Iterated map models of intracellular Ca2+ cycling dynamics in ventricular myocytes under periodic pacing have been developed to study the mechanisms of Ca2+ alternans. Two mechanisms of Ca2+ alternans have been demonstrated in these models: one relies mainly on fractional sarcoplasmic reticulum Ca2+ release and uptake, and the other on refractoriness and other properties of Ca2+ sparks. Each of the two mechanisms can partially explain the experimental observations, but both have their inconsistencies with the experimental results. Here we developed an iterated map model that is composed of two coupled iterated maps, which unifies the two mechanisms into a single cohesive mathematical framework. The unified theory can consistently explain the seemingly contradictory experimental observations and shows that the two mechanisms work synergistically to promote Ca2+ alternans. Predictions of the theory were examined in a physiologically-detailed spatial Ca2+ cycling model of ventricular myocytes.Intracellular calcium (Ca Action potential excitation and intracellular Ca2+ release are two tightly regulated processes+) current (INa) gives rise to the fast upstroke of the action potential, elevating the voltage to the plateau voltage. Then the L-type Ca2+ current  is activated, which maintains the long plateau. In the meantime, potassium (K+) currents (IK) are slowly activated, which repolarize the cell back to its resting potential. The Ca2+ brought in by L-type Ca2+ channels (LCCs) triggers a large amount of Ca2+ release from the SR and this release activity is enhanced by Ca2+ released from the SR, a process called Ca2+-induced Ca2+ release. Ca2+ released from the SR binds with myofilament (MyoF) to cause contraction. The SR is then replenished through Ca2+ reuptake via the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump. The Ca2+ that enters the cell via LCCs is extruded from the cell via Na+-Ca2+ exchange (NCX). These pumps maintain the Ca2+ gradient between the intracellular and extracellular space, and the intracellular Ca2+ homeostasis. With the presence of the Na+-K+ (NaK) pump, the gradients and homeostasis of Na+ and K+ are also maintained. Besides the normal heart rhythm, the complex regulation of membrane excitation and Ca2+ cycling can lead to various nonlinear dynamics in the heart that promote cardiac arrhythmias346Under normal conditions, the human heart contracts once every second or so to pump blood throughout the body. The contraction of the heart is caused by intracellular calcium Ca+ release2+ cycling system1011121315162+ are coupled via Ca2+-dependent ionic currents, alternans due to the electrical system will also result in alternans in the Ca2+ cycling system and vice versa. For Ca2+ cycling instability-induced alternans, two theories have been developedSeveral mechanisms of alternans have been shownet al.2+ release (FCR) curve is responsible for alternans, which was supported by a series of experiments from Eisner’s group1112132+ released from the SR and the SR Ca2+ content right before the release occurs. This SR Ca2+ content is called the diastolic Ca2+ load (DCL)  . This th2+ alternans262+ release and the properties of individual Ca2+ sparks. In a ventricular myocyte, the RyRs are clustered in the cell forming Ca2+ release units (CRUs) in conjunction with their proximate sarcolemmal ion channel clusters  to the whole-cell Ca2+ cycling dynamics. The model was derived based on the following properties of Ca2+ release. At any time, a CRU is in one of the three states . Ca2+ released from a primary spark may diffuse to the neighboring CRUs and recruit the recovered ones to firesecondary firings. After firing, a CRU remains refractory for a certain period of time. Due to the random opening properties of LCCs and RyRs, the primary sparks are probabilistic events. We assume that the probability of a CRU to fire as a primary spark during a pacing beat is α. Similarly, the secondary sparks are also probabilistic events, and we assume that the probability of a firing CRU to recruit a neighbor to fire during a pacing beat is γ. The recovery of the RyRs and SR refilling in a CRU gives rise to spark amplitude restitution36β. If there are a total of NT CRUs in the system, and at the kth beat, there are Nk sparks, then at the following [(k+1)th] beat, there are βNk unrecovered CRUs and (NT − βNk) recovered CRUs. The number of primary sparks in this beat is then α(NT − βNk), and thus (1 − α)(NT − βNk) recovered CRUs are available for recruitment. If a fraction f of these CRUs are recruited to fire, then the total number of sparks at (k+1)th beat is:In our previous studies26e states : recoverf depends on the number of sparks and the spatial pattern of the CRU states . An explicit form of function h will be derived later.where Inserting 2+ alternans, in which FCR and SR Ca2+ uptake were described by phenomenological functions of DCL. Here we express FCR and SR Ca2+ uptake as functions of spark number, which then links Ca2+ sparks to DCL. In the old models20212+ had to be used to keep 2+ of the cell is determined by the equation itself, i.e., the total Ca2+ is a solution of the model instead of a parameter, which changes in time.which links the number of sparks to DCL. Note that in previous studies20212+ cycling dynamics under periodic pacing. Since α and γ are functions of DCL (Lk), i.e., α = α(Lk) and γ = γ(Lk), 2+. By denoting nk = Nk/NT and lk = Lk/NT, f becomes a function of nk and lk, i.e.,α and γ are functions of lk, i.e., α = α(lk) and γ = γ(lk). Besides α and γ, β and ε are also functions of lk and/or pacing period T. These functions, as well as function h, will be defined in detail in the sections below.since For the purpose of our theoretical analysis, we rewrite whereandPrimary spark rate (α)—It is well known that the spark probability  increases with SR Ca2+ content41α is a sigmoidal function of DCL (lk), i.e.,lα is a parameter determining the half value of function α(lk) and sα is a parameter determining the slope of function α(lk). α0 describes the coupling fidelity between the proximate LCC cluster and the RyR cluster when the DCL is high, whose value lies between 0 and 1. α0 = 1 represents a perfect coupling between the LCC cluster and the RyR cluster, indicating that opening of the LCCs always causes the RyR cluster to fire. α0 = 0 means no coupling, indicating that opening of the LCCs always fails to cause the RyR cluster to fire. Physiologically, α0 is determined by the LCC open probability and conductance, the volume of the dyadic space, and the RyR sensitivity to Ca2+, etc.where CRU refractoriness (β)—The CRU refractoriness is determined by the recovery time of the RyRs. Here we simply assume that β only depends on the pacing period T in a sigmoidal function as:Tβ determines the half value of β and τβ determines the slope of the sigmoidal function.where Spark recruitment rate (γ)—No experimental data is available for γ. Again based on numerical simulationsγ is also a sigmoidal function of DCL (lk), i.e.,lγ is a parameter determining the half value of function γ(lk) and sγ is a parameter determining the slope of function γ (lk). γ0 describes the coupling between CRUs when the DCL is high, whose value lies between 0 and 1. γ0 = 0 indicates that a Ca2+ spark of a CRU will never cause its neighboring CRUs to fire, while γ0 = 1 indicates that a fired Ca2+ spark will always cause its neighboring CRUs to fire. Physiologically, γ0 is determined by the distance between CRUs, the cytosolic Ca2+ diffusion rate, and the RyR sensitivity to Ca2+, etc.where Spark amplitude and restitution (ε)—SR Ca2+ release and spark amplitude restitution have been measured and characterized experimentally36373843ε is a function of lk and pacing period T, i.e., ε = ε. We assume the following function for ε:lnadir is the SR Ca2+ content at the peak of the spark and r(T) is the spark amplitude restitution function. We set r(T) as36where τr is the time constant.where 2+ is released, which is determined by the DCL and the recovering status of the RyRs. Such a release-load relationship was first experimentally measured by Bassani et al.et al.1921g is a function of nk and lk asDuring a heartbeat, only a fraction of the SR free CaT→∞, according to Eqs 15 and 18, β = 0 and r = 1, all CRUs are recovered, thus the function g depends solely on lk since α, γ, and ε are functions of lk, i.e.,When γ0 = 0, then If there is no recruitment, i.e., g functions et al.g/lk) against the experimental data by Shannon et al.2+ cycling models342021In It’s generally assumed that the SERCA pump flux obeys a Hill-type kinetics as:vup is the maximum amplitude of the SERCA pump activity, Kup is the Ca2+ concentration at half SERCA activity, ci is the cytosolic Ca2+ concentration, and H is the Hill coefficient. Therefore, the amount of Ca2+ taken up by the SERCA pump depends not only on the amplitude of the cytosolic Ca2+ concentration but also on its time course of decay. Here we assume that ci decays exponentially: ci = cb + cpeλt−, where cb is the baseline Ca2+ and cb+cp is the peak Ca2+. cp is then proportional to the amount of Ca2+ released from the SR, i.e., cp = εnk+1. During a single beat, the amount of Ca2+ pumped back into the SR can be calculated aswhere H = 1 leads to:which for 2+ decays to the baseline, ci ~ cb, i.e., cpeλt− ≈ 0. Since cb ≪ cp∝εnk+1, we can then simplify Assume that at the end of the cycle, the Caυ describes the maximum SERCA pump activity and κ corresponds to the Kup of SERCA. The slope of this function iswhere g.which is a decreasing function of H~1.7 for physiological cellsH > 1, an explicit form for the function h may not be possible from H = 2), the function is too complex to be useful. However, it is generally true that h is an increasing function of g and its slope is a decreasing function of g, and therefore we use It was estimated We first performed a general linear stability analysis of the model to obtain the stability criterion. We then used numerical simulations of the iterated map model to investigate the nonlinear dynamics and make more specific theoretical predictions.nk+1 = nk = nss and lk+1 = lk = lss:The steady state of the iterated map model can be solved from the following equations, derived from nss and lss are the steady-state values of the number of sparks and DCL, respectively. The steady state can be graphically determined using where where andλ1| < 1 and |λ2| < 1, and becomes unstable when |λ1| > 1 or |λ2| > 1. Alternans occurs when λ1 < −1 or λ2 < −1. Applying this condition to The steady state is stable when both |i.e., the steady state is unstable as long as We first analyze two special cases:β = 0 and r = 1. β = 0 means no refractoriness of SR Ca2+ release and r = 1 means no spark restitution. Under this condition, g = g(lk) is solely a function of DCL (see (1)  DCL see . The modand the eigenvalues in 2+ uptake curve, independent of the 3R’s. Under this condition, alternans occurs when λ2 < −1, which leads to:Since there is no refractoriness, the instability is solely determined by the steepness of the FCR curve and that of the SR Ca2+ uptake function 2+ to be constant in the cell, which is not true since the cell is not a closed system for Ca2+. In the current model, the total amount of Ca2+ of the cell is not set as a parameter but a variable, and thus which can be also obtained from g = h, i.e., the SR always reuptakes exactly the same amount of Ca2+ that it releases within a beat. When lk+1 = lk. This corresponds to the experiments2324(2) nk, α , a large γ, and a very large β (>0.9), as shown in The condition for alternans is then F is needed for alternans  based on the experimental FCR data by Shannon et al. (the red curve in T < 450 ms). Refractoriness is a key parameter that promotes this behavior.Besides the general theoretical predictions, more specific predictions can be obtained by directly simulating the iterated map model. Depending on the choice of parameters, the model can exhibit alternans, different periodicities, as well as chaos. Alternans occurs at either fast or very slow heart rates. Here we choose the FCR function  and κ (Kup of SERCA). Reducing υ tends to suppress alternans at fast pacing rates but promote alternans at slower pacing rates , while othersτr ~ 450 ms). In τr . Adding the spark amplitude restitution into the model changed the onset of alternans, causing the FCR mediated alternans to occur at a longer T and the refractoriness mediated alternans to occur at a shorter T. For τr = 300 ms, the refractoriness mediated alternans is completely suppressed. Note that the magnitude of spark number alternans at fast pacing is increased for τr = 100 and 200 ms. Based on nk increases in the non-alternans regime as τr increases), and thus amplifies the amplitude of spark number alternans.To investigate the effects of spark properties on the mechanisms of alternans, we chose another set of control parameters in which strong alternans exists for both mechanisms . Reducinα  or the rα , showing Ca2+ alternans occurs at fast pacing (T < 450 ms). Reducing the SERCA pump amplitude vup by 50% attenuates the alternans amplitude, and causes alternans to occur at slower pacing (T < 750 ms), but also tends to suppress alternans at fast pacing. In general, this agrees with the iterated map results shown in Kup of the SERCA pump by 50% has a small effect on the onset of alternans while increasing the amplitude of alternans. Reducing both the amplitude and the Kup by 50% greatly changes the dynamics (T < 3250 ms). Second, high periodicity and irregular dynamics occur via a period-doubling bifurcation as T decreases. As T decreases even further, a stable period-1 state occurs, followed by a sudden transition to a period-3 state, and finally to irregular dynamics. This is very similar to the bifurcation sequence of the iterated map model shown in 2+ cycling model, the irregular dynamics is likely chaos since it only occurs for certain pacing periods and arises via period-doubling bifurcations. Moreover, the bifurcation diagram is very similar to the one obtained using the iterated map model in which the irregular dynamics is indeed chaos  plays an important role in promoting this mechanism of alternans by steepening the FCR function. At fast heart rates, however, the 3R’s, the increased FCR function slope, and the reduced SR Ca2+ uptake function slope work synergistically to promote alternans. The new model can consistently explain the seemingly contradictory experimental observations, as detailed below.In this study, we developed an iterated map model describing the intracellular Ca2+ release and miniwaves played important roles in the genesis of alternans, which indicates that spark recruitment (the γ factor) is important for alternans. Although a steep FCR curve was indicated to be responsible for alternans, it is unclear why dyssynchronous Ca2+ release and spark recruitment are also needed. On the other hand, in the 3R theory the roles of dyssynchronous Ca2+ release (corresponding to the α factor) and miniwaves (corresponding to the γ factor) are clear. However, since alternans in these experiments occurred at slow pacing rates (0.5 Hz), thus, it was likely that the RyRs had already or mostly recovered by the beginning of each beat. Alternans via the 3R theory requires a high probability of CRU refractoriness  when DCL remains constant from beat to beat. In general, the slopes of the FCR and the SR Ca2+ uptake functions work in synergy with the 3R’s to promote Ca2+ alternans and other complex dynamics in ventricular myocytes. The unification of the two mechanisms of Ca2+ alternans can provide novel insight into the identification of therapeutic targets for suppressing Ca2+ alternans. For example, reducing the slope of the uptake function (γ) can suppress both mechanisms of alternans, while reducing the refractory period can only suppress alternans caused by one of the two mechanisms. Suppressing Ca2+ alternans by reducing the slope of the uptake function has been demonstrated by experiments of SERCA2a overexpression19In conclusion, the iterated map model developed in this study provides a cohesive theory for CaHow to cite this article: Qu, Z. et al. A unified theory of calcium alternans in ventricular myocytes. Sci. Rep.6, 35625; doi: 10.1038/srep35625 (2016).


Most common forms of hair loss  are caused by aberrant hair follicle cycling and changes in hair follicle morphology. However, current treatments for alopecia do not specifically target these processes. Adipose-derived stromal vascular cells (ADSVCs) that can be harvested from fat cells are one of the latest breakthroughs in the aesthetic field. The potential use of stem cell-based therapies (SCBT) for the repair and regeneration of various tissues and organs offers a paradigm shift that may provide alternative therapeutic solutions, which can be applied to prevent hair loss. This study aimed to present clinical cases of SCBT for the treatment of alopecia areata by transplantation of ADSVCs in the scalp.6 cells into the scalp of the patient. Hair regeneration was assessed by three clinical tests: the pull test, hair quality, and hair density.Twenty patients (9 women and 11 men) were recruited to our retrospectively registered study. After lipoaspiration, autologous ADSVCs were generated and characterized before the injection of 4–4.7 ×  102, P < 0.0001), hair diameter  and pull-test values , untreated versus 6 months post-operative)]. Significant variation was observed between men and women only for hair diameter. No significant differences were observed with age.All patients experienced hair regeneration, increased hair growth and decreased pull test 3 and 6 months after the treatment with ADSVCs [hair density (85.1 ± 8.7 vs 121.1 ± 12.5 hair/cmThe obtained results prove the efficacy and the safety of the treatment, and satisfaction of the patients confirm the quality of the results. Hair loss is one of the most common complaints among all patients consulting a dermatologist and is usually associated with severe psychological disturbances, distress, and symptoms of depression. Most common forms of hair loss  are caused by aberrant hair follicle cycling and changes in hair follicle morphology. Cells with stem cell properties have recently been described in many integument appendages including feathers and teeth, but the hair follicle stands out as one of the best model systems for studying adult stem cells . Hair foThere are three phases of hair growth, which every hair follicle undergoes. The first phase is when follicles undergo extremely rapid epithelial cell division and execute exquisitely timed differentiation programs when in the growing (Anagen) phase. The second phase is when follicles growth stops during a certain period, the (Catagen) phase , 7. The The term “stem cell” is given to a cell which has the ability to self-renew as well as to differentiate into defined cellular subtypes. Multipotent stem cells are present in different adult tissues such as bone marrow, dental pulp, adipose tissue, etc. . MultipoIn the present study, we aimed to use autologous ADSVCs graft for the treatment of alopecia areata and to assess the safety and effectiveness of the transplantation. The clinical trial of 20 patients shows the use of ADSVCs for hair growth and improvement as a valuable treatment.n = 20, 38.3 ± 2.3 years, 9 women and 11 men) from the Middle East were enrolled in the study with no notable pathologic history in particular of hair diseases, with confirmed diagnostic of hair loss alopecia areata and all the selected subjects showed partial alopecia grade 1 or 2 at Ludwig Scale  at 37 °C for 30 min with shaking (250 rpm). The collagenase was inactivated by adding an equal volume of autologous serum, and the sample was centrifuged at 600 × g for 10 min. After centrifugation, the supernatant was discarded and the cell pellet was resuspended in NaCl 0.9%  and filtered through a 100 μm cell strainer  to remove debris. After centrifugation (300 RCF/5 min), 5 ml of the stromal vascular fraction were collected. All the processing must be realized within a maximal time of 90 min. The number of viable cells were determined manually (Trypan blue method) and validated on MACSQuant analyzer  (7AAD staining method). All the quality control tests and injections were done with the obtained fresh samples.The lipoaspirate  was diluFreshly isolated cells were characterized for ADSVCs surface protein expression  by flow 3; needle, 4 mm; gauge, 30; and depth, 4 mm [The ADSVCs were injected into the scalp of the patient according to the following procedure: (1) the upper frontal, biparietal, and upper pyramidal area were first treated with the aseptic chlorhexidine without local anesthesia; (2) to reach the hair follicle area, the injection into the scalp area was performed with the following attributes: syringe, 1 cmth, 4 mm ; (3) 0.2P values of less than 0.05 were considered significant.For descriptive statistics, mean and variance were reported when appropriate. SPSS version 21.0  was used for the statistical analysis. Comparisons between groups were performed using one-way ANOVA. Chi-square tests were applied to detect the difference in the rates between different groups. 6 cells were transplanted: in fact, 0.2 mL containing 0.160–0.188 × 106 cells were injected per spot . In agreement with Varma et al. [+/CD166+ cells in the 0.2 mL of transplanted sample per spot of injection, which prompted us to choose the minimum concentration of 160,000 cells/spot of injection . Importantly, to avoid any aggregation of the cells, which was observed in cases where the cell concentration was > 200,000 cells per 0.2 mL, and to maintain the minimum levels of CD105+ and CD166+ cells per injection, a total of 4.0–4.7 × 106 cells was delivered to the subjects.In this study, the effect of hair regenerative ADSVC therapy was evaluated in 20 patients  aged between 23 and 63 years old. First, based on a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT) published in 2013, which point out the minimal phenotypic criteria to characterize the uncultured SVF population from adipose tissue [a et al. , our res2) and hair diameter (μ), as well as the pull test (Table 2 in 100% of the subjects). All the patients showed a value superior to 0 for the pull test. In addition, no significant variations were observed with age.Second, we assessed the hair loss and growth which were determined as changes in hair density n/cm and hairP < 0.0001) with the injection of ADSVCs, especially 6 months after the treatment (80.8 ± 2.4 μ and 62.8 ± 1.7 μ vs. 60.5 ± 1.8 μ for 6 and 3 months postoperatively vs. preoperatively)  Fig. . In totaly) Fig. .Table 3CP < 0.0001) augmented after treatment with ADSVCs (121.1 ± 12.5 and 120.8 ± 12.6 vs. 85.1 ± 8.7 for 6 and 3 months postoperatively vs. preoperatively) (Table We can note that the hair density was significantly (y) Table . The meay) Table . The haiy) Table .Table 4CP < 0.0001) following ADSVC treatments (0.80 ± 0.17 and 0.90 ± 0.20 vs. 4.35 ± 0.33 for 6 and 3 months postoperatively vs. preoperatively). We could notice that the hair became stronger at 3 and 6 months postoperatively, leading to values of mainly 0 and 1 in the pull test .This study showed that the transplantation of autologous ADSVCs is safe and effective and can be considered an encouraging cell-based therapy for the treatment of alopecia and a nonsurgical hair loss treatment. Hair growth and thickness were markedly improved 6 months after the treatment Fig. .Fig. 4PhReports of the use of stem cells in the treatment of alopecia are rare. Researchers have utilized bone marrow/cord blood-derived stem cells or ADSVC-CM. However, to our knowledge, there is no study that investigated the effect of ADSVCs for the treatment of alopecia. Our results on the characterization of the isolated and transplanted cells agreed with the previously described immunophenotype , 21, 22.The injected ADSVCs in this study might release growth factors, thus promoting vascularization, encouraging new capillaries to form, increasing the production of hair, and improving the supply of blood to the scalp. This provides an ideal environment for the hair follicles to grow new, denser, and healthy hair. This research is the beginning of many series of studies that will be conducted in the future to improve the results and decrease the cost of the procedure to make it more efficient and affordable to patients. It is important to note that most cases of alopecia areata (approximately 80%) resolve spontaneously especially first cases of alopecia areata. It will be interesting to study the efficacy of ADSVCs transplantation for grade III and advanced cases of alopecia areata.In conclusion, treatment using ADSVCs appears highly effective for alopecia areata and may represent a new avenue of therapy for hair regeneration. ADSVC injection promotes good stability of the hair by increasing the hair density, the hair diameter, and decreasing the pull test to almost zero. Furthermore, patients must be very well selected depending on their lifestyle, the cause of hair fall and baldness grade to obtain a good result with this procedure.


We examined these properties of P2X receptors in mouse odontoblasts by patch-clamp recordings. K+-ATP, nonselective P2X receptor agonist, induced inward currents in odontoblasts in a concentration-dependent manner. K+-ATP-induced currents were inhibited by P2X4 and P2X7 selective inhibitors , while P2X1 and P2X3 inhibitors had no effects. P2X7 selective agonist (BzATP) induced inward currents dose-dependently. We could not observe P2X1, 2/3, 3 selective agonist (αβ-MeATP) induced currents. Amplitudes of K+-ATP-induced current were increased in solution without extracellular Ca2+, but decreased in Na+-free extracellular solution. In the absence of both of extracellular Na+ and Ca2+, K+-ATP-induced currents were completely abolished. K+-ATP-induced Na+ currents were inhibited by P2X7 inhibitor, while the Ca2+ currents were sensitive to P2X4 inhibitor. These results indicated that odontoblasts functionally expressed P2X4 and P2X7 receptors, which might play an important role in detecting extracellular ATP following local dental pulp injury.ATP modulates various functions in the dental pulp cells, such as intercellular communication and neurotransmission between odontoblasts and neurons, proliferation of dental pulp cells, and odontoblast differentiation. However, functional expression patterns and their biophysical properties of ionotropic ATP (P2X) receptors (P2X To prepare a Na+-free solution (Na+-free ECS), extracellular NaCl was substituted by equimolar tetraethylammonium chloride (TEA-Cl). For the extracellular Ca2+-free solution and the extracellular Na+- and Ca2+-free solution (Na+/Ca2+-free solution), extracellular Ca2+ was simply removed (0 mM) from the standard ECS or Na+-free ECS, respectively.A solution containing 136 mM NaCl, 5 mM KCl, 2.5 mM CaCl+-ATP), 2′(3′)-O-(4-Benzoylbenzoyl)adenosine 5′-triphosphate triethylammonium salt (BzATP), and αβ-methylene adenosine 5′-triphosphate (αβ-MeATP) were obtained from Sigma Aldrich Chemical Co. . Stock solutions for these agents were prepared in dimethylsulfoxide or MilliQ water , and later diluted to the appropriate concentrations in either extracellular solution or culture medium. Solutions and drugs prepared in an extracellular medium were applied to the cells by a rapid solution exchanging system .Pharmacological agents, 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro-1,4-diazepin-2-one (5-BDBD), NF110, NF449, KN62 were obtained from Tocris Bioscience . All the other reagents including adenosine 5′-triphosphate dipotassium salt dehydrate  were cultured in an alpha-minimum essential medium containing 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml fungizone  at 37°C with 5% COWhole-cell recordings were performed using a conventional patch-clamp recording configuration under voltage-clamp conditions. Patch pipettes (4–9 MΩ) were pulled from capillary tubes by using a DMZ universal puller , which were filled with an intracellular solution. The intracellular solution contained 140 mM KCl, 10 mM NaCl, and 10 mM HEPES (pH was adjusted to 7.2 by Tris). Whole-cell currents were measured using a patch-clamp amplifier . The current traces were monitored and stored using pCLAMP  after digitizing the analog signals at 10 kHz  and filtering the signals digitally at 3 kHz using pCLAMP. The data were analyzed offline by using pCLAMP and the technical graphics/analysis program ORIGIN . The solution temperature when measuring the whole-cell currents was maintained at 30°C.PP2X is the relative total ionic permeability after the activation of P2X receptor by the agonist (BzATP or K+-ATP), Pcontrol is 1.0 for the reversal potentials (Erevs) measured without any P2X receptor agonist in the ECS, ΔErev is the change in Erev by P2X receptor agonist, F is Faraday's constant, R is gas constant, and T is absolute temperature. The temperature was maintained at 30°C while measuring the ramp currents.We calculated the relative change in the total ionic permeability induced by the activation of P2X receptor by using the following Equation 1:P < 0.05 were considered significant.All data are presented as mean ± standard deviation (SD) of N observations, where N represents the number of cells tested or the number of experiments. Steel–Dwass multiple comparisons were used to determine nonparametric statistical significance. N = 6). Current amplitudes were normalized to these single cell capacitance values and expressed as current densities (pA/pF). Depolarized voltage steps from −100 to +80 mV at a holding potential (Vh) of −70 mV  evoked inward currents at Vh of −70 mV, in a concentration-dependent manner  of 52.9 μM (N = 6). A series of three times of repeated applications of 100 μM K+-ATP  elicited a significant desensitizing effect of current  and 48.9 ± 8.0% , respectively, over that at the first application , K+-ATP-induced peak inward current amplitudes , 100 μM K+-ATP-evoked currents decreased their peak amplitudes to 46.0 ± 9.2% , we could record only a residual small current component -evoked Na+ currents were slightly inhibited by application of 20 nM 5-BDBD to 84.7 ± 9.3%  at a Vh of −70 mV  relationship and analyze EP Figure  increase7 receptor, induced inward current in odontoblasts. Not only BzATP-induced currents but also K+-ATP-induced currents were sensitive to KN62, a selective antagonist for P2X7 receptors. In addition, the amplitudes of K+-ATP-induced currents were suppressed by 5-BDBD, a selective P2X4 receptor inhibitor  failed to induce any inward currents in odontoblasts at 100 μM concentration. Studies show that 10 μM αβ-MeATP activates P2X1, P2X2/3, P2X3, and P2X4/6 subtypes, while P2X4 receptors are less sensitive to αβ-MeATP action  receptors.In our previous study, we have shown that ATP, as intercellular-/neuro-transmitter, which was released from mechanically stimulated odontoblasts increased the intracellular Ca+-ATP-induced currents were increased by the removal of Ca2+, whereas they were decreased by the removal of Na+ from the extracellular medium. In addition, K+-ATP-induced currents were almost completely abolished in the Na+ and Ca2+-free ECS. Thus, the K+-ATP induced current was composed by Na+ and Ca2+ conductance. It has been well-known that P2X receptors show relative high Ca2+ permeability , while not by P2X7 receptor antagonist (KN62), indicating that P2X4 receptor in odontoblast has high Ca2+ permeability. These results are also in line with the results showing that P2X4 receptor showed highest relative Ca2+ permeability among the P2X family , the amplitude of K+-ATP-induced currents augmented compared to those in the standard ECS. These K+-ATP-induced Na+ currents were strongly inhibited by an antagonist of P2X7 receptors, but not of P2X4 receptors. Thus, the results indicated that the ionic permeability of P2X7 receptors in odontoblasts were also blocked by extracellular Ca2+, showing Ca2+ dependent block. In addition, the main ionic component of cation permeability for P2X4 receptor was Ca2+, whereas that for P2X7 was Na+. We could observe each residual K+-ATP-induced Ca2 and Na+ current component after P2X4 and P2X7 receptor inhibition. These were carried by other P2X receptor activation, including P2X2, P2X5, and P2X6 receptors; however, further study will be needed.The peak amplitudes of K50 of K+-ATP on the inward currents in odontoblasts was 52.9 μM. Comparing the EC50 of ATP for P2X1–P2X6 receptor activation, this value was of 5–50 times higher concentration than those reported by other studies, in which they have reported EC50 ranging from 1.0 to 10 μM (Jarvis and Khakh, 50 of K+-ATP in this study was in line with that for EC50 of ATP for P2X7 receptor activation (100 μM: Khakh et al., in vitro human tooth perfusion model (Egbuniwe et al., In previous studies, we showed that P2X receptors, as well as ionotropic glutamate receptors, could not mediate intercellular odontoblast-odontoblast communication (Sato et al., On the other hand, ATP that leaked from injured cells in dental pulp could activate P2X receptors in odontoblasts. The intracellular concentration of ATP is in the mM range (Imamura et al., 4 and P2X7 receptors, but not P2X1, P2X2/3, P2X3, and P2X4/6 receptors. P2X4 receptors have high Ca2+ permeability, whereas P2X7 receptors have Na+ conductance with a Ca2+ dependent block.In conclusion, odontoblasts functionally expressed P2X receptor subtypes of P2XYuta S carried out the measurement membrane currents. Yoshiyuki S, TS, and MT participated with design of the study. Yuta S and Yoshiyuki S performed the statistical analysis. Yoshiyuki S conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final 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.


Membrane invaginations called t-tubules play an integral role in triggering cardiomyocyte contraction, and their disruption during diseases such as heart failure critically impairs cardiac performance. In this review, we outline the growing understanding of the malleability of t-tubule structure and function, and highlight emerging t-tubule regulators which may be exploited for novel therapies.dyads, which generate Ca2+ sparks. Recent data have indicated that the dyadic anchoring protein junctophilin-2, and the membrane-bending protein BIN1 are key regulators of dyadic formation and maintenance. While the underlying signals which control expression and localization of these proteins remain unclear, accumulating data support an important role of myocardial workload.New technologies are revealing the nanometer scale organization of t-tubules, and their functional junctions with the sarcoplasmic reticulum called Although t-tubule alterations are believed to be a key cause of heart failure, the plasticity of these structures also creates an opportunity for therapy. Promising recent data suggest that such therapies may specifically target junctophilin-2, BIN1, and/or mechanotransduction. In mammalian cardiac myocytes, invaginations of the sarcolemmal membrane create an extensive network called the t-tubule system. These structures allow the cardiac action potential to propagate into the interior of the myocyte, initiating the process of excitation-contraction (EC) coupling. This role is afforded by precise control of the structure and function of t-tubules and their constituent ion transporters. Recent data have revealed an impressive malleability of t-tubules, giving new insight into how they are assembled and maintained, but also how they can be pathologically altered. In the present review, we outline these exciting new findings, and particularly highlight the emerging understanding of how t-tubule structure and function may be therapeutically targeted in diseases such as heart failure.In ventricular cardiomyocytes, t-tubules are present in a well-organized network. The majority of tubules in these cells are oriented transversely along z-lines, leading to the original designation of this system as the “transverse” or “t”-tubule network , 2. HoweT-tubules were long reported to be absent in atrial myocytes, and it was often hypothesized that the smaller, thinner size of these cells precluded a necessity for initiation of excitation-contraction coupling at internal sites . Recent dyads  face ryanodine receptors (RyRs) in the SR membrane, across a narrow 12–15 nm dyadic cleft [+ channels, this depolarization triggers LTCC opening and the resulting Ca2+ entry elicits SR Ca2+ release via the RyRs. This process, known as Ca2+-induced Ca2+ release (CICR), results in high cytosolic Ca2+ concentration and initiation of contraction as Ca2+ binds to the myofilaments. Precise positioning of LTCCs and RyRs is essential for efficient CICR. Both proteins are present in clusters [et al. reported that each dyad contains an average of 14 RyRs, grouped into a number of “super-clusters” [2+ spark, the fundamental unit of Ca2+ release in cardiomyocytes [2+ quarks [While the gross morphology of t-tubules in ventricular and atrial myocytes has been described in ever increasing detail, more attention has also been given to their nanometer scale organization and function. Of key importance for EC coupling are specialized junctions between the membranes of the t-tubules and sarcoplasmic reticulum (SR) known as ads Fig. . Within ic cleft . As the clusters  with an clusters . In recelusters” . Close lmyocytes . Opening+ quarks .Fig. 2T-2+ is recycled into the SR by the action of SERCA and extruded from the cell by the Na+-Ca2+ exchanger in the cell membrane. Like LTCCs, NCX is expressed at higher density in the t-tubules than in the surface sarcolemma, and along both transverse and longitudinal tubules [2+ removal following a Ca2+ spark [2+ influx which may trigger SR Ca2+ release from nearby RyRs [+ levels and, since the NCX is electrogenic, by depolarized membrane potentials.During relaxation of the cell, released Ca tubules , 29. Alt tubules , 30–32. 2+ spark . Howeverrby RyRs –37. Such2+ from nearby LTCCs or NCX, RyR Ca2+ release can be elicited by Ca2+ diffusion from neighboring dyads. In its most dramatic form, spontaneous Ca2+ waves of CICR can be observed to propagate across entire cardiomyocytes. However, in cells with low t-tubule density, smaller wave-like patterns of Ca2+ release also occur during the action potential, as Ca2+ diffuses from intact dyads into the gaps between them where non-dyadic or “orphaned” RyRs are present [2+ release across the cardiomyocyte, and thus the kinetics of the rising phase of the Ca2+ transient.Even in the absence of trigger Ca present –43 .A–C). Interestingly, t-tubule reorganization appears to not be restricted to left ventricular myocytes during heart failure, as similar findings have been reported in the right ventricle [Further evidence of t-tubule malleability has come from studies of cardiac pathology. A large body of evidence from a number of research groups , 49–57 hentricle , 64 and entricle , 65, 66.entricle , 16. Thu2+ release in failing cardiomyocytes  [2+ transient has been coupled to the slowed and decreased amplitude of contraction that is a hallmark of the failing heart [2+ release, including dispersion of RyR clusters which slows Ca2+ sparks [2+ release by re-shaping the action potential [+ levels occurs which, via NCX, leads to gain of SR Ca2+ content and release. Removal of NCX along with t-tubules diminishes this response [As in developing cells, a low density of poorly organized t-tubules promotes de-synchronized and protracted Cates Fig. a–c [38,ig. a–c) , 43. Thing heart , 68. Indng heart . Importa+ sparks . Loss ofotential –72. Furtresponse .2+ being extruded by NCX. For reasons which are not entirely clear, Ca2+ sparks occur almost exclusively at intact dyads where t-tubules are present [2+ spark generation at orphaned RyRs [2+ spark, DAD generation will be minimized since NCX is not located nearby [2+ wave, fewer nearby NCX proteins in t-tubules will mean that less Ca2+ is pulled away from the developing wave front, making its propagation more likely. T-tubule reorganization during heart failure may have similarly complex and opposing effects on the generation of early afterdepolarizations (EADs). Loss of Ca2+ and NCX currents along with t-tubules is expected to shorten the action potential, making phase 2 EADs less likely but phase 3 EADs more likely [In addition to negative effects of t-tubule reorganization on contractility in the failing heart, altered t-tubule structure is believed to have rather complex consequences for arrhythmia generation (reviewed in ). Delaye present , and sevned RyRs , 75. Furd nearby , 76. On e likely , 76. ShoThe striking similarity between t-tubule structure in developing and failing cardiomyocytes has led to speculation that t-tubule disruption during heart failure may be linked to re-emergence of the fetal gene program in this disease . Indeed,Junctophilin-2 (JPH2) is a structural membrane protein that anchors the sarcolemma to the SR –83 , and w2+ homeostasis and arrhythmia in heart failure, this protein is considered to be a promising therapeutic target. JPH2 overexpression has been observed to restore t-tubule structure and abnormal SR Ca2+ release in failing cardiomyocytes, attenuating disease progression [As there is now considerable evidence linking JPH2 downregulation to impaired Cagression , 97•. A gression . Howevergression , suggestgression , or otheThe membrane scaffolding protein BIN1, or amphiphysin-2, plays an important role in dyadic assembly and maintenance –101. TheConsistent with a key role of BIN1 in forming and sustaining dyads, genetic knockout was observed to be embryonically lethal, and cardiomyocyte-specific deletion to promote dilated cardiomyopathy . Downreget al., have suggested that t-tubule dismantling includes a less folded t-tubule membrane which increases arrhythmia susceptibility by allowing augmented ionic diffusion within the t-tubule lumen [The consequences of BIN1 loss during heart failure are proposed to include macroscale loss of t-tubule membrane and functional dyads, with expected negative consequences for EC coupling efficiency. However, nanometer scale alterations may also be critical. Hong le lumen •. FurtheGiven the role of BIN1 in organizing t-tubule membrane folds and dyads it may be a potential target for future therapy. While existing data suggest that increasing BIN1 levels in heart failure might simultaneously augment contractility and attenuate arrhythmogenesis, such studies have not yet been conducted. However, given what appears to be rather complex interaction between BIN1 and a variety of lipid and protein partners, therapeutic manipulation of this system may not be straight forward.1-receptor blockers [healthy hearts promoted t-tubule loss [Accumulating evidence supports a key role of workload as a dynamic regulator of t-tubular structure (reviewed in ). While blockers , 86, 114blockers . Interesule loss . Thus, tule loss .Workload is of course a rather nonspecific term, which may encompass many aspects of cardiac function. In an effort to more specifically identify signals which relate cardiac workload to t-tubular remodeling, we recently examined the role of ventricular wall stress •. We fouet al., Cardiovasc Res [Workload-dependent regulation of t-tubules has important therapeutic implications. As noted above, this mechanism has already been linked to the benefits of existing load-reducing drugs, but also likely contributes to the benefits of therapies such as ventricular assist devices . Quantitvasc Res ).2+ homeostasis, contractility, and arrhythmic potential. While loss and disorganization of t-tubules is now understood to be an important cellular-level contributor to heart failure, the malleability of this system may also be harnessed for therapy. Potential therapeutic targets include JPH2 and BIN1, which are critical for dyad formation and maintenance, and are downregulated in the failing heart. As recent data indicate that expression of these dyadic stabilizers is at least in part controlled by workload, future therapies may act to more effectively unload the failing heart and/or disrupt mechanotransduction.The above discussion has highlighted the impressive plasticity of cardiomyocyte t-tubular structure, and its central importance in regulating cellular Ca


Computer-assisted sperm analysis (CASA) parameters and penetration into viscous media were used to assess functional effects.Semen donors and patients were recruited in accordance with local ethics approval (13/ES/0091) from the East of Scotland Research Ethics Service REC1. Activities of CatSper and KSper were assessed by patch clamp electrophysiology. Sperm [Ca+ conductance or current reversal potential. hFF and progesterone  stimulated similar biphasic [Ca2+]i signals both in sperm populations and single cells. At a high hFF concentration (10%), the sustained (plateau) component of the [Ca2+]i signal was consistently greater than that induced by progesterone alone. In single cell recordings, 1% hFF-induced [Ca2+]i oscillations similarly to progesterone but with 10% hFF generation of [Ca2+]i oscillations was suppressed. After treatment to ‘strip’ lipid-derived mediators, hFF failed to significantly stimulate CatSper currents but induced small [Ca2+]i responses that were greater than those induced by the equivalent concentration of progesterone after stripping. Similar [Ca2+]i responses were observed when sperm pretreated with 3 μM progesterone (to desensitize progesterone responses) were stimulated with hFF or stripped hFF. hFF stimulated viscous media penetration and was more effective than the equivalent does of progesterone.hFF and progesterone significantly potentiated CatSper currents. Under quasi-physiological conditions, hFF (up to 50%) failed to alter membrane KN/A.in vitro study. Caution must be taken when extrapolating these results in vivo.This was an 2+]i signal and modulate the activation of CatSper. Simple in vitro experiments performed out of the context of the complex in vivo environment need to be interpreted with caution.This study directly demonstrates that hFF activates CatSper and establishes that the biologically important effects of hFF reflect, at least in part, action on this channel, primarily via progesterone. However, these experiments also demonstrate that other components of hFF both contribute to the [CaFunding was provided by MRC   and University of Abertay . Additional funding was provided by TENOVUS SCOTLAND (S.M.D.S.), Chief Scientist Office/NHS Research Scotland (S.M.D.S). C.L.R.B. is EIC of MHR and Chair of the WHO ESG on Diagnosis of Male infertility. The remaining authors have no conlicts of interest. Membrane potential was held at −92 mV between ramps (rev—to estimate resting Vm) and membrane conductance (Gm) were calculated as previously described . Membrane potential was held at 0 mV between ramps. Divalent (Ba2+) CatSper tail currents  with 488 nm (excitation) and 520 nm (emission) filters. After a control period, (30–60 s) stimuli were added using a multichannel pipetter as described by 2+]i responses to hFF and equivalent [P4] aliquots from the same fluo-4 loaded sample, tests were performed in parallel. Emission was background-corrected and normalized to the control (pre-stimulus) amplitude. To compare duration of P4 and hFF-induced transients, the half-duration (midpoint of the rising phase to midpoint of decay) was calculated. In desensitization experiments, cells were first stimulation with 3 μM P4 then, after a delay of 300 s, a second ‘test’ stimulus was applied in the continued presence of the desensitizing P4.Following swim-up, sperm (≈6 million/ml) were capacitated (3–5 h) then loaded with 4.5 μM Fluo-4 for 30 min, washed twice (700 g for 10 min) and resuspended in sEBSS.  by 98.6 ± 0.13% . Spermatozoa were exposed first to 1% charcoal-stripped hFF (ShFF) then to 1% hFF from the same sample incubated similarly but without dextran-coated charcoal. ShFF failed to stimulate ICatSper, , but subsequent application of hFF potentiated both inward and outward currents amplitude . Similarly, when tail currents were used to assess CatSper activation, hFF but not ShFF shifted voltage sensitivity to less positive potentials . The concentration of P4 present in 1% ShFF is 2–3 nM, which has been reported to increase CatSper currents  and also when progesterone was first dissolved in a mixture of 1% standard bath solution  and 99% Cs+ saline (to mimic ionic conditions in ShFF experiments), superfusion of sperm with 2 nM P4 significantly increased both outward and inward currents  and mean inward and outward currents were increased, but this effect was not significant (Table P > 0.1). Examination of [P4] concentrations showed that detectable effects of ShFF occurred only with hFF samples where the [P4] was unusually high .hFF contains, in addition to P4, prostaglandins  under quasi-physiological conditions . hFF did not alter resting membrane potential or outward membrane conductance indicating that hFF did not modulate/suppress Kons Fig. . Stimulaons Fig. .2+]i consisting of a transient followed by a plateau  and by an equivalent concentration of P4 alone . Analysis of these data pairs showed that at low concentrations of hFF (0.01–1%) the amplitudes of signals induced by hFF and P4 were similar  was consistently greater than that induced by an equivalent concentration of P4 . In cells stimulated with 10% hFF the [Ca2+]i transient also appeared longer than in cells from the same batch of Fluo-4 loaded sperm cells stimulated with an equivalent concentration of P4 .In agreement with previous reports hFF, similarly to P4, caused a dose-dependent, biphasic elevation of [Ca2+]i at the posterior head/neck showed transient responses in the vast majority of cells exposed to hFF, which resembled those induced by P4 alone . However, whereas 3 μM P4 was similarly effective .Similarly to population measurements, single cell imaging of i signals were similarly affected. Surprisingly, [Ca2+]i responses were always detected in cell populations stimulated with 1% ShFF, with the [Ca2+]i transient amplitude being 36.8 ± 1.8% of that in the parallel control (1% hFF) experiments . In 28 experiments where parallel recordings were carried out with ShFF and [P4] equivalent to that in ShFF, [Ca2+]i transient amplitudes were similar (P = 0.14). However, the subsequent [Ca2+]i ‘plateau’ was significantly greater with ShFF . After 5–10 min exposure to 1% ShFF or equivalent [P4], oscillations developed in ~20% of cells  which was significantly greater (P = 2.8×10−5) than the response to a second stimulation with 3 μM P4; Fig. Component(s) of hFF not removed by charcoal stripping contribute significantly to late/sustained components of hFF-induced  was small and not significant . Analysis of the kinematics  indicated this effect of hFF was primarily due to increased curvilinear velocity .To assess functional effects of hFF on motility, we measured hyperactivation and penetration into viscous medium. Both hFF (1 and 10%) and equivalent [P4] significantly stimulated penetration , depolarizing the membrane potential and potentially augmenting activation of CatSper. We could detect no effect of hFF on conductance or resting Vm even with 50% hFF  and their actions might be expected to result in a response to hFF smaller than that of an equivalent [P4] .[Ca2+]i responses to P4 resemble population responses , removing almost 99% of P4. In fluorimetric experiments the [Cater Fig. b. Furthei signal c–f. Thes2+]i transient induced by 1% ShFF appeared to be primarily a response to residual P4 (see above), when we investigated effects on patch-clamped sperm we observed no stimulation of CatSper currents, suggesting that other components of hFF modulate the response to P4. Two factors should be taken into account in interpreting these data. First, P4 applied by perfusion binds to the plastic perfusion tubing , thus comparison with fluorimetric [Ca2+]i assessment, where direct addition of ShFF to the well induced a significant [Ca2+]i response  failed to rescue stimulation of CatSper currents by ShFF FF Table ), when dnts Fig. a,c is ap2+ strongly stimulate hyperactivation    and University of Abertay . Additional funding was provided by Tenovus Scotland (S.M.D.S.), Chief Scientist Office/NHS Research Scotland (S.M.D.S).C.L.R.B. is EIC of MHR and Chair of the WHO ESG on Diagnosis of Male infertility. The remaining authors have no conflict of interest.Supplementary DataClick here for additional data file.Supplementary DataClick here for additional data file.Supplementary DataClick here for additional data file.Supplementary DataClick here for additional data file.Supplementary DataClick here for additional data file.Supplementary DataClick here for additional data file.


In fibers stretched beyond overlap, the decay of Ca2+ as measured by a change in fluo-3 fluorescence is significantly slower than the intensity decay of the meridional 1/38.5 nm−1 reflection of Tn; this is not simply explained by considering only the Ca2+ binding properties of Tn alone (Matsuo et al., 2+ binding properties of Tn in the context of the thin filament with and without cycling crossbridges. Calculations based on the model predict that the transient of Ca2+-bound Tn correlates with either the fluo-3 time course in muscle with overlapping thin and thick filaments or the intensity of the meridional 1/38.5 nm−1 reflection in overstretched muscle. Hence, cycling crossbridges delay the dissociation of Ca2+ from Tn. Correlation with the fluo-3 fluorescence change is not causal given that the transient of Ca2+-bound Tn depends on sarcomere length, whereas the fluo-3 fluorescence change does not. Transient positions of tropomyosin calculated from the time course of Ca2+-bound Tn are in reasonable agreement with the transient of measured perturbations of the Tn repeat in overlap and non-overlap muscle preparations.To explain disparate decay rates of cytosolic Ca Studies using both 45Ca2+ and fluorescence change techniques with native and covalently modified preparations, respectively, consistently demonstrate that the Ca2+ affinity of regulated actin is substantially lower than the Ca2+ affinity of isolated Tn , central (C), and myosin dependent (M) positions. Tn in association with Tm can interact with actin only when Tm is in position B , 2 (singly bound), or 3 (doubly bound). Affinity for actin is progressively reduced as i increases. Based on conservation of the mass for two partially overlapping subsystems (Tm and Tn), B + C + M = 1 for Tm and B + T = 1 for Tn.Coupled and uncoupled states of Tn are designated y Figure . CalciumM can arise by either rigor or cycling crossbridges, but cooperativity derives solely from cycling crossbridges, as seen in the records of Ca2+ binding measurements. With overlapping thin and thick filaments and ATP, M is a state in constant flux (steady-state) rather than at equilibrium; this is readily observed by Ca2+-dependent in vitro sliding of regulated actin filaments. Steady-state cooperativity is achieved if crossbridge turn-over generates additional opportunities (second chances) for M formation  acts on the C-M transition  performs adequately, any logistic function operating on C can be compatible with our model.In the system we describe,  et al., dM/dt=K0′M as a function of the population of strong binding myosin at any moment of steady-state is expressed by M formation is the product k−0. Ensemble size, n, expresses the number of Tm subunits acting in concert to form a ternary complex as described above. The orchestrating event could be a lateral stretch imposed on contiguous Tm subunits by an axial force acting on the thin filament  may have the following interpretations. The equilibrium potential of  may haveC-M are spontaneous processes governed by simple mass action , the relative abundances or probabilities of the other six states , α, and n that we hold constant  are varied to fit transient data. Slow and fast dissociation rates of Ca2+ from Tn  is calculated as a function of constant concentrations of Ca2+. With cycling crossbridges, the mathematical solution is a cooperative function of Ca2+ , respectively. Both solutions predict simple mass action (non-cooperative) calcium binding  by adjusting the rate constants  release Ca2+ faster than Ca2+-bound T states .The dissociation of Caes B2 + B release 2+-bound Tn and fluo-3 data differs dramatically in muscle fibers stretched beyond overlap. To simulate no overlap, we nullify k−0 in Equation (1), but otherwise preserve the rates established for the overlap condition . Previous results from amphibian fast skeletal muscle using inhibitors of SR Ca2+-ATPase have shown that the decay and not the peak in free Ca2+ depends on active Ca2+ transport  has the distinction of being a more general form of the Hill equation (Zot et al., M, is proportional to the fraction of maximum tension. This simplification is consistent with the proposal that tension bearing crossbridges are excluded from thin filament states C and B (Lehman et al., A second assumption of the model is that activated state, 2+ binding to the regulatory sites of TnC is uncoupled from the process of activation when the complex of Tm-Tn is in positions C and M. Consequently, we model the C position as favored at equilibrium. Tm occupies either C or B positions in reconstructions of skeletal and cardiac filaments, respectively, but the complex of Tm-Tn is positioned exclusively in C with Ca2+ present (Lehman et al., 2+ is required to release Tm-Tn from the B position. A possible, albeit more complicated, mechanism is for Ca2+ to have a second, independent action, namely, to perturb the equilibrium position of the Tm-Tn complex. This latter possibility is difficult to reconcile with an uncoupling mechanism.A third assumption of the model is that Ca2+ regulatory properties of Tn, we provide a satisfying explanation for the events of contraction arising from a transient of free Ca2+. The model presented here is consistent with previous experimental results showing non-cooperative Ca2+ binding to regulated actin in the presence or absence of rigor crossbridges and recapitulates the complex cooperative relationship between Ca2+-binding and force in the steady-state (Figure K1 alone is freely adjusted and K3 and K5 vary in a prescribed manner. A recent study shows that the model presented here can inform experiments that explain how a mutation in TnC alters the Ca2+ sensitivity of cardiac myofilaments associated with the hypertrophic state of the heart (Zot et al., 2+-insensitive mutant TnC on cooperative activation of skeletal muscle fibers (Zot et al., 2+-bound Tn, the model presented here is capable of guiding future experiments to uncover mechanisms by which mutations in excitation-contraction coupling lead to pathological conditions.Although, we do not address a specific myopathy, we suggest that results presented here can be extrapolated to mechanisms underlying disease. By employing a model that accounts for the CaEach of the authors contributed significantly to the conception and design of the work, drafting and revision of the manuscript, and final approval of the version to be published. Both authors agree to be accountable for all aspects of the work.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.


Here, an in silico drug screening and the following cell-based screening to measure the TRPV2 activation were carried out in HEK293 cells expressing TRPV2 using lead compounds (tranilast or SKF96365) and off-patent drug stocks. We identified 4 chemical compounds containing amino-benzoyl groups and 1 compound (lumin) containing an ethylquinolinium group as candidate TRPV2 inhibitors. Three of these compounds inhibited Ca2+ entry through both mouse and human TRPV2, with IC50 of less than 10 μM, but had no apparent effect on other members of TRP family such as TRPV1 and TRPC1. Particularly, lumin inhibited agonist-induced TRPV2 channel activity at a low dose. These compounds inhibited abnormally increased Ca2+ influx and prevented stretch-induced skeletal muscle damage in cultured myocytes from dystrophic hamsters (J2N-k). Further, they ameliorated cardiac dysfunction, and prevented disease progression in vivo in the same J2N-k hamsters developing dilated cardiomyopathy as well as muscular dystrophy. The identified compounds described here are available as experimental tools and represent potential treatments for patients with cardiomyopathy and muscular dystrophy.Transient receptor potential cation channel, subfamily V, member 2 (TRPV2) is a principal candidate for abnormal Ca A sub2+ concentration ([Ca2+]i) beneath the sarcolemma and within other cellular compartments has been reported in skeletal muscle fibres and cultured myotubes derived from Duchenne muscular dystrophy patients and mdx mice [2+]i in muscle fibre cells is regulated by multiple Ca2+-permeable channels, Ca2+ pumps, and transporters in the plasma membrane and sarcoplasmic reticulum, among which sarcolemmal Ca2+-permeable channels  and mechanosensitive, nonselective cation channels contribute to abnormal Ca2+ handling in dystrophic myocytes [2+ influx via the stretch-activated nonselective Ca2+ channel [2+ entry pathways whose activation results in perturbation of Ca2+ handling and subsequent muscular degeneration [2+]i and the observed 2-aminoethoxydiphenylborate (2-APB)-induced increase in [Ca2+]i i . Similar [Ca2+]i  in studi [Ca2+]i ) and in  [Ca2+]i . We obse [Ca2+]i , 19. Morneration . However2+]i under weak acidic conditions, was used to screen candidate compounds. Several TRPV2 inhibitors were identified using tranilast and SKF96365 . When the medium was replaced with high Ca2+ medium (5 mM) at neutral pH (7.4) containing TRPV agonist 2-APB, the increase in [Ca2+]i was very small  and non-transfected HEK293 cells were loaded with fura-2-AM and placed in low CaCll Figure . HoweverB Figure . The obsB Figure , indicat2+ response using a 96-well high-throughput plate-reader. Cells were placed in low CaCl2 (0.5 mM) medium, which was replaced with high CaCl2 medium (5 mM) with or without the TRPV agonist 2-APB and adjusted to various pH values. While no effects were observed at neutral extracellular pH, a marked increase in [Ca2+]i was detected under acidic conditions (pH < 7.0), concomitant with a pH drop from 7.0 to 6.5 , stretch-activated channels (streptomycin), and voltage-gated Ca2+ channels (diltiazem) at concentrations up to 100 μM did not inhibit the 2-APB-induced increase in [Ca2+]i in HEK293 cells expressing mTRPV2 . Using tranilast and SKF96365 as lead compounds, 350 drug candidates were selected from the chemical library and classified as series A or B, respectively. The 350 drug candidates were screened for the ability to inhibit Ca2+ entry through TRPV2 at a low concentration. A3, A48, A63, and B6   Figure . Other ti Figure .2+]i induced by a high concentration (5 mM) of extracellular CaCl2 was measured in CHO cells expressing hTRPV2 using a fluorescence ratio monitoring system. In this experiment, we used CHO cells instead of HEK293 cells, because the Ca2+ response induced by high (extracellular) Ca2+ is detected relatively easily in CHO cells. Ca2+ influx in response to CaCl2 exposure was observed in CHO cells expressing either mTRPV2 or hTRPV2 i in dystrophic myocytes from J2N-k, but not in normal myocytes from wild-type J2N-n hamsters  of the newly identified TRPV2 inhibitors effectively target hamster, mouse, and human TRPV2 isoforms.Perfusion with high extracellular Cas Figure , consist8 Figure . A 2-APBs Figure  and was s Figure . All TRPs Figure . These rTo assess the effects of the newly identified TRPV2 inhibitors on mechanical stretch-induced muscle degeneration, J2N-k myocytes were treated with a test agent for 30 min, after which cyclic stretch (20% elongation) was applied for 1 h. CK release was measured as an indicator of muscle damage. Treatment of myocytes with A3, A48, or A63 (30 μM), as well as tranilast (500 μM), reduced stretch-induced CK release from J2N-k myocytes by up to 70% Figure .in vivo, they were administered for 3 weeks to 9-week-old hamsters showing decreased cardiac function, after which echocardiography was performed  of lumin for the ion current was very similar to the IC50 for the Ca2+ response (<5 μM) under our experimental conditions. Lumin (15 μM) did not inhibit the L-type current  of control at 15 μM lumin. Lumin did not affect the increase in [Ca2+]i via TRPV1 or TRPC1 (unpublished observation). When lumin was administered to 9-week-old J2N-k hamsters for 3 weeks, the decrease in wall thickness was partially prevented, fibrotic area was reduced  was a reasonably effective way of measuring increases in [Ca2+]i mediated by TRPV2. Exposure to weak acidic medium in the presence of 2-APB also dramatically activated the TRPV2 current measured under voltage-clamped conditions using the whole cell patch clamp technique. As previously reported by Chung -3-methylbenzoic acid (A48) (S770000), and diltiazem were purchased from Sigma-Aldrich . Tranilast was obtained from Kissei Pharmaceutical . SKF96365 and ionomycin were obtained from Calbiochem . Ruthenium red was obtained from Wako Pure Chemical Industries Ltd. . 2-[(2E)-3--2-cyanoprop-2-enoylamino]benzoic acid (A3) (NATR020256) and 1-{2-[3-((1E)-2--2-azavinyl)indolyl]ethoxy}-3-methoxybenzene (B6) (NATR030155) were purchased from Vitas-M Laboratory, Ltd. . (2E)-3--N-(2-nitrophenyl)prop-2-enamide (A63) (OSSK-422912), 4,5-dimethoxy-2-(phenylureido)benzoic acid (A65) (OSSK-423626), and 1-indolizin-2-yl-4-methoxybenzene (B33) (OSSK-004571) were obtained from Princeton University. 4,4′-[3-[2-[1-ethyl-4(1H)-quinolinylidene]ethylidene]-1-propene-1,3-diyl]bis(1-ethylquinolinium) diiodide (lumin) was kindly provided by Hayashibara Biochemical Laboratories . Fura-2-acetoxymethylester (AM) was obtained from Dojindo Laboratories . All chemicals used in the study were of the highest available purity.Plasmid constructs of TRPV2, TRPV1, and transient receptor potential cation channel, subfamily C, member 1 (TRPC1) were generated using a PCR-based strategy from full-length cDNA sequences of mTRPV2 and hTRPV2, hTRPV1, and hTRPC1 cloned into the pIRES expression vector .2. After enrichment of myoblasts by preplating over several rounds, cells were seeded in culture dishes. Two days after seeding, the culture medium was replaced with Dulbecco's modified Eagle's medium (DMEM) containing 2% horse serum to induce myotube formation. Myocytes were analysed 2–4 days after the start of fusion.Myocytes were cultured from enzymatically dissociated muscles of J2N-n and J2N-k hamsters as previously described . Briefly3 and supplemented with 7.5% (v/v) foetal calf serum) and transfected with cDNA using Lipofectamine® 2000 (Invitrogen). Stable clones were isolated by puromycin selection .HEK293 and CHO cells were maintained in DMEM . Fura-2-AM fluorescence was measured using an Aquacosmos fluorescence image processor . The time-course data are presented as the ratio of fluorescence at an excitation wavelength of 340 nm to that at 380 nm. The increase in the fluorescence ratio induced by the TRPV2 agonist 2-APB or high extracellular Ca2+ (ΔR) was normalized to the initial ratio (R0) before stimulation. The fluorescence relative ratio (ΔR/R0) was plotted as the summary data in the fluorescence imaging analysis.[Caescribed . Briefly2. For stimulation with 2-APB (0.5 mM), the pH was adjusted to 6.5. All Ca2+ measurements were carried out at room temperature. Because the experimental conditions  for the microplate reader analysis were different from those used in the fluorescence imaging analysis, these experiments produced different 340/380 nm fluorescence ratios. For simplicity, the results are presented as normalized values (% maximum) from the microplate reader experiment.For screening, cells were plated in 96-well plates and the fura-2-AM fluorescence ratio at excitation wavelengths of 340 nm and 380 nm was monitored using a GENios Pro microplate reader  or POLARstar Omega . Cells were stimulated in BSS containing 5 mM CaCl2, 10 mM BAPTA, 10 mM HEPES, and 3 mM MgATP (adjusted to pH 7.2 with KOH). The extracellular solution contained 145 mM NaCl, 5 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 10 mM HEPES, and 10 mM glucose (pH was adjusted to 6.5 with NaOH). Because TRPV2 was reported to desensitize in the presence of extracellular Ca2+ [2+-free medium containing 1 mM EGTA to prevent desensitization. In the experiments confirming the pH dependence of the response, the pH was adjusted to 7.4. In the whole-cell recordings, the membrane potential of the cell was held at −60 mV, while voltage ramps of 400 ms from −100 mV to +60 mV were applied every 2 seconds. Currents through the electrode were recorded at 5 kHz and filtered at 2 kHz by an Axopatch 200B amplifier . The junctional potential was −11.7 mV. We did not compensate for the junctional potential in the calculations.TRPV2 currents were recorded under voltage-clamp conditions by the whole-cell patch-clamp technique . Patch plar Ca2+ , we used2+ currents, human Cav1.2/β2/α2δ1 calcium channel expressing CHO cells were purchased from ChanTest . The holding potential was held at −80 mV, while depolarizing pulses of 0 mV for 200 milliseconds were applied every 10 seconds. The composition of the extracellular solution was as follows: 145 mM NaCl, 4 mM KCl, 10 mM CaCl2, 10 mM HEPES, and 10 mM glucose (adjusted to pH 7.4 with NaOH). The composition of the intracellular solution was as follows: 112 mM CsCl, 27 mM CsF, 2 mM NaCl, 8.2 mM EGTA, 10 mM HEPES, and 4 mM MgATP (adjusted to pH 7.2 with CsOH). The data were analysed and plotted with Clampfit 9 . All experiments were performed at temperatures between 22° C and 25° C.For the measurement of L-type CaMechanical stretch was applied to myocytes using a chamber described in a previous study . A silicThe Namiki 2004 chemical library  was used to select 350 compounds via ligand-based virtual screening . The prein vitro colorimetric assay kit  according to the manufacturer's protocol The cTN-I level in the serum was measured, while quantitative immunoblotting and immunocytochemistry were performed as previously described [t-test and one-way ANOVA followed by Dunnett's test were used to analyse the data. Values of P < 0.05 (indicated as asterisks in the figures) were considered statistically significant. All cellular measurements were repeated at least 5 times. Representative data are shown.Creatine phosphokinase (CK) activity in the medium was determined using an escribed , 39. Pro