. Primary forms are defined when a secondary, or triggering, etiology cannot be determined . Seconda/EDFUNEV , 16. Tre/EDFUNEV .Despite its relative predominance throughout those with inducible urticarias, very few studies have fully characterized the disease by analyzing associated physical urticarias, prevalence of possible triggers, association with secondary allergic diagnoses and family history. The goal of this study was to review the current literature available on the subject and to analyze the characteristics of the disease in a cohort of patients in a local allergy clinic to look for possible trends and associations to fuel future research into this field.The study was taken in a mixed pediatric/adult practice allergy clinic in Vancouver, British Columbia, Canada operated by two attending physicians. Referrals are generally received by family physicians in addition to some from medical or surgical specialists. A chart review was undertaken to review patient visits from 2003 to 2016 using the electronic medical record system. Files were retrieved by searching the electronic database for the word “cold” and “urticaria”. The authors then manually reviewed the patient charts and included patients with a diagnosis of cold-induced urticaria in the study (see below). If patients had more than one consultation letter, the patient was only included once in the study. Relevant clinical and laboratory data was retrieved from the charts. This included demographic data, other types of co-existing physical urticarias, preceding/causative factors, associated symptoms, secondary allergic diagnoses, family history, laboratory values and treatment regimens.Patients were included in the main analysis if they met the following diagnostic criteria. They must have active symptoms at the time of consultation, duration of symptoms of longer than 6 weeks, test positive on cold provocation testing with the ice cube test and have a clinical history consistent with primarily cold-induced urticaria. Questions on history included the development of urticaria or anaphylaxis on exposure to cold environments, after consumption of cold foods and/or beverages or after immersion in cold water. Only patients considered to have primarily cold-induced urticaria  where included in the study. Cold provocation testing consisted of applying a melting ice cube in a thin plastic bag to the patient’s volar forearm for 5 min, and then observing for the development of a wheal. A positive test was defined as the formation of a wheal within 10 min after the ice cube stimulus was removed.® instrument. The instrument was placed vertically on the patient’s forearm and stroked once for a distance of approximately 60 mm. The test was considered positive if a pruritic palpable wheal of greater than or equal to 3 mm width was present within 10 min of provocation. Cholinergic urticaria was diagnosed based on the patient’s clinical history. A patient was considered to have a history of cholinergic urticaria if they developed urticarial lesions shortly after exercise, application of heat (such as a hot shower), and/or after consumption of hot and spicy foods or beverages. Provocation testing for cholinergic urticaria did not take place in this allergy clinic.The investigation into and diagnosis of other physical urticarias was initiated based on the patient’s clinical history. Dermatographic urticaria was diagnosed using Fric testData was analyzed using Microsoft Excel. Patients who met the diagnostic criteria for cold-induced urticaria as outlined above were included in the main analysis. To evaluate the effectiveness of cold provocation testing, we had a sub group of patients we analyzed with a clinical suspicion of cold-induced urticaria. These patients must have undergone provocation testing with a definitive result to be considered for analysis. Patients were considered to have a clinical suspicion of cold-induced urticaria if they had active symptoms at the time of consultation and had a clinical history consistent with primarily cold-induced urticaria (as outlined above).50 patients met the diagnostic criteria for cold-induced urticaria. The average age of patients seen at time of consultation was 31.3 years (median age of 28.5) years with a minimum age of 2 years and maximum of 62 years. 30% of the patients were male with 70% being female. Sixteen (32%) patients with cold-induced urticaria had evidence of co-occurring physical urticarias, including cholinergic (10% of patients) and dermographism (22% of patients). Two (4%) of the patients reported associated anaphylaxis from the cold and six (12%) patients had associated angioedema. Just over one half (52%) of patients had secondary allergic diagnoses with allergic rhinoconjunctivitis (34%) and asthma (14%) being the most common  were believed to have a preceding factor that may have triggered their cold-induced urticaria. One patient suffered a Hymenoptera sting with acute urticaria, after which the symptoms of cold-induced urticaria began. Two patients (4%) were believed to have their cold-induced urticaria triggered by an infection. The first patient was a 40-year-old male who had two episodes of infectious mononucleosis as a young adult, after which he developed cold-induced urticaria. The other patient was a 24 year-old female who suffered two episodes of pneumonia, and then developed cold-induced urticaria. Neither of these patients had viral loads or serology available to prove the theory. Three patients (6%) reported a family history of cold-induced urticaria Table .A total of 59 patients were identified as having a clinical suspicion of cold-induced urticaria and had cold provocation testing performed using the ice cube test. Of these patients, 84.7% tested positive and 15.3% tested negative.Twenty-two (44%) patients with confirmed cold-induced urticaria had laboratory testing to evaluate for cryoglobulins and/or cold agglutinins. Laboratory blood work was ordered for five patients, but the results could not be located due to either the patient not having blood drawn or the results not received in the allergy/immunology clinic. Of the 17 patients with resulted laboratory values, 16 patients were tested for cryoglobulins and 15 patients were tested for cold agglutinins. All patients tested for cryoglobulins and cold agglutinins had negative results.Pharmacologic treatment was prescribed for 49 patients (98%) with active urticaria. Of those patients with prescribed therapy, 22 patients (44%) were on regularly prescribed treatment, whereas 27 (54%) patients were on intermittent/as needed therapy. One patient did not have any prescribed pharmacotherapy and was managed with conservative measures alone. The majority of patients with cold-induced urticaria (90%) used non-sedating antihistamines as part of their treatment plan. Fewer patients were on other pharmacotherapies including omalizumab (2%), H2 receptor antagonists (6%) and sodium cromoglycate (2%). It appeared that patients on these second line therapies did not have control of their symptoms with non-sedating antihistamines alone. About one-third of patients (32%) with prescribed therapy had an epinephrine auto-injector  of the patients with cold-induced urticaria were felt to have a provoking factor (including infections or insect stings) for their cold-induced urticaria based on clinical history. This would be suggestive of secondary acquired cold-induced urticaria. However, these suspicions were not confirmed on laboratory testing. The remainder of patients were believed to have primary acquired cold-induced urticaria. Historically the majority 96%) of patients with acquired cold-induced urticaria have been shown to have primary cold-induced urticaria, with only rare occurrences of secondary cold-induced urticaria . The mos6% of patNLRP3 (CIAS1) gene that leads to overproduction of interleukin-1-beta (IL-1β). CAPS consists of three disorders including familial cold autoinflammatory (or urticaria) syndrome (FCAS/FCU), Muckle–Well syndrome (MWS) and neonatal-onset multisystem inflammatory disease (NOMID) also known as chronic infantile neurologic cutaneous articular syndrome (CINCA) [NLRP3 and other inflammatory diseases along with full evaluation of symptoms, lab tests , skin biopsy and complete medical history [In addition to acquired cold-induced urticaria, there are types of cold-induced urticaria that are felt to have a genetic component with familial transmission. This includes the cryopyrin-associated periodic syndrome (CAPS) which is a rare inherited autoimmune disorder with a mutation in  (CINCA) . Diagnos history . In our  history  also rep® measurements. Although there is no study comparing the sensitivity and specificity of each of these tests, both the ice cube test and TempTest® are considered acceptable for testing [® may be a better test as it allows for reproducible and standardized cold and hot provocation tests along with identification of temperature and stimulation time thresholds [Newly released consensus guidelines for the diagnosis and treatment of cold-induced urticaria were recently published in 2016 [resholds . In our Given that most cases of cold-induced urticaria are idiopathic, targeted treatment is often not possible. Avoidance of cold, such as immersion into cold bodies of water, is often recommended for both treatment and avoidance of a potentially more systemic reaction such as anaphylaxis. However, this advice is often not achievable or fully effective in managing symptoms . Non-sedCold-induced urticaria is a complex disease with significant overlap with other chronic inducible urticarias. A higher rate of atopic disorders has also been reported in this study which has not been reported by others. Idiopathic primary cold-induced urticaria continues to be most prevalent, however secondary causes such as due to cryoglobulinemia, infectious agents and insect stings can be identified in a cohort of patients. Although rare, in those with a family history of cold induced urticaria, a diagnosis of CAPS and other familial disorders should be entertained. Despite multiple different testing modalities currently available, diagnostic testing has been shown to have inconsistent results. The mainstay of treatment consists of cold avoidance techniques with non-sedating antihistamines being the most common pharmacotherapy employed. Other agents, including omalizumab and cyclosporine, are also available for those who do not respond to initial management. Although, this was a small chart review, we feel that this study adds to the growing body of knowledge that currently exists on cold-induced urticaria and reviews the current literature available to aid in diagnosis and management of this disease.