Inducible urticaria is a heterogeneous subgroup of chronic urticarias caused by awide variety of environmental stimuli, such as exercise, cold, heat, pressure,sunlight, vibration, and water. A new term, follicular traction urticaria, wassuggested as an unusual form of inducible urticarias. We report a patient whowas diagnosed with follicular traction urticaria. The prevalence of inducible urticaria in the generalpopulation is approximately 0.5%.A 29-year-old woman presented to our outpatient clinic with a history of pruriticwheals. The lesions appeared on the skin as 2 mm to 5 mm wheals one year before.Urticarial papules appeared 5-15 minutes after waxing or using an electric epilatoron the legs and forearms, but the reaction was spontaneously resolved within one totwo hours. Eyebrow plucking with tweezers did not provoke lesions but causederythema and mild pruritus. The patient reported no spontaneous urticaria attacks orother type of urticaria. No history of drug intake or systemic disease wasreported.Urticarial lesions could not be reproduced by stroking with a blunt object on thepatient's flexor and extensor surface of the forearm and back, or by skin tractionor gentle hair traction. Reddish follicular urticarial papules appeared on threetests: within 10 minutes on a hair removal test with wax on the right forearm;within 15 minutes on a hair removal test with an electric epilator on the leg; andwithin 20 minutes after a hair removal test with non-woven plaster on the leftforearm (3x5 cm area)  and 2. PHelicobacter pylori stool antigen test was positive anderadication treatment was given. After the therapy, although the controlHelicobacter pylori stool antigen was negative, the symptomswere not resolved. The reaction could be highly inhibited by 20 mg rupatadine 30–60minutes before the hair removal and 20 mg rupatadine after the hair removal.Laboratory assessment revealed a normal complete blood count, erythrocytesedimentation rate, C-reactive protein levels, antistreptolysin O levels, antibodytiters , total immunoglobulin E(IgE), as well as renal, thyroid, and liver functions. Serological tests forhepatitis B and C were negative. Chest X-ray, throat culture, and stool examinationfor parasites were normal. Urinalysis was normal and urine culture was negative.Only the 4 Two or more different subtypes of urticaria can coexist in thesame patient.1Inducible urticaria is a heterogeneous subgroup of chronic urticaria, in which whealsare elicited by exogenous physical stimuli. Skin stroking, cold, heat, pressure,sunlight, water, exercise, and vibration are the common triggers.et al.3 hasrecently reported a rare form of inducible urticaria, traction urticaria, in whichwheals were believed to be mostly triggered by skin traction. However, they couldalso reveal follicular wheals by gentle hair traction.3 Özkaya and Yazganoğlu reported asimilar case of follicular traction urticaria.2 The present report agrees with Özkaya andYazganoğlu's study revealing that hair traction was the triggering factor forthe wheals. However, no urticarial lesions could be elicited by stroking the patientwith a blunt object in our case. Based on a literature search, this is the secondcase of follicular traction urticaria reported in Turkey and the third case in theliterature. Shelley and Shelley5reported a case of follicular wheals caused by firm strokes with the broad edge of atongue blade. The authors differentiated the lesions from follicular tractionurticaria and labeled them as follicular dermatographism.Gallo 5 revealed thatfirm strokes release the antigen from the bloodstream to trigger focal urticaria insites with a high density of mast cells, namely around the hair follicle. Therationale was that the traction of hairs might release a different kind of antigenfrom the hair structure that interacts with IgE-sensitized mast cells around thehair follicle.Shelley and ShelleyFollicular traction should also be considered among the triggers of inducibleurticaria. Hair traction or skin stroking differentiates the type of the follicularwhealing. We believe that when the number of patients with follicular tractionurticaria increases, guidelines for a subtype of inducible urticaria shall becomposed.


The heart consists of millions of cells, namely cardiomyocytes, which are highly organized in terms of structure and function, at both macroscale and microscale levels. Such meticulous organization is imperative for assuring the physiological pump-function of the heart. One of the key players for the electrical and mechanical synchronization and contraction is the calcium ion via the well-known calcium-induced calcium release process. In cardiovascular diseases, the structural organization is lost, resulting in morphological, electrical, and metabolic remodeling owing the imbalance of the calcium handling and promoting heart failure and arrhythmias. Recently, attention has been focused on the role of mitochondria, which seem to jeopardize these events by misbalancing the calcium processes. In this review, we highlight our recent findings, especially the role of mitochondria (dys)function in failing cardiomyocytes with respect to the calcium machinery. The heart pumps more than 60 times per minute, it is an exquisite electromechanical organ, where every contraction is activated and triggered with the preceding bioelectrical stimulus. The electro-mechanical coupling can also be reversible whereby a mechanical insult or stretch can generate bioelectricity via the well-known phenomena of mechanoelectric feedback ,2.Mechanoelectric feedback fine-tunes heart contractions so that every single heartbeat can be adjusted in response to the previous contraction. When this mechanosensation-based tuning is lost, arrhythmias and electrical instability occur .2+ signaling [Several colleagues have investigated mechanoelectric feedback in large animals and at the cellular level; however, due to the lack of adequate technologies, subcellular study is a very recent matter of investigation ,5,6. Theignaling . The ‘caignaling ,12,13,14ignaling ,16, receignaling , impulseignaling , and sigignaling .Cardiac remodeling can be also metabolic during heart failure progression and aging ,21. ReceMitochondria, subsarcolemmal or perinuclear , are orgThese metabolic changes occurring in ischemic and failing heart are also directly or indirectly participating in the arrhythmogenic processes ,23. We hCardiac pathophysiological remodeling includes mitochondrial function ,32 and mHence, the following questions emerged: does the alteration of microdomain-related mitochondrial alignment play a role in the genesis of arrhythmia? Is mitochondrial metabolism involved? The answer to these questions are thought-provoking as (i) the role of mitochondria in heart failure is probably underestimated and (ii) many conventional technologies cannot selectively activate or interrogate mitochondrial mechanosensitivity at the sarcolemmal microdomain level.Whatever the underlying cause of heart failure—ischemic, hypertensive, or idiopathic dilated cardiomyopathy—this highly prevalent disease, once present, progresses slowly, even if current medical therapy appears to be able to slow down its exacerbations and above all to improve its mortality . ChangesEven if fatty acids generate more ATP than glucose, this process needs more oxygen and therefore ATP production to oxygen consumption ratio is higher than that for glucose utilization. Elevated FA oxidation reduces cardiac efficiency. Fatty acid beta-oxidation rise was seen as an attempt to compensate for increased energy demand in cardiomyocytes in the early phases of heart failure development . In adva2+-ATPase (SERCA) [The integrity of mitochondrial energetics allows fine calcium regulation and tuning by cardiomyocytes at the subcellular level. Appropriate calcium handling is necessary not just for efficient mitochondrial phosphorylation but also for keeping the reactive oxygen and nitrogen species (ROS and RNS) generation under close control (c.f. paragraphs below) ,40. In a (SERCA)   and type 2 inositol 1,4,5-trisphosphate receptors (IP3R2), the most important intracellular Ca2+ release channels in the heart [2+ release via RyR2 and/or IP3R2 channels determines mitochondrial Ca2+ accumulation. Both mitochondrial Ca2+ excess and deficiency may result in altered mitochondrial function [Mitochondrial calcium handling is fundamental in ATP generation, NAD/NADH balance , and in he heart   possibly to increase the mitochondria Call death .+/Ca2+ or H+/Ca2+ exchanger or mPTP) systems [Another channel responsible for mitochondrial calcium handling is the calcium uniporter (mCU)  which al systems .Cardiomyocytes can generate ROS mainly from mitochondria, where a small percentage  of electrons do not complete the whole transfer from the series of redox reactions of the electron transport chain leading to oxygen leaks as precursors of ROS formation. Other enzymes widely present in cardiac cells—such as NAD(P)H oxidase, xanthine oxidase, and uncoupled nitric oxide synthase—participate in ROS generation . In addiIn physiological conditions, the activity of enzymes that organize intracellular calcium handling (SERCA) can be modified by tyrosine nitration or cysteine oxidation related to RNS generation ,59. MitoDuring cardiac diseases, such as in experimental models of arterial hypertension and heart failure, as well as in heart failure patients ,56, high2+ in heart failure can partially depend on the oxidation of the RyR that increases sarcoplasmic calcium leakage during diastole and reduces intrasarcoplasmic calcium content [The increased concentration of intracellular Ca content . The dep content . Diastol content .We directly interrogated subsarcolemmal mitochondria by combining scanning ion conductance microscopy (SICM) together with optical mapping of impulse propagation, with the intent to apply a hydrojet pressure via the SICM nanopipette with nanometer precision to investigate the subcellular mechanisms underlying mechanically-induced intracellular calcium release in heart failure  as descr2+ transient during and after the release of the hydrojets, we observed that in AMC control cells the Ca2+ transient initiated and was confined around the mechanical stimulation site while in 16-week MI cells Ca2+ transient initiated and propagated in the entire cell  cardiomyocytes. Because HF cells denote unstriated areas compared to AMC cells  Figure A we obseire cell C followiKnowlton et al., demonstrate that localization of subsarcolemmal mitochondria is altered in heart failure, specifically because those organelles undergo a fission/fusion processes . To moniIn age-matched control (AMC) cardiomyocytes, TMRM-labeled mitochondria align preferentially with crests with periodic arrangements, and reflect regular arrangement of Z-grooves and T-tubule openings  as shown2+ efflux from the mitochondria has been observed by Belmonte et al. [2+ induced-Ca2+ release process. Interestingly, they observed an activation of mitochondrial Ca2+ induced-Ca2+ release both in atrial and ventricular cells. To note, this mechanical intervention does not result only in a mitochondrial Ca2+ efflux. Very recently, Prosser et al., were the first to demonstrate that a physiologic stretch rapidly activates reduced-form nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) to produce reactive oxygen species (ROS) in a microtubules-dependent process via X-ROS signaling [2+ transient (2+ propagation (observed in 69% of cases). Those data are not only in agreement with those found in HF cells  but also with commotio cordis experiments where ventricular fibrillation was provoked with a chest impact [Pressure flow inducing Cae et al.  independignaling ,70,71. Bransient A. Intereransient B,C and ii ; however, during myocardial infarction, such networks are destabilized by the upregulation of the tubulin protein family (not show here) [2+ for MiCai we have blocked LTCC with Nifedipine  . Among tow here) . The disow here)  is limitfedipine D; howeveIn this review, we have focused on mitochondria which seem to jeopardize cardiovascular function due to their displacement/relocation and fusion in failing cardiomyocytes.2+ signals could be accumulated to contribute to cardiomyocyte energy/signaling derangement.This review drew attention to the fact that mitochondria may play a more important role in a local environment. It is possible that, under certain pathological conditions, the local ROS, X-ROS, and Cai initiation and propagation) at single cell levels can be transposed to the mechanically induced arrhythmia encountered in vivo  [2+ waves underline arrhythmias, what we described here in terms of mitochondria relocation and Ca2+ handling may be involved as a novel arrhythmogenic substrate. Pharmacological action on cardiac mitochondria can be of upmost importance especially in the context of improving the function of a failing heart [2+ overload [Dissociation of excitation–metabolism coupling plus mitochondrial displacement during HF emphasizes a possible role of a ‘fine tuning’ role in cardiac physiology and pathology. Arrhythmia is a macroscale phenomenon; the mechanisms herein described  ,75,76. Iternans) . Thus, ang heart  when conoverload , low-dosoverload . In concoverload ,81,82. MProvokingly, when we talk about mitochondria—either on prokaryotic or eukaryotic cells—we always link those organelles with metabolisms. This is not the case as a recent publication in Nature demonstrates how mitochondria act as messengers between astrocytes and neurons . The sam


They include the Ca2+-selective Orai1 and members of the transient receptor potential canonical (TRPC) channels, which are activated by the endoplasmic reticulum Ca2+ sensor STIM1. The scaffolding protein Homer assembles SOC complexes, but its role in VSMCs is not well understood. Here, we asked whether these SOC components and Homer1 are present in the same complex in VSMCs and how Homer1 contributes to VSMC SOCs, proliferation, and migration leading to neointima formation. Homer1 expression levels are upregulated in balloon-injured vs. uninjured VSMCs. Coimmunoprecipitation assays revealed the presence and interaction of all SOC components in the injured VSMCs, where Homer1 interacts with Orai1 and various TRPC channels. Accordingly, knockdown of Homer1 in cultured VSMCs partially inhibited SOCs, VSMC migration, and VSMC proliferation. Neointimal area was reduced after treatment with an adeno-associated viral vector expressing a short hairpin RNA against Homer1 mRNA (AAV-shHomer1). These findings stress the role of multiple Ca2+ influx channels in VSMCs and are the first to show the role of Homer proteins in VSMCs and its importance in neointima formation.The molecular components of store-operated Ca Components of SOCs include the Orai family of channels and the transient receptor potential subfamily C (TRPC) channels, and both channels are gated by the ER Ca2+ sensor STIM14. STIM1 has an N-terminal, ER-resident EF hand that binds Ca2+ and a SAM domain that participates in clustering of STIM1 upon ER Ca2+ store depletion. The C-terminal, cytosolic portion of STIM1 consists of an inhibitory coiled-coil domain (CC1) that interacts with the V-shaped STIM1 Orai-activating region (SOAR)6. SOAR is followed by the C-terminal inhibitory domain (CTID) that allows access of the STIM1 regulator SARAF to SOAR, a serine/proline region, and a polybasic, lysine-rich domain (K-domain)7.Receptor-evoked Ca8 that functions as a highly Ca2+-selective, inward-rectifying channel and is the CRAC (Ca2+ release-activated Ca2+) pore-forming unit. Orai1 is activated by SOAR when STIM1 oligomerizes and accumulates at ER-PM junctions to bind to Orai1 after ER Ca2+ store depletion. The function and regulation of the TRPC channels by STIM1 have been documented in multiple cell types and for most TRPC channels. STIM1 interacts directly with TRPC1, TRPC4, and TRPC5, and indirectly with TRPC3 and TRPC6 by heteromultimerizing (a) TRPC1 with TRPC3 and (b) TRPC4 with TRPC6, allowing STIM1 to regulate TRPC3 and TRPC61. STIM1 SOAR interacts with both the N- (NT) and C-terminal (CT) TRPC1 coiled-coil domains (CCDs). When TRPC1 is in complex with TRPC3, cell stimulation facilitates the interaction of SOAR with the CT CCDs of TRPC1 and TRPC39. However, SOAR is not sufficient to activate TRPC channels, and gating of TRPC channels by STIM1 requires an additional electrostatic interaction of two conserved end lysines on STIM1 with two complementary aspartates/glutamates in the C-terminus of TRPC channels10.Orai1 is a hexamer of four transmembrane-spanning units3 receptors (IP3Rs) to form a TRPC1/3-Homer-IP3R complex11. This complex stabilizes TRPC channels and prevents their spontaneous activation and Ca2+ influx in resting cells. Homer also regulates the translocation and retrieval of TRPC3 to and from the plasma membrane in response to ER Ca2+ store levels and IP3R activity12. In agonist-induced platelet aggregation, Homer is involved in STIM1-Orai1 interaction mediating SOC entry (SOCE)13. Homers are scaffolding proteins that, in addition to TRPC channels and IP3Rs, bind to several Ca2+-signaling proteins 15. Homer1 was initially identified in neurons at the postsynaptic density to regulate dendrite morphogenesis and synaptic plasticity16. Homer1a, the founding isoform discovered as a neuronal immediate early gene, lacks the multimerizing coiled-coil domain and functions as a negative regulator of the long Homers17. Both Homers have EVH domains that bind to consensus proline-rich sequence (PXXF) present in target Ca2+-signaling proteins18.TRPC channels are also regulated by Homer proteins. We have previously shown that Homer binds to multiple family members of TRPC channels as well as to IP19. The subsequent extracellular matrix production and neointima formation result in narrowing of the lumen, leading to occlusive arterial disease. It is clear that STIM1 and Orai1 are critical components of SOCE in VSMC proliferation and migration, as revealed by their knockdown using siRNA21. TRPC1 has also been shown to be a significant component of proliferative VSMC SOCs since application of either siTRPC1 or anti-TRPC1 antibody attenuates SOCE23. In addition, TRPC3, TRPC4, and TRPC6 are also major components of Ca2+ influx in arterial smooth muscle cells24. Targeting individual Ca2+ influx channels for therapy may raise a problem due to redundancy and compensation. An alternative approach is to target a non-essential protein that is a common component of all channels, such as the scaffolding protein Homer1. However, how the Homer proteins function in SOCs and SOC-dependent activities in VSMCs is not known.Store-operated calcium entry (SOCE) is a major contributor of vascular smooth muscle cell (VSMC) phenotypic changes that lead to their proliferation and migration. In its normal state, VSMCs play a critical role in maintaining the integrity of the vessel wall and controlling vascular tone. VSMCs are normally quiescent, differentiated, and contractile. When the blood vessel wall is damaged from underlying factors, such as atherosclerosis, diabetes, and hypertension, VSMCs undergo pathological changes and dedifferentiate, proliferate, and migrate into the vessel lumenIn the present work, we examine if Homer1 is required for VSMC SOCE, migration, and proliferation leading to neointima formation and stenosis. We report that expression of Homer1, along with STIM1, Orai1, and TRPC channels, is increased in the neointima after balloon injury in the rat carotid artery. Knockdown of Homer1 significantly reduces SOCE, VSMC migration and proliferation, and neointima size. These findings identify a key role for Homer1 in SOC function in injured VSMCs, resulting in occlusive arterial disease and suggest Homer1 as a preferred therapeutical target.26, STIM1, Orai1, TRPC1, TRPC4, and TRPC6 expression levels are increased in injured medial and neointimal, proliferative VSMCs  , using right carotid arteries as negative control Fig. . Neointid’) Fig. . We alsod’) Fig. . Notablyels Fig. .Figure 12+ influx is toxic in many cells under different stressors, and it is expected that the cells will adapt to protect against the increased Ca2+ influx. An established native regulator of Ca2+ influx is Homer117. Homer1 complexes TRPC channels to IP3 receptors to keep the channel in the closed state11, and interacts with TRPC channel C-terminal PXXF motif located next to the residues mediating gating of TRPC channels by STIM111. Therefore, next, we examined if Homer1 expression levels increase, similar to STIM1, Orai1, and TRPC channels, and whether they interact with these proteins in the complexes. Homer1 protein was not detected in uninjured vessels, but its expression level markedly increases in injured VSMCs  after store depletion using the SERCA pump inhibitor cyclopiazonic acid (CPA) in the absence of external Ca2+. Cells treated with siSTIM1 and/or siHomer1 have reduced STIM1/Homer1 mRNA  compared to control (scrambled siRNA), similar to a reduction in SOCE seen in cells treated with siSTIM1 (54.1 ± 2.8%)  in injured VSMCs, contributes to SOCE. Rat aortic VSMCs were isolated and cultured to allow phenotypic modulation to proliferative and migratory state that occurs during culturing and is similar to what happens in balloon-injured VSMCs. Hallmarks of this change include elevated SOCE leading to this increased rate of proliferation and migration8%) Fig. . Notably8%) Fig. . VSMCs t2+ influx channel regulating PDGF-BB-induced VSMC migration. It is well established that receptor complexes are formed with specific complement of signaling proteins29. Our findings suggest that TRPC3 is the main Ca2+ influx channel activated by the PDGF receptors in VSMCs30.Next, we determined if Homer1 has any significant effects on VSMC migration. A scratch wound assay was done to determine VSMC migration in response to 100 ng/mL PDGF-BB. Cultured VSMCs were transfected with siHomer1, siSTIM1, siTRPC1, siTRPC3, siTRPC4, or siTRPC6 and incubated for 72 hrs, with the scratch wound made 15 hrs before analysis. Wound closures were analyzed postscratch to determine contribution from VSMC migration. Representative scratch wound analysis at 0 and 15 hrs postscratch are shown in Fig. VSMC proliferation was assessed in response to 10% serum in complete media. Cultured VSMCs were transfected with siHomer1, siSTIM1, siTRPC1, siTRPC3, siTRPC4, or siTRPC6, and plated onto 96-well plate. MTT assay was performed, and absorbance readings of solubilized formazan, whose amount of insoluble form is directly proportional to the cell number, were taken at 570 nm. Cells treated with siHomer1 proliferate significantly less (73.3 ± 4.2%) compared to control (scrambled siRNA) Fig. . Signifiin vivo situation by examining the role of Homer in neointima formation. After balloon injury on the left carotid artery, adeno-associated virus (AAV) encoding shScrambled (shScr) or shHomer1 was infused in the injured segment of the common carotid artery for 30 min and then aspirated out. Immunofluorescence staining was done on these injured arteries treated with the respective AAVs as well as on the right intact (uninjured) arteries. To assess the effects of Homer1 knockdown on neointima formation, we analyzed the (1) size of the neointimal area and (2) the immunofluorescence intensities for Homer1, Ki-67, and MHC expression levels normalized to DAPI (DNA fluorescence) stain (intensity ratio). Ki-67 is a marker for cell proliferation, while MHC is a marker for contractile smooth muscle cells. The results are shown in Fig. Homer1 stain: shHomer1: 0.02 ± 0.01 AU vs. shScr: 3.13 ± 0.49 AU; Ki-67 stain: shHomer1: 2.83 ± 0.96 AU vs. shScr: 6.39 ± 1.11 AU)  Fig. . Also, i2+ entry, migration, and proliferation, resulting in neointima formation. The biochemical and functional findings lead to several conclusions: (a) Homer1 expression levels are upregulated in neointimal VSMCs after balloon injury to carotid arteries; (b) Homer1 and STIM1 each interact with TRPC channels and Orai1 in these injured VSMCs; (c) knockdown of Homer1 in cultured VSMCs results in significant reduction of SOCE and in VSMC migration and proliferation; (d) knockdown of both Homer1 and STIM1 in cultured VSMCs results in an additive, significant reduction of SOCE; (e) in vivo knockdown of Homer1 in balloon-injured arteries reduces neointimal area and the expression of the cell proliferative marker, Ki-67, arising from VSMC migration and proliferation. Taken together, these data indicate the significance of Homer1 in occlusive arterial disease.In summary, this study identifies and establishes the role of Homer1 in vascular smooth muscle cell store-operated Ca13. Thus, upregulation of Homer1 and SOCE appears to be a feature of structural and functional changes in these two cell types. Future studies aimed at mechanisms of Orai and TRPC channel regulation by Homer should reveal the significance of Homer in VSMC SOCE. The additive effect of Homer1 and STIM1 knockdowns suggests that Homer1 can regulate SOCE independent of STIM1. The effects of Homer on SOCE and VSMC migration and proliferation may not be limited through its interactions with TRPC channels, Orai1, and IP3 receptors, and we cannot rule out the possibility of Homer interacting with other cellular proteins. However, it is likely that Homer mediates SOCE by forming a complex with the key SOC components: STIM1-Orai1-TRPCs-IP3Rs.That Homer1 binds to Orai1 in balloon-injured arteries and is required for VSMC SOCE suggests that Homer1 can regulate Orai1 as well as TRPC channel activity in migratory and proliferative VSMCs. Previously, Homer1 has been shown to interact with STIM1 and Orai1 in human platelets, and sequestration of Homer using a proline-rich peptide attenuates thrombin-induced SOCE and platelet aggregation3 receptors to ER-PM junctions in dendritic spines32. Consequently, Homer1 optimizes Ca2+ signaling, ER Ca2+ store refilling, and glutamate release33. In traumatic neuronal injury, downregulation of Homer1 is neuroprotective by suppressing glutamate excitotoxicity resulting from Ca2+ overload35. A similar protective mechanism and scenario seems to occur in injured arteries when Homer1 is downregulated, with the end result being suppression of VSMC SOCE, migration, and proliferation and reduction in neointima size.We have demonstrated that downregulation of Homer1 acts as a protective mechanism against neointima formation and occlusive arterial disease. In astrocytes and neurons, Homer1 has been shown to relocate ER tubules to junctional sites in proximity to the plasma membrane and to recruit IPIn conclusion, Homer1 downregulation can be considered for future therapeutic treatment of occlusive arterial disease.® antibodies from ThermoFisher used in this study include Donkey anti-Rabbit IgG (H + L) (488) (Cat #A21206), Donkey anti-Mouse IgG (H + L) (488) (Cat #A21202), Donkey anti-Rabbit IgG (H + L) (568) (Cat #A10042), and Goat anti-Rabbit IgG (H + L) (568) (Cat #A21069).All the siRNA used in the present study were obtained from Integrated DNA Technologies. Rat siRNA sequences were used and are listed in Supplementary Table 36. Briefly, an embolectomy was performed using a 2F Fogarty balloon (Edwards Lifesciences) that is inserted through a small arteriotomy in the external carotid artery and passed into the common carotid artery (CCA). The balloon was inflated at 2 atm pressure, and the catheter was partially withdrawn. The balloon was then deflated, and the catheter was inserted again through the CCA. This balloon injury procedure was done a total of three times. AAV-scrambled or AAV-Homer1 shRNA was infused in the injured segment of the CCA for 30 min and then aspirated out. After recovery from operation and anesthesia, the rats received a postoperative dose (10 mg/kg) of the oral analgesic Rimadyl (carprofen). Sham-operated animals were subjected to a similar procedure but without balloon insertion and injury.All procedures for maintaining the rats and for the balloon injury procedure were performed in accordance with NIH guidelines and regulations and approved by the Institutional Animal Care and Use Committee of the University of North Texas Health Science Center. Male Sprague-Dawley rats (~400–450 g) (Charles River Laboratories) were anesthetized with ketamine (100 mg/kg) via intraperitoneal injection, and balloon angioplasty was carried out as previously describedRats were anesthetized using 1–3% isoflurane, then perfused with 1X PBS and fixed with 4% paraformaldehyde. Right (uninjured) and left (injured) carotid arteries were dissected out and embedded in paraffin using Leica EG1150 embedder. Arteries were then sectioned (5 μm) on a Leica RM2255 microtome. Sections were air-dried, and H&E staining was performed using standard protocols.Rats were anesthetized using 1–3% isoflurane, then perfused with 1X PBS and fixed with 4% paraformaldehyde. Right (uninjured) and left (injured) carotid arteries were dissected out, fixed with 4% paraformaldehyde, and placed in cryoprotective embedding medium OCT (Tissue-Tek). Arteries were then frozen and sectioned (20 μm) at −20 °C in a Leica CM1950 cryostat. Sections were air-dried, and immunofluorescence staining was performed using standard protocols. Morphometric analysis of each arterial segment was performed using ImageJ software. Neointimal and medial areas were assessed by tracing and measuring the borders of the lumen and internal and external elastic laminas on anti-Ki-67-stained images taken from a 4× objective. Scale was converted from pixels to mm using a 1-mm scale bar, and these numbers (mm) were used to calculate the neointima/media ratio and the relative neointimal area (%). The average neointimal area of balloon-injured arteries treated with AAV-shScr was arbitrarily set to 100%, and the average neointimal area of shHomer1-injured arteries was calculated as a percentage of the area for AAV-shScr-treated injured arteries.g at 4 °C for 20 min. For the co-immunoprecipitation experiments, anti-STIM1 or anti-Homer1 antibody (1 μg) was added to cell extract (100 μl) and incubated for 2 h at 4 °C. Then, 50 μl of a 1:1 slurry of protein A Sepharose 4B (Sigma) or Protein G Plus Agarose (Pierce-ThermoFisher) beads were added to the antibody-extract mix and incubated for an additional 1 h at 4 °C. Beads were washed three times for 10 min with RIPA buffer, and proteins were released from the beads with 50 μl of SDS-loading buffer. Half of the eluted protein (25 μl) was loaded onto 8% or 10% tris-glycine SDS–PAGE gel under denaturing and reduced conditions. The gel was then transferred onto PVDF membrane for Western blot analysis.The carotid arteries were longitudinally cut, and the media and neointima were harvested after peeling off the adventitia. Tissues were lysed using 500 μl of RIPA buffer: 50 mM Tris-HCL, pH 8 containing 1 mM NaVO3, 10 mM NaPyrophosphate, 50 mM NaF, 150 mM NaCl, 0.2 mM EDTA, 1% Triton X-100, 0.5% Na-deoxycholate, and 0.1% SDS. The extracts were sonicated, and insoluble material was spun down at 30,000 × 2 and cervical dislocation. The descending aorta was dissected out and placed in ice-cold, sterile 1X PBS solution. After fat tissues, blood clots, and endothelium were removed in DMEM followed by 1X PBS wash, the artery was incubated in 0.6 mg/mL papain solution for 30 min at 37 °C, 5% CO2, and 100% humidity. The adventitia was stripped away to expose the vascular smooth muscle layer, which was subsequently cut up into pieces and incubated in a collagenase cocktail-DMEM solution containing 1 mg/mL each of collagenase II and H for 45 min at 37 °C, 5% CO2, and 100% humidity, with twice homogenization of VSMCs using syringe and 18G-1-1/2 needle in the incubation period. VSMCs were resuspended in 45% DMEM, 45% F12, and 10% FBS supplemented with L-glutamine, and poured through a cell strainer before incubation in 35-mm dish at 37 °C, 5% CO2, and 100% humidity.Rats were euthanized by CO® Transfection System (Invitrogen) was used to transfect siRNA and Homer1 cDNA into VSMCs, according to the supplier’s recommendation. Briefly, VSMCs were grown to 100% confluency in 35-mm dish . The cells were trypsinized, washed in 1X PBS solution, and resuspended in R buffer to a final volume of 120 μL. One μg of siRNA was mixed in with VSMCs to a final volume of 10 μL. For the Homer1 rescue experiment, 250 ng of Homer1 cDNA was added to the siHomer1-VSMC mix. Using a 10-μL Neon tip, the VSMC-siRNA mix was electroporated at Pulse Voltage: 1475 V; Pulse Width: 20 ms; Pulse Number: 2. The cells were then plated on fibronectin-coated coverslip (Ca2+ measurement), 35-mm dish (migration assay), or 96-well plate (proliferation assay), and incubated at 37 °C, 5% CO2, and 100% humidity for 72 hours before start of experiment. Passage #’s 8–15 were used for VSMCs in all groups, and results are compared within the same passage #’s.The Neon2. Cells attached to cover glass that formed the bottom of a perfusion chamber were perfused with prewarmed (37 °C) bath solution containing (in mm): 140 mm NaCl, 5 mm KCl, 1 mm MgCl2, 10 mm HEPES, 1 mm CaCl2, 10 mm glucose, adjusted to 310 mosm, pH 7.4. Chemicals were diluted in standard or Ca2+-free bath solution that contained 1 mm EGTA. [Ca2+]i was measured at the 340- and 380-nm excitation wavelengths. The emitted fluorescence was collected by digital camera (Nikon) at 510 nm and analyzed using the Elements software (Nikon). Results of SOCE from cells treated with siRNA are presented as a percentage of control (scrambled siRNA) 340/380 ratio. Passage #’s 8–15 were used for VSMCs in all groups, and results are compared within the same passage #’s.VSMCs were loaded with Fura-2 at 5 μm final concentration and incubated for 1 hour at 37 °C, 5% CO2 in culture media containing 0.4% FBS with or without 100 ng/mL PDGF-BB (R&D Systems). Brightfield images were captured at 10X magnification (Nikon) at 15 and 24 hours after scratch wound, and analyzed using ImageJ. The total area of migrated VSMCs in the wound was calculated and normalized to control (scrambled siRNA). Results of migration from cells treated with siRNA are presented as a percentage of control. Passage #’s 8–15 were used for VSMCs in all groups, and results are compared within the same passage #’s.After transfection with siRNA, VSMCs were cultured for 48 hours to form a monolayer. A 100-μL pipette tip was used to scrape the bottom of the dish, and the wound area was washed with 1X PBS. Cells were incubated at 37 °C, 5% CO® MTT Cell Proliferation Assay Kit (Invitrogen) was used to determine cell number according to the supplier’s recommendation. Briefly, 10 μL of 12 mM MTT were pre-mixed with 100 μL of phenol-red-free DMEM and, together, replaced the growth media. Cells were incubated at 4 hours, 37 °C, 5% CO2 so that the MTT can form the insoluble formazan, whose amount is directly proportional to the cell number. The formazan was then solubilized by addition of 100 μL of SDS-HCl solution provided in the kit and incubation for another 4 hours at 37 °C, 5% CO2 to form a color whose absorbance is read at 570 nm. Results of the proliferation from cells treated with siRNA are presented as a percentage of control (scrambled siRNA). Passage #’s 8–15 were used for VSMCs in all groups, and results are compared within the same passage #’s.After transfection with siRNA and plating onto 96-well plate, VSMCs were cultured for 72 hours. The Vybrantt-test or by one-way analysis of variance (ANOVA).Results are given as mean + SEM and significance was analyzed by Student’s Supplementary Information


V2.1 channel activity plays a key role in interneuronal communication and synaptic plasticity. SNAREs interact with a specific synprint site at the second intracellular loop (LII‐III) of the CaV2.1 pore‐forming α1A subunit to optimize neurotransmitter release from presynaptic terminals by allowing secretory vesicles docking near the Ca2+ entry pathway, and by modulating the voltage dependence of channel steady‐state inactivation. Ca2+ influx through CaV2.1 also promotes channel inactivation. This process seems to involve Ca2+‐calmodulin interaction with two adjacent sites in the α1A carboxyl tail (C‐tail)  site), and contributes to long‐term potentiation and spatial learning and memory. Besides, binding of regulatory β subunits to the α interaction domain (AID) at the first intracellular loop (LI‐II) of α1A determines the degree of channel inactivation by both voltage and Ca2+. Here, we explore the cross talk between β subunits, Ca2+, and syntaxin‐1A‐modulated CaV2.1 inactivation, highlighting the α1A domains involved in such process. β3‐containing CaV2.1 channels show syntaxin‐1A‐modulated but no Ca2+‐dependent steady‐state inactivation. Conversely, β2a‐containing CaV2.1 channels show Ca2+‐dependent but not syntaxin‐1A‐modulated steady‐state inactivation. A LI‐II deletion confers Ca2+‐dependent inactivation and prevents modulation by syntaxin‐1A in β3‐containing CaV2.1 channels. Mutation of the IQ‐like motif, unlike CBD deletion, abolishes Ca2+‐dependent inactivation and confers modulation by syntaxin‐1A in β2a‐containing CaV2.1 channels. Altogether, these results suggest that LI‐II structural modifications determine the regulation of CaV2.1 steady‐state inactivation either by Ca2+ or by SNAREs but not by both.Modulation of Ca Indeed, syntaxin‐1A/1B, SNAP‐25, and synaptotagmin‐1 specifically interact with CaV2.1 and CaV2.2 channels by binding to a synaptic protein interaction site (synprint) located within the intracellular loop connecting domains II and III (LII‐III) of the channels   and distinct human α1A constructs containing either a LI‐II deletion around the A454 residue, mutations in the IQ‐like region, or a CBD deletion. Our results reveal a cross talk between different pathways involved in the modulation of CaV2.1 inactivation, showing that regulation by syntaxin‐1A of the human CaV2.1 channel activity requires both the integrity of α1A LI‐II and the lack of a Ca2+‐dependent component in the channel steady‐state inactivation.To better understand the role of LI‐II in Ca2+ (CaV2.1) channel α1A subunit  was a gift from Professor J. Striessnig . cDNAs of the rabbit α2δ and rat β3 and β2a regulatory subunits, and syntaxin‐1A (subcloned into a pcDNA3 expression vector) were gifts from Dr. L. Birnbaumer  and Dr. J. Blasi . CaV2.1 α1A mutant subunits  were generated using site‐directed mutagenesis . All cDNA clones used in this study were sequenced in full to confirm their integrity.cDNA of the human voltage‐gated Caμg DNA/dish) , β3 or β2a, α2δ, and EGFP (transfection marker) cDNA constructs were used at a ratio of 1:1:1:0.3. When required, syntaxin‐1A was also cotransfected at the same ratio as CaV2.1 channel subunit cDNAs. Electrophysiological recordings were obtained from EGFP‐positive cells 24–48 h after transfection at room temperature (22–24°C).HEK 293 cells were transfected using a linear polyethylenimine (PEI) derivative, the polycation ExGen500  as previously reported  through WT or mutant CaV2.1 channels containing β3 or β2a regulatory subunits were recorded in the whole‐cell configuration of the patch‐clamp technique, using a D‐6100 Darmstadt amplifier . Pipettes had a resistance of 2–3 MΩ when filled with a solution containing (in mmol/L): 140 CsCl, 1 EGTA (intermediate Ca2+‐buffering condition) or 10 BAPTA (high Ca2+‐buffering condition), 4 Na2ATP, 0.1 Na3GTP, and 10 Hepes (pH 7.2–7.3 and 290–300 mOsmol/L). The external solution contained (in mmol/L): 140 tetraethylammonium‐Cl (TEACl), 3 CsCl, 2.5 CaCl2, 1.2 MgCl2, 10 Hepes, and 10 D‐glucose (pH 7.4 and 300–310 mOsmol/L). Previous work demonstrates that high levels of intracellular Ca2+ chelators  impair CaV2.1 inactivation in a similar way as when replacing extracellular Ca2+ by Ba2+  was used for pulse generation, data acquisition, and subsequent analysis.Ca2+ currents in response to a 50 ms  depolarizing test pulse (to +20 mV) from a holding of −80 mV, following 30‐sec steps to various holding potentials (conditioning pulses) between −80 and +20 mV   and slope factor (kinact) for steady‐state inactivation: Steady‐state inactivation was estimated by measuring peak Can represents the number of cells recorded for each experimental condition. Statistical significance was tested using one‐way Analysis of Variance (ANOVA) followed by a Bonferroni post hoc test. Differences were considered significant if P < 0.05. All statistical comparisons were performed using the GraphPad Instat software. All data are sampled from Gaussian  distributions (tested using the method Kolmogorov and Smirnov).Data are presented as the means ± SEM, and 2+ currents (ICa2+) through wild‐type (WT) CaV2.1 channel containing the regulatory β3 subunit (WTβ3), was measured at intermediate (1 mmol/L EGTA) and high (10 mmol/L BAPTA) intracellular Ca2+‐buffering conditions to evaluate its calcium dependency. In both conditions, syntaxin‐1A expression favored channel steady‐state inactivation, as indicated by a significant left shift of V1/2 inact to more negative voltages (by ~5–9 mV)   Ca2+‐dependent. On one hand, ICa2+ inactivation was reduced (with a significant ~8 mV right shift in the V1/2 inact) by increasing the buffering of intracellular Ca2+  by increasing intracellular Ca2+ buffering to abrogate the novel LI‐II deletion‐induced Ca2+‐dependent component of inactivation  presented a Ca2+‐dependent component   when increasing intracellular Ca2+ buffering  at the calmodulin‐binding IQ‐like motif  was only present when the Ca2+‐dependent component of the steady‐state inactivation of β2a‐containing CaV2.1 channels was removed, either by increasing intracellular Ca2+ buffering  site of the β2a‐containing CaV2.1 channel (ΔCBDβ2a) did not eliminate the Ca2+‐dependent component of ICa2+ steady‐state inactivation, and V1/2 inact was still significantly shifted to less negative values (by ~9 mV) when increasing intracellular Ca2+ buffering  under high intracellular Ca2+ buffering  regulatory β subunits through their interaction with the α interaction domain (AID) located at the first intracellular loop (LI‐II) of the CaV2.1 pore‐forming α1A subunit  of α1A , reduces voltage‐dependent inactivation  downstream the AID, around the A454 residue  did not abolish the Ca2+‐dependent component of cardiac channel inactivation , and the subsequent alteration of the interaction pattern between them and/or with their interacting partners , remains to be elucidated. However, there is evidence that make this hypothesis plausible since it has been reported that N‐tail, intracellular loop between domains III and IV (LIII‐IV) and C‐tail regions of CaV2.x or CaV1.2 α1 subunits modulate channel inactivation through direct and dynamic interactions with LI‐II, or indirectly via regulatory β subunits  structure of the rabbit CaV1.1 complex, containing the pore‐forming α1S and the regulatory α2δ1, β1a, and γ subunits, has been recently resolved with high, near‐atomic (3.6 Å) resolution   could not be resolved  α1 channel subunits, which do not contain the consensus synprint site, biochemically interact with syntaxin‐1A and SNAP‐25 at the carboxy‐terminal domain  determine the modulation of CaV2.1 steady‐state inactivation either by Ca2+ or by SNAREs but not by both.In conclusion, our data suggest that conformational modifications of The authors declare that no conflict of interests exists.In memory of Gemma G. Genè, PhD (1977–2017).


Training investigators for the rapidly developing field of implementation science requires both mentoring and scientific collaboration. Using social network descriptive analyses, visualization, and modeling, this paper presents results of an evaluation of the mentoring and collaborations fostered over time through the National Institute of Mental Health (NIMH) supported by Implementation Research Institute (IRI).Data were comprised of IRI participant self-reported collaborations and mentoring relationships, measured in three annual surveys from 2012 to 2014. Network descriptive statistics, visualizations, and network statistical modeling were conducted to examine patterns of mentoring and collaboration among IRI participants and to model the relationship between mentoring and subsequent collaboration.p = 0.001). More specifically, mentoring was significantly related to future collaborations focusing on new research (p = 0.009), grant submissions (p = 0.003), and publications (p = 0.017). Predictions based on the network model suggest that for every additional mentoring relationships established in 2012, the likelihood of a scientific collaboration 2 years later is increased by almost 7 %.Findings suggest that IRI is successful in forming mentoring relationships among its participants, and that these mentoring relationships are related to future scientific collaborations. Exponential random graph network models demonstrated that mentoring received in 2012 was positively and significantly related to the likelihood of having a scientific collaboration 2 years later in 2014  contains supplementary material, which is available to authorized users. Americans with mental disorders receive sub-optimal care due to formidable challenges in the implementation, sustainability, and scale-up of evidence-based treatments , 2. DissInvesting in the Future,” proposed a “phenotype” of the NIMH researcher of the future: transdisciplinary scientists, team players in a collaborative scientific enterprise, and translators [phenotypes needed to carry out the type of research needed to reduce the research-practice gap in mental health; it emphasized that “mentoring is essential” and called for “national mentoring networks,” to achieve NIMH objectives [Training investigators for the rapidly developing field of implementation science requires both mentoring and collaboration. The National Institute for Mental Health (NIMH) has prioritized team science and mentoring in all efforts to prepare the research workforce. A 2008 NIMH council workgroup report, “nslators . The repjectives . Mentorijectives , 7. Burnjectives , 10.Responding to the 2008 report, the National Institutes of Health (NIH) supported training programs in dissemination and implementation (D&I) research by placing heavy emphasis on mentoring, both from faculty to training participants and between trainees themselves. Since 2009, at least three training programs focused in implementation science have been pairing trainees with mentors with the goals of expanding the D&I research community and advancing the intellectual capital of this still developing field in the USA. The Implementation Research Institute was the first training institute focused on mental health implementation science . It was An interest in team science is increasing in many areas of science, but scientific collaboration and cross-disciplinary partnerships are of particular interest to D&I research , 15.  funding, 43 fellows in four cohorts were trained at IRI. Fellows attended two annual weeklong trainings at Washington University in St. Louis, traveled for a site visit on still-in-the-field implementation projects, attended implementation science conferences, and received research pilot funding. Further description of IRI can be found elsewhere .The fundamental design of IRI was to provide strong mentorship in D&I science to support new scientific collaborations, with an overall goal of stimulating IRI fellows’ production of D&I scholarly products such as papers, presentations at conferences, and grants. Thus, the program was designed as a learning collaborative in implementation science, for both fellows and faculty members . Based oMentoring at IRI was done by matching each fellow with one of the core faculty members. One innovative aspect of IRI, compared to the other training methods in D&I at the time, was the monthly mentoring via phone or video conference calls for a 2-year period by core faculty to IRI fellows. The requirement that each fellow also have a mentor at his or her home institution focused on grant writing and career development enabled the IRI core faculty mentors to focus specifically on the fellow’s development of research grants in implementation science.The central component in the training—the 5-day summer institute—was shaped to foster networking and collaboration. We required in-residence immersion for 5 days and nights and used small group sessions for faculty-to-fellow and fellow-to-fellow feedbacks on the scholarly products that fellows worked to advance during the week. We fostered informal networking by providing dinners in and off site, and convening IRI gatherings during the year, specifically at annual D&I conferences . While fThe design of our training was also guided by a team science approach . The gramentoring networks across three IRI cohorts;Describe and map the collaboration relationships among the IRI network (fellows and faculty) as they develop during the time of the IRI funding; andSimilarly describe and map the Explore and model the relationship between mentoring and subsequent scientific collaborations.This paper presents findings on the link between mentoring and new collaborations in implementation science, employing social network analysis. Social network analysis is suited to examine the development of collaboration in the IRI, given the centrality of mentoring and team science to NIMH-supported research training. This evaluation study seeks to answer the extent to which strong mentoring relationships are associated with increased scientific collaboration. Specifically, the goals of this paper are toThis is an evaluation study that examines the dynamics of mentoring and collaboration among participants of the IRI from 2012 to 2014. The study uses network data collected from all IRI participants during each summer in-person training workshop. Three types of network ties were collected: frequency of contact, receiving or providing mentoring, and type of scientific collaboration.Fellows (43 across four cohorts) were scholars who were selected through a competitive national application process and attended two annual training workshops. Core faculty were the core set of D&I senior scholars who directed and managed the IRI workshops and provided the most active and direct mentoring to the fellows. The institute had seven core faculty for the first 2 years (2012 and 2013); core faculty were increased to nine in 2014 with support from a National Institute on Drug Abuse (NIDA) supplement. Expert faculty were a larger set of researchers who had particular expertise relevant for IRI training. The set of core faculty remained mostly constant throughout the lifetime of the IRI initiative , while expert faculty generally attended only one of the annual workshops. Core faculty stayed in residence during each summer institute, but expert faculty members were invited to participate for 1 or 2 days and to be in residence for only one night [The IRI participants played one of three roles. ne night .An IRI participant survey was developed to collect network information from all IRI participants each year. The survey was introduced and described to the attending IRI members at each summer institute. Both current and former institute participants were invited to participate each year. The survey was administered with Qualtrics, an online web-based survey platform .Contact: “Please indicate which of the following people you know and how frequency you are in contact with them on the list below: (1) I don’t know this person, (2) I know this person but we have no contact, (3) I am in contact with this person yearly, (4) I am in contact with this person monthly, (5) I am in contact with this person weekly,” followed by a list of the names of all individuals who had participated in IRI so far. Only the names of the individuals participants indicated being in contact with at least yearly appeared in the next two questions. Mentoring: “On the list below please indicate which individuals have either mentored you or been mentored by you in the past year: (1) I mentored them, (2) They mentored me, (3) Neither.” Collaboration: “Please check any of the collaboration activities you have engaged in with the following individuals during the past year: (1) Worked together on developing new research, (2) Submitted a grant, (3) Presented research results, (4) Published a paper, (5) None of these.” Working together on developing new research was designed to capture the early stages of research collaboration, before formal grants are submitted or results are disseminated. Member pairs were considered linked if one or both indicated a relationship, following established social network data management protocols [The three network items were based on items that have been used successfully in past network studies  with slirotocols . Thus, lIn addition to the three network items, participants were asked to indicate what scientific discipline best described their scholarly work and expertise, based on the NIH Field of Training list . From anstatnet (version 2014.2.0), igraph (0.7.1), and ergm (3.24) packages in R  were used to build and test predictive models of network ties among the IRI fellows and core faculty . All net version 014.2.0, and ergm .24 packaA total of 94 people participated in IRI from 2012 to 2014, including 9 core faculty, 42 expert faculty, and 43 fellows. Response rates for the surveys were 80 % (2012), 76 % (2013), and 80 % (2014). Basic descriptive summaries of mentoring and collaboration suggest that the IRI design was working effectively. One hundred percent of IRI fellows reported receiving some type of mentoring each year, with the average number of received mentoring relationships ranging from 5.8 (in 2013) to 6.5 (2012). Similarly, all fellows reported some type of collaboration with others  for each of the 3 years. Average number of collaborations ranged from 6.5 in 2012 to 8.0 in 2014.Figure across disciplines relative to within disciplines (53 %). This interdisciplinary tendency increased slightly in subsequent waves . (Detailed results not reported here but are available from the authors.)Table Figure Figure One of the primary assumptions of the IRI program is that scholarly productivity of IRI fellows will be accelerated by fostering mentoring relationship built during IRI activities, and a primary mechanism by which mentoring affects productivity is via establishment of new and effective scientific collaborations. In this section, we report analytic results focusing on the relationship between mentoring and collaboration among IRI participants.Simple bivariate correlations between mentoring received and reported total collaborations range from a low of 0.37 (for mentoring and collaborations reported in 2013) to a high of 0.70 for mentoring received in 2012 and collaborations reported in 2014. Figure These simple network and bivariate analyses are suggestive, but we can test our hypothesis of mentoring leading to collaboration more explicitly by using new stochastic network modeling techniques. Exponential random graph models are a relatively new technique that combines maximum likelihood estimation with network simulations to be able to build statistical models of networks . ERGMs aSame discipline captures the homophily effect of discipline (when both IRI participants have the same discipline) on the likelihood of observing a collaboration tie. Role-fellow captures the simple main effect of role, in this case being an IRI fellow. Finally, GWESP  is a network structural term that captures the patterns of transitivity in the observed network. Transitivity is the common social pattern of closure, where if one person is connected to two other people , then there is an increased probability that the two other people are also connected. Local structural covariates such as GWESP are typically included in ERGMs as they improve their stability and increase the fit of the models to the observed data [Table ved data . Model 2p = 0.001). In addition to interpreting the patterns and sizes of the fitted parameters, the quality of an ERGM can be assessed by examining its goodness-of-fit. Goodness-of-fit diagnostics were run to assess how closely a set of 1000 simulated networks based on model 2 match the observed IRI 2014 collaboration network on four different network characteristics: minimum geodesic distance (compactness), the edgewise shared partner distribution, the degree distribution, and the triad census (pattern of triangles). For a good fit, the value of the statistic (such as number of nodes with degree = 1) of the observed network should fall within an empirical 95 % confidence interval calculated from the simulated model-based networks. Out of 61 network statistics, 58 of them fall within the confidence bounds, suggesting that model 2 fits the data well; and that with just five predictors, our ERGM is able to produce predicted networks that look very much like the 2014 IRI collaboration network. The results of models 1 and 2 both show that there is a positive discipline homophily effect on collaboration; collaboration is more likely to be observed between two IRI members from the same discipline . Also, being an IRI fellow reduces the likelihood of collaboration—this simply reflects the high level of collaboration among the small number of core faculty. The GWESP term is also significant, suggesting that there is transitivity among the IRI members. After controlling for these covariates, model 2 finds that mentoring received in 2012 has a significant and positive relationship with the likelihood of collaboration in 2014  probability range. Figure These results demonstrate the utility of scientific training that explicitly connects mentoring by experienced implementation scientists with subsequent scientific collaboration among trainees and other scientists. First, the network analysis demonstrates that the IRI developed a tight-knit community of D&I research scholars. The program was successful in forming mentoring relationships among IRI participants, with mentoring ties strongest between core faculty and IRI fellows, consistent with program design. To our knowledge, the IRI was the first implementation science researcher training program that provided direct mentoring activities over an extended period of time . This novel training approach can serve as a model for other federally funded scientific training initiatives.Investing in the Future (2008): that mentoring is one of the elements essential for the development of a successful research career, and mentoring should be directed toward developing researchers who can work collaboratively [Second, we found evidence for a connection between mentoring and scientific collaboration of all types. The SNA results demonstrate a sizeable impact of the mentoring relationships on future scientific collaborations, with every additional mentoring relationship predicting increases in new scientific collaborations on grants, presentations, and publications. These findings support a key principle in the NIMH National Advisory Council’s Workgroup on developing the research workforce, ratively . CollaboStudies of the outcomes of large research and training initiatives, such as the TTURCs, the Transdisciplinary Research on Energetics and Cancer centers (TREC), and the Clinical and Translational Science Awards (CTSA), have steadily moved from early descriptive and case study work to more recent attention to core aspects of team science , 32. InvThe study reported here has a number of notable design and analysis strengths, including longitudinal data collection, multiple overlapping cohorts, measuring scientific collaboration both generally and specifically , and using statistical network modeling to establish the quantitative relationship between mentoring and collaboration. This is the first study, to our knowledge, to model the relationship between mentoring and scientific collaboration. Moreover, it reflects the utility of network analysis for studying mentoring and collaboration specifically and for enhancing D&I science more generally .However, a number of design and analytic limitations should be kept in mind when interpreting these results. The mentoring and collaboration measures were based on self-report and there is the potential for some social desirability bias. In fact, when asked about mentoring relationships, we did not (a) control for context  because the entire IRI network—alums and fellows—responded to the same survey at the same time nor (b) ask for mentoring frequency. Future studies may disentangle when and how often mentoring occurs to understand the details of the mentoring process and how it affects collaboration among members of the network. This was an observational study, so the causal relationship between mentoring and collaboration cannot be firmly established absent a control group.  More specifically, we cannot specify the extent to which collaboration ties were the result of mentoring within IRI or the result of other mentoring that occurred outside of IRI as well as other non-mentoring aspects of the program such as instruction in D&I research methods. The small number of core faculty involved in IRI across all the years raises questions of generalizability and scalability. Would the same relationships be found with a different set of faculty? Also, although we found that scientific collaborations among IRI fellows and core faculty were slightly more likely to be interdisciplinary in nature over the course of the trainings, this was not examined in detail in the statistical models that were focused on mentoring and collaboration. This will be examined in more detail in future work.The strong scientific collaboration network and high impact mentoring demonstrated in the first few years of the IRI provide an important foundation for future training. The IRI renewal, recently awarded by the NIMH, will support 5 years (2016–2021) of training deliberately designed as a mentoring collaborative. We will continue to assess the development and impact of mentoring ties, extending our analysis to examine not only collaboration but scientific productivity and other markers of career success, such as leadership to the field of D&I science.


Lactobacillus and Bifidobacterium have been successfully used to promote growth and prevent diseases. Previous reports have demonstrated that Bacillus subtilis (B. subtilis) was a potential probiotic for animals. In this research, 180 B. subtilis were isolated from the soil, identified, and investigated in vitro. Furthermore, five B. subtilis were selected and mixed to investigate their effect on growth performance, immune response, intestine microbiota, and disease resistance in rabbits. Rabbits with a diet of 106 CFU g−1 mixed B. subtilis exhibited the best growth performance and higher serum IgG and IgA than controls (P < 0.05). Moreover, dairy with B. subtilis can promote the balance of intestinal flora. The major proinflammatory factor and β-defensin were upregulated compared with the controls. After 7 weeks of feeding, the survival rate of the rabbits fed with B. subtilis was significantly higher than those in the control groups postinfected with Escherichia coli. At the same time, this study detected higher expression of β-defensin and reduced bacteria contents of the heart and cecal contents from the diet mixed with B. subtilis compared with the control groups. In conclusion, dietary supplementation with B. subtilis for rabbits could improve growth performance, intestinal homeostasis, and immune organ index and enhance innate immune response as well as disease resistance. These findings showed that the induction of β-defensin by B. subtilis might be an interesting new therapeutic strategy to strengthen innate defense mechanisms.Probiotics such as  Bacillus spp. are considered to be suitable probiotics (Bacillus subtilis (B. subtilis) DJM-51 isolated from soil demonstrated strong antagonism toward the tomato pathogen Clavibacter michiganense subsp. michiganense ATCC 7429  infection and rabbit Clostridium perfringens disease  and incubated at 37°C for 24–72 h. Suspected colonies were subjected to Gram stain and biochemical tests, including an arabinose biochemical tube test, glucose biochemical tube test, mannitol biochemical tube test, 3% NaCl gelatin biochemical tube test, and MR-VP biochemical tube test. Following those tests, 16S rRNA sequencing was performed with universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′), and the sequences were blasted in NCBI.E. coli, was previously isolated from clinically infected rabbits developing acute diarrhea and stored by the Environmental Microbiology Laboratory at Shandong Agricultural University.The bacterial pathogen, enterohemorrhagic B. subtilis. Overnight cultures of B. subtilis were centrifuged at 3,000 × g for 10 min, washed twice with phosphate buffer saline, and resuspended in a new liquid medium (108 CFU mL−1). The new media were adjusted to pH 2.5 by adding a hydrochloric acid solution with 1% pepsin or to pH 7.2 with 1% trypsin and incubated at 37°C. At 0, 1, and 2 h of incubation, 100 μL of the cultures were removed and spread on modified nutrition agar for cell number estimation. Respectively, the bacterial suspensions were inoculated into modified nutrition broth with 0.3% bile salt and cultured at 37°C for 24 h. Optical density at 600 nm (OD600) was measured and compared to a control culture without bile salts.This study was designed as in a model gastrointestinal tract experiment to investigate the survival of B. subtilis strains were centrifuged at 6,000 × g for 15 min, and then the supernatant was assayed for protease activity according the SB/T 10317-1999  was incorporated into agar (1% w/v) plates, which were mixed for uniformity and poured onto plates to solidify. Overnight cultures of B. subtilis (108 CFU mL−1) were transferred to holes (5-mm diameter) punched into the agar palates. The plates were then incubated as anaerobic or aerobic at 37°C for 24 h, depending on the target strain, and the antimicrobial ability was recorded as being in the inhibition zone . The formula of the basic diet for each group is shown in Table The healthy weaned New Zealand White rabbits (35 days) were kept in the same environment with sufficient room, feed, and ventilation. The rabbits were fed with a basal diet supplemented with 0, 102.Rabbit kidney (RK-13) cells were grown in DMEM  containing 10% fetal bovine serum  at 37°C in 5% COThe cecal contents bacterial DNA of each sample was extracted using HiPure Stool DNA Kit B  following the manufacturer’s instructions. The DNA extractions were quantified by ultraviolet spectroscopy and amplified using universal primers of 341F (CCTACGGGNGGCWGCAG) and 806R (GGACTACHVGGGTATCTAAT) to target the V3–V4 domain of bacterial 16S rRNA. The amplicons were normalized, pooled, and sequenced on the Illumina GAIIx platform.The raw Illumina fastq files were quality-filtered, de-multiplexed, and analyzed using quantitative insights into microbial ecology. Sequences with more than one ambiguous nucleotide or within correct barcodes or primers were removed. The Ribosomal Database Project classifier was used to classify tags into different taxonomies against Greengenes Database (version 20101006) with confidence threshold of 0.5. The software Mothur was used to cluster tags of more than 97% identity into operational taxonomic units (OTUs), and then the abundances of OTUs were calculated.TransZol Up , and 1 µg total RNA was reverse-transcribed with HiScript® II Q Select RT SuperMix for qPCR (+gDNA wiper) . Quantitative real-time PCR (qRT-PCR) oligonucleotide primers for immune-related genes and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed using Primer 3 software (http://bioinfo.ut.ee/primer3-0.4.0/), based on the published GenBank sequence  and the Applied Biosystems 7500 Fast Real-Time PCR System . The qRT-PCR conducted in a total volume of 20 µL and the amplification steps consisted of 94°C for 30 s, 40 cycles of denaturation at 94°C for 5 s, and extension 60°C for 34 s, and a dissociation curve analysis. The purified PCR products were cloned into the pMD18-T and sequenced to verify correct amplification. Each sample was performed in triplicate. Data were calculated based on the 2−ΔΔCt method. The relative expression mRNA was normalized to GAPDH.Total RNA was extracted from the spleen and jejunum of rabbits using ce Table . qRT-PCRU test or the Kruskal–Wallis test. The survival rate of rabbits was analyzed using the Kaplan–Meier method. Statistically significant differences required that P < 0.05.Statistical analyses were performed with SPSS 19.0, and one-way analysis of variance was used to identify differences among the groups. When variances were not homogeneous, the data were analyzed by the non-parametric Mann–Whitney Bacillus strains with the typical volcano shape were selected as subjects for biochemical experiments. In total, 180 B. subtilis were isolated from the soil and confirmed by 16S rRNA sequencing. Five strains  exhibited excellent probiotic properties in vitro, including artificial gastric juice, intestinal juice, and bile salt tolerance (Table B. subtilis were susceptible to the common antibiotics tested (data not shown). The 16S rRNA sequence of five B. subtilis was deposited in GenBank (Genbank no. KY750231-235).According to colony morphology and the results of microscopic examination, Gram-positive ce Table . BYS2, B6 CFU g−1B. subtilis showed the best growth performance and were heavier (P < 0.05) than the control diet rabbits. Rabbits fed a diet with 107 CFU g−1B. subtilis showed higher growth performance than the controls, but the difference was not significant (P > 0.05). However, no significant differences were observed between rabbits fed with 105 CFU g−1B. subtilis diets and the controls after 4, 5, 6, and 7 weeks of feeding  serum IgG and IgA than the controls. However, the serum IgA of rabbits fed with 107 CFU g−1B. subtilis were significantly different (P < 0.05) than the controls after only 5 and 6 weeks of feeding  was increased after 5 weeks of feeding. The percentage of Bacteroides  and Clostridium  were significantly decreased after 5 and 7 weeks of feeding.High-throughput sequence analysis of bacterial 16S rRNA V3–V4 region was conducted on cecal contents of rabbits after 5 and 7 weeks of feeding. As shown in Figure B. subtilis, the expressions of innate immune-related genes were examined in the spleen and jejunum of rabbits fed with the probiotic compared to the controls after 5 and 7 weeks of feeding. Figure P < 0.05). The expression of NOD-like receptor (NLR) C3 and NLRX1 was generally higher in the jejunum than in the spleen  in the jejunum after 7 weeks of feeding. The antiviral proteins OAS showed no significant difference , which were not obviously changed after stimulation with killed cells.To further explore the induction of innate immunity by B. subtilis was significantly higher than the controls after the E. coli challenge. At 1, 2, and 3 days postinfection (dpi), the survival rate of rabbits fed B. subtilis was 79.2, 57.8, and 50.6%, respectively, which were higher than the control group  E. coli content in their cecal contents compared to the controls at 3 dpi.As shown in Figure P < 0.05; Figure P < 0.05; Figure P < 0.05; Figure P < 0.05; Figures P < 0.05; Figure As shown in Figure Bacillus cultures were examined microscopically, and no more than 5% of free spores were observed. Therefore, the remarkable resistance of five B. subtilis strains to gastric intestinal juice and bile salt conditions can not be due to the presence of spores. Bacillus sp. can produce certain essential nutrients and extracellular enzymes, as well as provide necessary growth factors to promote host growth  promoted animal growth and immune regulation (Bacillus circulans (2 × 106 CFU g−1 diet) significantly enhanced the growth performance and non-specific immunity levels and disease resistance of fish . PRRs have been implicated in pathogen control and elimination and have been recently identified as key mediators of inflammatory and immune responses. There are few reports about NLRs in rabbits, and only the predicted sequences are available in Genebank. At present, the full lengths of some NLRs in rabbits have been obtained, and their biological functions (no published data) have been verified. In this study, the dietary administration of ytokines , 35. A pytokines . Olive f defense . IL-1β i defense . The maiodentium , 40. It odentium . In thisB. subtilis. On the one hand, defensins can directly act on pathogenic microorganisms and more importantly participate in the innate immunity as well as the initiation and regulation of adaptive immune response. Previous study has demonstrated that lactobacilli and the VSL#3 bacterial mixture restored intestinal barrier function through the upregulation of human-beta defensin 2 via induction of proinflammatory pathways of the nuclear factor-κB (Bacillus showed stronger ability in induce innate immunity than the killed cells when used the same dose in the RK-13 cells. To the best of our knowledge, this research is the first report on the innate immune response to B. subtilis in rabbits.Previous studies have suggested that several probiotics induce innate immunity through β-defensin 2 induction in Caco-2 cells . The antactor-κB . HoweverB. subtilis was higher than that of the controls after they were challenged with E. coli. This conclusion was supported by increased body weights, serum immunoglobulin, and immune organ index and enhanced expression of major innate immunity genes involved in initiating and regulating immune response against E. coli. It is worth noting that β-defensin was significantly upregulated in the B. subtilis feeding group postinfection with E. coli, especially DEFB 114, DEFB 134, and DEFB 135. Given that β-defensin has a strong antibacterial ability, the higher expression of β-defensin may be the most important reason for disease resistance. Meanwhile, the present results showed that the number of E. coli in the cecal contents significantly declined with a diet of B. subtilis during the test days. As various reports have shown, several bacilli have established antimicrobial properties against various Gram-positive and Gram-negative pathogenic bacteria (E. coli in vitro and therefore possess potent immunostimulatory capacities. It is also important that the intestinal flora is mainly composed of anaerobic bacteria, and relatively few aerobic bacteria. B. subtilis can inhibit the growth of pathogenic E. coli by consuming oxygen in the intestine and regulating the balance of intestinal flora. Previous research showed that B. subtilis PY79hr can inhibit colonization and persistence of Salmonella enteritidis, C. perfringens, and E. coli (B. subtilis may prevent the overgrowth of E. coli, enhance the resistance to E. coli invasion of the intestinal tract, and ameliorate disease processes. As a result, a diet including B. subtilis decreased the concentration of E. coli in vivo. B. subtilis may be competitive exclusion approaches in the control of infection with enteric pathogens.In return, the survival rate of rabbits fed bacteria . In this E. coli . In the B. subtilis that were selected for in vitro experiment showed good probiotic potential in rabbits. Enhancing the disease resistance of rabbits fed with B. subtilis may due to the probiotic’s influence on the improved growth performance, serum immunoglobulin, immune organ index, intestinal homeostasis, and immune response of rabbits, as well as its antibacterial benefits. The data from this study enhance the understanding of the mechanism of action between probiotics and the host, allowing for the development of probiotic-based strategies to prevent pathogenic infection.In conclusion, the five MG and FW designed and conducted the study, performed most of the experiments, and wrote the manuscript. GH and RL performed the calculation with support from QQ. FW and NL collected samples. TC and LW discussed the results and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We monitored mPTP opening using the calcein/cobalt fluorescence technique in adult rat and wild type or cyclophilin D (CypD) knock-out mice cardiomyocytes. Cells were either treated with Ca2+ ionophores or subjected to hypoxia followed by reoxygenation. The ionophores induced mPTP-dependent swelling in isolated mitochondria. A23187, but not ionomycin, induced a decrease in calcein fluorescence. This loss could not be inhibited by CypD deletion and was explained by a direct interaction between A23187 and cobalt. ETH129 caused calcein loss, mitochondrial depolarization and cell death but CypD deletion did not alleviate these effects. In the hypoxia-reoxygenation model, CypD deletion delayed both mPTP opening and cell death occurring at the time of reoxygenation. Thus, Ca2+ ionophores are not suitable to induce CypD-dependent mPTP opening in adult murine cardiomyocytes. Hypoxia-reoxygenation conditions appear therefore as the most reliable model to investigate mPTP opening in these cells.Opening of the mitochondrial permeability transition pore (mPTP) plays a major role in cell death during cardiac ischaemia-reperfusion. Adult isolated rodent cardiomyocytes are valuable cells to study the effect of drugs targeting mPTP. This study investigated whether the use of Ca Its opening causes mitochondrial swelling, loss of membrane potential, ATP depletion and outer membrane permeabilization leading to cell death. The precise components of the pore remain putative. Recent data provided evidence in support of a role for the F1F0 ATP synthase in pore formation6 although there is no agreement in the mechanism of pore formation. Nevertheless, a common agreement considers that the soluble protein cyclophilin D (CypD) located within the mitochondrial matrix is the main regulator of mPTP as CypD lowers the Ca2+ threshold required to elicit mPTP opening. Indeed, excess intramitochondrial Ca2+ is a crucial factor to trigger mPTP opening7. This is the reason why numerous studies have used Ca2+ ionophores to induce mPTP opening in a variety of cells11. This is a useful approach to study the effect of drugs interacting with mPTP and the search for new mPTP inhibitors remains a major pharmacological objective.Necrosis is a prevalent form of cell death that contributes to cell injuries in a number of diseases. This is particularly relevant during cardiac ischaemia-reperfusion2+ ionophores, A23187, ionomycin and ETH129 might represent useful tools to study mPTP opening/inhibition in excitable cells such as cardiomyocytes where Ca2+ fluxes have a critical role. On one hand, A23187 and ionomycin are carboxylic acid ionophores which equilibrate Ca2+ concentrations across biological membranes and thus between the different compartments of the cell in an electroneutral manner12. On the other hand, ETH129 is a neutral compound which binds Ca2+ with a high specificity over other divalent cations. The complex formed carries a positive charge and permeates membranes in response to negative membrane potential14. We used the calcein loading CoCl2 quenching technique15 to monitor mPTP opening and compared the effect observed in adult cardiomyocytes isolated from rat as well as wild type and CypD knock-out mice. We observed that among the three ionophores tested, ionomycin was without effect on calcein or TMRM fluorescence and that A23187 and ETH129 modified the distribution of calcein in cardiomyocytes independently from CypD and thereby are not valid tools to monitor mPTP in isolated cardiomyocytes. In contrast, hypoxia-reoxygenation was responsible for a CypD-dependent mPTP opening as demonstrated by the loss of mitochondrial calcein and the subsequent cell death. This setting appeared as the most suitable model to investigate mPTP opening in cardiomyocytes.The aim of this study was to investigate whether three Ca2+ influx and no swelling could be observed after addition of a high Ca2+ concentration (200 µM). A23187 (1 µM) and ionomycin (10 µM) allowed Ca2+ to equilibrate across mitochondrial membranes and triggered a slow mitochondrial swelling  and antimycin A (1 µM) in order to inhibit mitochondrial respiration. In these conditions, the lack of mitochondrial membrane potential limited Caing Fig. . This sw2+ influx in cells. Mitochondrial compartment of isolated adult cardiomyocytes were preloaded with Rhod-2 at 37 °C for 40 min, and then supplemented with 0.5 µM A23187 or 10 µM ionomycin. Figure 2+ confirming the ionophoretic properties of the drugs.Then, we verified the ability of both ionophores to induce mitochondrial Ca2+ ionophores to induce mPTP opening in isolated rat adult cardiomyocytes. The cells were co-loaded with 1 µM calcein in the presence of CoCl2 and TMRM and incubated at 37 °C. After 5 min incubation, the addition of 0.5 µM A23187 resulted in an immediate decrease in calcein fluorescence . Hence, we hypothesized that the decrease in calcein fluorescence was the consequence of an interaction between A23187 and cobalt instead of Ca2+. The experiment of Fig. 2+ influx was inhibited when cardiomyocytes were pre-treated with 1 mM CoCl2. Therefore in our experimental conditions (Tyrode’s medium containing 1.3 mM Ca2+) A23187 might transport cobalt across mitochondrial membranes inducing the quenching of calcein fluorescence inside mitochondria without triggering mPTP opening.To study the mechanism of calcein release from mitochondria, we performed experiments in a Tyrode’s medium devoid of Ca187 Fig. . Moreove187 Fig. . As EGTA2+ ionophores to induce mPTP opening in cardiomyocytes was confirmed using ionomycin. Although like A23187, high concentrations of ionomycin have the capacity to elicit a CypD-dependent mitochondrial swelling in isolated cardiac mitochondria  led to cell death (Tcell death50 = 56 ± 16 min)  Fig. . All thei.e., suggesting a CypD-independent mPTP opening process.To confirm this effect, the same experiments were duplicated in cardiomyocytes isolated from CypD knock-out mice. In these cells the absence of CypD was assumed to inhibit mPTP opening. Figure 2+. The absence of Ca2+ in the incubation medium did not modify the effect of 20 µM ETH129 on calcein and TMRM fluorescence  in order to deplete sarcoplasmic reticulum Ca2+ stocks22. In these conditions, the effect of ETH129 was associated with a delay in calcein loss as compared to control cells , ETH129 did not modify mitochondrial membrane potential. Treatment of energised mitochondria with increasing concentrations of ETH129 (10–50 µM) resulted in a concentration-dependent depolarization and at 50 µM, ETH129 immediately collapsed membrane potential as effectively as FCCP Fig. . Thus, E1/2 = 63 ± 17 min) that preceded propidium iodide labelling of cells by a few minutes  and propidium iodide labelling. For instance, after 120 min reperfusion, whereas 80% of cardiomyocytes were still alive in the CypD deleted cells, nearly all the cells were dead in wild type cells. This demonstrates that these experimental conditions allow to monitor a CypD-dependent mPTP opening, leading to cell death in mouse cardiomyocytes.The conditions required to induce mPTP opening are those encountered by a mitochondria when an ischemic organ is subjected to reperfusion and this can be mimicked in cells using a sequence of hypoxia-reoxygenation. Figure tes Fig. . The del2 quenching technique based on the monitoring of the mitochondrial fluorescence of calcein allowed the investigation of mPTP opening at the single cell level24. This technique has been used to evaluate mPTP opening in different cells and pathological situations29 but also to investigate the regulation of the pore. Different biophysical methods or pharmacological agents modulating oxidative stress or Ca2+ fluxes have been used to induce mPTP opening31.Numerous experimental protocols have been described to monitor mPTP opening in mitochondria isolated from different organs but the demonstration of its occurrence in cells is more difficult. The development of the calcein loading CoCl2+ ionophores to induce mPTP opening in cardiomyocytes which would represent a simple tool to identify potential mPTP inhibitors. We used freshly isolated murine adult cardiomyocytes because they are fully differentiated and can be electrically paced, i.e., a more physiological setting than cultured cells. We investigated the effects of two families of Ca2+ ionophores. The first one includes A23187 and ionomycin which belong to the carboxylic acid ionophores. Such compounds allow the equilibration of Ca2+ concentrations across biological membranes by exchanging one Ca2+ for 2 H+ in an electroneutral manner. The second one, ETH129, is a neutral compound which binds Ca2+ with a high specificity over other divalent cations. The complexed form of ETH129, which carries a positive charge, permeates membranes in response to negative potential. Several studies demonstrated that ETH129 transports Ca2+ across biological membranes14 and induces mPTP opening in mitochondria isolated from Saccharomyces cerevisiae22 but to our knowledge, ETH129 has never been used in mammalian cells.In the present study, we have evaluated the use of Ca33. Our experiments suggest that A23187 is able to transport cobalt across membranes and therefore the decrease in calcein fluorescence is due to mitochondrial cobalt entry and not to mPTP opening in our experimental conditions. However a number of studies have used these drugs to trigger mPTP35 and in the present study, isolated de-energised cardiac mitochondria treated with A23187 or ionomycin underwent a CsA-sensitive swelling. A possible explanation for this discrepancy is that the mitochondrial matrix Ca2+ concentration reached in the presence of these drugs is not sufficient to induce mPTP opening in cardiac mitochondria. Indeed, experiments performed with isolated cardiac mitochondria demonstrate that they are very resistant to Ca2+ overload and able to take up huge Ca2+ concentrations before mPTP opening in contrast to mitochondria issued from other organs such as liver36. The greater resistance of cardiac mitochondria to Ca2+ overload might be linked to the contractile function of the heart which constantly involves large Ca2+ fluxes. It is also known that a moderate increase in intramitochondrial Ca2+ concentration influences energy production by stimulating several Kreb’s cycle enzymes. Therefore, cardiac mitochondria should be able to handle larger amount of Ca2+ to supply sufficient energy for contractile adaptation. This hypothesis is in accordance with previous data showing that interfibrillar mitochondria, which produce ATP for contractile function, are more resistant to Ca2+ overload than subsarcolemmal mitochondria37.Our data demonstrate that A23187 and ionomycin were unable to trigger mPTP opening in cardiomyocytes. They did not alter mitochondrial membrane potential or cell shape. The immediate and large calcein loss from cardiomyocytes challenged with A23187 was unrelated to mPTP opening. Indeed, this calcein loss could not be inhibited either by CsA treatment or CypD deletion. These results also rule out the possibility that calcein loss could be due to transient mPTP opening2+ stores as sarcoplasmic reticulum depletion with ryanodine greatly delayed calcein fluorescence decrease. These results support the existence of a rapid and permanent mPTP opening and are consistent with the ability of ETH129 to elicit mPTP opening in isolated mitochondria. Nevertheless, neither CsA treatment nor CypD deletion limited the decreases in calcein and TMRM fluorescence induced by ETH129 in cardiomyocytes. This shows that the effect of ETH129 is independent from a CypD-dependent mPTP opening process. However, it is well-established that CypD only facilitates mPTP opening. mPTP can form and open in the absence of CypD or in the presence of CsA when the inducing stimulus is sufficient38. This is probably the case with ETH129 which provokes a fast and high mitochondrial Ca2+ overload and concomitantly uncouples mitochondrial membrane potential. Indeed membrane depolarization was shown to facilitate mPTP opening although the mechanisms involved remain uncertain40.Contrasting with electroneutral ionophores, ETH129 induced a delayed and concentration-dependent hypercontracture of cardiomyocytes. This was associated with a loss of mitochondrial membrane potential preceding calcein loss by a few minutes. After calcein loss, cells quickly take up propidium iodide dye evidencing cell death. This might rely on intracellular Ca2+ overload was the first factor described to activate mPTP opening41 and is still considered as essential. However, other cellular regulatory factors such as oxidative stress, adenine nucleotide depletion or elevated phosphate concentrations are involved and proceed by enhancing the sensitivity of the mPTP to Ca2+4. This is why a cellular model that takes into account these regulatory factors seems more relevant to study mPTP opening. Indeed, the reoxygenation of hypoxic cardiomyocytes is clearly associated with a CypD-dependent mPTP opening. This protocol has the advantage to combine Ca2+ overload with other factors such as oxidative stress which are known to enhance the mPTP sensitivity to Ca2+. Moreover, the cellular conditions generated by the hypoxic period  facilitate mPTP opening during the reoxygenation phase.This study also reveals that hypoxia reoxygenation is an excellent model to highlight mPTP opening in cardiomyocytes. Matrix Ca2+ ionophores are useful tools to induce mPTP opening in some cells. However, they cannot be used to trigger a CypD-dependent mPTP opening in cardiomyocytes either because the mechanism of Ca2+ transport at the level of the mitochondrial membrane is not effective enough  or the agent is too powerful (ETH129) and leads to a non-CypD regulated mPTP opening corresponding to what we observed during severe hypoxia-reoxygenation. In addition, the results of the present study demonstrate that the hypoxia-reoxygenation model is a suitable setting to highlight and investigate mPTP opening in cardiomyocytes.In conclusion, CaAll compounds were purchased from Sigma-Aldrich  unless specified.Ppif−/− mice) were obtained from Jackson Laboratories . Animals were housed in an air-conditioned room with a 12 h light–dark cycle and received standard rodent chow and drinking water ad libitum. All animal procedures used in this study were in accordance with the Directives of the European Parliament (2010/63/EU-848 EEC) and were approved by the Animal Ethics Committee Afssa/ENVA/Universite Paris Est Creteil .Male C57BL/6J mice (8 to 10 week-old) and male Wistar rats (250–300 g) were purchased from Janvier . CypD knock-out mice  using a Polytron homogeniser. Samples were then further homogenised for five consecutive times in a Potter-Elvehjem glass homogeniser at 1500 rpm. The homogenate was centrifuged at 1000 g for 5 min at 4 °C. The supernatant was centrifuged at 10000 g for 10 min at 4 °C. The mitochondrial pellet was resuspended in homogenization buffer without EGTA and protein concentration was determined using the Advanced protein assay reagent .Male Wistar rats were used for the preparation of cardiac mitochondria as previously described540) using a Jasco V-530 spectrophotometer equipped with magnetic stirring and thermostatic control. In a first set of experiments, mitochondrial swelling induced by A23187 and ionomycin was measured in deenergised mitochondria. Experiments were carried out at 30 °C in a swelling buffer . Mitochondria (0.5 mg/mL) were incubated in the presence of rotenone, antimycine (1 µM each) and 200 µM Ca2+. After 30 s, swelling was induced by addition of 1 µM A23187 or 10 µM ionomycin.Mitochondrial swelling was assessed by measuring the change in absorbance at 540 nm (A2PO4 (pH 7.4 at 30 °C) in the presence of pyruvate/malate (5/5 mM), ruthenium red (10 µM) and 200 µM Ca2+. After 30 s, swelling was induced by the addition of 10 µM ETH129. When used, cyclosporin A (CsA) was introduced at the beginning of the incubation period.In a second set of experiments, the effect of ETH129 on mitochondrial swelling was measured in energised conditions. Mitochondria (0.5 mg/mL) were incubated at 30 °C in a respiration buffer containing 100 mM KCl, 50 mM sucrose, 10 mM HEPES, 5 mM KHMitochondrial membrane potential was evaluated by the uptake of the fluorescent dye rhodamine 123 which accumulates electrophoretically into energised mitochondria in response to their negative inner-membrane potential. Rhodamine 123 (0.2 µM) and the respiratory substrates pyruvate/malate (5/5 mM) were added to respiration buffer contained in a cuvette maintained at 30 °C. Fluorescence was monitored over time using a Jasco FP 6300 spectrofluorometer . After 20 s of recording, mitochondria (0.4 mg/mL) were added in the cuvette and the effect of increasing concentrations of ETH129 on the membrane potential was examined after addition of the compounds at 120 s. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone  was added at the end of the experiment to depolarise mitochondrial membrane potential.2/5%CO2 containing 133 mM NaCl, 4.7 mM KCl, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4, 12 mM NaHCO3, 10 mM KHCO3, 10 mM HEPES, 30 mM taurine, 0.032 mM phenol red, 5.5 mM glucose, 10 mM 2,3butanedionemonoxime pH 7.4 to wash out blood. After 2 min of perfusion, liberase , trypsin EDTA (14 mg/100 ml) and 12.5 μM Ca2+ were added to the buffer and the heart was perfused for approximately 8–9 and 13–15 min for mice and rats, respectively. The heart was placed into a beaker in the same buffer containing 10% bovine serum albumin pH 7.4 at 37 °C to stop the digestion. Ventricles were then cut into small fragments and cells isolated by stirring the tissue and successive aspirations of the fragments through a 10 ml pipette. After 10 min the supernatant was removed and the remaining tissue fragments were re-exposed to 10 ml of the same buffer. Then, the cells were suspended in the same buffer and Ca2+ was gradually added to 1 mM into an incubator at 37 °C. Finally, the cardiomyocytes were suspended in culture medium M199. They were seeded on 35 mm Petri dishes pre-coated with 10 μg/ml sterilised laminin and incubated for 90 min before being used.Ventricular cardiomyocytes were isolated from mice and rats by an enzymatic technique. The heart was retrogradely perfused for 15 min at 37 °C with a stock perfusion buffer bubbled with 95%O2 which results in calcein fluorescence localised in mitochondria24. Cells were loaded with 1 μM calcein-AM for 30 min at 37 °C in 2 ml of M199, pH 7.4, supplemented with 1 mM CoCl2 and then washed free of calcein and CoCl2 and M199 was replaced by a Tyrode’s buffer . When the mitochondrial membrane potential was analysed, cardiomyocytes were loaded with 20 nM tetramethylrhodamine methyl ester (TMRM) for 30 min at 37 °C and then rinsed with the Tyrode’s solution. To monitor mitochondrial Ca2+, cardiomyocytes were loaded with 5 µM rhod-2 AM  for 40 min at 37 °C and then washed free of rhod-2 AM. When cell death was studied, propidium iodide (5 μM) which permeates only the damaged cells was introduced in the Tyrode’s buffer. To induce mPTP opening, calcium ionophores were added to the medium after 5 min stabilization, and remained present until the end of the measure  and CoClure Fig. . Cardiom2) for each cardiomyocyte and a fluorescence background corresponding to an area without cells was subtracted. For comparative purposes, the fluorescence intensity minus background was normalised according to the maximal fluorescence value .In normoxic experiments, images were acquired every 1 or 5 min. In hypoxia-reoxygenation experiments, images were acquired every 5 min during hypoxia, every min during the first 5 min following reoxygenation and then every 5 min. The illumination times were of 25 ms , 70 ms (propidium iodide), 50 ms (TMRM) and 70 ms (rhod-2). Images were analyzed using a digital epifluorescence imaging software . Fluorescence was integrated over a region of interest (≈80 μmmPTP50]) and to induce a 50% increase in propidium iodide fluorescence (time to 50% cell death [Tcell death50]) were determined. When cell contracture was evaluated, cells were considered hypercontracted when they exhibited an irreversible loss of the rod-like shape  contained in a single field. In hypoxia-reoxygenation experiments, the time of reoxygenation necessary to induce a 50% decrease in calcein fluorescence , N2 (74%) and CO2 (5%) maintaining an O2 concentration of 21%. Oxygen in the perfusate was measured in the chamber using a fibre optic sensor system . Cardiomyocytes were paced to beat by field stimulation .Mouse cardiomyocytes were placed into a thermostated (37 °C) chamber  which was mounted on the stage of an IX81 Olympus microscope  and were perfused with the Tyrode’s solution at a rate of 0.5 ml/min. The chamber was connected to a gas bottle diffusing a constant stream of O2 concentration of 1–2%. This medium was the Tyrode’s solution (bubbled with 100% N2) supplemented with 20 mM 2-deoxyglucose and subjected to a constant stream of N2 (100%). At the end of the ischemic period, reoxygenation was induced by rapidly restoring the Tyrode’s flow and 21% O2 in the chamber  followed by Newman-Keuls’ post test. Significance was accepted when p < 0.05.The data are reported as mean ± S.E.M. Statistical significance was determined using either Student’s two-tailed unpaired