Recently, a transgenic rabbit with rhodopsin Pro 347 Leu mutation was generated as a model of retinitis pigmentosa (RP), which is characterized by a gradual loss of vision due to photoreceptor degeneration. The purpose of the current study is to noninvasively visualize and assess time-dependent changes in the retinal structures of a rabbit model of retinal degeneration by using speckle noise-reduced spectral-domain optical coherence tomography (SD-OCT).Wild type (WT) and RP rabbits (aged 4–20 weeks) were investigated using SD-OCT. The total retinal thickness in RP rabbits decreased with age. The thickness of the outer nuclear layer (ONL) and between the external limiting membrane and Bruch's membrane (ELM–BM) were reduced in RP rabbits around the visual streak, compared to WT rabbits even at 4 weeks of age, and the differences increased with age. However, inner nuclear layer (INL) thickness in RP rabbits did not differ from that of WT during the observation period. The ganglion cell complex (GCC) thickness in RP rabbits increased near the optic nerve head but not around the visual streak in the later stages of the observation period. Hyper-reflective change was widely observed in the inner segments (IS) and outer segments (OS) of the photoreceptors in the OCT images of RP rabbits. Ultrastructural findings in RP retinas included the appearance of small rhodopsin-containing vesicles scattered in the extracellular space around the photoreceptors.In vivo imaging of RP rabbit retinas by using SD-OCT is a powerful method for characterizing disease dynamics and for assessing the therapeutic effects of experimental interventions.In the current study, SD-OCT provided the pattern of photoreceptor degeneration in RP rabbits and the longitudinal changes in each retinal layer through the evaluation of identical areas over time. The time-dependent changes in the retinal structure of RP rabbits showed regional and time-stage variations.  Retinitis pigmentosa (RP) is an inherited retinal disorder characterized by a progressive loss of visual function due to degeneration of rod and cone photoreceptors and eventual atrophy of the entire retina in vivo detection and monitoring of changes in retinal architecture without sacrificing animals.Optical coherence tomography (OCT) devices allow non-invasive detection of retinal architecture, including quantitative measurements of retinal thickness and longitudinal observation of the retinal architecture In mouse models of retinal degeneration, Fischer The purpose of this study was to visualize the time-dependent changes in photoreceptors, elucidate the pattern of changes in each retinal layer around the visual streak in identical eyes of RP rabbits by using SD-OCT, and assess the visual functions by electroretinography (ERG).We first investigated whether the retinal structures of WT rabbits could be clearly visualized using SD-OCT. Vertical OCT images, which passed through the center of the ONH , permittNext, we examined a 20-week-old RP rabbit that expressed mutated rhodopsin . The outAs observations revealed that photoreceptor damage was severe around the visual streak, we were encouraged to investigate the time-dependent changes in the photoreceptors of identical RP rabbits beneath the visual streak with SD-OCT and compared them with those of the WT rabbits . At 4 weIn the current SD-OCT study, there were additional findings in the photoreceptor layers. In the sections of WT rabbits, the reflectivity of IS and OS was low compared to that of the ELM and IS/OS lines. In contrast, the IS and OS were highly reflective in RP rabbits, and almost equivalent to the ELM and IS/OS lines throughout the study ages .To compare the SD-OCT data with those from the histological examination, histological sections of the age-matched RP and WT rabbits were prepared . The numP = 0.010, unpaired t-test) as early as 4 weeks. The a-wave amplitude was reduced with RP rabbit aged .In the SD-OCT examinations, the retinal thickness in the RP rabbits appeared to decrease with age. Therefore, we quantitatively measured the mean total retinal thickness around the visual streak in the WT and RP rabbits  and S2. OCT examination showed that the photoreceptors were most severely damaged at the visual streak, approximately 3 mm ventral to the ONH We first evaluated the thickness of the ONL where the nuclei of photoreceptors are located . In WT rP = 0.037, unpaired t-test). Areas that exhibited a difference in ONL thickness between WT and RP rabbits expanded with age. At 20 weeks, the ONL thickness in RP rabbits was significantly smaller than that of WT rabbits in each of the 7 areas examined , ganglion cell layer (somas of ganglion cells), and IPL. To determine the influence of photoreceptor degeneration on the inner retina, GCC thickness was measured  and 5D. In summary, the decrease in the ONL and ELM–BM thickness in RP rabbits was first detected in the areas approximately 3.0 mm ventral to the lower edge of the ONH . Thinning of the IS and OS (measured as the ELM–BM thickness) followed thinning of the ONL. In contrast, the INL thickness in RP rabbits did not change throughout the observational period of 4 to 20 weeks. The GCC thickness in RP rabbits increased in areas away from the visual streaks but close to the ONH in the later phase of observation .In this study, we examined time-dependent changes in photoreceptor degeneration in identical RP rabbits, and compared the pattern of changes in individual retinal layers between WT and RP rabbits for the first time by using SD-OCT. In RP rabbits, we observed regional differences in the degree of photoreceptor loss. That is, the ONL (ONL: the somas of photoreceptors) in RP rabbits was thinnest beneath the visual streak, where the densities of rod and cone photoreceptors were the highest in WT rabbits, and the photoreceptors of RP rabbits were relatively preserved in the area near the ONH. The current observations by using SD-OCT revealed longitudinal changes in the RP rabbit retina that were fairly consistent with a previous histological study of the RP rabbits To elucidate the unique “highly reflective IS and OS" feature of the outer photoreceptor layer during photoreceptor degeneration in RP rabbits, an electron microscopy study was conducted on 4- or 20-week-old RP rabbits. We detected vesicles around the photoreceptors and loss of most of the IS and OS. We speculate that these destructive structures in RP rabbits cause the hyper-reflectivity seen in the outer photoreceptor layers (between ELM and BM) on SD-OCT images .The vesiAleman et al. reported the following disease sequence in human and murine RP caused by mutation of the rhodopsin gene: ONL diminution with INL thickening, amalgamation of residual ONL with the thickened INL, and progressive retinal remodeling with eventual thinning seen in OCT This study has some limitations. The area imaged with SD-OCT was quite restricted such that the degeneration in RP retinas obtained with SD-OCT did not always correlate with the total retinal function. Furthermore, OCT or ERG could not be performed on rabbits younger than 4 weeks as their eyelids had not yet opened.in vivo time-dependent changes in the retinal structures were seen layer-by-layer in RP rabbits by using SD-OCT. These changes in the retinal structure had regional and temporal variations not only in the outer retina but also in the inner retina of RP rabbits. This study demonstrates that in vivo imaging with SD-OCT can facilitate the characterization of morphological disease dynamics and serve as a powerful tool for developing new treatments, such as gene therapy, intraocular devices, and neuroprotective treatments, in rabbit models of RP.In conclusion, despite these short comings, This study was conducted in accordance with the Association Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. All the protocols were approved by the Institutional Review Board of the Kyoto University Graduate School of Medicine (MedKyo11229).New Zealand White rabbits  and RP rabbits with rhodopsin P347L mutation  Rabbit eyes were fixed overnight in a mixture of 10% neutral buffered formalin and 2.5% glutaraldehyde and then transferred to 10% neutral buffered formalin. The tissues were trimmed, embedded in paraffin, sectioned vertically through the optic nerve (superior-inferior), and stained with hematoxylin and eosin. The retina beneath the visual streak was examined and compared between 10- and 20-week-old WT and RP rabbits.ERG was performed to assess the visual function of WT and RP rabbits at 4, 6, 10, and 20 weeks. Pupils were dilated with tropicamide (0.5%) and phenylephrine (0.5%) eye drops. Rabbits were dark-adapted for more than 60 min before anesthetization with an intramuscular injection of ketamine (25 mg/kg) and xylazine (2 mg/kg). ERG was recorded using a gold loop corneal electrode with a light-emitting diode . A reference electrode was placed in the mouth, and a ground electrode was attached to the ear. Stimuli were produced with a light-emitting diode stimulator (Mayo Corp.). The ERG response signals were amplified, digitized at 10 kHz with a band-pass filter of 0.3 to 500 Hz and analyzed . Two steps of stimulus intensities  were used for evaluating rod and mixed cone and rod responses. The b-wave amplitude of the rod response and the a-wave amplitude of the mixed cone and rod response were analyzed.Multiline OCT , which was customized based on a Spectralis HRA+OCT Multiline OCT uses an 870-nm super-luminescent diode as a light source. The scan rate of the SD-OCT was 47,000 A-scans per second, with an axial resolution of ∼7 µm.After ERG recording, rabbits were placed on a platform such that the visual streaks, which were approximately 3 mm ventral to the ONH, were located at the center of the image. The SD-OCT machine used in this study was To measure the total retinal thickness at the visual streak, a volume scan image was obtained . The linTo measure and assess the thickness of each retinal layer, vertical OCT images, which passed through the center of the ONH and included the visual streak, were obtained by averaging 100 B-scans. To measure the thickness of the ONL, ELM–BM, INL, and GCC, the boundary lines between the OPL and ONL, ELM and BM, IPL and INL, and the vitreoretinal interface and IPL were manually delineated in a masked fashion . The thiThe enucleated rabbit eyes were fixed in the same manner as the H&E stain. The eyes were subsequently fixed in 1% osmium tetroxide for 90 min. The retina was dehydrated through a graded series of ethanol (50–100%), cleared in propylene oxide, and embedded in epoxy resin. Ultrathin sections were cut by using an ultramicrotome and stained with uranyl acetate and lead citrate. For ultrastructural immunohistochemistry, the enucleated RP rabbit eyes were fixed in 4% paraformaldehyde and 0.05% glutaraldehyde for 4 h. The fixed retina was cut with a microslicer  into sections with a thickness of 65 µm. The sections were incubated with mouse monoclonal anti-rhodopsin antibody (Ret-P1 (sc-57433), Santa Cruz, California, U.S.A.) and subsequently, with gold-conjugated Fab fragment of goat anti-mouse IgG , followed by silver enhancement . The stained sections were observed by transmission electron microscopy .t-test by using PASW Statistics version 18.0 . The level of statistical significance was set at P<0.05.Data from WT and RP rabbits were analyzed with an unpaired Figure S1Ultrastructure of photoreceptors in 20-week-old WT and RP rabbits.  Ultrastructure of photoreceptors in 20-week-old WT rabbits. The inner segments of photoreceptors (IS) and the outer segments of photoreceptors (OS) were regular and dense.  Ultrastructural changes in 20-week-old RP rabbits. The IS and OS were mostly absent, and the residual IS and OS were less organized than those in WT rabbits. In the magnified image (D), many small vesicles (arrowheads) appeared to be cleaved from the IS into the extracellular space around the photoreceptors (arrows).(TIF)Click here for additional data file.Figure S2Measurement of mean total retinal thickness. (A) An infrared image on volume scan mode of SD-OCT. In the volume scan mode, the region ventral to the optic disc, including visual streak  was imaged. (B) One of the 19 horizontal OCT sections on volume scan mode. The lines of the vitreoretinal interface and the Bruch's membrane are manually delineated at each horizontal section (C). (D) The retinal thickness map constructed from the volume scan OCT images. Total retinal thickness was measured within the red circle shown (E). The diameter of the red circle was 1 mm, and the center was 3 mm ventral to the inferior edge of the ONH .(TIF)Click here for additional data file.Figure S3Measurement of the thickness of individual retinal layers. Four vertical OCT sections that pass through the center of the ONH and visual streak are shown. On each section, the boundary lines between each retinal layer were manually delineated. The ONL, ELM–BM, INL, and GCC thicknesses were evaluated in 0.5-mm segments as a function of the distance from the inferior optic disc margin up to 4.0 mm ventral to the inferior edge of the ONH. ONL, outer nuclear layer; ELM, external limiting membrane; BM, Bruch's membrane; INL, inner nuclear layer; and GCC, ganglion cell complex.(TIF)Click here for additional data file.


We hypothesize that there is a differential regulation of intracellular CaL channels was evaluated by patch clamp and Western blotting. The function of ryanodine-sensitive Ca2+ releases in response to caffeine were assessed by a laser confocal microscope. Our results indicated that simulated microgravity increased the functions of CaL channels and ryanodine-sensitive Ca2+ releases in cerebral VSMCs, whereas, simulated microgravity decreased the functions of CaL channels and ryanodine-sensitive Ca2+ releases in small mesenteric VSMCs. In addition, 3- or 7-day recovery after removal of suspension could restore the functions of CaL channels and ryanodine-sensitive Ca2+ releases to their control levels in cerebral and small mesenteric VSMCs, respectively.Sprague-Dawley rats were subjected to 28-day hindlimb unweighting to simulate microgravity. In addition, tail-suspended rats were submitted to a recovery period of 3 or 7 days after removal of suspension. The function of CaL channels and ryanodine-sensitive Ca2+ releases in cerebral and small mesenteric VSMCs may be responsible for the differential regulation of intracellular Ca2+, which leads to the altered autoregulation of cerebral vasculature and the inability to adequately elevate peripheral vascular resistance in postspaceflight orthostatic intolerance.The differential regulation of Ca Postspaceflight orthostatic intolerance has been regarded as one of the major adverse effects of microgravity exposure and there are still no effective countermeasures 2+ in vascular smooth muscle cells (VSMCs) is an important determinant for functional and structural adaptations in vasculature 2+ influx from the long-lasting voltage-dependent Ca2+  channels in plasma membrane and Ca2+ releases from the ryanodine receptors (RyRs) in sarcoplasmic reticulum (SR) are likely to play the essential roles in controlling intracellular Ca2+2+ influx by opening CaL channels. The increased concentration of intracellular Ca2+ ([Ca2+]i) subsequently activates RyRs and produces the transient local Ca2+ release events in micromolar concentrations (Ca2+ sparks) from SR, which in turn activate nearby Ca2+-activated K+ (KCa) channels in plasma membrane, leading to membrane hyperpolarization, inhibition of CaL channels, and thereby favoring vasodilation by reducing the Ca2+ influx L channels in plasma membrane and RyRs in SR are important mediators to control arterial excitation-contraction coupling and subsequent structural remodeling by handling intracellular Ca2+. It has been demonstrated that hypertension L channels or RyRs.Intracellular Ca2+ in rat cerebral VSMCs L channels 2+ in rat small mesenteric VSMCs by reducing the function of ryanodine-sensitive Ca2+ releases associated with the downregulation of RyR2 mRNA and protein expression L channels in rat small mesenteric VSMCs L channels in plasma membrane and ryanodine-sensitive Ca2+ releases from SR may account for the changes of intracellular Ca2+ in cerebral and small mesenteric VSMCs of simulated microgravity rats. Furthermore, these results also indicated that CaL channels and ryanodine-sensitive Ca2+ releases may play an important role in the differential adaptations of cerebral and small mesenteric arteries in simulated microgravity rats. However, it is not clear whether the alterations of CaL channels in plasma membrane are accompanied by the changes of ryanodine-sensitive Ca2+ releases from SR in cerebral or small mesenteric VSMCs of simulated microgravity rats, respectively. In addition, it is not clear whether the function of CaL channels and ryanodine-sensitive Ca2+ releases could recover after removal of suspension. The purpose of the present work was 1) to investigate the function of CaL channels by comparing the whole-cell currents, protein expressions of α1C-subunit in cerebral and small mesenteric VSMCs isolated from control rats, simulated microgravity rats, and rats after removal of suspension, and 2) to assess the functions of ryanodine-sensitive Ca2+ releases by measurements of intracellular Ca2+ in response to caffeine in cerebral and mesenteric VSMCs isolated from the same groups as described above. Taken together, the present study provided initial evidences that there is a differential regulation and recovery in the functions of CaL channels and ryanodine-sensitive Ca2+ releases of cerebral and small mesenteric VSMCs from simulated microgravity rats, respectively, which may provide a novel mechanism for cerebral and small mesenteric arterial adaptations during microgravity.Our previous work reported that 28-day simulated microgravity increased the concentration of intracellular Cathe Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health , with approval from Committee on the Ethics of Animal Experiments of the University of Fourth Military Medical University (Permit Number: 10001). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Unless otherwise stated, all chemicals and reagents used in this study were obtained from Sigma Chemical Company .All animal procedures described in this study were performed in adherence with Experiment I was designed to investigate the functions of CaL channels and ryanodine-sensitive Ca2+ releases in cerebral and small mesenteric VSMCs isolated from control rats, simulated microgravity rats, and tail-suspended rats submitted to a recovery period of 3 days after removal of suspension. Experiment II was designed to investigate the function of CaL channels in small mesenteric VSMCs isolated from simultaneous control rats and tail-suspended rats submitted to a recovery period of 7 days after removal of suspension. In Experiment I, a total of 90 male Sprague-Dawley rats, 7–9 weeks of age and weighing ∼200 g, were randomly assigned into 3 groups (n = 30/group): 28-day simultaneous control (CON-28 d), 28-day tail-suspension (SUS-28 d), and tail-suspended rats submitted to a recovery period of 3 days at the end of 28-day suspension (SUS+Rec-3 d). In Experiment II, 20 male Sprague-Dawley rats, 7–9 weeks of age and weighing ∼200 g, were randomly assigned into 2 groups (n = 10/group): 35-day simultaneous control (CON) and tail-suspended rats submitted to a recovery period of 7 days at the end of 28-day suspension (SUS+Rec-7 d). The SUS rats were maintained in an about −30° head-down tilt position with their hindlimbs unloaded and caged individually in a room maintained at 23°C on a 12∶12-h light-dark cycle. The CON rats and rats submitted to a recovery period were maintained in individual cages and were treated similarly except for the tail suspension. All the animals received standard lab chow and water ad libitum. Animals were anesthetized with pentobarbital sodium (50 mg/kg ip) and killed by exsanguinations via the abdominal aorta. The wet weight of left soleus and the length of left femur were measured to confirm the efficacy of deconditioning and monitor any effect on growth.The tail-suspended, hindlimb-unweighting rat model, which was described in detail previously 2, 0.42 Na2HPO4, 0.44 NaH2PO4, 4.2 NaHCO3, and 10 HEPES, equilibrated with 95% O2 and 5% CO2 at pH adjusted to 7.4 with NaOH. The cerebral arteries  and small mesenteric arteries with its branches were dissected and cut into 1–2 mm length. The cerebral arterial segments were digested for 18 min  at 37°C with the solution contained 4 mg/ml papain , 2 mg/ml dithioerythritol , 1 mg/ml bovien serum albumin (BSA), and 5 mM taurine in PSS. Vessel segments were then transferred to enzyme-free PSS containing 1 mg/ml BSA and 5 mM taurine at room temperature for 10 min and triturated with a flame-polished pipette to disperse VSMCs. Isolated VSMCs were suspended in Ca2+-free PSS containing 1 mg/ml BSA and 5 mM taurine and stored at 4°C for use within 8 h.Enzymatic isolation of single VSMC was carried out as previously described 2, 2 CaCl2, 10 HEPES, and 10 glucose, equilibrated with 95% O2 and 5% CO2 at pH 7.4 adjusted with NaOH. The pipette solution contained (in mM) 143 KCl, 1 CaCl2, 1 MgCl2, 3 EGTA, and 10 HEPES, equilibrated with 95% O2 and 5% CO2 at pH 7.2 titrated with KOH.Patch-clamp recordings were performed as previously described L channel currents were measured with the conventional voltage clamp configuration. Cell capacitance (Cm) and access resistance (Ra) were estimated from the capacitive current transient evoked by applying a 20-mV pulse for 40 ms from a holding potential of −60 mV to −40 mV. The cell was held at −40 mV and then stepped in 10-mV increments from −30 to +60 mV. Voltage steps were 250 ms in a duration and 2-s intervals were allowed between steps. Currents were filtered at 0.5 kHz and digitized at 4 kHz. Nonspecific membrane leakage and residual capacitive currents were subtracted using the P/4 protocol. Currents were sampled and averaged while the current amplitude was stabilized. Barium (Ba2+) was used rather than Ca2+ as the charge carrier to increase unitary currents and to minimize Ca2+-dependent run-down. Currents were normalized to Cm to obtain the current densities. To obtain the I–V curve of CaL, the current densities were plotted against the corresponding command potentials. Two kinds of external solutions were used, i.e., solution A and B. Solution A was used while making a gigaohm seal between the recording pipette and cell surface. It contained (in mM) 130 NaCl, 5.4 KCl, 1 MgCl2, 10 BaCl2, 10 HEPES, and 10 glucose, equilibrated with 95% O2 and 5% CO2 at pH 7.4 adjusted with NaOH. After a seal of 2 GΩ was obtained, the perfusion fluid was changed to solution B before current recording. It contained (in mM) 75 Tris-Cl, 50 BaCl2, 10 HEPES, and 10 glucose, equilibrated with 95% O2 and 5% CO2 at pH 7.4 titrated with Tris base. The pipette solution contained 150 CsCl, 1 MgCl2, 10 EGTA, 5 HEPES, 5 Na2ATP, and 5 Na2 creatine phosphate, equilibrated with 95% O2 and 5% CO2 at pH 7.2 titrated with CsOH. In the present study, extracellular application of 5 µM Bay K 8644 (the specific agonist of CaL) and 0.1 µM nifedipine (the specific blocker of CaL) were used to identify the properties of CaL as describe before Whole-cell CaL channel α1C-subunit . The membrane then incubated for 45 min with Infrared (IR)-labeled secondary antibodies (LI-COR) in PBS-T containing 0.01% SDS. A monoclonal mouse antibody raised against the structural protein β-actin (Sigma) was used as a lane-loading control. The bound antibody was detected by the Odyssey infrared imaging system (LI-COR), and the densities of the immunoreactive doublet bands at 200 and 240 kD associated with anti-α1C subunit of CaL channel were expressed as a percentage of the β-actin density for each lane. Densitometry analysis of bands was performed by Scion image .Protein samples of cerebral and small mesenteric arteries were prepared according to the published methods 2+ releases from SR in cerebral and small mesenteric VSMCs, respectively, we investigated the average changes of intracellular Ca2+ fluorescence intensity in response to caffeine. The intracellular Ca2+ was measured with Ca2+ indicator, Fluo-3-acetoxymethyl ester , as previous described 2+-free balanced salt solution (BSS) in the following composition (mM): 126 NaCl, 5 KCl, 0.3 NaH2PO4, 10 HEPES, 1 MgCl2, 10 glucose, 1 EGTA, equilibrated with 95% O2 and 5% CO2 at pH 7.4 adjusted with NaOH 2+-free BSS was administrated to the cell and a period of 3 min was recorded. To avoid any laser-induced change in Ca2+ signaling, each cell was scanned only once. Also, only one cell was scanned in each scanning time to avoid cell density-induced changes in fluorescence intensity. The average fluorescence intensity was used to indicate the changes of intracellular Ca2+ and the maximal increase of Ca2+ fluorescence intensity was used to indicate the function of ryanodine-sensitive Ca2+ releases from SR in VSMCs as describe before In an attempt to establish whether simulated microgravity affects the function of ryanodine-sensitive CaL channel current densities in different groups, followed by a S-N-K-Post Hoc. Student's t-test was used to determine significant differences of body weights, soleus weights, femur lengths, and the maximal increases of Ca2+ fluorescence intensity in different groups. A value of P≤0.05 was considered to be statistically significant.Except the data of body weight are given as means ± SD, all other data are expressed as means ± SE. One-way ANOVA was used to determine the differences of CaExperiment I and Experiment II, suggesting a normal growth rate during and after simulated microgravity. However, after 28-day tail-suspension, the wet weights of left soleus in SUS-28 d rats were about 58.7% less than that in CON-28 d rats, indicating the deconditioning effects of simulated microgravity. In addition, the wet weights of left soleus in SUS+Rec-3 d rats were 24.3% less than that in CON-28 d rats, which means 3-day recovery after removal of 28-day tail-suspension could only partially recover the mass reduction of atrophic soleus due to simulated microgravity. There were no significant difference in the wet weights of left soleus between CON-35 d and SUS+Rec-7 d rats, suggesting a 7-day recovery after removal of 28-day tail-suspension could completely recover the mass reduction of soleus.As summarized in For cerebral VSMCs, cell capacitance (Cm), an index of cell membrane area, did not show significant differences among CON-28 d , SUS-28 d , and SUS+Rec-3 d rats . Correspondly, there were neither significant changes in Cm of small mesenteric VSMCs among CON-28 d , SUS-28 d , and SUS+Rec-3d rats . These results suggested that simulated microgravity did not affect the cell surface areas of cerebral and small mesenteric VSMCs, which were consistent with our previous report L currents of cerebral VSMCs in SUS-28 d rats showed larger inward components of trace as compared with that recorded in CON-28 d  curves which were further expressed in terms of current densities were shown in the right panel of I–V curves further showed that simulated microgravity significantly increased the CaL current densities in cerebral VSMCs  forms of the Caectively . As the 2+]i and was followed by a sustained increase in [Ca2+]i that was above the basal values in the continued presence of caffeine  was used to diminish Ca2+ influx through voltage-dependent Ca2+ channels in plasma membrane. As compared with that of CON-28 d rats, 28-day simulated microgravity significantly induced a higher resting [Ca2+]i level in cerebral VSMCs  evoked a transient peak increase of [Cacaffeine . In presal VSMCs  and a loic VSMCs , respectectively . In addial VSMCs . In contic VSMCs . These r2+ by its effects on CaL channels in plasma membrane and the ryanodine-sensitive Ca2+ releases from SR, i.e., simulated microgravity increased the functions of CaL channels and ryanodine-sensitive Ca2+ releases in cerebral VSMCs, whereas, simulated microgravity decreased the functions of CaL channels and ryanodine-sensitive Ca2+ releases in small mesenteric VSMCs. In addition, 3- or 7-day recovery after removal of suspension could restore the functions of CaL channels and ryanodine-sensitive Ca2+ releases to their control levels in cerebral and small mesenteric VSMCs, respectively. These differential regulations of CaL channels and ryanodine-sensitive Ca2+ releases in cerebral and small mesenteric VSMCs may be responsible for the altered autoregulation of cerebral vasculature and the inability to adequately elevate peripheral vascular resistance.The principal and novel finding of this work is that simulated microgravity differentially regulates the intracellular CaThere is a blood pressure gradient from the head to the feet in humans at 1 G in the upright posture. When exposed to microgravity, all gravitational blood pressure gradients are lost. Thus, blood vessels in cerebral circulation are exposed to higher than normal 1-G blood pressure, whereas vessels in lower body regions are exposed to lower than normal upright 1-G blood pressure. Therefore, the differential adaptations in cerebral and hindquarter arteries during real/simulated microgravity have been postulated to be a basical process of vascular autoregulation in response to sustained elevation or reduction in local transmural pressures, which may contribute to cardiovascular dysfunction and ultimately lead to the post spaceflight orthostatic intolerance L channels of the Cav1.2 gene family are the principal Ca2+ influx pathway in VSMCs and participate in multiple physiological functions, including excitation-contraction coupling, cell apoptosis, and cell proliferation in small resistance vessels. CaL channels are heteromeric complex composed of a central pore-forming α1C subunit and regulatory β, γ, and α2δ subunits in VSMCs. The pore-forming α1C subunit (about 190–240 kDa) is considered to confer most functional properties to the CaL channel. It is currently believed that membrane depolarization of VSMCs associated with elevated blood pressure permits extracellular Ca2+ entry through CaL channels, whereas, membrane hyperpolarization of VSMCs associated with lower blood pressure inhibits extracellular Ca2+ entry through CaL channels L channels regulate a tonic level of vascular tone and contribute to the dynamic autoregulation in the vascular beds. The abnormalities of CaL channels are regarded as part of the extensive biological and morphological adaptations during the pathogenesis of vascular diseases. By whole-cell and Western blotting, the present work demonstrated that simulated microgravity depolarized cerebral VSMCs , and thus can contribute to the overall rise in the concentration of intracellular Ca2+2+ activator site for Ca2+, whereas ruthenium red is a frequently used inhibitor. At concentrations of >5 mM, Caffeine activates all RyR isoforms to cause Ca2+ release from the SR 2+ in response to high mM concentrations of caffeine reflects the amount of calcium within the SR. Structural and functional studies indicate the important role of the SR and any alteration of ryanodine-sensitive Ca2+-releases from the SR could consequently alter contractile responsiveness of arterial smooth muscle cells in cardiovascular diseases 2+ releases  in cerebral VSMCs  in small mesenteric VSMCs  and the RyRs. In the present study, the increased Ca2+ transients were mainly due to activate RyRs because caffeine may either direct or indirect inhibition of IP3Rs 3Rs has been also reported to evoke localized Ca2+ transients and contribute to the global elevation of intracellular Ca2+3Rs during simulated microgravity in the further work.First, the present work showed that 3-day recovery after removal of suspension could completely restore the effects of simulated microgravity on the function of ryanodine-sensitive Ca of rats . Howeveral VSMCs  and partic VSMCs . Only 7-ic VSMCs . What is2+ determined by its effects on the functions of CaL channels and ryanodine-sensitive Ca2+ releases in cerebral and mesenteric VSMCs of rats, respectively. However, 3- or 7-day recovery after removal of simulated microgravity could restore these functions to their control levels in cerebral and mesenteric VSMCs, respectively. Our results suggested that differential regulation of intracellular Ca2+ might be the initiating factors leading to the functional and structural adaptations in the vessel caused by altered distribution of pressures and flows within the vasculature during microgravity.Taken together, the present work showed that simulated microgravity differentially regulated the intracellular Ca


The stored Ca2+ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca2+, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca2+. We review here biochemical and biophysical evidence demonstrating that release of bound Ca2+ into the lumen of SR requires Ca2+/H+ exchange at the low affinity Ca2+ sites. Rise of lumenal Ca2+ above its dissociation constant from low affinity sites, or reduction of the H+ concentration by high pH, prevent Ca2+/H+ exchange. Under these conditions Ca2+ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca2+pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.The Ca In muscle cells, this transport activity plays an important role in lowering cytosolic Ca2+ as required for relaxation of contractile elements, and storing transported Ca2+ in the lumen of SR for subsequent release and contractile activation (Carafoli Sarcoplasmic reticulum (SR) membrane vesicles, originally referred to as “relaxing factor”, were first isolated from skeletal muscle by Ebashi and Lipmann , and HasCarafoli . GeneralCarafoli .2+ within the transmembrane region of the enzyme , leading to a new cycle.SERCA1 is a 996 amino acid membrane bound protein  steady state experiments in which oxalate is used for complexation of lumenal Ca2+ (Martonosi and Feretos b) pre-steady state experiments in which lumenal Ca2+ has yet to rise  while Ca2+ in the outer medium remains sufficiently high (μM) for ATPase activation  of active transport may be considered to be an intrinsic feature of the pump, if the ATPase reaction sequence allows an alternate pathway leading to hydrolytic cleavage of Pi without vectorial displacement of Ca2+  is likely to participate in Ca2+/H+ exchange  , vectorial translocation of bound Ca2+ in the direction of the SSM electrode is prevented, even though K+ is present in high concentration and may neutralize acidic residues at alkaline pH. This indicates a requirement for specific H+ binding at the Ca2+ sites, in order to obtain Ca2+ release.Further evidence for Ca+ exchange at the transport sites following phosphoenzyme formation occurs in Ca2+ ATPases, but does not occur in copper ATPases  preventing phosphoenzyme formation ). This indicates that if exchange is limited due to low H+ concentration, Ca2+ is less likely to dissociate from the phosphoenzyme. On the other hand, while Ca2+ translocation is reduced, steady state ATPase activity is increased as the pH is raised, and continues after maximal levels of Ca2+ uptake are reached. It is apparent that alkaline pH reduces Ca2+/H+ exchange and dissociation of bound Ca2+, whereby the phosphoenyme bypasses the Ca2+ release step and proceeds to hydrolytic cleavage of Pi. Therefore, reduction of the Ca2+/ATP transport ratio can be produced either by a high Ca2+ concentration or a low H+ concentration in the lumen of the vesicles.The importance of Ca2+/H+ exchange and a pathway for slippage of the Ca2+ pump, is given in Scheme A diagram of the sequential steps in the ATPase mechanism, derived from the original reaction diagram of de Meis and Vianna  and modi2+, yielding E1 · 2Ca2+. Utilization of ATP yields ADP · E1 ~ P · 2Ca2+, followed by release of ADP and utilization of the phosphorylation potential to change vectorial orientation and affinity of the Ca2+ sites. Bound Ca2+ is then released into the lumenal medium in exchange for H+. Hydrolytic cleavage of nH+ · E2-P and transition of nH+ · E2 to E1 finally yields closure of the lumenal gate and exposure of the Ca2+ sites to the cytosolic medium. Formation of E1 · 2Ca2+ then starts a new cycle.In the diagram on Scheme 2+ is higher than its dissociation constant, or lumenal H+ is too low to sustain exchange, Ca2+ release and formation of nH+ · E2 are prevented. Interference with completion of the ATPase cycle would then cause reversal to E1 ~ P · 2Ca2+ . On the other hand .Strong evidence for the role of protons and the nHand Fig. , further2+ binding, phosphoenzyme formation, occlusion and then dissociation of bound Ca2+, which are mechanically linked to specific bending and rotation patterns of each headpiece domain. These movements provide an explanation for the long range linkage of phosphorylation and Ca2+ binding domains, including the roles of critical amino acids in substrate binding, catalytic reactions, and Ca2+ transport. They also demonstrate that the states included in the reaction diagram are structurally distinct, and possess specific features that are functionally relevant. It is of interest that movement of M1-M2 causes displacement of membrane helix M4L from M5 and M6, thereby opening the lumenal gate and allowing outflow of Ca2+, following Ca2+/H+ exchange. The lumenal gate is then closed upon cleavage of phosphate and dissociation of H+, when reverse rotation of the A domain is accompanied by upward displacement of M4L and reduction of the space between the M4 and M6 helices.High resolution crystal structures of most states  comprising the ATPase reaction sequence have been obtained, and are described in detailed reviews (Toyoshima 2+ ATPase (SERCA1) of (at least human and rabbit) fast twitch skeletal muscle, and produces uncoupling of ATP utilization and Ca2+ transport, with a consequent thermogenic effect , a 31 amino acid polypeptide  of the Ca2+ sites on lumenal orientation would be significantly affected. The physiological interest of PLB and SLN is related to the reversibility of their effects upon phosphorylation catalyzed by signaling kinases (Koss and Kranias Both SLN and PLN reside within a groove surrounded by transmembrane helices M2, M4, M6 and M9, as shown by crystallographic studies (Toyoshima et al. 2+ pump uncoupling, an important question is whether uncoupling of SERCA1 interferes with reduction of cytosolic Ca2+ below the level allowing contractile relaxation of muscle fibers. The evidence presented above indicates that the Ca2+ pump is perfectly coupled when the SR lumen Ca2+in is low, even if cytosolic Ca2+out is relatively high. In the light of this information, we consider that relaxation of a muscle twitch occurs in less than 1 s and, within this time, lumenal Ca2+ does not reach a concentration higher than its dissociation from E2-P · 2Ca2+, as shown in experiments performed with rabbit native SR vesicles (Fig. 2+ is reduced to a level producing contractile relaxation, such a Ca2+ level will be also low with regard to SERCA activation, and the ATPase would then proceed at very low rates or remain inactive. Therefore during relaxation, the pump remains quiescent, mostly in the Mg2+ bound E1 state (Toyoshima et al. 2+ were to become higher than its dissociation constant from E2-P · 2Ca2+, and cytosolic Ca2+ were to remain above the level required for full ATPase activation. This may occur upon prolonged muscle activity, if cytosolic Ca2+ is maintained relatively high by multiple action potentials and Ca2+ flux through plasma membrane voltage sensitive channels, as expected in shivering thermogenesis. Alternatively, a rise of intracellular pH above 7.0, may affect intracellular Ca2+ signaling, as recently reported for G protein signaling (Isom et al. 2+ rise to high levels, while cytosolic Ca2+ remains sufficiently high. Most importantly, it was reported that in brown fat, in addition to uncoupling of the mitochondrial respiratory chain, uncoupled SERCA contributes to non shivering thermogenesis (de Meis et al. Considering the possibility of Ca


Despite increasing recognition that mentoring is essential early in medical careers, little is known about the prevalence of mentoring programs for medical students. We conducted this study to survey all medical schools in Germany regarding the prevalence of mentoring programs for medical students as well as the characteristics, goals and effectiveness of these programs.A definition of mentoring was established and program inclusion criteria were determined based on a review of the literature. The literature defined mentoring as a steady, long-lasting relationship designed to promote the mentee's overall development. We developed a questionnaire to assess key characteristics of mentoring programs: the advocated mentoring model, the number of participating mentees and mentors, funding and staff, and characteristics of mentees and mentors . In addition, the survey characterized the mentee-mentor relationship regarding the frequency of meetings, forms of communication, incentives for mentors, the mode of matching mentors and mentees, and results of program evaluations. Furthermore, participants were asked to characterize the aims of their programs. The questionnaire consisted of 34 questions total, in multiple-choice (17), numeric (7) and free-text (10) format. This questionnaire was sent to deans and medical education faculty in Germany between June and September 2009. For numeric answers, mean, median, and standard deviation were determined. For free-text items, responses were coded into categories using qualitative free text analysis.We received responses from all 36 medical schools in Germany. We found that 20 out of 36 medical schools in Germany offer 22 active mentoring programs with a median of 125 and a total of 5,843 medical students  enrolled as mentees at the time of the survey. 14 out of 22 programs (63%) have been established within the last 2 years. Six programs (27%) offer mentoring in a one-on-one setting. 18 programs (82%) feature faculty physicians as mentors. Nine programs (41%) involve students as mentors in a peer-mentoring setting. The most commonly reported goals of the mentoring programs include: establishing the mentee's professional network , enhancement of academic performance  and counseling students in difficulties .Despite a clear upsurge of mentoring programs for German medical students over recent years, the overall availability of mentoring is still limited. The mentoring models and goals of the existing programs vary considerably. Outcome data from controlled studies are needed to compare the efficiency and effectiveness of different forms of mentoring for medical students. Mentoring is key to a successful career in medicine -4. MentoLittle is known about the availability and structure of mentoring programs for medical students. The most recent systematic review of the literature  includedIn 2000, Woessner et al.  criticizTherefore, we sent an electronic survey to all 36 medical school deans in Germany between June and October 2009. The principal objective of our study was to attain an overview of the existing mentoring programs for medical students in Germany. We aimed to assess the prevalence of mentoring for medical students as well as to compare and categorize the structure and goals of existing mentoring programs. We further designed the study to characterize mentors, mentees, and the mentoring relationships these programs form.The term "mentor" has been assigned a multitude of varying definitions in the literature -24. FollBased on these defining characteristics we established the following inclusion criteria for programs to be included in our analysis: (1) The program is designed to include medical students as mentees, regardless of whether the program is offered to the entire university or exclusively to medical students. (2) The program establishes a relationship between a mentor and one or several mentees, or alternatively between a clearly defined number of mentors and a single mentee or a group of mentees. (3) These mentoring relationships have an intended minimum duration of one year. (4) The program is designed not merely to foster skills or knowledge, but to encourage the overall development of mentees.We designed a questionnaire to assess key characteristics of mentoring programs, participating mentors, mentees and their relationships (See Appendices 1 & 2 for instructions to participants and the survey questionnaire). Our questionnaire solicited the following information about each program: name, web URL (if applicable), advocated mentoring model (one-on-one or group mentoring), the program's goals and intended duration, the number of participating mentees and mentors, as well as, funding and staff. We then asked about the characteristics of participating mentees  and mentors . We further sought to characterize the program's mentor-mentee relationships regarding the frequency of meetings, forms of communication, incentives for mentors, the mode of matching mentors and mentees, and results of program evaluations. The questionnaire consisted of a total of 34 questions in multiple-choice (17), numeric (7) and free-text (10) format.The LMU Medical School Ethics Committee waived ethical approval for the study. The data was collected and stored securely and analyzed anonymously.23. Additionally, we searched the webpages of all medical faculties for the terms "mentor*" and "mentoring program" to identify further contacts to interview.We distributed an electronic survey generated from our questionnaire between June and September 2009. We contacted the deans' office of all 36 medical schools in Germany by e-mail and asked them to complete our online survey or to forward the invitation to whom it concerned. If this failed to obtain a response, we contacted medical education professionals at the faculties in question through the network of Master of Medical Education graduatesAfter completion of the survey, we excluded programs from the analysis that did not meet the inclusion criteria previously specified , increase in knowledge or capability (level 2), change of behavior as a result of the learning experience (level 3), and the impact of this change in behavior (level 4) .We received a total of 39 responses from the 36 medical schools in Germany, which identified a total of 25 active programs. Among the responders were 4 deans of student's affairs, 19 deans' office staff members, 11 mentoring program coordinators and 5 other medical education specialists. Disclosure of the program's budget was voluntary which resulted in missing data. Two programs were unable to specify the population size from which mentees were recruited. Four programs could not specify the number of potential mentors. Three programs did not disclose the average number of meetings between mentors and mentees per year. For all other items a complete data set was obtained.Three programs were excluded from our data analysis as they failed to meet certain defined inclusion criteria: one program only has an intended duration of six months, one program solely promotes mentees' research development, and one program focuses exclusively on the transfer of medical knowledge  have a website containing information about the program. Nine of these 13 websites were found to specify the goals and structure of the program as well as provide substantial and relevant information for potential mentees.Aspects of free-text responses were categorized into (a) declared goals of the programs, (b) means by which the program aims to achieve these goals and (c) intended character of the mentoring relationships. The majority of programs intend to build and expand mentees' networks at the medical school such as among faculty and peers  offer mentoring in a one-on-one setting  include medical students from all 6 curricular years, 7 programs (32%) are exclusively for preclinical year students and 6 (27%) are limited to clinical year students  than in group-mentoring programs (9.9 mentees per mentor). Most programs  allow mentees to choose their mentors, while the remaining 12 programs (55%) assign mentors to mentees . In total, at the time of the survey 587 medical students  are enrolled in one-on-one mentoring programs with more than half of them  in one program. In the one-on-one structure, personal meetings between mentors and mentees take place an average of 4 times per year . Three one-on-one programs allow mentees to choose their mentor (2 of which offer online mentor profiles), while 3 programs assign mentors to their mentees (2 of which use profile questionnaires for matching mentors and mentees).Eleven programs (50%) reported receiving funding from their respective university. Eight programs (36%) are funded by tuition fees and 5 programs (23%) by third party funds. Nine programs (41%) employ an average of 1.1 physicians or scientific staff members to administrate the mentoring program. Student assistants run 11 programs (50%). Five programs (23%) employ a secretary or other non-scientific member of staff. Additionally, we asked participants to voluntarily provide their program's annual budget. Several programs did not provide this information. For some who did, the budget seems implausibly low relative to the employed staff. Therefore, the budget required to run mentoring programs for medical students cannot be legitimately analyzed from our data set.15 programs (68%) reported conducting evaluations on a regular basis. Two programs reported that results had been submitted for publication in a scientific journal. Evaluations are performed through online or paper surveys, interviews or feedback meetings. Most evaluations focused on mentee and mentor satisfaction corresponding to Kirkpatrick's level 1. Four programs evaluated topics discussed between mentors and mentees and what mentees perceived as the impact of their relationship (Kirkpatrick's level 2). One program found that geographical distance had no impact on the quality of the mentoring relationship. None of the evaluations measured the efficacy of mentoring in altering behavior or achieving objective outcome criteria (Kirkpatrick's levels 3 & 4).Our data show that there has been a dynamic emergence of mentoring programs for medical students over recent years in Germany. Out of 22 mentoring programs with personally allocated mentors at German medical schools, 14 have been established within the last 2 years. Despite methodological differences, it is remarkable that in the study of Woessner et al. in 2000 ,19. HoweAlthough we used clearly defined inclusion criteria, we found mentoring programs with diverse declared goals. This heterogeneity reflects the multifaceted notion of mentoring. In a recent review on mentoring programs for medical students by Frei et al. , the proThe mentoring programs in our study represent various forms of mentoring ranging from one-on-one to group mentoring, and from peer-mentoring to mentoring by senior faculty members. To establish characteristics of successful mentoring programs, it would be necessary to correlate the structures of various mentoring programs with their outcome. However, as outlined above, the outcome levels on which the programs in our study are evaluated are insufficient to establish this correlation. Evaluations of mentoring programs published in the literature are also rarely based on validated questionnaires ,19. TherOne of the reasons for this lack of research certainly lies in the difficulties associated with measuring the effectiveness of mentoring in medicine. Currently available instruments for the evaluation of formal mentoring programs in medicine, such as the tools developed by Morzinski et al.  or Berk Besides focusing on the benefits for student mentees, the evaluation of medical student mentoring programs should also address the benefits for mentors and the educational institution. It has been shown recently that mentoring medical students led to personal and professional development of the mentoring physician . By offeIt has been hypothesized in the literature that there is a lack of mentoring for medical students in most countries ,19. IndeOur study has assessed the prevalence of formal mentoring programs for medical students in Germany. However, for two main reasons, only limited conclusions can be drawn from this. Firstly, the enrolment rates differed greatly between programs. This raises the question whether all programs were truly available to all eligible students. Differences in publicity of the program, recruitment and selection of mentees may account for different enrollment rates. These factors were not sufficiently measured by our survey and need to be taken into account when assessing the true availability of the programs in our study to the eligible population of students. Secondly, students might form satisfying mentoring relationships outside or in the absence of formal mentoring programs or might simply not desire mentoring.However, our data does provide some indirect evidence that there is an unmet demand for mentoring among medical students. Firstly, 16 out of 36 medical schools do not offer any mentoring program for medical students to date. Considering evidence that medical students encounter severe difficulties finding a suitable mentor outside formal programs , it seemTo our knowledge, there are no similar studies that could be used as a reference for international comparison. Meta-analyses of the literature ,19 cannoIn conclusion, our study assessed the availability of mentoring programs for medical students in Germany. We found that despite the emergence of numerous programs over the past few years only a limited number of medical students are enrolled in formal mentoring programs and only a small percentage of those receive mentoring in a one-on-one mentoring setting.To our knowledge, our survey instrument represents the first detailed questionnaire developed for a cross-sectional study of mentoring programs covering key characteristics of formal mentoring programs. Elements of our survey may be used to develop a standardized survey instrument that will need to be validated to be used in further national or international studies.Higher-level outcomes of mentoring programs as well as the quality of the mentoring process need to be assessed with validated instruments in order to appraise their long-term impact on medical students' professional development and behavior. Based on this data, mentoring programs for medical students could be improved and managed to maximize their benefit for mentees, mentors and medical schools.The authors declare that they have no competing interests.FGM participated in the conception and design of the study and questionnaire, carried out the data acquisition, participated in the analysis and interpretation of data and wrote the manuscript. KD participated in the conception and design of the study and questionnaire, participated in the analysis and interpretation of data and critically reviewed the manuscript. PvdB participated in the conception and design of the study and questionnaire, participated in the analysis and interpretation of data, prepared the figures and critically reviewed the manuscript. SS participated in the conception and design of the study and questionnaire, participated in the analysis and interpretation of data and critically reviewed the manuscript. SN participated in the analysis and interpretation of data and critically reviewed the manuscript. MRF participated in the conception and design of the study and questionnaire, participated in the analysis and interpretation of data and critically reviewed the manuscript. All authors read and approved the final manuscript.Dear Sir or Madam,You have been referenced as a contact person for a mentoring program at your university. We are conducting a study about mentoring programs for medical students at German medical schools. The study will provide an overview of existing mentoring programs for medical students in Germany. We would like to ask you to participate in our study. You can complete our survey online by clicking on the link provided below. Completing the questionnaire will take you approximately 15 minutes. You can save your answers at any time and proceed later. If you prefer to answer our questions on the phone, please let us know.not understand tutoring , career advising offices which do not establish a steady mentor-mentee-relationship or supervision for specific  projects.All mentoring programs that enroll medical students as mentees should be included into the study. By mentoring we understand a steady relationship between a mentor and one or several mentees, or alternatively between a clearly defined number of mentors and a single mentee or a group of mentees designed to foster the development of mentees. By mentoring we do If your university offers more than one mentoring program that involve medical students as mentees, simply complete the questionnaire multiple times. If any questions arise during completion of our survey, please do not hesitate to contact us.Yours sincerely,[FREE TEXT]1.1 Which university do you respond for? [YES/NO] 1.2 At your university, is there a mentoring program that involves medical students as mentees? [FREE TEXT]1.3 What is the name of the mentoring program? MEDICAL FACULTY/ENTIRE UNIVERSITY/OTHER]1.4 Is the mentoring program designed exclusively for students at the faculty of medicine or the entire university? [MC: [FREE TEXT]1.5 Is there a website that offers information about your program? If yes, please provide URL. [FREE TEXT]1.6 What are the goals of your mentoring program? one mentor - one mentee /one mentor - multiple mentees (group mentoring)/multiple mentors - one mentee /multiple mentors - multiple mentees (group mentoring)/other]1.7 Please specify the ratio of mentors and mentees in a typical interaction within your program . [MC: [NUMERICAL]1.8 How long has your program been running (in years)? third party funding/university funds/tuition fees/other]1.9 How is your mentoring program funded? [MC: numerical]1.10 What is the annual budget of your mentoring program? [[scientific staff/physicians [numerical]/secretaries [numerical]/student assistants [numerical]/other [numerical]]1.11 How much staff is employed primarily for the administration of the mentoring program? [YES/NO]1.12 Is the participation in your program voluntary for mentees? [YES/NO]1.13 Is the participation in your program voluntary for mentors? NUMBER OF ACTIVE MENTEES IN THE PROGRAM [NUMERICAL]/NUMBER OF ACTIVE MENTORS IN THE PROGRAM [NUMERICAL]/SIZE OF POPULATION FROM WHICH MENTEES ARE RECRUITED (INCLUDING PARTICIPATING MENTEES) [NUMERICAL]/SIZE OF POPULATION FROM WHICH MENTORS ARE RECRUITED (INCLUDING PARTICIPATING MENTORS) [NUMERICAL]]1.14 How many mentees and mentors currently participate in your mentoring program? [[ALL STUDENTS/STUDENTS OF PRECLINICAL YEARS ONLY/STUDENTS OF CLINICAL YEARS ONLY/OTHER]1.15 What is the mentees' educational level? [NO/FEMALE STUDENTS ONLY/STUDENTS WITH FOREIGN BACKGROUND ONLY/OTHER]1.16 Is the mentoring program designed exclusively for a specific subgroup of students? [ACADEMIC PHYSICIANS/PHYSICIANS AT NON-ACADEMIC HOSPITALS/PHYSICIANS IN PRIVATE PRACTICE/NON-PHYSICIAN UNIVERSITY SCIENTISTS/SCIENTISTS OUTSIDE THE UNIVERSITY/OTHER]1.17 What is the mentors' professional profile? [STUDENTS (PEER MENTORING)/RESIDENT PHYSICIANS OR RECENT GRADUATES/ATTENDING PHYSICIANS OR MULTIPLE YEARS OF WORK EXPERIENCE/PROFESSORS/OTHER]1.18 What is the mentors' educational level? [NUMBER OF INTENDED MEETINGS [NUMERICAL]/NUMBER OF ACTUAL MEETINGS [NUMERICAL]]1.19 How many meetings between mentors and mentees are intended by the program and how many actually take place annually? [PERSONAL MEETINGS/EMAIL/PHONE/OTHER]1.20 Which forms of communications are regularly used between mentors and mentees? [FREE TEXT]1.21 Which topics are typically discussed between mentors and mentees? [NONE/FINANCIAL COMPENSATION/RECOGNITION AS WORKING HOURS/REDUCED TEACHING OBLIGATIONS/SPECIFIC TRAINING FOR MENTORS/GREATER CHANCE OF PROMOTION/CERTIFICATES/OTHER] 1.22 Are there any extrinsic incentives for becoming a mentor in your program? [NUMERICAL]1.23 What is the financial compensation for mentors (in euro per mentee and year) MATCHED BY PROGRAM/MENTEE CHOOSES MENTOR/MENTOR CHOOSES MENTEE/OTHER]1.24 How are mentors and mentees matched? [MC: [PRESELECTED GROUP/ENTIRE POPULATION]1.25 (only if 4.1 is "mentee chooses mentor") Does the mentee choose his/her mentor from a preselected group of potential mentors (mentors recruited by program) or from the entire population of potential mentors (mentors recruited by mentee)? NONE/PAPER-BASED MENTOR PROFILES/ONLINE MENTOR PROFILES/MENTORING-SPECIFIC GET-TOGETHER EVENTS/PERSONAL INTERVIEW OF MENTEES AND/OR MENTORS BY THE PROGRAM/OTHER]1.26 (only if 4.1 is "mentee chooses mentor") Are there any auxiliary resources for the selection of a mentor by the mentee? [MC: NONE/PAPER MENTEE PROFILES/ONLINE MENTEE PROFILES/MENTORING-SPECIFIC GET-TOGETHER EVENTS/PERSONAL INTERVIEW OF MENTEES AND/OR MENTORS BY THE PROGRAM/OTHER]1.27 (only if 4.1 is "mentor chooses mentee") Are there any auxiliary resources for the selection of a mentee by the mentor? [MC: RANDOMLY/TAKING SPECIFIC CRITERIA INTO ACCOUNT]1.28 (only if 4.1 is "matched by program") How is the matching performed? [MC: [FREE TEXT]1.29 (only if 4.5 is "specific criteria") Which criteria are used to match mentors and mentees? [FREE TEXT]1.30 How are mentors recruited for the program? [YES/NO] 1.31 Is an evaluation of the mentoring relationships performed? [FREE TEXT]1.32 How is the evaluation performed? [FREE TEXT]1.33 What are key results of your evaluation? YES/NO + FREE TEXT COMMENT]1.34 Have results of your evaluation been published? If so, where? [[FREE TEXT]1.35 Besides the mentoring relationships, does your mentoring program include special events ? If so, please describe. [FREE TEXT]1.36 Please provide your name for further questions. [FREE TEXT]1.37 Please provide your phone number and/or e-mail address. [FREE TEXT]1.38 Would you like to be acknowledged in a publication of this study? The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6920/11/68/prepubSupplementary table S1. Supplementary table S1 lists the names and institutions of all programs excluded from (1a) and included in the study (1b).Click here for file


STIM proteins target Orai1-3, tetrameric Ca2+-permeable channels in the plasma membrane. Structure-function analysis revealed the molecular determinants and the key steps in the activation process of Orai by STIM. Recently, STIM1 was found to be expressed at high levels in skeletal muscle controlling muscle function and properties. Novel STIM targets besides Orai channels are emerging.Stromal interaction molecules (STIM) were identified as the endoplasmic-reticulum (ER) Ca2+/calcineurin/nuclear factor of activated T cells (NFAT)-dependent morphogenetic remodeling programs and to support adequate sarcoplasmic-reticulum (SR) Ca2+-store filling. Also in our hands, STIM1 is transiently up-regulated during the initial phase of in vitro myogenesis of C2C12 cells. The molecular targets of STIM1 in these cells likely involve Orai channels and canonical transient receptor potential (TRPC) channels TRPC1 and TRPC3. The fast kinetics of SOCE activation in skeletal muscle seem to depend on the triad-junction formation, favoring a pre-localization and/or pre-formation of STIM1-protein complexes with the plasma-membrane Ca2+-influx channels. Moreover, Orai1-mediated Ca2+ influx seems to be essential for controlling the resting Ca2+ concentration and for proper SR Ca2+ filling. Hence, Ca2+ influx through STIM1-dependent activation of SOCE from the T-tubule system may recycle extracellular Ca2+ losses during muscle stimulation, thereby maintaining proper filling of the SR Ca2+ stores and muscle function. Importantly, mouse models for dystrophic pathologies, like Duchenne muscular dystrophy, point towards an enhanced Ca2+ influx through Orai1 and/or TRPC channels, leading to Ca2+-dependent apoptosis and muscle degeneration. In addition, human myopathies have been associated with dysfunctional SOCE. Immunodeficient patients harboring loss-of-function Orai1 mutations develop myopathies, while patients suffering from Duchenne muscular dystrophy display alterations in their Ca2+-handling proteins, including STIM proteins. In any case, the molecular determinants responsible for SOCE in human skeletal muscle and for dysregulated SOCE in patients of muscular dystrophy require further examination.Here, we will focus on the role of STIM1 in skeletal-muscle structure, development and function. The molecular mechanism underpinning skeletal-muscle physiology points toward an essential role for STIM1-controlled SOCE to drive Ca However, these features may be dependent on the cell type and/or levels of STIM2, since basal [Ca2+] as well as the thapsigargin-releasable Ca2+ did not differ between wild-type mouse embryonic fibroblast (MEF) cells and MEF cells deficient for STIM2 [2+], markedly more than STIM1 overexpression. Consistent with these findings, small reductions in ER [Ca2+] caused STIM2, but not STIM1, translocation and redistribution to punctae, with subsequent activation of Ca2+ influx  . In this2+] [2+] . Knockdoor STIM2 . Similar+ influx . While bepletion ,39, STIM/- cells ,39. Fromignaling . Neverth2+-signaling function. Loss of STIM1/Orai1 signaling in patients results in severe T-cell immunodeficiency and concomitant viral, bacterial and fungal infections , but required the translocation of STIM1. This study therefore points out that STIM1 may be an important integrator molecule that mediates cross-talk between Ca2+-dependent signaling and cAMP-dependent signaling. Recently, STIM1-mediated store-operated cAMP signaling has been implicated in the downstream effects of Ca2+ signaling induced by eicosapentaenoic acid, an omega-3 polyunsaturated fatty acid present in fish oil . The mechanism involves the direct binding of CAD or SOAR to a region encompassing aa 1809-1908, located in the C-terminus of Cav1.2.In addition to the well-described Orai targets of STIM1, two very recent studies identified the voltage-operated Caof STIM1 ,59. STIMImportantly, while CAD or SOAR activates Orai1-mediated currents, they inhibit Cav1.2-mediated currents. The interaction of STIM1 with the C-terminus of Cav1.2 is critical for its inhibition. This process is independent of functional Orai1-channel activity, but Orai1 may help STIM1-mediated Cav1.2 inhibition by trapping STIM1 in punctae that contain Cav1.2 channels, thereby recruiting STIM1 in the vicinity of Cav1.2 channels. In addition, STIM1 expression seems to regulate the plasma-membrane level of Cav1.2 . Overexp2+-influx pathways may be the molecular switch that accounts for the reciprocal regulation of voltage-gated Ca2+ influx (inhibition) and store-operated Ca2+ influx (activation) [2+] in response to membrane depolarization, although immune cells  also express voltage-gated Ca2+ channels   . This mae fibers . Interes2+ influx may be beneficial and holds potential for future therapeutic strategies to tackle muscular dystrophy either by targeting STIM1/Orai1 or TRPC channels, since both channel complexes have been shown to be implicated and/or up-regulated in Duchenne muscular dystrophy models [Independently of the mechanism, preventing Cay models ,106. Ally models ; ii) bloy models ; iii) iny models .2+ influx may be critical for targeting this pathology, inhibiting Ca2+ release from the ER has also been shown to be beneficial [3R-mediated Ca2+ release [2+ overload, thereby protecting dystrophic muscle cells against Ca2+-dependent apoptosis [2+ influx as well as ER Ca2+ release.Furthermore, while reducing Caneficial . For ins release ,110 and poptosis . Hence, 2+ influx as an early event in the development of muscular dystrophy has been obtained from mouse models. We need to keep in mind that the need and physiological role of SOCE might be different for mouse and human skeletal muscle. For instance, a micro-array analysis of human skeletal-muscle biopsies from control patients and Duchenne muscular dystrophy patients  indicated that the STIM1-mRNA levels were not increased, but rather tended to decrease in the muscle of the dystrophic patients, while Orai1-mRNA levels were not significantly changed ; TRPC: canonical transient receptor potential.CRAC: CaThe authors declare that they have no competing interests.GB conceived the experiments, analyzed the data and wrote the manuscript. SK performed the experiments, analyzed the data and wrote the manuscript. JPD, HDS, LM and JBP discussed the data and revised the manuscript.


Voltage- and Ca2+-dependent L-type Ca2+ channels (LCCs) allow for Ca2+ entry into the myocyte, which then binds to nearby ryanodine receptors (RyRs) and triggers Ca2+ release from the sarcoplasmic reticulum in a process known as Ca2+-induced Ca2+ release. The highly coordinated Ca2+-mediated interaction between LCCs and RyRs is further regulated by the cardiac isoform of the Ca2+/calmodulin-dependent protein kinase (CaMKII). Because CaMKII targets and modulates the function of many ECC proteins, elucidation of its role in ECC and integrative cellular function is challenging and much insight has been gained through the use of detailed computational models. Multiscale models that can both reconstruct the detailed nature of local signaling events within the cardiac dyad and predict their functional consequences at the level of the whole cell have played an important role in advancing our understanding of CaMKII function in ECC. Here, we review experimentally based models of CaMKII function with a focus on LCC and RyR regulation, and the mechanistic insights that have been gained through their application.Excitation-contraction coupling (ECC) in the cardiac myocyte is mediated by a number of highly integrated mechanisms of intracellular Ca One such signaling pathway is that involving the Ca2+/calmodulin (CaM)-dependent protein kinase (CaMKII), which targets and functionally regulates a number of proteins that are central to cardiac excitation-contraction coupling (ECC), and has been identified as a promising new target for antiarrhythmic therapy in patients with heart failure  and ryanodine receptor (RyR) regulation, and the mechanistic insights that have been gained through their application.Cardiac electrophysiology is a discipline with a rich and deep history dating back more than a half-century. Much of our understanding of fundamental biological mechanisms in this field comes from experimental research coupled with integrative mathematical modeling. The goal of this modeling has been to achieve a quantitative understanding of the functional mechanisms and relationships that span from the level of molecular structure and function to integrated cardiac myocyte behavior in health and disease. Advances in computational techniques and hardware have led to the recent development of many sophisticated large-scale models of heart tissue, in which these electrophysiological myocyte models are the fundamental building blocks. Some of the most fundamental advances in computational cell biology, including formulation of dynamic models of voltage-gated ion channels, mitochondrial energy production, membrane transporters, intracellular Ca2+. The junctional SR (JSR) is the portion of the SR most closely approximating  via voltage-gated LCCs and triggers the opening of RyRs, and hence JSR Ca2+ release, is known as Ca2+-induced Ca2+ release (CICR) and comprises the initial transduction event of ECC. Recent imaging studies have demonstrated that dyadic clefts are small, where junctions contain an average of 14 RyRs, with some clusters incompletely filled with RyRs, and with a large fraction of clusters closely spaced within 20–50 nm, suggesting that smaller clusters may act together to function as a single site of CICR  and Ca2+ transients, and detection of local Ca2+ release events known as Ca2+ sparks  to the CaMKII regulatory domain in response to beat-to-beat transient increases of intracellular Ca2+ concentration. In cardiac myocytes, an activated CaMKII molecule can undergo autophosphorylation by neighboring subunits at threonine amino acid residues in its regulatory domain, which allows the kinase to retain activity even upon dissociation of Ca2+/CaM  and the SR Ca2+-ATPase (SERCA). The model predicted that CaMKII plays a critical role in the rate-dependent increase of the cytosolic Ca2+ transient by increasing ECC gain, but that it does not play a significant role in rate-dependent changes of AP duration. Similar results were obtained in the human cardiac AP model of O'hara et al. (+ current (INa), and the transient outward K+ current (Ito1). The HRd model was later updated and used to investigate the role of altered CaMKII signaling in the canine infarct border zone , and that disruption of the regulatory system itself  leads to the most aberrant physiological phenotypes. Livshitz and Rudy . This model identifies Ca2+ alternans as the underlying mechanism for electrical alternans, both of which were eliminated with CaMKII inhibition, suggesting this as an antiarrhythmic strategy. Zang et al. , it clearly demonstrates that the multiple targets of CaMKII interact to produce net alterations of integrated cellular function which are difficult to predict from knowledge if its functional effects on isolated individual targets. Saucerman and Bers  but not in the cytosol is a key factor that would influence the way in which local signaling mechanisms would be incorporated into models that followed that included detailed reconstruction of dyadic Ca2+ dynamics. In one such model, Soltis and Saucerman . Recently, Morotti et al.  correlates well with a CaMKII-mediated shift in modal gating of LCCs . Following incorporation into the canine cardiac myocyte model , as shown in Figure 2O2 . The simulated increase in ICaL via oxidized CaMKII corroborates the experimental findings of Song et al. (in silico model of H2O2 exposure discussed here predicts that the fraction of LCCs gaiting in mode 2 will shift from 5% in control (absence of ROS) to 7% upon this increase in ROS. Using this model, the simulated diastolic RyR leak also increases in response to oxidative stress (~44%) primarily as an indirect consequence of increased Ca2+ influx via LCCs. Wagner et al. . They, however, provide additional evidence indicating that their observed increase in Ca2+ leak does not require the presence of CaMKII, suggesting an important role for CaMKII-independent mechanisms of ROS-mediated alteration of cardiac ECC as well.Along these lines, Foteinou et al.  have recg et al.  who demor et al.  recently2+ mismanagement leads to decreased scavenging resources and accounts for ROS overflow as is believed to occur in heart failure (Hill and Singal, As these ROS-mediated effects need to be further examined, future studies will focus on establishing quantitative links between cellular ROS and redox balance, CaMKII activity and function, ECC, and whole-cell electrophysiology. Recently, Gauthier et al. ,b develo2+ ions, CaM, CaMKII, LCCs, and RyRs interact in the cardiac dyadic junction both at rest and during triggered ECC events. On an intermediate scale, models have predicted the consequences of normal and abnormal CaMKII signaling on whole-cell Ca2+ cycling, effects of ROS imbalance, AP shape, and the generation of cellular arrhythmias such as EADs. Incorporation of these cellular models into higher scale tissue simulations has provided important insight into the relationship between CaMKII function, electrical wave conduction velocity, and the emergence of arrhythmogenic substrates in diseased tissue. The continuum of biological scales spanned by these models allows for the development of multiscale approaches whereby we can predict and understand the emergence of macroscale phenotypes as a consequence of CaMKII-mediated molecular signaling events. A great deal of evidence now implicates CaMKII as a nexus point linking heart failure and arrhythmias (Swaminathan et al., 2+ leak and/or increased likelihood of spontaneous Ca2+ release events and DADs, as the key mechanism underlying arrhythmogenesis. Others (Hashambhoy et al., 2+ levels and AP properties, including the appearance of EADs. These mechanisms may co-exist in heart failure, and it is likely that both play an important role. Moreover, CaMKII modulation of other targets such as PLB, INa, and K+ currents, many of which have been incorporated into the above cell models, adds additional layers of complexity to the task of interpreting and predicting the mechanisms by which CaMKII signaling alters cardiac function in health and disease. As a result of the breadth and diversity of CaMKII targets, strategies that involve broad inhibition of CaMKII activity will impact and alter the function of many cellular subsystems, with complex consequences that may not all be beneficial. As mathematical models of CaMKII signaling in the cardiac dyad and beyond are further developed, they will play a key role in identifying target-specific therapeutic strategies for improving myocardial function and reducing arrhythmias.Integrative modeling of cardiac ECC, cell signaling, and myocyte physiology has played a critical role in revealing mechanistic insights across a range of biological scales. With respect to CaMKII function at the smallest scale, models have shed light on how CaThe 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.


Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are known to have electrophysiological properties similar to mature adult cardiomyocytes. However, it is unclear if they share similar calcium handling property. We hypothesized that Ca2+ sparks in human induced pluripotent stem cell (hiPSCs)-derived cardiomyocytes (hiPSC-CMs) may display unique structural and functional properties than mature adult cardiomyocytes.Ca2+ sparks in hiPSC-CMs were recorded with Ca2+ imaging assay with confocal laser scanning microscopy. Those sparks were stochastic with a tendency of repetitive occurrence at the same site. Nevertheless, the spatial-temporal properties of Ca2+ spark were analogous to that of adult CMs. Inhibition of L-type Ca2+ channels by nifedipine caused a 61% reduction in calcium spark frequency without affecting amplitude of those sparks and magnitude of caffeine releasable sarcoplasmic reticulum (SR) Ca2+ content. In contrast, high extracellular Ca2+ and ryanodine increased the frequency, full width at half maximum (FWHM) and full duration at half maximum (FDHM) of spontaneous Ca2+ sparks.Ca, spontaneous Ca2+ sparks were detected in hiPSC-CMs. The Ca2+ sparks are predominately triggered by L-type Ca2+ channels mediated Ca2+ influx, which is comparable to sparks detected in adult ventricular myocytes in which cardiac E-C coupling was governed by a Ca2+-induced Ca2+ release (CICR) mechanism. However, focal repetitive sparks originated from the same intracellular organelle could reflect an immature status of the hiPSC-CMs.For the first time The process involves entry of Ca2 through L-type Ca2+ channel that activates the ryanodine receptors (RyRs)-mediated Ca2+ release from sarcoplasmic reticulum (SR) and resulting in intracellular Ca2+ transients 2+ sparks, a local and transient Ca2+ release originating from a single RyR or a cluster of RyRs, constitute the elementary events of cardiac E-C coupling 2+ transients are believed to represent the recruitment and summation of many Ca2+ sparks after an increase in opened L-type Ca2+ channels 2+ release channel is tightly linked to the gating of L-type Ca2+ channel and plays a key role in the intracellular Ca2+-handling in cardiac myocytes 2+ sparks may reflect the organizational maturity of RyRs in the cardiomyocytes Excitation-contraction (E-C) coupling in the adult mammalian heart is governed by the Ca2+ transients in hiPSC-CMs are dependent on the CICR mechanism in which the key calcium-handling proteins include RyR2, SERCA2a and L-type Ca2+ channels 2+ sparks and different properties of Ca2+ sparks 2+ sparks in hiPSC-CMs remain unclear.Human induced pluripotent stem cells (hiPSCs) can be generated by somatic reprogramming of fibroblasts with a set of transcription factors and differentiated into multiple cell lineages, including cardiomyocytes 2+ sparks in hiPSC-derived cardiomyocytes play instrumental roles in their structural and functional maturity. To better understand regulation of E–C coupling in hiPSC-CMs, we performed confocal laser scanning microscopy in conjunction with the Ca2+ fluorescent indicator, fluo-4, to determine the relationship between occurrence of spontaneous Ca2+ sparks and activation of L-type Ca2+ channels. The dynamic parameters of Ca2+ spark unveiled novel fundamental characteristics of Ca2+ handling and regulation in hiPSC-CMs.We hypothesized that the Cain vitro and teratoma formation in vivo. hiPSCs were maintained on mouse embryonic fibroblasts (MEF) feeder in hESC medium . Unless specified, all culture reagents were from Invitrogen.Human induced pluripotent stem cells (hiPSCs) were generated in our laboratory from human neonatal dermal fibroblasts  via retroviral-based ectopic expression of Yamanaka factors  Embryoid bodies (EBs) were generated after mechanical dissection of hiPSCs and maintained in suspension culture in a cardiomyogenic medium or CARM which contains DMEM High Glucose 485 mL, L-Glutamine 5 mL, NEAA 5 ml, Selenium Transferrin 5 mL (Sigma) and 2-mercaptoethanol 3.5 µL supplemented with 5 µM SB 203580 (Sigma), a specific p38-MAPK inhibitor Detail description of immunocytochemistry assay of hiPSCs is described in 2 1.8, MgCl2 1, glucose 10, HEPES 10, pH 7.40 with NaOH) under current-clamp conditions by an Axopatch 200B patch clamp amplifer . Patch pipettes, with a resistance of 2–4 MΩ, were pulled by a horizontal puller , and filled with an internal solution containing (in mM): KCl 130, MgCl2 1, MgATP 3, EGTA 10, and HEPES 10, pH 7.20 with KOH. In addition, the effect of isoproterenol  was tested on ventricular-like hiPSC-CMs.Dissociated hiPSC-CMs were cultured on 3.5 cm glass-bottom dishes . The spontaneous action potentials (AP) were recorded from hiPSC-CMs in normal Tyrode’s solution ((in mM): NaCl 140, KCl 5.4, CaCl2+ transients and Ca2+ sparks were recorded in ventricular-like hiPSC-CMs using a LSM-710 laser scanning confocal microscope  with a 40×, 1.3 numerical aperture oil immersion objective and axial resolutions of 1.5 µm 2+ load was estimated by rapid application of 10 mM caffeine with a multi-channel rapid application system.Ca2+ ([Ca2+]i) concentration was determined at the end of Ca2+ imaging experiment by the ionomycin method  was 3.8±0.7 in hiPSC-CMs. To observe spread patterns of Ca2+ transients of hiPSC-CMs, transverse line-scan images of Ca2+ transient were performed. As shown in 2+ increased first at the periphery of the cell before propagating towards the centre of the cell with a mean time delay of 46±15 ms (n = 7) (2+]i was performed as described in  (n = 7) b. Calibr2+, and then Ca2+ quickly dropped homogeneously to resting levels in adult rat cardiomyocytes . The average amplitude of Ca2+ transients (F/F0) was 3.5±0.6  at different times. To better characterize the spatial and temporal properties of Ca2+ sparks, line-scan imaging was carried out to monitor Ca2+ dynamics at 3 ms resolution in hiPSC-CMs. Fluorescence (the ratio of fluorescence to background fluorescence (F/F0)) profiles of Ca2+ sparks (bottom) were shown in 2+ sparks shown in 2+ sparks, even during the occurrence of spontaneous Ca2+ transients. In adult rat cardiomyocytes, repetitive Ca2+ sparks were seldom observed  (As shown in ll = 31) .2+ sparks. An overlay of 160 original Ca2+ sparks was shown in 2+ sparks , spatial size  or duration  of spontaneous Ca2+ sparks. 0, FDHM and FWHM of Ca2+ sparks which we deduced the relationship between the amplitude and size distributions of Ca2+ sparks and the population of Ca2+ sparks from their histogram plots. The mean values for F/F0, FWHM and FDHM were 1.64±0.04, 2.31±0.03 µm and 30.9±0.6 ms, respectively. Ca2+ sparks between hiPSC-CMs and adult ventricular myocytes (nspark = 302) have similar characteristics of Ca2+ sparks  on the rate of occurrence of spontaneous Ca2+ sparks was observed. As presented in 2+ channels by nifedipine significantly reduced the frequency of occurrence of Ca2+ sparks without affecting F/F0, FDHM and FWHM of Ca2+ sparks  which indicated that L-type Ca2+ channels blockade did not affect SR Ca2+ load in hiPSC-CMs.SR Ca2+ influx is an important trigger for SR Ca2+ release. To observe effect of extracellular Ca2+ concentration on Ca2+ sparks, 5 mM CaCl2 was applied in extracellular solution. 2+ sparks before and after the application of 5 mM CaCl2. It is clear that the frequency of Ca2+ sparks was 5.4±0.8 sparks/100 µm.s in control, significantly increased to 10.4±0.5 sparks/100 µm.s after application of 5 mM CaCl2  in the presence of 5 mM CaCl2 , respectively (2+ sparks in the presence of 5 mM CaCl2 (1.48±0.02) was significantly lower than those in control (1.51±0.04) (*P<0.05) (2+ concentration resulted in an increase in big spark populations.CamM CaCl2 . The hisectively . However*P<0.05) . The res2+ sparks are local and transient calcium release events from a cluster of RyRs in the SR. Delineating the properties of RyRs in hiPSC-CMs is thus a matter of fundamental importance to Ca2+ sparks. In the present study, the spark frequency FDHM and FWHM showed significant increase (P<0.05), whereas F/F0 was not significant changed after application of 50 nM ryanodine , when compared with control (2+ sparks in hiPSC-CMs.Ca control . These r2+ spark is an infrequent and stochastic elementary event of Ca2+ release 2+ sparks are often associated with the transverse tubules (TTs) at the Z-disk of a sarcomere where RyRs and L-type Ca2+ channels colocalize 2+ sparks may originate from the same RyR cluster 2+ sparks emerged at the same sites were observed in hiPSC-CMs. In contrast, such phenomenon has rarely been reported in adult quiescent ventricular myocytes In adult cardiac myocytes, Ca2+ sparks in hiPSC-CMs were not associated with Ca2+ transients or Ca2+ wave propagation throughout the cells  Ca2+ properties were observed among the cardiomyocytes (with same post cardiac differentiation time point) derived from all 4 lines, including the one presented in this study . Nevertheless, long-term following up studies were not performed due to low yield of cardiac differentiation.Electrophysiological property of pluripotent stem cell-derived CMs may vary due to culture duration of hiPSC-CMs 2+ sparks and documented their fundamental characteristics in hiPSC-CMs. We found that the Ca2+ sparks in hiPSC-CMs share similar temporal and spatial properties with adult cardiomyocytes. Moreover, RyRs are functioning in hiPSC-CMs and a majority of spontaneous Ca2+ sparks is L-type Ca2+ channel dependent. However, the Ca2+ sparks in hiPSC-CMs appear to be stochastic with a tendency of repetitive occurrence at some sites. Such phenomenon might be attributed to a heterogeneous array of RyRs due to the lack of T tubules or immature T-tubule system in hiPSC-CMs.In summary, we identified spontaneous CaFigure S12+]i by using ionomycin.Measurement of [Ca (A) Representative line scan (X-T) image of Ca2+ transients before and after the application of ionomycin and EGTA. (B) The fluorescent intensity profiles of Ca2+ transients in A. (C) The Ca2+ concentrations of spontaneous Ca2+ transients were calculated by using equation: [Ca2+]I = Kd[(F−Fmin)/(Fmax−F)]. Abbreviations: Kd, the dissociation constant value of a fluorescence; F, the measured fluorescence value; Fmax, the fluorescence value with 2 µM ionomycin; Fmin, the fluorescence value with Ca2+-free bath solution containing 5 mM EGTA.(TIFF)Click here for additional data file.Figure S22+ transients in rat cardiomyocytes.The characteristics of Ca A representative line-scan (X-T) image of Ca2+ transient recorded from field stimulated rat cardiomyocyte (top) and the corresponding intensity profiles (bottom) of Ca2+ transient. nrat = 5, ncell = 12. Abbreviations: F/F0, fluorescence (F) normalized to baseline fluorescence (F0).(TIFF)Click here for additional data file.Figure S32+ sparks in rat cardiomyocytes.The characteristics of spontaneous Ca (A) A representative line-scan (X-T) image of Ca2+ sparks recorded from rat cardiomyocytes. (B) A typical Ca2+ spark from the cells indicated by arrow in A. (C) The three-dimensional surface plot of the Ca2+ spark in B. (D) The spatial width of Ca2+ spark. (E) The duration of Ca2+ spark. Abbreviations: F/F0, fluorescence (F) normalized to baseline fluorescence (F0).(TIFF)Click here for additional data file.Figure S42+ transients in hiPSC-CMs.Effects of 5 µM nifedipine on spontaneous Ca Representative line scan (X-T) images (top) and the corresponding intensity profiles (bottom) of Ca2+ transients before and after the application ofnifedipine. nrat = 5, ncell = 13. Abbreviations: F/F0, fluorescence (F) normalized to baseline fluorescence (F0); s, seconds.(TIFF)Click here for additional data file.Table S1The percentages of hiPSC-CM subtypes and the action potential properties.(DOCX)Click here for additional data file.Table S22+ sparks in rat cardiomyocytes.Spatio-temporal properties of Ca(DOCX)Click here for additional data file.Table S32+ sparks in hiPSC-CMs derived from additional hiPSC lines derived from 3 healthy subjects.Characteristics of spontaneous Ca(DOCX)Click here for additional data file.Text S1Materials and Methods S1, Results S1, Discussion S1, References S1.(DOCX)Click here for additional data file.