We have previously shown that the 18-kDa mitochondrial translocator protein of the outer mitochondrial membrane (TSPO) can modulate mitochondrial Ca2+ uptake. Experiments were designed to test the role of the TSPO in a murine pressure-overload model of HF induced by transverse aortic constriction (TAC). Conditional, cardiac-specific TSPO knockout (KO) mice were generated using the Cre-loxP system. TSPO-KO and wild-type (WT) mice underwent TAC for 8 weeks. TAC-induced HF significantly increased TSPO expression in WT mice, associated with a marked reduction in systolic function, mitochondrial Ca2+ uptake, complex I activity and energetics. In contrast, TSPO-KO mice undergoing TAC had preserved ejection fraction, and exhibited fewer clinical signs of HF and fibrosis. Mitochondrial Ca2+ uptake and energetics were restored in TSPO KO mice, associated with decreased ROS, improved complex I activity and preserved mitophagy. Thus, HF increases TSPO expression, while preventing this increase limits the progression of HF, preserves ATP production and decreases oxidative stress, thereby preventing metabolic failure. These findings suggest that pharmacological interventions directed at TSPO may provide novel therapeutics to prevent or treat HF.Heart failure (HF) is characterized by abnormal mitochondrial calcium (Ca Current therapies aim at treating the systemic neurohormonal changes that occur during HF2, but none have been successfully developed to directly address cardiomyocyte function and viability. Understanding some of the key myocyte abnormalities in HF and developing specific therapies toward reversing these abnormalities can improve cardiac function and survival. Several abnormalities related to mitochondrial function have been observed in HF, including impairment of intracellular calcium (Ca2+) homeostasis4, reduced cellular respiration6, increase in the production of reactive oxygen species (ROS), and impaired mitochondrial quality control (mitophagy)7, all contributing to reduced contractile function8 and reduced myocyte survival9. These events all involve, in part, the proper communication between Ca2+ and mitochondria10.Congestive heart failure (HF) is still one of the leading causes of death, despite advances in therapeutic options2+ through the voltage-gated Ca2+ channels triggers Ca2+-induced Ca2+ release from the ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR). Areas with high, localized concentrations of Ca2+, known as microdomains, form at the junction between the SR and mitochondria, and result in mitochondrial Ca2+ uptake12. In the normal heart, Ca2+ enters the mitochondria through the voltage-dependent anion channel (VDAC) on the outer mitochondrial membrane (OMM)13 before it passes through the mitochondrial Ca2+ uniporter (MCU) in the inner membrane14. It is known that mitochondrial Ca2+ serves as a regulator of energy production15. However, during HF, mitochondrial Ca2+ uptake is typically reduced16, which can impact the production of energy and contribute to the pathogenesis of HF.During excitation-contraction coupling, the influx of Ca17. In addition to regulating steroidogenesis18, there is evidence that TSPO is involved in modulating oxidative stress19, and, critically, mitochondrial physiology and metabolism20. These latter effects include modulation of Ca2+ transport21, regulation of transcription of genes involved in mitochondrial electron transport chain, and thus ATP production22, and mitochondrial quality control23.The 18-kDa translocator protein (TSPO) is a five transmembrane domain protein that is primarily localized in the OMM and ubiquitously expressed throughout the body with elevated expression in steroid-synthesizing tissues, such as adrenal glands, gonads and the brain, as well as in the heart23, a ubiquitous channel located on the OMM which regulates the energy balance of mitochondria, serving as a common pathway for metabolite exchange between mitochondria and the cytoplasm24. Alterations in VDAC function, expressed as open-probability (Po), can impact oxidative phosphorylation by controlling the rate of adenine nucleotide exchange across the OMM25. Recent evidence has shown that expression of the TSPO is increased during stress conditions30, with the degree of expression reflecting the severity of the condition31. Thus, conditions which increase TSPO expression, such as HF, can modulate metabolism and affect cell survival through its effect on VDAC Po; however, no data exist on the role played by TSPO during HF development. Our previous work showed that altering TSPO activity with a pharmacologic ligand protoporphyrin IX (hemin) decreased Ca2+ uptake in mitochondria isolated from cardiac tissue by decreasing the open probability of VDAC21, which raises the possibility that TSPO may play a role in mitochondrial Ca2+ handling.The above effects of TSPO could be explained via modulation of VDAC2+ entry and systolic function; and thus b) genetic modulation of the TSPO could normalize mitochondrial Ca2+ uptake and prevent the abnormalities in cardiac structure and function evident in HF, specifically by normalizing cellular energetics and redox balance inside cardiomyocytes. These hypotheses were tested using a cardiomyocyte-specific, conditional knockout of the TSPO in a murine model of pressure-overload HF. Our studies show for the first time that TSPO-KO animals were protected from the progression of HF when compared to wild-type (WT) controls. In concordance with this protection, TSPO-KO mice had elevated activity of the mitochondrial complex I which led to the reduction of the oxidative stress in TSPO-KO TAC mice, shifting redox balance toward a more reduced state and preserving the mitochondrial membrane potential (ΔΨm). Accordingly, the TAC-induced decline in mitochondrial Ca2+ uptake was prevented in TSPO-KO vs. WT mice, with correspondent preservation of ATP generation. Furthermore, the signaling cascade activating the removal of damaged mitochondria via mitophagy was significantly impaired in WT TAC and restored in TSPO KO cardiomyocytes, thus contributing to improved mitochondrial function. Together, these data suggest that TSPO can be a potential pharmacological target to correct the abnormalities in mitochondrial Ca2+ transport and mitochondrial bioenergetics associated with HF.The objectives of the present study were to test the hypotheses that: a) HF increases expression of TSPO, with a concomitant decrease in inward mitochondrial CaloxP system as described in Methods and illustrated in Fig. 32. DNA was taken from tails, amplified using polymerase chain reaction, processed through DNA electrophoresis, and visualized under UV lighting  in 11-12 week-old mice. Representative images in Fig. To determine if TSPO has an adaptive or maladaptive effect on the development of pressure-overload HF, the anatomic and physiologic responses to pressure-overload were assessed weekly by echocardiography monitoring in unconscious mice. Sham animals, both WT and KO, did not exhibit morphologic or performance changes in the 8-week observation period after sham surgery. After TAC surgery, the increases in wall thickness after one week were similar in the WT and KO animals Fig. , which fin vivo data indicate that knocking out TSPO limited HF development and preserved cardiac function with pressure overload.In parallel with the differences in wall thickness and ventricular dimensions, WT TAC animals developed reduced systolic function. As seen in Fig. To further characterize the severity of HF development, anatomical and histological analyses were conducted. Consistent with the echocardiographic findings, measurement of heart weight-to-tibial length (HW:TL) ratios revealed greater TAC-induced cardiac mass (due to greater dilation) in WT compared to KO animals  ratio  than their corresponding sham group , was similar in myocytes from WT and TSPO-KO mice under sham conditions . Moreover, after TAC, mitochondrial Ca2+ uptake in KO myocytes was 7.7–9.5-fold higher than that in WT  exhibited higher mPTP opening at any given [Ca2+], but no differences in mPTP activity were seen between TSPO-KO and WT TAC myocytes  has been associated with Cates Fig. . These d2+] Fig. , so thisols Fig. ; as high2+ entry into the mitochondria can regulate energy production via activation of several key dehydrogenases16, and enhancing mitochondrial Ca2+ in the context of HF can maintain NADH oxidation3 and, hence ATP production6. To assess substrate-dependent [ATP] changes in intact cells under substrate conditions which activate complex I 37. Substrate addition led to an increase in ATP level above baseline in all groups, which was significantly higher in KO sham cardiomyocytes compared to WT sham cardiomyocytes in the presence of pyruvate/glutamate (p < 0.01), but not in the presence of methyl-succinate. This finding suggests that complex I mediated respiration drove significantly more ATP production in KO sham cardiomyocytes vs. WT. With methyl-succinate to drive complex II mediated respiration, there was no difference between WT and KO shams , TSPO ablation resulted in restoration of the mitochondrial reserve capacity in TSPO-KO TAC cardiomyocytes utilizing substrate for either mitochondrial complex I or II  stimulation  at basal conditions, significantly more oxidized than WT sham . In both WT and KO sham cardiomyocytes, FAD/FADH2 levels were well maintained during 4 Hz FS and ISO stimulation , whereas KO TAC mice had preservation of complex I activity (similar to KO sham). Importantly, we found that complex I activity after TAC was much higher in TSPO-KO vs. WT mice  was significantly decreased in WT TAC cardiomyocytes, but not in TSPO-KO TAC , confirming that the inability of mitochondria to maintain ΔΨm in HF was related to impairment of the mitochondrial complex I. In TSPO-KO TAC mice, basal levels of ΔΨm were significantly closer  to that observed in sham controls . We conclude that TSPO ablation improved mitochondrial oxidative phosphorylation via restoration of the electron flux flow at the level of the mitochondrial complex I.Furthermore, we found that mitochondrial membrane potential  accumulates on the mitochondrial surface, recruiting the E3 ubiquitin ligase (Parkin) to the outer mitochondrial membrane from the cytosol. Parkin then polyubiquitinates mitochondrial membrane proteins, marking them for degradation and recognition by the adaptor protein SQSTM1/p62. SQSTM1/p62 binds to the microtubule-associated protein 1A/1B-light chain 3 (LC3) on the autophagosome and to the ubiquitinated mitochondrial proteins, tethering the autophagosome to the mitochondrionWe evaluated mitophagy by monitoring mitochondrial Parkin accumulation and LC3-mediated autophagosome formation in cardiac myocytes using confocal microscopy. As shown in Fig. 29 and under stress conditions in other tissues30. Pressure overload for 8 weeks in WT animals resulted in the expected changes in cardiac morphology, function, and metabolism associated with HF . First, we demonstrated that TAC resulted in a 3-fold increase in expression of the TSPO in WT animals Fig. , an upresee Figs . These iSecond, this study demonstrated that cardiac-specific conditional KO of TSPO substantially abrogated the deleterious effects of TAC. By preventing TSPO upregulation during TAC, there was well-preserved left ventricular function . In addition, TSPO-KO decreased associated HF markers, with lower levels of ventricular dilation, pulmonary congestion, ANP levels and extents of cardiac fibrosis and hypertrophy.2+ uptake  reduced Ca2+ uptake in isolated mitochondria21, as was shown with another TSPO ligand PK1119545. Upregulation of TSPO expression during HF coincided with a decrease in mitochondrial Ca2+ uptake, which was normalized in TSPO-KO mice  on the IMM47. A recent study13 demonstrated that reducing Ca2+ flux via VDAC severely affected heart contractility in zebra fish, while interventions to increase Ca2+ flux through the OMM restored normal contraction of the heart. These data highlight the possible impact of OMM proteins such as VDAC and TSPO in the regulation of mitochondrial Ca2+ uptake and cardiac function.As a modulator of VDAC2+ uptake could lead to most of the mitochondrial sequelae , but we cannot rule out additional primary TSPO effects. Better maintenance of mitochondrial and energetic function by TSPO-KO, as seen here during TAC, could maintain cardiac function and slow the progression to HF48. Notably, the TSPO-KO TAC mice (vs. WT TAC) had higher respiratory reserve values and higher activity of the mitochondrial complex I, indicating preservation of mitochondrial bioenergetics. These findings agree with those of Gatliff et al.49 who showed that CF35 cells with siRNA-induced TSPO knockdown showed greater mitochondrial ATP production compared to control cells, while an overexpression of TSPO decreased ATP. Taken together, our data show that TSPO-KO mice were resistant to the progression of HF, and this protective effect was most likely due to a combination of enhanced mitochondrial Ca2+ uptake and energy production.The remarkable beneficial effects of the TSPO-KO  are consistent with TSPO upregulation being a central amplifier of the progression from compensated hypertrophy (seen in TSPO-KO mice after TAC) to more full-blown HF (seen in WT TAC). A primary effect of TSPO overexpression to inhibit mitochondrial Ca2+ overload under acute ischemia/reperfusion50. With ischemia/reperfusion, significant Ca2+ overload during reperfusion, mediated by sarcolemmal Na+/Ca2+ exchanger51, can result in opening of the mitochondrial permeability transition (mPTP), leading to mitochondrial swelling, membrane rupture and myocyte death. In contrast to the detrimental effect of Ca2+ overload under this acute pathologic challenge, the better maintained physiological mitochondrial Ca2+ uptake during chronic TAC in TSPO-KO promoted improved mitochondrial energetics and cardiac function. Notably, mPTP, which is a key turning point in above ischemia/reperfusion damage, was not influenced by TSPO-KO . This “Redox-optimized ROS balance” hypothesis59 postulates that the physiological steady state in cardiac mitochondria is tuned to an intermediate redox state that prevents excessive ROS formation at the ETC under highly reduced conditions62, but also maintains anti-oxidative capacity under highly oxidized conditions63. Our data fit with this concept, as basal FAD/FADH2 ratio was 0.56 under sham conditions, with minimal ROS generation. During HF, however, Ca2+ uptake was depressed and FAD/FADH2 was severely oxidized, with a concordant increase in ROS generation. TSPO ablation restored normal Ca2+ flux to mitochondria, improved complex I activity, shifting redox index toward an “intermediate state” and reduction in ROS generation , nor to human or large animal models. TSPO has numerous reported effects on steroid transport and lipid metabolism2+ uptake and complex I-mediated respiration leading to the normalization of ATP production oxidative stress and mitochondrial quality control. These findings are the first observations that modulation of the mitochondrial outer membrane translocator protein could be a target for the prevention or treatment of HF.Taken together, these experiments have shown that HF induced by pressure overload resulted in increased expression of TSPO. Abrogating this increase by genetic ablation resulted in cardioprotection reflected by preserved ventricular function, cellular histology and clinical signs of heart failure. This cardioprotective effect was associated with preserved mitochondrial CaAll protocols were in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health  and approved by the University of California Davis Institutional Animal Care and Use Committee.67. A 5-mm thoracotomy was made and the transverse aorta was isolated and a suture looped around a blunt 27-gauge needle, yielding a transverse aortic diameter of approximately 0.4-mm. The sham procedure was identical except that the aorta was not ligated.C57Bl/6J mice were obtained from The Jackson Laboratory . The aorta was surgically constricted as previously describedTspo gene had normal expression until Cre-mediated deletion of exons 2 and 3, induced by a single intraperitoneal injection of 40 mg/kg of tamoxifen (dissolved in sunflower oil and 100% ethanol at a 10:1 ratio). This recombination created a frame-shift mutation and a premature stop, which rendered the Tspo gene inactive. Exons 2 and 3 in the TSPO gene were flanked with loxP sites. This vector construct was inserted into C57BL/6 embryonic stem cells, which were subsequently screened through polymerase chain reaction (PCR) and DNA electrophoresis. Mice that had loxP sites were bred with each other to form TSPOflox/flox mice. Homologous TSPOflox/flox mice were then bred with mice containing the Tg(Myh6-cre)1Jmk/J gene. The Tg(Myh6-cre)1 Jmk/J gene controls the expression of Cre recombinase by the mouse myosin, heavy polypeptide 6 (Myh6). Animals that were homologous for TSPOflof/flox and had the inducible Cre recombinase were considered to be KOs.TSPO-floxed mice were produced in conjunction with the Mouse Biology program at UC Davis. TSPO-floxed mice were bred with mice containing the Tg(Myh6-cre)1Jmk/J gene , in order to generate inducible, cardiac-specific, conditional KO mice. To genetically determine if TSPO was floxed, DNA was taken from tails, amplified using polymerase chain reaction, processed through DNA electrophoresis, and visualized under UV lighting. The TM cDNA synthesis kit . qRT-PCR was performed with commercial and customized TaqMan probes  and cDNA synthesized from 1 µg of RNA using the iScriptVentricular function was monitored using 2D echocardiography on a weekly basis using a Visualsonics Vevo 2100 echocardiography machine and a MS 550D probe (22–55 MHz). Animals were anesthetized using isoflurane (0.75–1%). 2D echocardiography was performed at weekly intervals until the mice were ready to be sacrificed after 8 weeks of sham or TAC operation. Ejection fraction was determined from 2D images by manual determination of endocardium and epicardium from end-diastolic and end-systolic images.Eight weeks after surgery, mice were anaesthetized with isoflurane and sacrificed by cervical dislocation. Hearts were excised, weighed and immediately fixed by perfusion in a retrograde manner with 4% paraformaldehyde in phosphate-buffered saline (PBS) followed by immersion fixation in buffered 4% paraformaldehyde. Hearts were kept at 4 °C before the tissue was processed. Lungs were extracted, blotted dry, and weighed. Increased cardiac mass was assessed by comparing the ratios between heart weight-to-tibial length. Pulmonary congestion was assessed by comparing lung weight-to-tibial length ratios.Extracted hearts were rinsed with PBS, placed in 10% Neutral Buffered Formalin, and paraffin-embedded. Hearts were sliced into 5 µm sections and collagen fibers were stained with Picro-Sirius Red stain. Images were taken with an Olympus BH-2 Bright-field Microscope using the 20x objective lens. Quantification of fibrosis was performed using ImageJ software.38. Briefly, mice were anesthetized by inhalation of isofluorane. When the reflexes were absent, hearts were excised, placed into the Langendorff system and perfused with a Ca2+-free washing solution, followed by an enzyme solution containing Liberase TM . Freshly isolated ventricular myocytes were stored at room temperature and used within 8 hours of isolation.Left ventricular myocytes were isolated using a Langendorff perfusion system68. Coverslips with the attached cells were placed in a perfusion chamber, and then permeabilized with 50 µg/ml Saponin in the intracellular solution containing (in mM): 135 KCl, 0 NaCl, 20 HEPES, 5 pyruvate, 2 glutamate, 2 malate, 0.5 KH2PO4, 0.5 MgCl2, 15 2,3-butanedione monoxime, 5 EGTA, and 1.86 CaCl2 to yield a free [Ca2+]i of 100 nM with pH 7.2. After permeabilization, the bath solution was changed to the same intracellular solution but without saponin, and extramitochondrial Ca2+ ([Ca2 +]em) was elevated to initiate mitochondrial Ca2+ uptake. X-Rhod-1 was excited with the 543-nm line of a green HeNe laser, and emitted fluorescence was measured at 552–617 nm. Mitochondrial X-Rhod-1 fluorescence intensity (F) in each experiment was normalized to the level of fluorescence recorded before stimulation (F0) but after cell permeabilization. Changes in [Ca2+]m are expressed as ΔF/F0, where ΔF = F − F0.Myocytes plated on laminin-coated coverslips were loaded with X-Rhod-1 AM for 40 minutes at 37 °C1) in 67 mM sucrose, 50 mM Tris/HCl, 50 mM KCl, 10 mM EDTA, and 0.2% BSA with a pH = 7.2, homogenized with a glass Teflon pestle, and subsequently centrifuged at 700 g for 10 min at 4 °C. The supernatant was collected and then spun at 8000 g for 10 min at 4 °C. The pellet was then resuspended in ice-cold buffer (IB2) containing 250 mM sucrose, 3 mM EGTA/Tris, and 10 mM Tris/HCl with a pH = 7.2, followed by centrifugation at 8000 g for 10 min at 4 °C. Mitochondria were then resuspended in IB2. Protein concentration was measured using the Bradford Assay method.Mitochondria were isolated from left ventricles using a standard differential centrifugation method. Briefly, minced hearts were suspended in mitochondrial isolation buffer  concentration using the fluorescent dye mag-fluo-4 68. Since free [Mg2+]i is kept constant within a rather narrow range, ATP hydrolysis leads to concomitant increase in free [Mg2+]i as measured with fluorescent Mg2+ indicators such as mag-fluo-4. Therefore, an increase in mag-fluo-4 fluorescence indicates a decrease in ATP concentration. For ATP measurements myocytes were loaded with 10 µM mag-fluo-4  for 30 min at 37 °C. All data from these measurements are expressed as R = 1 − F/F0.ATP measurements were performed indirectly via the free magnesium  for 40 min at 37 °C38. Opening of mPTP resulted in the loss of mitochondria-trapped calcein (620 Da) and a decrease of fluorescence. At the end of each recording 10 μg/ml of the pore-forming antibiotic alamethicin was applied to provide a control measure for maximum calcein release from mitochondria. Loss of mitochondrial calcein induced by elevating Ca2+]em was quantified as the rate of decline of fluorescence (d(ΔF)/dt) calculated from the linear fit to the initial decrease of calcein fluorescence. The rate of decline was normalized (%) to the basal decline of calcein fluorescence addition (taken as 100%) before [Ca2+]em elevation.mPTP activity was monitored in permeabilized myocytes loaded with 5 μM calcein/AM  for 30 min at 37 °C68. Changes in MitoSox Red fluorescence intensity (F) were normalized to the level of fluorescence recorded prior to stimulation (F0), and expressed as ΔF/F0.ROS production was measured in intact myocytes loaded with 0.5 μM MitoSox Red  was measured to evaluate mitochondrial redox state68. Data are presented as the ratio of oxidized FAD to reduced FADH2 (FAD/FADH2) calculated as (F − Fmin)/(Fmax − Fmin) where F is the fluorescence intensity, and Fmin is the fluorescence obtained after addition of 4 mM NaCN , taken as 0%. Fmax is the fluorescence obtained after addition of 10 μM FCCP , taken as 100%.Flavin adenine dinucleotide (FAD)-linked protein autofluorescence (λm) were followed using the potential-sensitive dye tetramethylrodamine methyl ester 68. Cells were exposed to 5 nM TMRM for 30 min at 37 °C prior to experiments. All solutions contained 5 nM TMRM during recordings.Changes in mitochondrial membrane potential  kits for these two complexes. Mitochondria were isolated using the standard procedure stated above. After isolation, proteins were extracted from the mitochondria using the provided detergent solution. Twenty micrograms of proteins were then loaded into wells pre-coated with antibodies specific for Complex I. Assay time, reagents and buffers added, were followed exactly as stated in the protocol booklets for each complex.69. Freshly isolated myocytes were plated on sterile, laminin-coated glass coverslips in PC-1 medium, supplemented with penicillin and streptomycin (50 μg/ml). PINK1, Parkin, SQSTM1/p62, TOMM22 and accumulation of LC3-II were also monitored by Western blot.Adenoviral gene transfer in isolated myocytes that were kept in short-term culture  was used to express mCherry-Parkin and GFP-LC3 to monitor Parkin accumulation in mitochondria and LC3-mediated autophagosome formationPrimary antibodies for TSPO , COX IV , PINK1 Abcam ab23707), SQSTM1/p62 (Abcam ab56416), TOMM22 (Abcam ab57523), LC3 , Parkin (Santa Cruz Biotechnology sc-133167) and β-Actin (Santa Cruz Biotechnology sc-47778) were diluted to a concentration of 1:1000, and secondary antibodies were conjugated with Horseradish Peroxidase was diluted to a concentration of 1:2000. Signals were detected using an Alpha FluorChem Imaging Systems 8900 and bands were quantified relative to housekeeping protein  and α-actinin  were the primary antibodies. Images were taken using a Zeiss LSM 700 Confocal Laser Scanning Microscope.Hearts were fixed in 4% PFA and then embedded in paraffin. 5 micrometer sections were cut and these sections were deparaffined in a series of xylene and alcohol concentrationst test or two-way ANOVA, followed by Tukey post-test when appropriate. A value of p < 0.05 was considered statistically significant. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.All data are expressed as mean ± standard error. Statistical significance of differences between experimental groups was determined using Student paired Supplementary File


Escalating energy security, burgeoning population and rising costs of fossil fuels have focussed our attention on tapping renewable energy sources. As the utilization of food crops for biofuel production culminates into food vs. fuel dilemma, there is an intensive need for alternatives. Production of biofuels from lignocellulosic biomass owing to its profuse availability and high holocellulose content is a promising area for research.In the present study, pineapple leaf, an agro-industrial waste was pretreated with laccase to enhance the enzymatic digestibility of the substrate for improved production of reducing sugar. Variables determining enzymatic delignification of pineapple leaf waste have been optimized by response surface methodology based on central composite design. Maximum delignification of 78.57%(w/w) resulted in reducing sugar of 492.33 ± 3.1 mg/g in 5.30 h. The structural changes in pineapple leaf waste, after laccase treatment, were studied through Fourier transformed infrared spectroscopy, X-ray diffraction and Scanning electron microscopy. Specific surface area, pore volume, and pore diameter of the substrate were studied using the Brunauer–Emmett–Teller and Barrett–Joyner–Halenda methods and found a significant increase in the aforementioned parameters after delignification.Laccase mediated delignification of pineapple leaf waste is a cleaner sustainable process for enhanced production of reducing sugar which can accomplish the demand for biofuels. Lignocellulosic biomass is a potential renewable resource for biofuel production due to wide availability, low cost and rich holocellulose content. Predominant lignocellulosics that have been employed for bioenergy production are oil palm frond , poplar A major impediment in the utilization of lignocellulosic biomass as a substrate for ethanol production is the presence of lignin that resists chemical or enzymatic digestion of cellulose and hemicellulose polymers . Lignin The mechanism involved in various pretreatment processes specifically pertaining to the alterations in physical structure and chemical composition of biomass should be understood completely. The ultimate goal of any pretreatment process is to enhance the accessibility of biomass to the enzymes for efficient generation of reducing sugar at low enzyme dose. The conventional pretreatment methods result in the formation of furfurals, hydroxymethyl furfurals, acetic acid and formic acid etc., that inhibit the subsequent steps of bioethanol production. During enzymatic delignification, enzyme acts specifically and release phenolic intermediates which act as natural mediators enhancing the delignification process . High yiOwing to the aforementioned advantages of enzymatic pretreatment, laccase mediated delignification of pineapple leaf waste was carried out in the present study. Laccase is an extracellular, multi-copper enzyme which degrades lignin through molecular oxygen and oxidizes different aromatic and non-aromatic compounds present in lignin . Till daRecently, multivariate statistical techniques are predominantly being used for optimization of process parameters. Factorial designs viz., central composite and Box–Behnken are used, for evaluating the effect of one factor at several levels of other factors in addition to the interaction among them, which is not possible in one-factor-at-a-time . In the Ananas comosus, Giant Kew variety) leaf waste was collected locally from the farm of IIT Kharagpur, India. It was chopped, sun dried, pulverised to particle size of 0.2 mm and used in the present study. Lignin, cellulose and hemicellulose content of raw and delignified pineapple leaf wastes were estimated using standard protocols  × 100, where I002 represents maximum peak intensity (2θ) between 22° and 23° and Iam represents minimum peak intensity (2θ) between 18° and 19° for cellulose I [The crystallinity of raw and delignified samples of pineapple leaf waste was determined through X-ray diffraction (XRD) studies using CoKSpecific surface area, pore volume and pore diameter of pineapple leaf waste sample were studied using the Brunauer–Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH) method. The adsorbate suitable for evaluation of porosity of pineapple leaf waste was nitrogen at 77 K using Quantachrome Autosorb Automated Gas Sorption System apparatus. The 0.2 mm particle sized pineapple leaf waste sample was degassed for overnight at 60 °C to remove the moisture from the pores.Biochemical characterisation of pineapple leaf waste clearly indicate that it was rich in cellulose  and hemicellulose  with significant lignin  content. The reported composition of cellulose , hemicellulose  and lignin  of pineapple leaf were in close proximity with that obtained in the present study . For effThe elemental analysis Table  depicts A range of various parameters were considered for studying their effect on delignification of pineapple leaf waste. The percentage delignification and its corresponding residual lignin for all the parameters were shown in the Fig. Using the experimental data of enzymatic delignification and various interaction terms of experimental variables, a second order polynomial equation was fitted for the process which is represented as:F value (27.23) and very low P value (< 0.001) thus specifying the significance of the model. High R2 value (0.96) of laccase delignified pineapple leaf waste demonstrates the robustness of the model. No significant difference between the R2 and adjusted R2 values was observed, indicating that the regression model is reliable and competent for the process. It is noted from the coefficients of terms in the regression equation that the individual terms of incubation time, pH, temperature and square terms of pH have profound influence on the percentage of delignification.In the above equation, units for solid loading, incubation time, temperature and enzyme concentration are % (w/v), h, °C and IU/mL respectively. Analysis of variance (ANOVA) of the quadratic equation for the delignification (%) of pineapple leaf waste has been summarized in Table The 3D response surface plots are graphical representation of the regression equation. The response surface plots pertaining to the conditions for enzymatic delignification of pineapple leaf waste were shown in Fig. Figure Bambusa bambos where the maximum delignification of 84% was obtained at pH 6.9, 35 °C, liquid:solid ratio 6:1, incubation time 8 h, and enzyme concentration 400 IU/mL [From the analysis of response surface plots, the optimum conditions for enzymatic delignification were found to be solid loading 25% (w/v), incubation time 5.30 h, 45 °C, pH 6.16, enzyme concentration 582.82 (IU/mL). Under these optimum conditions, the predicted delignification was 77.56% which was close to the experimental delignification 78.57%. Similar results were reported in 00 IU/mL . It is n00 IU/mL . ThroughEnergy density plays a very important role not only in determining the efficacy of lignocellulosic feedstock as a potential biofuel producer but also for validating the efficiency of the process involved in the biofuel generation viz. the delignification process. A low energy density feed stock is less energy efficient to convert into biofuel than that of high energy density feedstock. The energy density of the raw and enzymatically delignified pineapple leaf waste was found to be 17.13 ± 0.65 kJ/g and 15.95 ± 0.5 kJ/g respectively. The reduction in the energy density of delignified pineapple leaf waste compared to the raw substrate may be due to lignin degradation which is in accordance with the reported work where energy density of the lignocellulosic feedstock (Kans grass) was reduced after enzymatic delignification process . There eThe changes in structure of pineapple leaf waste after enzymatic delignification under optimum conditions from RSM were assessed through SEM studies. The delignification process creates cracks and pores on the substrate due to the removal of lignin which was clearly observed in the SEM images of raw and delignified biomass Fig. . The del−1 (OH stretching of lignin), 2919 cm−1 (C–H stretching of lignin), 1735 cm−1 (C=O stretching of hemicellulose), 1629 cm−1 (C=O stretching vibration in conjugated carbonyl of lignin), 1538 cm−1 and 1515 cm−1 (C=C stretching vibrations of aromatic rings of lignin), 1454 cm−1 , 1430 cm−1 (C–H in-plane deformation of lignin), 1376 cm−1  1251 cm−1 (syringyl ring breathing and C–O stretching in lignin and xylan), 1050 - 1150 cm−1 (C–OH stretching vibration of the cellulose and hemicelluloses), 1037 cm−1  and 665 cm−1 (aromatic C–H bending of lignin) after enzymatic delignification process [FTIR has been used to explore the changes of the functional groups and compositional modifications in laccase delignified substrate against the raw substrate with significant changes in the absorption spectrum of delignified substrate Fig. . Notable process –29. The Lantana camara where an increase in crystallinity of enzymatically delignified sample (25.21%) was observed over that of the raw sample (19.57%) [The cellulose accessibility to cellulase for efficient saccharification is governed by various factors like cellulose crystallinity, lignin and hemicellulose content, its distribution in the substrate and porosity . XRD pro(19.57%) .Fig. 5X-The cellulose, hemicellulose and lignin contents of raw and delignified pineapple leaf waste under optimized conditions are provided in Table To determine the effect of delignification on production of reducing sugar, saccharification was performed with raw and delignified pineapple leaf waste. The reducing sugar (mg/g) from raw and delignified substrates was found to be 189.0 ± 8.89 and 492.33 ± 3.1 respectively with a significant increase of 2.6-folds. The results show that enzymatically delignified pineapple leaf waste can produce considerably high amount of reducing sugar after saccharification.2/g respectively, that showed an increase in the surface area by 1.78-folds. The obtained results from the present study are substantially higher than the reported work where surface area analysis of raw and ionic liquid pretreated oil palm empty fruit bunch was 0.55 m2/g and 0.75 m2/g [2 adsorption and desorption techniques.One of the important parameters that affect the hydrolysis rate of cellulose is specific surface area. The absorption of cellulase on the surface of cellulose is mandatory for the hydrolysis reaction to occur as the extent of hydrolysis depends on the surface area accessible to the enzymes. Based on BET analysis Table , surface.75 m2/g . BJH anaThere exist a direct relation between the pore volume or interior surface area of the substrate and the extent of hydrolysis by cellulase . EnhanceThe prime objective of the delignification process is to remove lignin that acts as barrier to cellulose which is major contributor of reducing sugar. Conventional pretreatment process not only leads to a significant amount of hemicellulose loss along with lignin but also results in formation of intermediates such as furfurals and hydroxymethylfurfurals that may act as inhibitors for the fermentation process. Besides, conventional pretreatment processes are also associated with equipment corrosion, high cost, high water requirement, excess use of chemicals, formation and incorporation of salts into the biomass and enormous waste production. In order to overcome these issues, enzymatic delignification of biomass was carried out in the present study. Enzymatic delignification is an eco-friendly process which can work under mild operating conditions. Laccase being specific, acts on lignin moieties of the substrate and has less effect on the structure and composition of cellulose and hemicelluloses. Laccase mediated delignification enhances saccharification efficiency of cellulase thereby leading towards cleaner production of biofuel  such as Biochemical characterisation of pineapple leaf waste shows the presence of high cellulose and hemicellulose with significant lignin content. Laccase mediated delignification process degrades lignin up to 78.57% (w/w) which is accompanied by an increase in cellulose crystallinity of 6.46%. This substantiates the effective action of laccase on pineapple leaf waste during delignification process which results in increased production of reducing sugar (492.33 ± 3.1 mg/g). Structural characterization as well as surface area and porosity analysis of the substrate further corroborate the enzymatic delignification process. The energy density studies of pineapple leaf waste prove that it is a competent substrate for biofuel production. In the perspective of cleaner production, enzymatic delignification of biomass is a green, sustainable and eco-friendly process for improved production of reducing sugar that contributes for efficient biofuel production. The agro-industrial waste utilization for generation of value added products is not only an effective way of waste removal from the environment but also economical.


Accurate mitochondrial function and Ca2+ signaling are of utmost importance for adequate heart function and crucial to maintaining the cardiovascular health. Due to the highly sensitive nature of these processes in the heart, this review article highlights the current knowledge regarding sex dimorphisms in the heart implicating the importance of E2 and ERs in the regulation of cardiac mitochondrial function and Ca2+ ion channels, thus the contractility. In particular, we provide an overview of in-vitro and in-vivo studies using either E2 deficiency; ER deficiency or selective ER activation, which suggest that E2 and ERs are strongly involved in these processes. In this context, this review also discusses the divergent E2-responses resulting from the activation of different ER subtypes in these processes. Detailed understanding of the E2 and ER-mediated molecular and cellular mechanisms in the heart under physiological and pathological conditions may help to design more specifically targeted drugs for the management of CVDs in men and women.Numerous epidemiological, clinical, and animal studies showed that cardiac function and manifestation of cardiovascular diseases (CVDs) are different between males and females. The underlying reasons for these sex differences are definitely multifactorial, but major evidence points to a causal role of the sex steroid hormone 17β-estradiol (E2) and its receptors (ER) in the physiology and pathophysiology of the heart. Interestingly, it has been shown that cardiac calcium (Ca Cardiovascular Diseases (CVDs) are one of the top age-associated chronic diseases with growing importance due to the dramatic increase in life expectancy  and are 2+) is a key player in the regulation of myocardial contraction and the deregulation of Ca2+ signaling due to the alteration of Ca2+ ion channels function in cardiomyocytes is highly associated with the development of cardiac diseases, such as heart failure  have a lower rate of CVDs and cardiac death, compared to women without HRT  and the Heart and Estrogen/Progestin Replacement Study (HERS I and II) showed that HRT has no obvious beneficial effect on CVDs, and may actually increase the risk and events of CVDs in postmenopausal women (Kronos Early Estrogen Prevention Study (KEEPS) and the Early vs. Late Intervention Trial with Estradiol (ELITE) addressed the question of the so-called “timing hypothesis.” They showed significant beneficial cardiovascular effects in women who initiated HRT in the early postmenopause vs. late menopause period , which catalyzes the aromatization of androgens to E2, or E2 resistance, caused by a point mutation in the ERα gene (ESR1), the following have been reported: increased total cholesterol level, the development of insulin resistance, impaired glucose tolerance, type 2 diabetes mellitus, and impaired vasodilatation  and Estriol (E3) to the group of sex steroids called Estrogens. Thereby, E2 is the predominant and most biologically active form . Estrogemitogen-activated protein kinases ERK1/2 and -p38  as well as phosphoinositide 3-kinase-serin/threonine-specific kinase B (PI3K/AKT), which in turn phosphorylate ERs  and beta (ERβ), which are members of the nuclear receptor superfamily  40). Bo. Bo40). late ERs –47 or otlate ERs  48–51).–51.mitoglate ERs  52, 53), 53mitogBoth ERs are localized in different cardiac cells such as cardiomyocytes, endothelial cells, smooth muscle cells, and cardiac fibroblasts in human hearts from both sexes , 55. StuRecent reports showed that E2 can signal through a third protein, the G-protein-coupled estrogen receptor (GPER), formerly known as GPR30, a membrane receptor with seven transmembrane spanning domains , 69. GPEStudies showed that mutations in the genes coding for ERα and ERβ are associated with differences in heart morphology, such as increased left ventricular mass and wall thickness , 87. FurFor ERβ, the SNP variant rs1271572 was associated with increased risk of MI in Spanish men , while RThe reasons for the inconsistency in data regarding the SNPs within the genes of ERα and ERβ could be due to the limited power within the studies, differences in methodology and study population . DespiteThe physiology of E2-actions through its multiple receptors is diverse and highly complex. The detailed understanding of their effects and underlying molecular mechanisms are essential for future therapeutic applications in humans. In order to clarify remaining questions regarding the functions of each individual receptor within the heart, different mouse models with a deficiency or overexpression of ERα, ERβ, and GPER have been generated , 102.At the basal level, male and female whole body ERα-deficient (ERKO)-mice are obese and insulin resistant . They alnppa and nppb, but no fibrosis development was observed  revealed variations in the expression of genes involved in metabolism, cell growth and differentiation, muscle architecture, and relaxation compared to WT-mice . Furtherobserved . In agreobserved . OverallInterestingly, the use of csERα-OE mice demonstrated that ERα provides cardioprotection in female mice by enhancing neovascularization and impairment of cardiac remodeling in response to cardiac ischemic injury . All togIn contrast to ERKO-mice, male and female ERβ-deficient (BERKO)-mice show a mild metabolic phenotype characterized by increased cortical bone formation and loss of trabecular bone . In addiSeveral studies in BERKO-mice demonstrate the relevant role of ERβ in male and female mice after cardiac injury. The lack of ERβ significantly decreased post-ischemic cardiac recovery and therefore myocardial function in female, but not male, mice . In OVX 2+-ATPase 2a (SERCA2a) expression, suggesting less reduction in diastolic Ca2+-reuptake into the sarcoplasmic reticulum post-MI. Most of these functional parameters were improved in both sexes by csERβ-OE; however, the effect on LV volumes and ejection fraction was more pronounced in males than females. This was possibly due to reduced cardiac remodeling with lower cardiac fibrosis and lower expression of fibrosis markers , which was observed particularly in male csERβ-OE hearts after MI.In a mouse model with a cardiomyocyte specific ERβ-overexpression (csERβ-OE), under basal conditions there were no observed differences in heart weight, morphology, and function in males and females . InteresThere are several studies stating the phenotype of mice lacking GPER . The stuA recent study in mice with a cardiomyocyte-specific GPER-KO (csGPER-KO) revealed under basal conditions adverse alterations in cardiac structure and impaired systolic and diastolic function in both sexes, in comparison to WT-mice, with more profound increases in LV dimensions, and wall-thinning among male KO-mice . Using DAlthough studies with transgenic ER mice failed to provide a clear consensus regarding the physiological and pathological roles of ERs, they suggest that each of the ER subtypes play a protective role in the heart.2+ is a critical regulator of myocardial function. Ca2+ regulates contraction, and deregulation of Ca2+ signaling has been associated with cardiac dysfunction and pathology such as arrhythmias and heart failure (2+ levels are tightly regulated via the excitation-contraction (EC) coupling pathway , through the opening of SR Ca2+ release channels, known as ryanodine receptors . This process is termed Ca2+-induced Ca2+ release. The combination of Ca2+ influx via the L-type Ca2+ channels and Ca2+ release from SR leads to the formation of cytosolic Ca2+ transients. The binding of cytosolic Ca2+ to the myofilaments then initiates cardiomyocyte contraction. Subsequent relaxation occurs by removal of Ca2+ from the cytosol mainly via the following mechanisms: (I) The SERCA2a re-uptakes the cytosolic Ca2+ back into the SR; the activity of this channel being modulated by its endogenous inhibitor phospholamban (PLN); (II) The Na+/Ca2+ exchanger (NCX) extrudes the Ca2+ out of the cells; (III) The mitochondrial Ca2+ uniporter transports Ca2+ into the mitochondria  compared to male patients  is the most abundant cardiac L-type Ca2+ channel which triggering cardiac contraction by regulation of ICa, L in cardiomyocytes  itself  of Caactivity –159, and) itself –163. Schon rates . In a reon rates . Here, tNCX) are significantly higher in cardiomyocytes from female humans, rats, and rabbits compared to their male counterparts  -mediated phosphorylation of RyR2 being lower in female cardiomyocytes than in male cardiomyocytes (2+ release/Ca2+ current) in female rat and mice cardiomyocytes, which results from decreased size and duration of Ca2+ sparks by RyR2  in the heart . It is cith CVDs , 186. E2ith CVDs .2) was significantly higher in female mice compared to males  and enhanced release of cytochrome c accompanied by dramatic increase in ROS formation, which leads to cell death via the induction of apoptosis pathways  in cardiomyocytes, and consequently to the depletion of ATP production (2+ retention capacity, and the GPER-mediated cardioprotective effect of post-ischaemic E2 is related to a decrease in mPTP sensitivity to Ca2+ overload, a process which is mediated via activation of the MEK/ERK/GSK-3β axis.Here again, using ER deficient mice could be shown that these E2 effects are mediated by ERs. Male ERKO hearts subjected to I/R showed an accumulated Caoduction . Using Eoduction . By contoduction . HoweverThese data suggest that depending on the time period of E2-treatment, sex, and species different ERs can be activated by E2, which mediate the mitochondrial-dependent cardioprotective effect of E2 against I/R injury.2+ ion channels and mitochondrial function by E2 and ERs could be, at least partly, responsible for differences in cardiovascular disease incidence and outcomes. However, further attempts toward a more detailed understanding of E2 and ERs roles in the heart are needed to develop new drugs that target the beneficial effects on CVD in both sexes.In the past, most clinical and animal studies did not include both sexes or differentiate between sexes in the data analysis. This might be the possible reason that our understanding of the molecular and cell-based mechanisms underlying sex-based differences in cardiovascular system are still incomplete so far. A more thorough understanding of underlying sex-dimorphic mechanisms in cardiac health and disease is required to effectively treat patients with CVDs. The presented data in this review support the concept that sex specific regulation of cardiac CaAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Recent advances in microscopy techniques have allowed for optical recordings of local Ca2+ fluxes and action potentials from multiple sub-cellular domains within cardiac cells with unprecedented spatial and temporal resolution. Since then, sub-cellular local information of the spatio-temporal relationship between Ca2+ release and action potential propagation have been unlocked, providing novel mechanistic insights in cardiac excitation-contraction coupling (ECC). Here, we review the promising perspectives arouse from repeatedly probing Ca2+ release at the same sub-cellular location while simultaneously probing multiple locations at the same time within a single cardiac cell. We also compare the results obtained in three different rodent models of cardiac diseases, highlighting disease-specific mechanisms. Slower local Ca2+ release has been observed in regions with defective action potential conduction in diseased cardiac cells. Moreover, significant increment of Ca2+ variability (both in time and in space) has been found in diseased cardiac cells but does not directly correlate with local electrical defects nor with the degree of structural aberrations of the cellular membrane system, suggesting a role for other players of the ECC machinery. We finally explore exciting opportunities provided by the technology for studying different cardiomyocyte populations, as well as for dissecting the mechanisms responsible for subcellular spatio-temporal variability of Ca2+ release.Asynchronous Ca The first pumps Ca2+ back into the SR consuming ATP, the latter extrudes the cation in the extracellular space, exploiting the electrochemical gradient of Na+, with a stoichiometry of 3Na:1Ca  . During  (RyR) 2 . This me (RyR) 2 . When a  couplon . This init (CRU)  and actiit (CRU) . During  trigger . After e 3Na:1Ca . This bamalities . To ensuhronized .2+ transient desynchronization can be experimentally reproduced in myocytes acutely detubulated through osmotic shock  EPPTEA, a fluorinated voltage-sensitive dye (VSD) (2+ transients in isolated cardiomyocytes at different non-contiguous domains. Isolated cardiac cells were field stimulated at room temperature (i = 1 to 10)  as the ratio between the standard deviation and the mean of a specific parameter, namely the time to peak of Ca2+ release.In the last few decades, several techniques have been developed in order to get functional information of the TATS in isolated cardiomyocytes. In particular, optical methodologies have met the need to reduce invasiveness of traditional methods and have undergone a wide evolution. The major limiting factor to optically record fast physiological events, like AP and Can (RAMP)  microsco fashion ,b, 2016cye (VSD) : the RAMperature  to reach) Figure . The len2+ transients originating close to SS and TTs during steady-state stimulations in control ventricular cardiomyocytes  and t-tubules (TTs) , as wellmyocytes . These r1.The spatial CV of time to peak , that is the ratio of the standard deviation to the mean between TTi = 1 to 10 recorded during a single activation .2.The beat-to-beat CV of time to peak (btb CV of TTP), that is the ratio of the standard deviation to the mean between consecutive activations  calculated at a specific membrane site .i in control rat ventricular cardiomyocytes could be related to heterogeneous functional or structural characteristics at different calcium release sites. Non-homogeneous phosphorylation levels of RYR2 or DHPRs in space, for instance, or variability in the dyadic cleft dimensions/structure could lead to a large spatial CV even in control cells. This hypothesis was denied when we observed a quantitatively similar variability of Ca2+ release in time, i.e., when we compared consecutive activations at the same single t-tubule site. In control rat ventricular cardiomyocytes btb CV of TTP was also in the order of 25%  reside within the volume probed by each scanned line (5–10 CRUs in ∼10 μm3) , that acutely promotes RyR2 hyperphosphorylation at Protein Kinase A (PKA) sites, thus prolonging the duration of RyR2 opening (RyR2 ton) and enhancing RyR2 cytosolic and luminal Ca2+ sensitivity , and further experiments with a direct RyR2 agonist would be helpful to define the reciprocal role of the two mechanisms (Ca2+ channels vs. RyR2 PKA-mediated phosphorylation).Regarding the mechanisms underlying the observed spatio-temporal variability, we first hypothesized that the presence of a non-negligible spatial CV  . A typicchannels . During channels . This lositivity . As expesitivity  was simi2+ and electrical activity was then explored in three models of cardiac disease characterized by TATS morpho-functional remodeling.The ability of our method to probe the spatiotemporal relationship between CaAll animal handling and procedures were performed in accordance with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and conformed to the principles and regulations as described in the editorial by We compared the following pathological settings:•a rat model of ischemic heart failure (HF-ischemic), in which myocardial infarction was induced by ligation of the left anterior coronary artery as previously described ; the ani•a rat model of left ventricular hypertrophy induced by spontaneous hypertension . Notably we analyzed the late-stage of the disease, when LV dilation and failure occurred. Thus, this model represents a second example of overt heart failure (HF-SHR) .•a mouse model of hypertrophic cardiomyopathy (HCM), expressing a human mutated cardiac troponin T , associated with high risk of sudden cardiac death and severe diastolic dysfunction in patients . Mice emIn order to quantify the loss of TATS transverse components (TT) from confocal images of isolated cardiomyocytes, we used AutoTT, a software developed specifically for quantitative analysis of the TATS architecture . Cardiom2+ channels and RyR2   sealed-off intracellular membrane structures. Alternatively, t-tubules could be truly depleted from the “diseased” cells with a real loss of membrane domains and associated proteins. Discrimination between these possibilities would be crucial to acquire new insights about pathological t-tubular turnover, but rather difficult to explore experimentally. We instead focused our attention on the function of the “residual” remodeled TATS elements. Besides a reduction of transverse components, remodeled TATS show: (1) a reduction in the number of t-tubular openings on the surface (mouths) , (2) a gand RyR2 . Recentland RyR2 . There imp steps , but the reduced . Increasty in HF , and thi(CaMKII) . Similar(CaMKII)  but chan(CaMKII)  and CaMK(CaMKII)  that occIn addition to phosphorylation , RYR2 is2+ channels and, more importantly, impaired RyR2 function in the CRUs may lead to a general disruption of local Ca2+ release synchronicity in remodeled TATS.Thus, while arrays of RyR2s normally tend to open and close together, RyR2 functional uncoupling in failing myocytes is proposed to result in a greater open probability during diastole and desynchronised channels opening during systole. Increased dyadic cleft distance associated with impaired Ca2+ transient TTPs are prolonged and both the spatial and btb CV (of TTPs) are increased  and uncoupled (AP-) t-tubules. Ca2+ transient TTPs were significantly prolonged in AP- as compared to corresponding AP+ in HF-ischemic and HCM myocytes. These results strongly suggest that the Ca2+ transient detected at AP- tubules originates from the Ca2+ signal propagating from neighboring electrically coupled sites (AP+). This phenomenon was confirmed by a follow-up work, in which we employed a β-adrenergic agonist in HF-ischemic myocytes  Figure  in diseamyocytes . At vari2+ release variability measured with our technique is determined by RyR2 functional state rather than propagative Ca2+-cascade from functioning AP+ sites to AP-. Again, this result has been confirmed by acute treatment of HF-ischemic myocytes with β-adrenergic agonist  and in time (btb CV). We found that, in control cardiomyocytes, space and btb CV of TTP are similar one other (approximately ∼25%) and are both reduced by half upon isoproterenol treatment. Starting from the consideration that a relative small number of RyR2 are located in the scanned regions, our results in control cardiomyocytes suggest that the local variability of TTP may be tightly related to the stochastic opening of RyR2s and is influenced by the channel functional state. The beat-to-beat variability at single membrane domains strongly exclude the presence of systematic structural or functional factors underlying the variability in space. The stochastic nature of Ca2+ release is further confirmed by the fact that mean local Ca2+ transients, resulting from averaging 10 consecutively measurements, perfectly overlap in terms of amplitude and kinetics between SS and TT. In our view, the fact that an average “in time” abolishes the spatial variability is a further demonstration that the mechanisms underlying this variability is the stochasticity of Ca2+ release.We employed the RAMP microscope to simultaneously and consecutively measure local TATS activity and obtain information regarding Ca2+ fluxes of “residual” structurally remodeled TATS in three pathological conditions  and found a number of common changes as well as disease-specific alterations: prolonged local Ca2+ transients TTPs and markedly increased spatial and temporal variability of TTPs. Despite different numbers, we found a population of “residual” t-tubules failing to propagating AP in all three pathological settings. We found two main results. The first (expected) was an increased spatial and btb CV of TTPs in disease remodeled TATS. The second (unexpected) was that the electrical coupling of t-tubules (AP+ and AP-) does not influence the variability of Ca2+ release.We next studied the electrical function and associated Ca2+ release is increased, the coexistence of “in parallel” myofibril layers with different timing of initial activation would result in reduced force development and overall slower kinetics. However, we can speculate that the increased temporal variability of Ca2+ release may generate also the coexistence of “in series” myofibrils with different timing of activation, eventually promoting arrhythmias by favoring AP alternans. These new pro-arrhythmogenic mechanisms that occur in remodeled t-tubules may be added to those previously described  and absent (AP-), respectively, exclude a major role of Ca2+ channels function and indicates that the main player in the local variability of Ca2+ release is the RyR2 functional state. While RyR2 function resulting from targeted single post-translational modification  has been largely studied in channels re-expressed in bilayers, the complex RyR2 alterations that occur in cardiac diseases cannot be reproduced in vitro. Preserving the manifold context of pathological settings is therefore essential to accurately investigate disease-related channel alternations. Thanks to our RAMP approach, a novel set of information may become accessible, allowing for unprecedented exploration of RyR2 function in living cardiomyocytes. Distinct modulation of different subpopulations of RyR2 clusters, those coupled to the t-tubules (dyadic RyR2) and those non-coupled (corbular RyR2), has been recently highlighted in a number of studies .A number of mechanisms may contribute to the increased spatial and btb CV of TTPs of remodeled TATS, including dyadic structural changes and alterations of Ca studies , 2016. C studies , while P studies , 2016. I studies . As an eIn the absence of abundant transverse membrane invaginations, as in atrial cells or in disease-remodeled ventricular cardiomyocytes, we can configure the presence of at list other three populations of RyR2 in addition to those mentioned above:- RyR2s that are coupled to axial tubules, well represented in atrial cells.- RyR2s that are “orphaned” of their t-tubules because of t-tubule disruption in disease-associated structural remodeling.- RyR2s that are coupled to non-propagating (AP-) t-tubules, as observed in disease-remodeled myocytes and described in detail above.2+ release with the RAMP technique may provide significant insights to clarify the functional characteristics of these RyR2 populations. For instance, in mouse atrial myocytes, Ca2+ release from the SR in axial tubules is approximately two times faster at the center of the cell as compared to Ca2+ release at the surface. Rapid Ca2+ release correlated with colocalization of highly phosphorylated (highphos) RyR2 clusters at junctions between axial tubules and SR junctions (2+ signaling has been recently reported across different species (2+ release. We can speculate that the spatio-temporal variability of Ca2+ release could be reduced at the highphos RyR2 clusters and our approach may help establishing the role of axial tubules, highphos RyR2 clusters, and non-junctional Ca2+ release. RAMP measurements can be extended to the intact tissue in order to study subcellular Ca2+ release spatio-temporal variability in various cells at the same time (2+ release that may occur in human cardiomyocytes. Though, direct RAMP measurements of Ca2+ release in human t-tubules are needed, and may represent the most exciting perspective for this technology.The study of subcellular spatio-temporal variability of Caunctions . Novel e species , possiblame time . These same time . Loss ofame time . We beliMS analyzed the data. MS, CC, CF, FP, and LS wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The handling Editor declared a shared affiliation, though no other collaboration, with several of the authors CF, FP, and LS at the time of review.


Here we show that phosphatidylinositol -bisphosphate (PIP2) regulates TMEM16A channel activation and desensitization via binding to a putative binding site at the cytosolic interface of transmembrane segments (TMs) 3–5. We further demonstrate that the ion-conducting pore of TMEM16A is constituted of two functionally distinct modules: a Ca2+-binding module formed by TMs 6–8 and a PIP2-binding regulatory module formed by TMs 3–5, which mediate channel activation and desensitization, respectively. PIP2 dissociation from the regulatory module results in ion-conducting pore collapse and subsequent channel desensitization. Our findings thus provide key insights into the mechanistic understanding of TMEM16 channel gating and lipid-dependent regulation.The calcium-activated chloride channel (CaCC) TMEM16A plays crucial roles in regulating neuronal excitability, smooth muscle contraction, fluid secretion and gut motility. While opening of TMEM16A requires binding of intracellular Ca The calcium-activated chloride channel (CaCC) TMEM16A plays crucial roles in regulating neuronal excitability and muscle contraction. Here authors show that phosphatidylinositol -bisphosphate (PIP2) regulates TMEM16A channel activation and desensitization via binding to a putative binding site. As the founding member of the transmembrane protein 16 (TMEM16) family, TMEM16A proteins are the bona fide pore-forming subunits of CaCC5. Aberrant upregulation of TMEM16A has been found in various pathological conditions, including head and neck squamous carcinomas, gastrointestinal stromal tumors, breast cancer, asthma and chronic obstructive pulmonary disease2. Owing to its importance in physiology as well as its therapeutic potential, there is an urgent need to understand the molecular underpinnings of TMEM16A–CaCC activation and regulation.Ca2+- and voltage-dependent activation and nonselective anion permeability5. Whereas it requires membrane depolarization for activation under sub-micromolar cytosolic Ca2+, TMEM16A becomes constitutively open under saturating Ca2+ concentrations (above 1–2 µM). Structurally, TMEM16A channels adopt an architecture of a “double-barreled” homodimer with each monomer consisting of ten transmembrane (TM) helices with cytoplasmic N− and C-terminal domains11  was first suggested to regulate channel activation and inactivation of both TMEM16A and TMEM16B CaCCs26. However, this hypothesis has been challenged, as purified TMEM16A alone was sufficient to form Ca2+-activated chloride channels27. Immunoprecipitation experiments also showed a lack of TMEM16A–CaM interaction, and exogenous manipulations of CaM exerted no effects on TMEM16A channel activation and permeability28.In addition to Caion Fig. . This phied Fig. 7,9. Whil2), an important signaling lipid29 that also acts as a key regulator of numerous ion channels31, was recently proposed to regulate TMEM16A channel gating34. This hypothesis was further bolstered by the observation that TMEM16F, a dual function TMEM16 ion channel and lipid scramblase35, requires PIP2 for its ion channel activity36. Despite these advances, the precise roles of PIP2 in modulating TMEM16A channel gating are poorly defined, and the structural determinants that govern this lipid-dependent regulation of TMEM16A channel gating remain to be elucidated.Phosphatidylinositol -bisphosphate  following 5 min of channel activation is ~3.95 µM, a value comparable to those reported in other well-characterized PIP2-regulated ion channels such as KCNQ and Kir channels40.In addition to its important roles in a plethora of cellular signaling processesa2+ Fig. . The eff8 PIP2 molecules can strongly impede TMEM16A desensitization. The effect of full-length PIP2 on attenuating TMEM16A desensitization, however, is much more sustained than that of diC8 PIP2 upon removal from the cytosolic perfusion P3 significantly attenuates desensitization while PI(4)P exerts minimal effect on the channel, consistent with a recent study34  but not EGTA solution can partially restore TMEM16A activity . We found that PIP2 depletion by poly-L-lysine 46 results in almost complete inhibition of the channel, which is rapidly reversed via exogenous diC8 PIP2 application  curve  in TMEM16A. Previous functional and structural studies have unambiguously demonstrated that basic lysine and arginine residues at the membrane-cytosol interface usually form the PIP2 binding sites via electrostatic interactions with the phosphate headgroups and phosphodiester linkage phosphate of PIP249. Neutralization of these residues should reduce the channel’s PIP2 binding affinity, thereby accelerating channel rundown. Guided by the atomic structures of a mouse TMEM16A9, we conducted a systematic alanine mutagenesis scanning of all basic residues located approximately between the inner leaflet and the cytosol interface  in saturating 100 µM Ca2+ . Different from R451 and K567, R482 resides in an unresolved short peptide in the C-terminus of TM2–3 loop preceding TM39. Based on our atomistic simulations, R482 has a relatively lower interaction probability of 0.60 ± 0.10 with PIP2, likely owing to the flexibility of this region. Instead of interacting with the phosphate groups of PIP2, R575 appears to form hydrogen bonds with the inositol ring and the phosphodiester linkage of PIP2 with a high probability (0.86 ± 0.08)  confirms that a full-length PIPing Fig. . The sixIP2 Fig. , forming08) Fig. .Fig. 4St2 , which is located at TM4–5 linker 56 did not alter TMEM16A desensitization. As diC8 PIP2 dose-dependently attenuates TMEM16A desensitization, likely by stabilizing the open conformation of the pore  of functional TMEM16A channels following NFA washout , a classical CaCC blockerout Fig. . This resee Fig. . To elim2+ binding site and our proposed putative PIP2 binding site . Depletion of PIP2 rapidly converts the O1-PIP2 state to a transient desensitized state (D1) that can either be reversed back to the O1-PIP2 by exogenous PIP2 or be overcome by more Ca2+ or depolarizing voltage to adopt a fully open state (O2)  state, which is resistant to reactivation by Ca2+, voltage, and exogenous PIP2. Our functional characterizations using the large anion SCN−, the CaCC blocker NFA, and pore mutations suggest that a “pore collapse” mechanism may underlie this high Ca2+-induced desensitization in TMEM16A. This collapsed, nonconducting D2 state was likely captured in the recent Ca2+-bound TMEM16A structures in which the channel’s steric gate adopts an apparently constricted conformation that precludes ion permeation9. Taken together, our findings illuminate a critical structural framework for the mechanistic understanding of lipid-dependent modulation of channel gating in TMEM16 ion channels.We herein propose a simplified “two-module” model of the ion permeation pore to explain the mechanism of how PIPxt Figs. c and 7. 2+-binding module and the PIP2 regulatory module, both of which appear to form a structural repeat54 . Our current study together with previous reports32 strongly support the notion that PLC-induced PIP2 hydrolysis not only leads to TMEM16A channel activation via the IP3 signaling pathway, but concomitantly the subsequent reduction in plasma membrane PIP2 concentration could provide a negative feedback to turn off TMEM16A. We believe that this built-in PIP2-dependent “brake” allows fine-tuning of TMEM16A–CaCC’s activities during Ca2+ signaling. Further studies are needed to interrogate the physiological relationship between TMEM16A and PIP2 and whether this autonomous regulatory mechanism exists in other TMEM16 proteins.In both nonexcitable and excitable cells, TMEM16A–CaCC can be activated by intracellular Ca14  tagged with a C-terminal eGFP or mCherry (in an N1 or a PUNIV vector) was used for all experiments. Single point mutations were generated using QuikChange site-directed mutagenesis kit (Agilent). Primers for PCR were purchased from IDT DNA Technologies  and laminin (Sigma) coated coverslips placed in a 24-well plate (Eppendorf) reaching 40–60% confluency were transiently transfected using X-tremeGENE 9 DNA transfection reagent (Sigma) at a 1:2 (m:v) DNA:transfection reagent ratio. Typically, 200 ng of TMEM16A DNA was used for each well of a 24-well plate to yield sufficient channel expression for recordings.HEK293T cells, authenticated and tested negative for mycoplasma by Duke Cell Culture Facility (ATCC CRL-11268), were grown in DMEM supplemented with 10% FBS (Sigma), 1% penicillin and streptomycin (Gibco), and supplied with 5% CO2, adjusted to pH 7.3 (NaOH), and the bath solution  contained 140 mM NaCl, 10 mM HEPES, adjusted to pH 7.3 (NaOH). Internal free Ca2+ solutions (<100 µM) were made by adding CaCl2 at concentrations calculated using WEBMAXC online software (http://maxchelator.stanford.edu/webmaxc/webmaxcE.htm) to an EGTA-buffered solution containing (in mM): 140 NaCl and 10 HEPES, 5 EGTA, adjusted to pH 7.3 (NaOH). Free Ca2+ concentrations of EGTA-buffered Ca2+ solutions were subsequently measured by a spectrometer using the ratiometric Ca2+ dye Fura-2 (ATT Bioquest), and using a Ca2+ standard curve generated from a Ca2+ calibration buffer kit (Biotum). Solutions containing 100 µM Ca2+ or more were made by directly adding CaCl2 into a solution containing (in mM): 140 NaCl and 10 HEPES, pH 7.3 (NaOH).All electrophysiology recordings were carried out at room temperature within 24–48 h after transfection. Inside-out patch clamp recordings were performed on cells expressing fluorescently-tagged TMEM16A channels (mCherry or eGFP) with glass electrodes pulled from borosilicate capillaries (Sutter Instruments). Pipettes were fire-polished using a microforge (Narishge) and had a resistance of 2–3 MΩ. After formation of gigaohm seal, inside-out patches were excised from cells expressing TMEM16A constructs of interest. The pipette solution  contained (in mM): 140 NaCl, 5 EGTA, 10 HEPES, 2 MgCl2+ concentrations to the cytoplasmic side of the excised patches.Application of the intracellular solution to excised inside-out patches was performed using a pressurized perfusion apparatus  in which the perfusion outlet was directly positioned in front of the pipette tip containing the excised membrane patch. TMEM16A channels were activated by focal perfusion of solutions containing the desired Ca2+, current responses were elicited by voltage steps of −80 mV and +80 mV lasting 100 ms each at an inter-sweep interval of 5 s, and the membrane was held at 0 mV. Steady state currents measured at −80 mV or +80 mV voltage steps were used for analysis.For the rundown protocol in saturating Ca2+ concentration-dependent recordings, the membrane was held at +60 mV, and TMEM16A’s outward currents were elicited by perfusion of EGTA-buffered Ca2+ solutions of various free Ca2+ concentrations. Quasi-steady state currents were measured for each Ca2+ application and normalized to the peak current elicited by 100 µM Ca2+ to construct the dose-dependent curve.For Ca8 PI(4)P, diC8 PIP2, diC8 PIP3 , and full-length brain PIP2 (Avanti) were first dissolved in water to generate stock solutions of 5 and 0.5 mM for diC8 lipids and full-length lipids, respectively. All lipid stock solutions were kept at −80 °C for long-term storage. On the day of recordings, phosphoinositide lipids were diluted in recording solutions containing desired free Ca2+ concentrations, sonicated on ice for 5–10 min, and loaded into the pressurized perfusion chamber. All phosphoinositide-containing solutions were used within 2 h.For phosphoinositide experiments, diC2-mediated rescue experiments in sub-micromolar Ca2+, inside-out patches from cells expressing mTMEM16A were perfused with the desired sub-micromolar Ca2+ and +80 and −80 mV steps lasting 200 ms each were applied to elicit outward and inward currents, respectively. The inter-sweep interval was 5 s and the membrane was held at 0 mV. Channel activity was measured for 20–30 s to establish a baseline. Membrane PIP2 depletion was then induced by ~15–20-s application of Ca2+-free (5 mM EGTA) solution containing 100 µg/ml PLL or a solution with sub-micromolar Ca2+ solution containing 100 µg/ml PLL to induce desensitization. diC8 PIP2-containing solutions with the desired sub-micromolar Ca2+ were then applied to rescue channel activity. Steady state currents at the end of each +80-mV depolarization step were used for analyses.For PIP2 depletion on the voltage-dependent activation of TMEM16A, a two-step procedure was used to construct G–V relationships. First, using inside-out patches, we obtained maximal channel activation of TMEM16A (under 100 µM Ca2+) using a voltage step protocol consisting of +120 mV depolarization step and −60 mV repolarization step, the tail current from which was used to calculate the Gmax. To assess the effects of PLL, the membrane patch was first treated with PLL (100 µg/ml) for 15–17 s before 100 µM Ca2+ was applied to obtain the Gmax. Subsequently, the voltage-dependent activation of TMEM16A was quickly obtained by applying sub-micromolar Ca2+ concentrations of 0.255 and 0.387 µM Ca2+ using an I–V protocol in which the membrane was held at −60 mV, and voltage steps from −120 to +120 mV were applied to elicit channel opening. In both control and PLL-treated conditions, the tail currents measured at the −60-mV repolarization step in response to various voltages under 0.255 or 0.387 µM Ca2+ were normalized to the tail current measured at −60 mV under 100 µM Ca2+ to obtain the G/Gmax–V curves.To probe the effects of PIP−, both the extracellular (pipette) and intracellular (perfusion) solutions were symmetric in permeant ions (Cl− and SCN−) and contained either 140 mM Cl− (control) or 28 mM Cl− and 112 mM SCN−(SCN−). All intracellular solutions also contained 100 µM Ca2+. In experiments testing the effects of SCN− on TMEM16A’s PIP2 sensitivity, internal and external solutions were symmetric and contained 28 mM Cl− and 112 mM SCN−, and the internal solution contained 100 µM Ca2+ with various concentrations of diC8 PIP2 . The normalized currents measured at 5 min were used to construct the dose-dependent curves showing effects of SCN− on TMEM16A’s PIP2 sensitivity.For experiments with SCN−) both contained (in mM): 140 NaCl, 5 EGTA, 10 HEPES, adjusted to pH 7.3 (NaOH) and osmolarity of ~300 (D-mannitol). The intracellular solution (perfusion) also contained 100 µM Ca2+ to activate TMEM16A, and inward and outward currents were elicited by a repeated ramp protocol from −100 to +100 mV. Intracellular solution was then replaced by perfusion with a solution (low 14 mM NaCl) containing (in mM): 14 NaCl, 5 EGTA, 10 HEPES, adjusted to pH 7.3 (NaOH), 100 µM Ca2+ and osmolarity of ~300 (adjusted with D-mannitol). The reversal potential (Erev) was determined as the membrane potential at which the current was zero. The shift in Erev was calculated by subtracting the Erev of intracellular 14 mM NaCl from the Erev measured in symmetric 140 mM NaCl.For reversal potential measurements, inside-out configuration was used for rapid exchange of the intracellular solution (bath solution). First, TMEM16A channels were activated in which the pipette solution and perfusion solution (symmetric 140 mM ClAll electrophysiology recordings were low-pass filtered at 5 kHz (Axopatch 200B) and digitally sampled at 10 kHz (Axon Digidata 1550 A) and digitized by Clampex 10 (Molecular Devices).2+, 100 µM Ca2+-elicited currents measured at −80 or +80 mV were normalized to the peak current amplitude, and the half-decay time (t1/2) was calculated by fitting the channel current decay with a curve using a custom-written MATLAB script.All offline data analysis was performed using Clampfit, Microsoft Excel, and MATLAB (MathWorks). To quantify the extent of rundown in saturating Ca2+ dose-dependent concentrations (EC50), normalized Ca2+-induced currents were fit into a nonlinear regression curve fit of four parameters with the equation:max denotes current normalized to the max current elicited by 100 µM Ca2+, [Ca2+] denotes free Ca2+ concentration, H denotes Hill coefficient, and EC50 denotes the half-maximal activation concentration of Ca2+.For quantification of CaCl/PNa was calculated using the Goldman–Hodgkin–Katz equation:rev is the measured shift in reversal potential (in mV); PNa and PCl are the relative permeabilities of TMEM16A toward Na+ and Cl−, respectively; [Na]o and [Na]i are external and internal sodium concentrations, respectively; [Cl]o and [Cl]i are external and internal chloride concentrations, respectively; F is the Faraday’s constant , R is the gas constant (8.314 J mol−1), and T is the absolute temperature (298.15 K at 25 °C).The permeability ratio P8 PIP2 under sub-micromolar Ca2+, PIP2-mediated current rescue ratio was calculated byIBaseline is the steady state current before PLL application, IPLL is the steady state current during PLL treatment, and IPIP2 is the peak current following PIP2 application.In current rescue experiments by diCG/Gmax–V curves were fit with a Boltzmann equation:G/Gmax is the normalized channel conductance, z is the number of equivalent gating charges, Vm is the membrane voltage, V1/2 is the voltage at which G/Gmax is 0.5, F is the Faraday’s constant, R is the gas constant, and T is the absolute temperature.The normalized 2+-bound state (PDB 5OYB) was used in all molecular docking and atomistic simulations. The sequence in the cryo-EM structure has four additional residues (448EAVK451) compared with the construct used in the current experimental studies. This segment was included in all simulations to minimize any unnecessary disruption of the origin cryo-EM structure. Several short loops in cytosolic domain  are absent and they were rebuilt using the ProMod3 tool with Swiss-PBD server. The backbone of missing residues was first modeled with the Swiss-PDB fragment library, and the side chains were then constructed from a rotamer library63. The N− and C-terminal segments (M1-P116 and E911-L960) as well as a long loop in the cytosolic domain (Y131-V164) are also absent in the cryo-EM structure. These regions are presumably dynamic and thus not included in simulations. The residues before and after the missing part were capped with either an acetyl group (for N-termini) or a N-methyl amide (for C-termini). To minimize the effects of missing residues on the cytosolic domain, the backbone of structured parts of the cytosolic domain  was harmonically restrained with a force constant of 1 kcal/(mol.Å2) during all simulations described below.The cryo-EM structure of mouse TMEM16A in the Ca2+-bound state of TMEM16A. In the first set, the full−length PIP2 molecule was first docked into the putative binding site identified from mutagenesis scanning using Autodock Vina64. Briefly, docking was carried out over a search space of 29 × 32 × 57 Å covering the putative PIP2 binding site formed by the intracellular portions of TMs 3, 4, 5, and TM2–3 linker. The docked structure was then used to initiate multiple atomistic simulations in explicit membrane and water to examine the stability of PIP2 in the proposed binding site and atomistic details of PIP2-TMEM16A interactions. The TMEM16A structure (with or without PIP2) was first inserted in model POPC lipid bilayers and then solvated in TIP3P water using the CHARMM-GUI web server65. The second and third sets of simulations involved the Ca2+-bound, but PIP2-free state TMEM16A. In the second set, 5% of randomly chosen POPC molecules were replaced with full−length PIP2 molecules, to examine spontaneously rebinding of PIP2. In the third set of simulations, 20 diC2 PIP2 molecules (head group) were randomly placed in solution, to explore possible binding sites of PIP2 head group alone. All final solvated systems were neutralized and 150 mM KCl added. The final simulation boxes contain about ~600 lipid molecules (POPC and/or PIP2) and ~70,000 water molecules and other solutes, with a total of ~316,000 atoms and dimensions of ~150 × 150 × 135 Å3. The CHARMM36m all-atom force field66 and the CHARMM36 lipid force field67 were used. PIP2 parameters were adopted from a previous study68. All simulations were performed using CUDA-enabled Amber1469. Long-range electrostatic interactions were described by the Particle Mesh Ewald (PME) algorithm with a cutoff of 12 Å. Van der Waals interactions were cutoff at 12 Å with a smooth switching function starting at 10 Å. The lengths of hydrogen-containing covalent bonds were constrained using SHAKE and the MD time step was set at 2 fs. The temperature was maintained at 298 K using the Langevin dynamics with a friction coefficient of 1 ps−1. The pressure was maintained semi-isotopically at 1 bar at both x and y  directions using the Monte Carlo barostat method71.Three sets of atomistic simulations were performed, all involving Ca2). In the last equilibration step, only protein heavy atoms were harmonically restrained, and the system was equilibrated 10 ns in under NPT  conditions. All production simulations were performed under NPT conditions. Three independent 400-ns simulations for the PIP2-bound state of TMEM16A in pure POPC bilayers. For spontaneously binding simulations, four simulations of 200–700 ns were performed PIP2-free TMEM16A in either mixed POPC-PIP2 bilayers or in a POPC membrane with free, soluble PIP2 headgroups. Snapshots were saved every 50 ps for analysis. For analysis of PIP2-TMEM16A contacts, only 100–400 ns trajectories were included. Salt–bridge interactions between PIP2 and basic residues in the putative TMEM16A binding site were considered formed whenever the minimum distance between any atom of the ammonium (Lys) or guanidinium (Arg) moieties and oxygen atoms in 1-/4-/5- phosphate group was no >5 Å. The pore profile of the putative ion permeation pathway was calculated using program HOLE72. The PMF of water was calculated directly from the histograms of water distribution along the membrane normal. The 3D water density distribution near the ion permeation pathway was calculated using a cubic grid with a resolution of 0.5 Å.All systems were first minimized for 5000 steps using the steepest descent algorithm, followed by a series of equilibration steps where the positions of heavy atoms of the protein/lipid were harmonically restrained with restrained force constants gradually decreased from 10 to 0.1 kcal/. Sequence alignment was performed in UniProt online software .PDB coordinate files were downloaded from the Protein Data Bank website t-test was used for single comparisons between two groups (paired or unpaired), and one-way ANOVA was used for multiple comparisons. Comparisons yielding p-values < 0.05 are considered to be statistically significant. Data in summary graphs are represented as mean ± standard error of the mean (SEM), and each data point represents an independent recording experiment. Symbols *, **, ***, and **** denote statistical significance corresponding to p-value <0.05, <0.01, <0.001, <0.0001, respectively.All statistical analyses were performed in Prism software (GraphPad). Two-tailed Student’s Further information on research design is available in the Supplementary InformationSupplementary Movie 1Supplementary Movie 2Reporting SummaryDescription of Additional Supplementary FilesPeer Review FileSource Data


Recent research has shown that it is also complemented by stromal interaction molecule (STIM) protein-mediated store-operated Ca2+ entry (SOCE). SOCE is described as Ca2+ flow into cells in response to the depletion of endoplasmic reticulum Ca2+ stores. The present review summarizes recent studies that indicate a relationship between neuronal SOCE that is mediated by STIM1 and STIM2 proteins and glutamate receptors under both physiological and pathological conditions, such as neurodegenerative disorders. We present evidence that the dysregulation of neuronal SOCE and glutamate receptor activity are hallmarks of acute neurodegenerative diseases  and chronic neurodegenerative diseases . Emerging evidence indicates a role for STIM proteins and glutamate receptors in neuronal physiology and pathology, making them potential therapeutic targets.Neuronal calcium (Ca Ale cells ,39,40, cle cells ,42, and le cells . STIM-me neurons . Althoug neurons ,41,44, a neurons  and cort neurons ,44. Prev neurons  STIM iso+ levels ,47,48. H+ levels ,49. For + levels ,47. Receresponse .2+ levels, participates in the regulation of spine morphogenesis, influences neuronal Ca2+ dynamics during synaptic excitation, and regulates gene expression [2+ content activates STIM2-mediated SOCE, which supports a constant level of the Ca2+-CAMKII complex and stabilizes mushroom spines that participate in memory storage [2+ entry pathway, which regulates gene expression and proliferation of neural progenitor cells (NPCs). Both the suppression and deletion of STIM1 or Orai1 significantly alleviate the proliferation of embryonic and adult NPCs [STIM1 knockdown also decreases the proliferation and early differentiation of human NPCs [2+ influx via TRPCs [2+ influx, which triggers the recruitment of TRPC1 into the PM where it is activated by STIM1. TRPC1 is thought to modify the initial Ca2+ signal that is caused by Orai1 activation [2+ release from the ER, which in turn causes STIM1 aggregation and inhibits L-type VGCCs, thus enhancing ER spine content and stabilizing mushroom spines [V1.3, which is essential for the protection of dopaminergic neurons in the substantia nigra region [2+ shortage is an early event in neurodegeneration associated with UPS inhibition observed in these diseases. The above results deliver some better insight into the contribution of STIM proteins in neurodegeneration mechanisms.New evidence suggests that neuronal STIM-mediated SOCE preserves ER Capression . Althougpression , STIM2 wpression ,52. Low  storage . In turn storage . SOCE isult NPCs . STIM1 kman NPCs . In addiia TRPCs . TRPC1 ativation . Moreovetivation ,57. Accotivation . The infm spines . In turna region . Loss ofa region . Importaa region ,62. This3-dependent Ca2+ release from the ER and TRPC3-mediated slow excitatory postsynaptic currents were impaired. The disruption of these two pathways abolished cerebellar motor behavior [2+ influx not only via Orai and TRPCs, but also through AMPARs [2+ influx. Additionally, the induction of SOCE by thapsigargin (TG) results in both direct and indirect AMPAR activation. We also found that both STIM1 and STIM2 proteins cooperate with GluA1 and GluA2 subunits of AMPARs. Although these interactions occur mainly in pyramidal neurons, they may also occur in non-pyramidal cells [Increasingly more research is focusing on the influence of STIM proteins on glutamate receptors. Ng et al. showed that the activation of group I mGluRs stimulates STIM1 oligomerization and its transport to the PM . This isbehavior . Our stuh AMPARs . AMPAR aal cells . Garcia-al cells . STIM2 ial cells .Stim1/Stim2 conditional knockout (cKO) exhibited an enhancement of LTP, which was associated with increases in the phosphorylation of GluA1, the transcriptional regulator CREB, and L-type VGCCs at PKA sites [Stim genes in the forebrain results in spatial memory impairments, similar to such impairments that are caused by NMDAR blockade [3, which in turn interacts with IP3R, causing Ca2+ release from ER stores and the activation of SOCE. SOCE inhibitors decrease NMDA-dependent Ca2+ influx and synaptic plasticity in the hippocampus [Importantly, both serine residues, Ser-845 and Ser-831, play a pivotal role in LTP and LTD  . Yap et KA sites . The ablblockade . Baba etblockade . The actpocampus . Neverthpocampus . Therefopocampus . Other spocampus ,72.Stim2 cKO mice [Stim2 cKO mice [S)-3,5-dihydroxyphenylglycine (DHPG)-induced LTD. The SOCE inhibitor YM-58483 disrupts DHPG-induced LTD and the maintenance of DHPG-mediated cytosolic Ca2+ signals. These results suggest that SOCE is essential for the activation of downstream mGluR effectors. On the other hand, Majewski et al. found that mGluR signaling is impaired in STIM1-overexpressing mouse hippocampal neurons, with no changes in LTP or basal synaptic transmission [In contrast to LTP, LTD is mediated by the endocytosis of AMPARs from synaptic sites. A decrease in NMDA-mediated LTD was recently reported in cKO mice . AlthougcKO mice . Mice wicKO mice . In primcKO mice . The inhsmission . In thessmission . L- and smission ,75, and smission ,57. Thersmission .2+ levels , which in turn triggers downstream lethal cascades and is considered to be the main cause of neuronal apoptotic death after TBI. Ca2+ overload in TBI can be driven by both Ca2+ release from the ER and Ca2+ influx from the extracellular space through glutamate receptors, VGCCs or SOCE [2+ accumulation [2+ release from ER stores [3R- and/or RyR-dependent rapid Ca2+ release from intracellular stores. The latter mechanism operates as slow Ca2+ influx from the extracellular space. Since STIM1 is a Ca2+ sensor located in the ER that is known to play a role in neuronal injury, the influence of this protein on mGluRs has become an interesting focus of research. Hou et al. reported that mGluR-dependent IP3R- and/or RyR-mediated Ca2+ release from the ER after traumatic neuronal injury (TNI) in STIM1-deficient mice was significantly alleviated [2+; therefore, mGluR1-mediated signaling cannot induce Ca2+ release from intracellular stores. This mechanism indicates that STIM1 might link mGluR1 with its downstream effectors, which is consistent with previous findings [2+ homeostasis. This neuronal protection was associated with the inhibition of ER Ca2+ release via a mGluR1-dependent pathway and a reduction of STIM1 expression (with no effect on the expression of STIM2 or Orai1) and SOCE-mediated Ca2+ influx [2+ release and reducing SOCE after STIM2 knockdown was associated with a reduction of Ca2+ overload. Since STIM2 is considered to support the development of secondary brain injury, the authors speculated that it may be a potential therapeutic target for the treatment of TBI [Traumatic brain injury (TBI) generally results from a jolt or blow to the head, but it may also be caused by a penetrating head injury . It caus or SOCE . Interes or SOCE . Numeroumulation ,80. The R stores ,81 and iR stores . The forleviated . In the findings . The dow+ influx . Therefo+ influx ,83. In c+ influx . The dowt of TBI .2+ ions  and initiates ischemic brain damage and cell death [2+ accumulation during cerebral ischemia [2+ transport to the ER is inhibited, which triggers persistent STIM2 activation and SOCE-mediated Ca2+ accumulation [2+ influx by increasing the release of glutamate and activating ionotropic receptors [2+ inflow may rapidly increase Ca2+ concentrations to a dangerous level. Additionally, the lack of STIM2 decreases Ca2+ overload during an ischemic challenge. In acute hippocampal slices and hippocampal neurons in culture that were isolated from Stim2 KO mice, SOCE is diminished and ER content is lower, so these animals are better able to survive hypoxic conditions. Stim2 KO mice are better protected against cerebral ischemia compared with wildtype mice [2+ concentrations [Stim1 siRNA. The suppression of STIM1 in the early stage of ischemia attenuates neuronal death by inhibiting SOCE-induced neuronal apoptosis [Cerebral ischemia is a pathological condition that is caused by the insufficient supply of oxygen and nutrients to the brain, which may result from embolism, thrombosis, or systemic hypoperfusion . Under lll death  , which is translated to polyglutamine (polyQ) in the huntingtin protein (HTT) [2+ homeostasis [2+ levels, was a significant pathogenic event in HD [2+ concentrations also resulted from Ca2+ efflux via RyRs and IP3R1 [3R1 after mGluR1 activation, increasing its sensitivity to IP3, which persistently reduced ER Ca2+ levels [2+ concentration overactivated synaptic SOCE and enhanced STIM2 expression, which resulted in the disruption of dendritic spines [3Rs or STIM2 was shown to normalize SOCE and prevent dendritic spine loss, thus resulting in neuroprotective effects [3R1 [2+ homeostasis is considered a pathological hallmark of HD, and these compounds may be leading molecular structures for the treatment of HD [Huntington’s disease is an autosomal dominant neurodegenerative disorder that results from the expansion of a CAG repeat in the huntingtin gene (in (HTT) ,105. Thein (HTT) . Neurodein (HTT) ,108. Huneostasis . Studiesnt in HD   and chronic neurodegenerative disorders . Nevertheless, changes in neuronal SOCE may be in opposite directions in different pathological conditions. SOCE appears to be neuroprotective in AD, whereas the blockade of this process may be neuroprotective in other neurological disorders, such as TBI, cerebral ischemia, and HD. In TBI, cerebral ischemia, and HD, neuronal ER Cativation . In TBI,tivation . In conttivation ,94,95. Srfilling . AlthougIn conclusion, the present study reveals that the restoration of physiological neuronal STIM-dependent SOCE and the normalization of STIM protein expression and glutamate receptor activity may confer potential beneficial effects for the treatment of both acute and chronic neurodegenerative diseases. Nevertheless, this approach has limitations . Undoubtedly, further studies are required in this field.