Rabbits with naturally high levels of cholesterol ester transfer protein (CETP), unlike rodents, have become an interesting animal model for the study of lipid metabolism and atherosclerosis, as they have similarities to humans in lipid metabolism, cardiovascular physiology and susceptibility to develop atherosclerosis. Rodents, such as mice, are not prone to atherosclerosis as they lack the mass and activity of CETP, as a key player in lipoprotein metabolism. Recently, APOE-knockout in rabbits has been shown to promote atherosclerosis and associated premature IVD degeneration that mimic the symptoms of atherosclerosis and structural changes of IVDs in humans. Here we examined whether APOE-knockout promoted IVD degeneration in rabbits is associated with imbalanced inflammatory catabolic activities, as the underlying problem of biological deterioration that mimic the symptoms of advanced IVD degeneration in humans. We analysed in lumbar nucleus pulposus (NP) of APOE-knockout rabbits the cell viabilities and the intracellular levels of inflammatory, catabolic, anti-catabolic and anabolic proteins derogating IVD matrix. Grades of IVD degeneration were evaluated by magnetic resonance imaging. NP cells were isolated from homozygous APOE-knockout and wild-type New Zealand White rabbits of similar age. Three-dimensional cell culture with low-glucose was completed in alginate hydrogel. Cell proliferation and intracellular levels of target proteins were examined by MTT and ELISA assays. Alike human NP cells of different disc degeneration grades, NP cells of APOE-knockout and wild-type rabbits showed significantly different in vivo cell population densities (p<0.0001) and similar in vitro proliferation rates. Furthermore, they showed differences in overexpression of selective inflammatory and catabolic proteins (p<0.0001) similar to those found in human NP cells of different disc degeneration grades, such as IL-1β, TNF-α, ADAMTS-4, ADAMTS-5 and MMP-3. This study showed that premature IVD degeneration in APOE-knockout rabbits was promoted by the accumulation of selective inflammatory catabolic factors that enhanced imbalances between catabolic and anabolic factors mimicking the symptoms of advanced IVD degeneration in humans. Thus, APOE-knockout rabbits could be used as a promising model for therapeutic approaches of degenerative disc disorders. Intervertebral disc degeneration is one of the main causes of low back pain. It is characterized by structural deterioration and unfavourable changes in molecular phenotype of IVD cells that enhance expression levels of inflammatory cytokines, such as interleukin beta IL-1β) and tumour necrosis factor alpha (TNF-α). Inflammatory cytokines have been described to induce inflammatory catabolic processes in IVDs and promote accelerated degradation of the extracellular matrix β and tum. Success2) that adversely affects the activities as well as the survival of IVD cells, especially NP cells in the middle of the IVDs. Accordingly, nutrient impairment is considered as one of the major factors of IVD degeneration  were ascertained in NP tissues of APOE-knockout rabbits. The recorded mean numbers of NP cells were 11400 ± 681 for APOE-knockout rabbits and 19619 ± 406 for wild-type rabbits (p<0.0001). As compared to the mean values of the wild-types the amount of NP cells in APOE-knockout rabbits was reduced by about 41.7%  (To determine the ut 41.7% . However= 0.018) .Since inflammatory cytokines can enhance the expression of catabolic factors and promote an inflammatory catabolic metabolism of the IVD matrix, we determined the levels of inflammatory and catabolic proteins in NP cells of APOE-knockout and wild-type rabbits.Enhanced expression levels of the inflammatory cytokines TNF-α and IL-1β as well as their receptors TNF-α R1 and IL-1 R were detected in NP cells of APOE-knockout rabbits. The mean expression levels of TNF-α and TNF-α R1 in NP cells of APOE-knockout rabbits amounted 138.8 ± 3.516 pg/ml and 96.46 ± 2.253 pg/ml respectively; and the respective mean levels for the wild-type rabbits were 63.35 ± 2.016 pg/ml and 54.13 ± 2.127 pg/ml (P<0.0001) . The meaThe catabolic factor ADAMTS-4 and ADAMTS-5 showed high mean expression levels in NP cells of both APOE-knockout and wild-type rabbits. However, the mean levels in NP cells of APOE-knockout rabbits were considerably higher. The mean levels of ADAMTS-4 and ADAMTS-5 were 4017 ± 149.7 pg/ml and 3278 ± 136 pg/ml in APOE-knockout; and 1222 ± 58.28 pg/ml and 1433 ± 60.29 pg/ml wild-type respectively (P<0.0001) .MMPs showed differentiated patterns of expressions in NP cells of APOE-knockout rabbits as compared to that in wild-type. MMP-1 and MMP-3 showed enhanced mean expression levels in NP cells of APOE-knockout rabbits, whereas the mean expression levels of MMP-2, MMP-7, MMP-8, MMP-10 and MMP-13 remained unaffected. The recorded mean expression levels for MMP-1 and MMP-3 were 135.1 ± 2.93 pg/ml and 4967 ± 220.2 pg/ml in APOE-knockout; and 60.32 ± 1.1684 pg/ml and 827.4 ± 34.07 pg/ml in wild-type respectively (P<0.0001) . The resSince decreased expression levels of the anti-catabolic factors (TIMPs) can lead to enhanced NP matrix proteolysis, we determined the levels of TIMP-1, TIMP-2, TIMP-3 and TIMP-4 in NP cells of APOE-knockout rabbits. A greatly enhanced level of TIMP-1 was determined in NP cells of APOE-knockout rabbits as compared to that in wild-types. The mean expression levels were 10226 ± 100 pg/ml and 3770 ± 70.39 pg/ml respectively (P<0.0001) . A moderThe expression of several anabolic factors were examined because growth factors could have crucial roles in the development, growth and maintenance of intervertebral discs. From all tested anabolic factors only TGF-β1, IGF-1 and BMP-2 could be detected exclusively in NP cells of APOE-knockout rabbits with mean levels of 51.06 ± 1.518 pg/ml, 63.01 ± 1.615 and 43.34 ± 1.121 pg/ml respectively. Although the minimum detectable level for all tested anabolic factors were very low, the concentration levels of BMP-4, BMP-6 and BMP-7, FGF-2 and TGF-β3 were not traceable in NP cells of both APOE-knockout and wild-type rabbits . NP cellThe levels of the major IVD matrix proteins aggrecan, collagen II and collagen I were examined in NP cells of APOE-knockout and wild-type rabbits. An extremely decreased concentration level of aggrecan was detected in NP cells of APOE-knockout rabbits with mean concentration level of 24526 ± 2165 pg/ml, and the recorded mean concentration level in wild-type was 65087 ± 2552 pg/ml (P<0.0001) . MoreoveDegenerative intervertebral disc disease is described as a biological disintegration process of IVD matrix that can be promoted by various factors that may cooperate with each other, such as genetical, nutritional and inflammatory factors. It has been previously shown that adult IVDs have significantly lower cell population density than IVDs of children. This is due to the fact that the concentration levels of glucose and oxygen decrease in the adult IVDs, as blood supply and diffusion decrease during aging. The declined nutrient supply into adult IVDs promotes harsh changes of the cell environment predominantly in the center of the IVDs , 40–42.in vivo gene therapeutic approaches. For that reason, we examined the cell proliferation and the protein expression pattern that affect the NP tissue in APOE-knockout rabbits, which were recently characterized by structural deterioration of the IVD matrix that mimics the symptoms of advanced IVD degeneration in humans [Degeneration of IVDs becomes apparent as early as the second decade of life. Comparable to IVD degeneration, atherosclerosis also starts to develop early in adult age. In almost the same manner the intensification of IVD degeneration and atherosclerosis in the abdominal aorta and aortic bifurcation, takes place between 44 and 64 years of age , 43–44. n humans .in vitro and in vivo studies that displayed the critical effect of nutrient deprivation on the maintenance of IVD cell viability and anabolic processes [in vitro culture conditions equivalent proliferation rates  and ADAMTS-5 (2.3 fold) in NP cells of APOE-knockout rabbits . The resIn addition to that, our data exhibited imbalances between the imperceptible levels of anabolic factors (Tab.1) and the critically enhanced levels of catabolic factors in NP cells of APOE-knockout rabbits, which highlight a decrease in stimulation of matrix synthesis and an increase in promotion of matrix degradation. In line with this, we detected significantly diminished levels of aggrecan and collagen II in NP cells of APOE-knockout rabbits .In conclusion, APOE-knockout in rabbits could induce accelerated IVD degeneration that could be characterized by molecular and structural changes mimicking the symptoms of advanced IVD degeneration in humans. The results apparently point out that degenerative change in IVDs could be caused not only by loss NP cells but also by unfavourable phenotypic changes in NP cells. NP cells of APOE-knockout rabbits exhibited enhanced levels of inflammatory catabolic cytokines, imbalances between catabolic and anti-catabolic factors along with imbalances between catabolic and anabolic factors, that emulate the results found in human NP cells of degenerative IVDs.in vivo model, as they can be used in studies of both disc disorder and atherosclerosis. Moreover, they can bypass the standard surgical interventions that are commonly applied for induction of accelerated disc degeneration in other research animals, and so contribute to the 3Rs.Therefore, APOE-knockout rabbits could be used as a promising animal model for therapeutic approaches of degenerative disc disorders. They can be seen as an advantageous APOE-knockout rabbits can be used as a new model to set up a stable, less immunogenic and non-pathogenic ex vivo gene therapy system to enhance the regeneration potential of NP cells and advance the treatment strategies of degenerative disc disorders. For that degenerative NP cells can be isolated from NP tissue of APOE-knockout rabbits and transduced in culture by recombinant AAV6 carrying the relevant therapeutic genes identified here, as we recently established in degenerative human NP cells –52. The S1 Checklist(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


In secretory organs, such as the pancreas, salivary gland, and lacrimal gland (LG), [Ca2+]i elevation in acinar cells triggers fluid secretion, which plays vital roles in the maintenance of functional health across the life-course. It is important to understand the secretory mechanism of secretory organs, but lack of analytic systems available for living animals limits the scope of research to gain deeper insights into the precise mechanism of secretion. We established an intravital imaging system for specific cell types of secretory organs to monitor the [Ca2+]i changes using mouse line expressing Yellow Cameleon 3.60, a genetically encoded Ca2+ indicator. Elevation of [Ca2+]i in specific cell types of secretory organs could be monitored after cholinergic stimulation ex vivo and intravitally. We found that a marked attenuation of LG [Ca2+]i response to cholinergic stimulation was induced under pathological conditions by postganglionic denervation. Intravital Ca2+ imaging in secretory organs will broaden our understanding of the cellular mechanisms in animal models of secretory diseases.Intracellular calcium ([Ca Intracellular Ca2+ concentration ([Ca2+]i) is regulated by intracellular release from the endoplasmic reticulum store or influx through a variety of Ca2+ channels in response to stimulation by neurotransmitters and a variety of hormones2.Intracellular Ca2+]i elevation in the secretory acinar cells is the key trigger for secretion of a mixture of water and proteins synthesized in the acinar cells5. The fluid mixture produced by secretory organs is secreted onto the epithelial surface out of the organs, and is necessary for maintenance of epithelial homeostasis and body metabolism5. The dysfunction of secretory organs leads to various diseases, such as diabetes, dry mouth, and dry eye8, by poorly understood mechanisms. Evaluation of intracellular Ca2+ signaling under normal physiological and pathological conditions is an effective approach to elucidate the mechanisms underlying the diseases caused by dysfunction of secretory organs.In secretory organs, such as the pancreas, salivary gland (SG), and lacrimal gland (LG), i elevation12. Upon binding of free Ca2+, the Ca2+ responsive element calmodulin (CaM) of YC alters the efficiency of the two fluorescent proteins CFP and YFP. The other well-recognized GECI is GCaMPs, a single fluorophore probe based on a circularly permuted green fluorescent protein, their monitoring of Ca2+ signaling is depending on fluorescence intensity. Compared to GCaMPs, YCs have less dynamics range and photostability but the advantages of them are less sensitive to motion artifact and expression level differences because of rationing of two fluorescent proteins14.In recent studies of Ca2+ signaling in living animals15. This mouse line has been demonstrated to be useful in monitoring the Ca2+ signaling in lymphoid tissues  and intestinal gut epithelial cells15. However, no studies have demonstrated intravital imaging of Ca2+ signaling in specific secretory organs of YC3.60 transgenic mice.Previously, a transgenic mouse line conditionally expressing YC3.60 was established to monitor the long-term, spatiotemporal Ca2+ signaling within secretory organs in a cell-type-specific manner using YC3.60 transgenic mice in combination with two-photon microscopy. This visualization system opens a wide range of new possibilities in the study of intravital secretory activities or behaviors.In this manuscript, we describe a useful and efficient visualization system to monitor the Ca2+ imaging in secretory organs, we examined the tissue morphology and distribution of YC3.60 expression in various secretory organs. There were no obvious differences in tissue morphology between YC3.60 and wild-type mice.  surrounding the basal surface of acinar cells are stellate-shaped cells and have a contractile function, which helps expelling secretory components from acinar cells to the excretory duct18.Acinar cells are polarized epithelial cells that play an essential role in the production of secretory components19.Secretory organs are composed primarily of acinar cells, and MEC have also been found in almost all secretory organs with notable exception of the pancreasTo examine the localization of the YC3.60 probe in secretory organs, we performed immunofluorescent staining of calponin Fig.  and wheaIn the pancreas, calponin-positive cells were not observed and fluorescence from the YC3.60 probe was observed in the cytoplasm of acinar cells. In the salivary gland, calponin-positive cells which are stellate in shape were observed. The fluorescence of the YC3.60 probe was localized in calponin-positive cells and parts of the cytoplasm of acinar cells. Calponin-positive cells were also observed in the lacrimal gland (LG). The fluorescence of YC3.60 probes co-localized with calponin-positive cells in the LG.Figure 18. These results indicate that localization of the YC3.60 probe uniquely exhibits a cell-type-specific pattern in secretory organs of YC3.60 transgenic mice.Our results of the distribution of MEC positively stained with the anti-calponin antibody in various secretory organs corresponded with previous reports20. Parasympathetic stimulation increases outflow of pancreatic fluid, saliva, and tears through the intracellular Ca2+ mobilization due to the activation of muscarinic acetylcholine receptors (mAChRs) in the pancreas, SG, and LG, respectively22.The autonomic nervous system regulates the secretory functions of the pancreas, SG, and LG2+ imaging in secretory organs ex vivo and intravitally, we established the ex vivo and intravital Ca2+ imaging systems. Figure ex vivo and intravital two-photon Ca2+ imaging system.To confirm whether the genetically encoded YC3.60 probe can be used in spatiotemporal Ca2+ ATPase pump23 and the mAChR agonist acetylcholine (ACh) using isolated pancreases from YC3.60 transgenic mice by using our ex vivo imaging system.We initially evaluated the changes in FRET ratios induced by the calcium ionophore ionomycin; cyclopiazonic acid (CPA) which is an inhibitor of the endoplasmic reticulum CaFigure There were no changes in the fluorescence of CFP and YFP before ionomycin stimulation . 10 seconds after the ionomycin stimulation, a gradual decrease in the extent of CFP fluorescence and an increase in YFP fluorescence were observed when compared to that before stimulation. The fluorescence changes of CFP and YFP were sustained long after the application of ionomycin to the isolated pancreas. Ratiometric FRET images remained unchanged before ionomycin stimulation and began to change in response to ionomycin stimulation. Corresponding to the dynamics of CFP, YFP, and FRET images, ionomycin stimulation induced a notable increase in the FRET ratio accompanying inverse changes in CFP and YFP intensities  in both acinar cells and MEC of SG, and the increased FRET ratio gradually returned to the baseline after removal of ACh Fig. . In bothIn presence of the mAChR antagonist atropine, the FRET ratio elevation induced by ACh was inhibited compared to the FRET ratio following ACh stimulation alone at the same dosage in the SG Fig. . The relWe also analyzed the FRET ratio dynamics in the LG MEC. After stimulation with ionomycin, the FRET ratio in LG MEC transiently increased to a higher level, then dipped and returned to a sustained plateau. The AUC value of the LG MEC was significantly higher after ionomycin stimulation compared to before stimulation Fig. .After the stimulation with CPA, the FRET ratio in the MEC of LG peaked and gradually returned to the baseline Fig. .2+]i in the MEC of Fura2-loaded LG from guinea pigs17. Additionally, atropine mostly inhibited FRET ratio elevation induced by ACh in the LG MEC compared to the control.ACh stimulation induced a transient increase of the FRET ratio in the LG MEC, and the increased FRET ratio gradually returned to the baseline after removal of ACh Fig. . For the2+ responses to physiological stimulation in secretory organs.These data confirmed that YC3.60 enables the monitoring of Ca2+ indicators (GECIs) using two-photon microscopy is an advantageous strategy to study the physiological function of specific organs when compared to the strategies using synthetic Ca2+ indicators, because GECIs allow for long-term and cell-type-specific imaging without potential damage to the target organs15.Intravital imaging of transgenic mice carrying genetically encoded Ca2+ signaling along with secretory function, we analyzed the relationship between Ca2+ mobilization in specific types of cells of secretory organs and fluid secretion from corresponding secretory organs following cholinergic stimulation intravitally. Bethanechol (Bet) is a parasympathomimetic choline carbamate that selectively stimulates muscarinic receptors without any effect on nicotinic receptors30. Unlike acetylcholine, Bet is not hydrolyzed by cholinesterase31. Therefore, it has a long duration of action in our intravital imaging analysis.To examine whether YC3.60 transgenic mice in combination with two-photon microscopy are useful in visualizing the intracellular CaFigure In the pancreas, the ratiometric FRET ratio image was unchanged before Bet stimulation and changed to a warm color 15 seconds after intravenous injection of Bet Fig. . The chaIn the SG, Bet changed the FRET ratio image to a warm color 15 seconds after the injection Fig. . The FREIn the LG, a change of the FRET ratio image was observed immediately after the injection of Bet Fig. . The warSupplementary movie 2+ imaging of these secretory organs were similar to the results of ex vivo Ca2+ imaging.Corresponding to the dynamics of ratiometric FRET images, intravenous injection of Bet induced dramatic increases in FRET ratio of the pancreas, SG, and LG  as a dry eye disease model to investigate the Ca2+ signaling in the MEC of LG under the dry eye conditions.The autonomic nervous systems consist of sympathetic and parasympathetic nerves that innervate the LG, and the parasympathetic innervation of the LG plays an essential role in the regulation of LG tear secretory functionFigure Two-photon imaging of the LG showed an extensive decrease in the number of stellate calponin-positive MEC depending on the time after PGDL compared to the sham-operated side Fig. .2+ responsivity to ACh stimulation in the MEC of LG, isolated LG from PGDL-treated YC3.60 transgenic mice was stimulated with ACh at a dosage of 10, 50, 100, and 1,000 nM . Oshima et al. established a mouse line ubiquitously expressing the YC-Nano 15 probe under control of the CAG promotor and elaborated the association of Ca2+ signaling and exocytosis of pancreatic acinar cells28.Pancreatic acinar cells are a classic model of exocrine organs for investigation of secretory pathways mediated by Ca2+ sensitivity. YC-Nano15 is YC variant with a longer linker containing 5 amino acids, and has high sensitivity to Ca2+ 35. Since YC 3.60 has a short linker (2 amino acids) and a point-mutated CaM, its Ca2+ sensitivity is lower than YC-Nano1535.YCs are composed of a linear combination of CFP, calmodulin (CaM), a linker peptide, the CaM-binding peptide of myosin light-chain kinase (M13), and YFP. The flexibility of the linker peptide between CaM and M13 in the YCs is critical for determining Ca2+ response to 5−10 nM ACh but higher Ca2+ response to 50−500 nM ACh  were used in this study. YC3.60 transgenic mice was obtained by crossing CAG-Cre mouse in which the Cre recombinase gene is under control of the CAG promotor  and YC3.60flox mouse in which neomycin phosphatase transfer gene is inserted between CAG promotor and YC3.60 gene15. All procedures were performed in accordance with the Association of Research and Vision in Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmology and Vision Research and approved by the Animal Experimentation Ethics Committee of Keio University School of Medicine, Tokyo, Japan. All animals were quarantined and acclimatized for 1 week prior to the experiments under the same general conditions , water and food were available ad libitum).Eight-week-old male Yellow Cameleon 3.60 (YC3.60) transgenic mice, a mouse line expressing the CaYC3.60 transgenic mice were euthanized with an overdose of pentobarbital sodium and the secretory organs including the pancreas, salivary gland, and lacrimal gland (LG) were dissected.39, at room temperature (RT) for 30 minutes. WGA-stained secretory organs were washed three times with a saline solution  for 3 minutes.For visualization of the cell surface, these secretory organs were stained with phalloidin-labeled wheat germ agglutinin  which binds to lectins and acts as a marker for the cell surface17 . After being washed with PBS, secretory organs were incubated with Alexa Fluor 555-conjugated donkey-anti rabbit secondary antibody  for 3 hours at room temperature (25 ± 5 °C) and washed with PBS.For visualization of MEC, the secretory organs dissected from YC3.60 mice were fixed in 4% paraformaldehyde (PFA) at 4 °C overnight. After fixation, secretory organs were blocked with 1% bovine serum album (BSA) in PBS containing 0.25% Triton X-100 for 1 hour at room temperature and then incubated overnight at 4 °C with a rabbit polyclonal antibody against calponin, a specific MEC markerWGA-stained or immuno-stained secretory organs were observed with an upright two-photon microscope  equipped with a water immersion objective lens  which was connected to a femtosecond laser source, Ti:sapphire laser . The excitation wavelength for YC3.60 probe, WGA, and Alexa-555 was 830 nm, and the emission was simultaneously detected through a band-pass filter for YC3.60 (510−550 nm) and for WGA and Alexa 555 (575−630 nm). Fluorescence images at a depth of approximately 200 µm from the surface of each secretory organ were reconstructed from 200 images acquired at z-step sizes of 1 µm using Imaris software .YC3.60 transgenic mice were euthanized with an overdose of pentobarbital sodium. Each secretory organ  dissected from YC3.60 mice was transferred to round coverslips that were mounted on the bath region of a perfusion chamber and continuously perfused with a saline solution through polyethylene tubes connected to a Masterflex peristaltic pump  at a flow rate of 0.8 mL per minutes.2+ mobilization, atropine was applied to each secretory organ 2 minutes before the application of ACh.Solutions of ionomycin, a calcium ionophore ; cycropiazonic acid ; and acetylcholine  were diluted to the desired concentrations with the saline solution and used as stimulants. Atropine, an antagonist for muscarinic acetylcholine receptors , was prepared in a saline solution before use. Ionomycin, CPA, and ACh were applied to each secretory organ for 4 minutes, 4 minutes and 1 minute, respectively. For evaluation of the effect of atropine on ACh-induced Ca2+ signals were observed with a two-photon microscope equipped with a 25× water-immersion objective lens by measuring the change in FRET (fluorescence resonance energy transfer) ratio that was calculated as the ratio of YFP to CFP fluorescence intensity. An excitation wavelength of 830 nm was used for FRET imaging. The two-photon excited fluorescence images of CFP and YFP were acquired in separate channels through dichroic mirrors and emission filters, BP460-500 and BP520-560, respectively. In the pancreatic secretory lobe, SG and LG, the fluorescence images were acquired at approximately 1 frame/second for 8 minutes at depth-intervals of 2 µm from the surface of each secretory organ to a maximum depth of 10 µm. Fluorescence images were reconstructed using Imaris software and analyzed with MATLAB software . In the pancreatic acinar cells, the fluorescence images were acquired at approximately 1 frame/500 ms for 8 minutes. Images were analyzed with the Aqua Cosmos software .CaYC3.60 transgenic mice were anesthetized with an intraperitoneal injection of urethane (1.2 g/kg). For imaging of the pancreas, mice were placed in a dorsal position. Abdominal skin and a small portion of the peritoneum located at left inferior of the sternum were incised, and then the pancreas located on the left side of the stomach was exteriorized. For imaging of the SG, the SG was surgically exteriorized after incision of the skin of the anterior neck spaces between submandibular lymph nodes and the sternum. For imaging of the LG, mice were placed in a lateral position, the skin on the right temporal side of the head was incised and the LG was exteriorized. After each secretory organ was exteriorized, a custom-built metal ring was attached to the peritoneum, sternocephalicus muscle, and masseter muscle using a cyanoacrylate-based glue  for imaging of the pancreas, SG, and LG, respectively. Each exteriorized secretory organ was placed on round coverslip which is located on a custom-buit metal ring. Secretory organs were mounted with water-soluble O.C.T. compound for protecting against desiccation and then sandwiched with round coverslip for eliminating the effect of organs movement due to breathing. During intravital imaging, mouse was placed on a heating-pad and body temperature was maintained at 37 °C with thermos-controller .Solution of bethanechol , an agonist of the muscarinic ACh receptor, was prepared in saline solution before use. Bet was intravenously injected into jugular vein to deliver a volume up to 100 µL.Changes in the FRET ratio were observed with a two-photon microscope equipped with an XL Fluor 4 × /340 NA 0.28 objective lens. The fluorescence images were acquired at approximately 1 frame/second for 8 minutes, at depth intervals of 5 µm from the surface of each secretory organ to a maximum depth of 50 µm. Acquired fluorescence images were reconstructed using Imaris software and analyzed with MATLAB software.2+]i in the each secretory organ, minimum FRET ratio under stimulation with Ca2+-free saline solution containing 2 mM EGTA (Rmin) and the maximum FRET ratio under stimulation with saline solution containing 10 µM ionomycin (Rmax) were measured. FRET ratios were converted to [Ca2+]i using in situ dissociation constant (Kd) (250 nM for YC3.60) and Hill coefficient 14 with standard ratiometric equation9; [Ca2+]i = Kd[(R − Rmin)(Rmax − R)−1](1/n). Empirical value of  in the pancreas, SG, and LG is , , and , respectively.To estimate the [Ca40 by phenol red thread , which was placed on the temporal side of the conjunctiva between the limbus and the outer canthus for 15 seconds. The length of the moistened area from the edge was measured to within 0.5 mm.YC3.60 transgenic mice were anesthetized with urethane . Tear secretion was measured using a modified Schirmer testThe Bet solution (1 mg/kg) was used as a stimulant and intravenously injected through the jugular vein. Measurements of tear secretion were obtained 1 and 2 minutes before and 0.5, 1, 2, 3, and 5 minutes after injection of Bet.41 under a stereomicroscope for 15 seconds. To eliminate the saliva secretion from parotid gland, cleaning tissue paper was fitted to oral cavity opposite the maxillary second molar where the excretory duct of the parotid gland opens42. Measurements of saliva were obtained 2 minutes before, and 1, 3, and 5 minutes after intravenous injection of Bet (1 mg/kg).Saliva secretion in YC3.60 transgenic mice was measured using phenol red thread under anesthesia by urethane . The oral cavities were held open using a mouth gag. The retained saliva in the mouse was removed before measurement. Phenol red thread was placed at the sublingual caruncle, a mucosal fold located behind the lower incisors on the floor of the mouth37. In brief, mice were placed in a prone position, and the skin on the right temporal side of the head was incised under deep anesthesia using a combination anesthetic containing medetomidine (0.75 mg/kg), butorphanol (5 mg/kg), and midazolam (4 mg/kg). The post-ganglionic nerve bundle was detached from the blood vessels at the caudal root site of the ventral surface of the LG and denervated under a stereomicroscope. PGDL was performed only on the right LG; the left LG remained as a control.PGDL was performed according to a previous reportex vivo Ca2+ imaging was performed 1 and 3 days after PGDL. Intravital Ca2+ imaging and measurement of tear secretion was performed 3 days after PGDL.Immunohistochemistry of calponin and Statistical analyses were performed with the JMP12 software . Comparison between the two groups was determined by an F-test followed by a Student’s t-test for parametric variables and Mann-Whitey U test for non-parametric variables. Multiple comparisons were performed by a one-way ANOVA followed by parametric Dunnett test or non-parametric Steel test. Differences between the measurement variables were considered significant if the resultant P-value was 0.05 or less.Supplementary informationSupplementary Movie 1Supplementary movie 2Supplementary movie 3Supplementary movie 4Supplementary movie 5Supplementary movie 6Supplementary movie 7Supplementary movie 8


Acute myeloid leukemia (AML), caused by the abnormal proliferation of immature myeloid cells in the blood or bone marrow, is one of the most common hematologic malignancies. Currently, the interactions between malignant myeloid cells and the immune microenvironment, especially T cells and B cells, remain poorly characterized.In this study, we systematically analyzed the T cell receptor and B cell receptor (TCR and BCR) repertoires from the RNA-seq data of 145 pediatric and 151 adult AML samples as well as 73 non-tumor peripheral blood samples.We inferred over 225,000 complementarity-determining region 3 (CDR3) sequences in TCR α, β, γ, and δ chains and 1,210,000 CDR3 sequences in B cell immunoglobulin (Ig) heavy and light chains. We found higher clonal expansion of both T cells and B cells in the AML microenvironment and observed many differences between pediatric and adult AML. Most notably, adult AML samples have significantly higher level of B cell activation and more secondary Ig class switch events than pediatric AML or non-tumor samples. Furthermore, adult AML with highly expanded IgA2 B cells, which might represent an immunosuppressive microenvironment, are associated with regulatory T cells and worse overall survival.Our comprehensive characterization of the AML immune receptor repertoires improved our understanding of T cell and B cell immunity in AML, which may provide insights into immunotherapies in hematological malignancies. Acute myeloid leukemia (AML), caused by the abnormal proliferation of immature myeloid cells in the blood or bone marrow (BM), is the most common acute leukemia in adults and the second most common in children . For manT cell and B cell are key components of the adaptive immunity. With the development of ICB therapy, the antitumor properties of infiltrating T cells have been well confirmed in many solid tumors such as melanoma and non-small cell lung cancer . Upon biIn this study, we characterized TCR and BCR repertoires in both pediatric and adult AML by detecting and analyzing the CDR3 sequences in TCR α, β, γ, and δ chains and B cell immunoglobulin (Ig) heavy (IgH) and light  chains from the RNA-seq data in AML patients and non-tumor donors. We investigated the clonal expansion patterns of T cells and B cells in the AML microenvironment and described the differences between AML and non-tumor samples. We also compared the differences between pediatric and adult AML samples and identified the association of tumor immune receptor repertoires with clinical outcome. These results provided insights into the immune receptor repertoires and T/B cell functions in AML.We previously developed a computational algorithm TRUST , 24–26 thttps://portal.gdc.cancer.gov/, Jun 2017). RNA-seq reads have been previously aligned to hg38 human reference genome using STAR2 [https://www.ncbi.nlm.nih.gov/sra, PRJNA263846) and successfully processed using the GDC mRNA analysis pipeline (https://docs.gdc.cancer.gov/Data/Bioinformatics_Pipelines/Expression_mRNA_Pipeline). The limited available clinical annotation on these normal samples only allowed categorical information such as male/female and children/adults to be parsed out. Since the maturity of the adaptive immunity is dependent on age, especially in early age, the pediatric AML samples were further divided into infants  and children  group in the downstream analyses. Control samples were not divided due to the lack of age information.Our study investigated a total of 296 primary AML samples  to all the AML and non-tumor RNA-seq samples. Formatted txt files with CDR3 calls were used in the downstream analyses, in which the est_lib_size column represents the number of reads mapped to TCR/BCR region. The number of total sequencing reads was obtained from each bam file using samtools [To characterize the immune receptor repertoires, we applied TRUST3.0.1  in each sample. The clonotype diversity of T/B cells was estimated by TCR/BCR CDR3s per kilo TCR/BCR reads (CPK)  in each LVGW and RDMWIVGW were considered matches) were collected. Each motif with 3 or more sequences was considered a B cell cluster. This approach provided flexibility in detecting amino acid changes from non-synonymous mutations, yet maintained low computational complexity.To identify B cell lineage clusters in each sample, we extracted an octamer starting from the first position (not counting the starting "C") in each complete IgH CDR3 as motifs. All the IgH CDR3 sequences  which contain amino acid matches to the motif with 0-1 mismatch   was defiWilcoxon rank-sum test was used to compare the differences between TCR/BCR CPK, γδ CDR3 fractions, and SHM rates among AML and non-tumor groups. Spearman’s rank correlation was used to check the association among αβ, γδ, or IgH and IgK/IgL CDR3 calls, and partial Spearman’s rank correlation was used to check the association between different Ig isotype fractions in the AML and non-tumor groups. Survival analyses were visualized using Kaplan–Meier curves, and the statistical significance was estimated using Log-rank test. Details for the other analyses were described in supplementary methods  and non-tumor samples 170,000). Despite deeper sequencing coverage of AML than non-tumor samples  CDR3s from the AML  and non-tumor  samples  and BCR  CDR3 from the bulk RNA-seq data from both pediatric and adult AML samples as well as non-tumor controls. The human immune system evolves with age, as exposures to multiple self and foreign antigen challenges promote the maturation of immune-related cells and organs . We founNotably, we found that pediatric AML with highly expanded IgA1 B cells and adult AML with highly expanded IgA2 B cells, which might represent an immunosuppressive microenvironment, are associated with worse overall survival. Recent studies reported that IgA-producing plasma cells can function as potent immunosuppressors through the secretion of PD-L1 in prostate  and liveIn summary, our comprehensive analyses of TCR and BCR CDR3 sequences from AML RNA-seq samples provided the first overview of the immune receptor repertoires in both pediatric and adult AML microenvironments. We found a higher clonal expansion of both T cells and B cells in the AML microenvironment. In addition, adult AML samples have a significantly higher level of B cell activation and more secondary Ig class switch events than pediatric AML or non-tumor samples. Furthermore, we found that pediatric AML with highly expanded IgA1 B cells and adult AML with highly expanded IgA2 B cells are associated with worse overall survival. The identified TCR/BCR repertoires and the observed associations from this work provide useful resources and insights into the future development of novel immunotherapies for hematological malignancies.Additional file 1: Table S1. Clinical characteristics of AML samples. Table S2. Clinical characteristics of non-tumor samples.Additional file 2: Figure S1. In silico validation on the accuracy of TRUST for assembling TCR and BCR from RNA-seq data by using single cell RNA-seq data of CD45 positive white blood cells from pre-treatment melanoma patients. Figure S2. Overview of TCR CDR3s in AML and non-tumor samples. Figure S3. Comparison of TCR CPK and γδ T cell fraction between PB and BM samples in pediatric AML. Figure S4. Overview of BCR CDR3s in AML and non-tumor samples. Figure S5. Comparison of BCR CDR3s between PB and BM samples in pediatric AML. Figure S6. Violin plot showing the expression level of AICDA is significantly higher in adult AML than pediatric AML. Figure S7. Boxplots showing the normalized number of IgA/IgG/IgM + IgD CDR3s in AML and non-tumor groups. Figure S8. Kaplan-Meier curves showing the survival difference relative to IgA fraction in pediatric and adult AML. Figure S9. Distribution of 9 Ig isotypes across adult AML IgA2 low , medium  and high  group. Figure S10. The enriched GO terms with IgA2 fraction in adult AML. Figure S11. Boxplot showing the expression level of PD-L1 in pediatric AML IgA1 high/low group and adult AML IgA2 high/low group. Figure S12. t-SNE plots showing the expression level of different gene makers in normal and AML  peripheral blood single cell RNA-seq data.Additional file 3. Supplementary methods.Additional file 4. Processed data for both AML and non-tumor samples.


To generate novel rabbit models with a large-fragment deletion of either LDL receptor (LDLR) and/or apolipoprotein (apoE) genes for the study of hyperlipidemic and atherosclerosis.CRISPR/Cas9 system directed by a multiple sgRNAs system was used in rabbit embryos to edit their LDLR and apoE genes. The LDLR and apoE genes of founder rabbits were sequenced, and their plasma lipids and lipoprotein profiles on a normal chow diet were analyzed, western blotting was also performed to evaluate the expression of apolipoprotein. Sudan IV and HE staining of aortic were performed to confirm the formation of atherosclerosis.Six knockout (KO) rabbits by injection of both LDLR and apoE sgRNAs were obtained, including four LDLR KO rabbits and two LDLR/apoE double- KO rabbits. Sequence analysis of these KO rabbits revealed that they contained multiple mutations including indels, deletions, and substitutions, as well as two rabbit lines containing biallelic large fragment deletion in the LDLR region. Analysis of their plasma lipids and lipoprotein profiles of these rabbits fed on a normal chow diet revealed that all of these KO rabbits exhibited remarkable hyperlipidemia with total cholesterol levels increased by up to 10-fold over those of wild-type rabbits. Pathological examinations of two founder rabbits showed that KO rabbits developed prominent aortic and coronary atherosclerosis.Large fragment deletions can be achieved in rabbits using Cas9 mRNA and multiple sgRNAs. LDLR KO along with LDLR/apoE double KO rabbits should provide a novel means for translational investigations of human hyperlipidemia and atherosclerosis. Hyperlipidemia is the major risk factor of atherosclerosis . To studNew Zealand White (NZW) rabbits were purchased from the Animal Genetic Engineering Laboratory at Yangzhou University. Rabbits were allowed access to food and water ad libitum. All animal studies were conducted according to the approval of the Animal Care Committee of the Yangzhou University.http://www.ncbi.nlm.nih.gov/) and designed the CRISPR/Cas9 sgRNAs (sgRNA) using the tool on the website of http:// crispr.mit.edu. After screening, we obtained six sgRNA targeting loci: four for the LDLR gene  by anchoring to exons 2 and 7 respectively and two for apoE gene (apoE-sgRNA5 and -sgRNA6) by anchoring to intron and exon 1 as shown in Fig. To design KO loci of rabbit LDLR and apoE genes, we obtained sequences from the Library of the National Center for Biotechnology Information (NCBI)  at every 12 h (for six iterations) from 8 AM to 8 PM . At the last time injection with FSH, the donors and recipients were injected with 10 IU human chorionic gonadotropin (HCG) simultaneously, and then, the donors were mated with fertile male rabbits. At 18–20 h later, embryos were flushed out using PBS and collected for microinjection. A mixed solution containing Cas9 mRNA (40 ng/ul)and multiple sgRNAs (13 ng/ul) was microinjected into the cytoplasm of embryos under a Leica inverted light microscope. The injected embryos were transferred into M2 cushion fluid and incubated at 38 °C, 5% COGenomic DNA was extracted from a small piece of ear biopsy using the phenol- chloroform extraction method. sgRNA target sites were amplified by PCR using the primers shown in Table s in vivo or in vitro for studying phenotypic effects of genetic perturbations. However, off-target effects are an inherent risk in this technology [http://crispr.mit.edu/). The primers are listed in Table The CRISPR/Cas9 system has enabled efficient modification of genechnology . We screEDTA plasma was collected from rabbits that were fasted for 16 h. Plasma total cholesterol (TC), TG, LDL-C and HDL-C were measured using enzymatic assay kits . Plasma apoE, apolipoprotein B (apoB), and apolipoprotein A-I (apoA-I) were detected by Western blotting. The primary antibodies used are as follows: goat anti-apoE , sheep anti-apoA-I , and goat anti-apolipoprotein B (apoB)  polyclonal antibodies (Abs). Immunocomplexed proteins were identified by reaction with horseradish peroxidase-conjugated donkey anti-goat IgG  and donkey anti-sheep IgG  polyclonal Abs.Plasma lipoprotein profiles were analyzed using agarose gel electrophoresis. The protocol has been described in detail by our published protocols .We selected two rabbits among six KO founder rabbits for pathological analysis, which were 5♀ founder with LDLR/apoE double-KO and 7♂ founder with large fragment deletion. The aortic trees were isolated and opened out and, after fixing in formalin for 24 h, they were stained with Sudan IV . For histological analysis, serial paraffin sections were stained with hematoxylin-eosin (HE) and immunohistochemically stained with monoclonal antibodies against either macrophages  or a-smooth muscle actin for smooth muscle cells .As shown in Fig. To verify whether multiple sgRNAs can cause any off-targeting effects, we examined 30 POTS in total, Primers are listed in Table On a normal chow diet, six founder KO rabbits at the age of 12 weeks exhibited hyperlipidemia (except 1♂) and their plasma TC, TG, LDL-C levels were significantly higher than those of WT rabbits Fig. c. Among Elevated levels of lipoproteins can drive the development of atherosclerosis in humans and animals. We obtained six KO rabbits including four LDLR KO  rabbits and two LDLR/apoE double KO rabbits. Because our previous article  introducLDLR gene [Hyperlipidemia is a common genetic disorder inherit by genetic mutations mostly in LDLR, apoB-100, or proprotein convertase subtilisin kexin type 9 (PCSK9) loci which can result in xanthoma, atherosclerosis, coronary atherosclerotic heart disease (CAD), and other cardiovascular diseases . The majDLR gene , but apoDLR gene  to the aDLR gene , the aniSaccharomyces cerevisiae [Escherichia coli [The CRISPR/Cas9 system was developed to edit the genome, and even the utility of it for gene therapy in humans has been recognized and extensively investigated . With threvisiae , Escherihia coli , mouse, hia coli  and humahia coli . In 2016hia coli  first dehia coli  showed tLDLR gene consists of 18 exons and 17 introns with a length of approximately 45 kb [LDLR, a transmembrane cellular protein, plays a crucial role in the receptor-mediated pathway of lipoprotein metabolism . The humly 45 kb . These ely 45 kb . It can ly 45 kb . Therefoly 45 kb . Based oly 45 kb ; their sly 45 kb , 36.Although we have successfully generated LDLR and LDLR/apoE double KO rabbit model with the spontaneous hyperlipidemia, but their genetic mutations are different from patients and also their plasma lipids are significantly higher than patients, which may limit their subsequent clinical translational applications. And for providing a reliable basis for hyperlipidemia therapy, the further research on pathogenesis of our models should be performed. In addition, the off-target effect in CRISPR/Cas9 system is a critical concern. However, this side effect was not found in our study that we supposed the Web-based algorithms may miss the potential off-target sites with less sequence similarity or such effect didn’t appear. In all, our animal models with its typical atherosclerosis and spontaneous hyperlipidemia provide novel models for the study of familial hyperlipidemia and cardiovascular disease.We generate the LDLR/apoE double KO rabbits and achieved an ~ 3 kb large gene fragment deleted from LDLR gene in the rabbit model. The LDLR KO and LDLR/apoE double-KO rabbits will be useful for further studies investigating spontaneous hyperlipidemia and related diseases.


Phenethyl isothiocyanate (PEITC) from cruciferous vegetables can inhibit the growth of various human cancer cells. In previous studies, we determined that PEITC inhibited the in vitro growth of human glioblastoma GBM 8401 cells by inducing apoptosis, inhibiting migration and invasion, and altering gene expression. Nevertheless, there are no further in vivo reports disclosing whether PEITC can suppress the growth of glioblastoma. Therefore, in this study we investigate the anti-tumor effects of PEITC in a xenograft model of glioblastoma in nude mice. Thirty nude mice were inoculated subcutaneously with GBM 8401 cells. Mice with one palpable tumor were divided randomly into three groups: control, PEITC-10, and PEITC-20 groups treated with 0.1% dimethyl sulfoxide (DMSO), and 10 and 20 μmole PEITC/100 μL PBS daily by oral gavage, respectively. PEITC significantly decreased tumor weights and volumes of GBM 8401 cells in mice, but did not affect the total body weights of mice. PEITC diminished the levels of anti-apoptotic proteins MCL-1 (myeloid cell leukemia 1) and XIAP (X-linked inhibitor of apoptosis protein) in GBM 8401 cells. PEITC enhanced the levels of caspase-3 and Bax in GBM 8401 cells. The growth of glioblastoma can be suppressed by the biological properties of PEITC in vivo. These effects might support further investigations into the potential use of PEITC as an anticancer drug for glioblastoma. Aside from surgery, chemotherapy with the alkylating agent temozolomide and radiotherapy are first-line therapies for glioblastoma, but their survival benefits are short-lived and the tumors may develop resistance to therapies . This moThere are no reports in the available literature disclosing that PEITC inhibits the growth of glioblastoma in vivo. In the present study, we first investigated the anti-tumor effects of PEITC in a xenograft model of glioblastoma in nude mice.The body weights of each group were measured every 3 days, and the results are shown in Ectopic tumor-bearing nude mice were treated with vehicle and PEITC at different concentrations for 21 days, and they were anesthetized with 1–3% isoflurane every 1 week during scanning. The efficacy of the treatment was evaluated by bioluminescent imaging (BLI) A. PhotonThe tumor volume of each mouse was measured every 3 days during treatments for 21 days, and six representative tumors from three groups were extracted as shown in luc2 cells in vivo.All samples were observed under microscopy at ×100 magnification after immunohistochemical (IHC) staining. Five regions of each slide were randomly selected for photographing A. ResultLiver was collected from every mouse of every group after treatment, embedded in paraffin, sectioned into 5 μm-thick slices, deparaffinized, and stained with hematoxylin and eosin (H&E). Liver specimens from PEITC-treated and control groups revealed similar hepatocyte arrangement in hepatocytes and lobular architectures .luc2 cells in vivo.There were no significant differences in mice liver between PEITC-treated and control groups, so there was no obvious hepatic cytotoxicity after PEITC treatment GBM 8401/luc2 cells-bearing mice  ) vector using JetPEI™ transfection reagent  [luc2/CMV]) was obtained from Promega . Stable clone was selected by two-week treatment of 200 μg/mL hygromycin and validated by a Xenogen IVIS imaging system . Luciferase expressing stable clone was finally named as GBM8401/luc2.We transfected GBM 8401 cells with the plasmid of pGL4.50 luciferase reporter  . The plaFoxn1nu/CrlNarl nude mice were bought from the National Laboratory Animal Center, Taipei, Taiwan. All studies followed the National Institutes of Health Guidelines for Animal Research, and were approved by the Institutional Animal Care and Use Committee of Taipei Medical University (number: LAC-2017-0248). GBM8401/luc2 cells (1 × 107) in 150 μL mixture containing serum-free RPMI and Matrigel (2:1) were subcutaneously inoculated into the right hind legs of the 30 mice [2 [3, mice were randomized into three different treatment groups (n = 10 for each group), including vehicle, PEITC-10 group, and PEITC-20. The vehicle group was treated with 110 μL phosphate-buffered saline (PBS) plus 10 μL DMSO by gavage daily for 21 days. PEITC-10 and PEITC-20 groups were treated with PEITC 10 μmole/100 μL PBS/day and PEITC 20 μmole/100 μL PBS/day for 21 days, respectively. We monitored the tumor growth with bioluminescent imaging (BLI) and caliper. The body weights and tumor volumes of mice were measured 3 times per week after treatment. Finally, livers and tumors extracted from mice were prepared for pathologic examination and immunohistochemical (IHC) staining on day 21. Tumor weights of mice were also recorded. The flow chart of our experimental protocol is summarized in Six-week-old male athymic CAnN.Cg- 30 mice . We meas mice [2 . After tMice tumor growth was also monitored by BLI once per week during treatment progress. Intraperitoneal injections of 150 mg/kg of D-luciferin  were administered to mice from each group 15 min before BLI scanning. During the scanning procedure, mice were anesthetized at 1–3% isoflurane dosage and emitted photons were recorded by a Xenogen IVIS imaging system 200 as described previously . The acqMice were sacrificed on day 21, and tumors and livers were extracted. Tumors and livers were both fixed with 4% paraformaldehyde (PFA) at 4 °C for 24 h. Paraffin-embedded tumor tissues and liver tissue were sliced at 5 μm thickness by Bio-Check Laboratories Ltd. . The IHC and hematoxylin and eosin (H&E) staining protocols were performed according to the manufacturer’s recommendations. For immunohistochemical staining, primary antibodies including MCL-1, XIAP, Bax, and active Caspase-3 antibodies were added, respectively. An H&E staining kit from Bio-Check Laboratories Ltd. was used to stain slices. All slices were finally mounted with Prolong Gold Antifade reagent  and were imaged by microscopy-based TissueFAXS platform  at 100× magnification . Positivp < 0.05 or less.All data were represented with the mean ± standard error. One-way ANOVA with Newman–Keuls multi-comparison test was used for the comparison between PEITC-treated and control groups, and between PEITC-treated groups. Difference between the means was considered significant if


CSRP3) is a key regulator of striated muscle function, and its mutations can lead to both hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) in patients. However, due to lack of human models, mechanisms underlining the pathogenesis of MLP defects remain unclear. In this study, we generated a knockout MLP/CSRP3 human embryonic stem cell (hESC) H9 cell line using CRISPR/Cas9 mediated gene disruption. CSRP3 disruption had no impact on the cardiac differentiation of H9 cells and led to confirmed MLP deficiency in hESC-derived cardiomyocytes (ESC-CMs). MLP-deficient hESC-CMs were found to develop phenotypic features of HCM early after differentiation, such as enlarged cell size, multinucleation, and disorganized sarcomeric ultrastructure. Cellular phenotypes of MLP-deficient hESC-CMs subsequently progressed to mimic heart failure (HF) by 30 days post differentiation, including exhibiting mitochondrial damage, increased ROS generation, and impaired Ca2+ handling. Pharmaceutical treatment with beta agonist, such as isoproterenol, was found to accelerate the manifestation of HCM and HF, consistent with transgenic animal models of MLP deficiency. Furthermore, restoration of Ca2+ homeostasis by verapamil prevented the development of HCM and HF phenotypes, suggesting that elevated intracellular Ca2+ concentration is a central mechanism for pathogenesis of MLP deficiency. In summary, MLP-deficient hESC-CMs recapitulate the pathogenesis of HCM and its progression toward HF, providing an important human model for investigation of CSRP3/MLP-associated disease pathogenesis. More importantly, correction of the autonomous dysfunction of Ca2+ handling was found to be an effective method for treating the in vitro development of cardiomyopathy disease phenotype.Muscle LIM protein (MLP,  CSRP3, is primarily expressed in cardiomyocytes and skeletal muscle cells and consists of 194 amino acids with two LIM domains1. These LIM domains mediate interactions with a variety of proteins in different subcellular regions, including the cytoplasm and the nucleus. In the cytoplasm of cardiomyocytes, MLP has been found in the sarcomeres, intercalated disks, and costameres of striated myocytes. In addition, MLP can directly affect F-actin cytoskeleton dynamics by enhancing CFL2-dependent F-actin depolymerization2 and stabilizing actin filaments into bundles3. These studies suggest that MLP plays an integral role in sarcomeric structure. Previous studies have shown that mutations  in CSRP3 are associated with hypertrophic cardiomyopathy (HCM)5 and dilated cardiomyopathy (DCM)6 in humans. In addition, MLP has been shown to be significantly downregulated in chronic human heart failure (HF)7. In mouse models, MLP-deficient mice develop dilated DCM with hypertrophy after birth, and are the first transgenic animal model of HF1. However, the underlying molecular mechanisms of CSRP3 mutation-induced cardiomyopathy are not well understood.Muscle LIM protein (MLP), encoded by 8, phenotypic results have shown significant discrepancies between identical CSRP3 mutations in animals and humans. For example, MLPW4R/+ transgenic mice are observed to develop features of HCM and HF8, whereas MLPW4R/+ is a known to be a benign polymorphism in humans carrying the mutation9. Efforts to obtain primary human cardiac tissue to resolve these discrepancies have not been successful due to difficulties in obtaining and culturing human cardiac tissue in vitro. Advances in disease modeling using human pluripotent stem cells (PSCs) in recent years have provided a novel pathophysiological approach to study the mechanics of disease and discover new targets for pharmacological intervention10.Despite the existence of genetically modified mouse models for MLP deficiencyCSRP3−/− hESC-H9 cells, to elucidate the mechanism of cardiomyopathy and HF caused by MLP defects. We report here that MLP-deficient human embryonic stem cell-derived cardiomyocytes (hESC-CMs) recapitulate the phenotypes of HCM and HF. Mechanistically, impaired calcium handling is a major mechanism underlying the pathogenesis of MLP-associated HCM and HF, which can be prevented by pharmaceutical blockade of calcium entry into the cell.We thus created a MLP-deficient cardiomyocyte model using from 11. After selection with puromycin, the resistant colonies were expanded for PCR screening verification. Among 12 clones, we found one homozygous CSRP3 knockout line with 1-nucleotide insertion resulting in a frame-shifted coding sequence with premature stop codon at 14th amino acid, resulting in homozygous null alleles  and found no off-target mutations in CSRP3−/− hESCs by DNA sequencing (data not shown)12.To generate the human PSC model of MLP deficiency, we designed a single-guide RNA targeting exon 3 of CSRP3 using the epiCRISPR/Cas9 gene editing system Fig. . H9 hESCles Fig. . CSRP3−/ogy Fig. . We furtCT4 Fig.  and had CT4 Fig. . FinallyCT4 Fig.  and diffCT4 Fig. . Moreove−/− lines (MLP KO) and the parental H9 cells (WT) toward cardiomyocyte lineages (hESC-CMs) using small molecule-based protocols  showed both the WT and MLP KO derived cells contained close to 85% positive cTnT cells by flow cytometry at day 10 post differentiation . Double immunostaining for MYL2 and MYL7 showed both 83% of WT and MLP KO hESC-CMs were positive for the ventricular marker MYL2+  and nonsubjective flow cytometry method to measure hESC cardiomyocytes size via flow cytometry measurement of forward scatter using calibration spheres for cell size determination21. As previously reported22, only a small number of (<7%) WT hESC-CMs were observed to be binuclear or multinuclear, while MLP KO hESC-CMs exhibited significantly higher percentages of bi- or multi-nucleation (about 17%) at day 30 post differentiation  calcium sensor into the AAVS1 locus of the WT and MLP KO H9 hESC lines as previously reported24  at days 15, 22, and 3035. Beginning at day 30 post differentiation, MLP KO hESC-CMs exhibited a significant decrease in mtDNA/nDNA ratio compared with WT hESC-CMs  activity, a “metabolic sensor”, that can be activated by ATP depletion and increased AMP/ATP ratio 30 Fig. . These f37, have been shown to aggravate HCM and induce HF in HCM models of disease38. Deletion of the β2-adrenergic receptor39 and overexpression of a β-adrenergic receptor kinase 1 inhibitor40 has been shown to prevent the development of cardiomyopathy in MLP-deficient mice. We thus examined whether treatment with ISO could accelerate the development of HCM disease phenotype in MLP KO hESC-CMs. One week of 10 μM ISO treatment from the day 15 of cardiac differentiation resulted in markedly increased calcium release amplitudes of WT hESC-CMs and significantly decreased calcium release amplitudes in MLP KO hESC-CMs  for 10 days beginning at day 20 of cardiac differentiation was found to significantly improve impaired calcium handling function in MLP KO hESC-CMs, including increased rate of calcium uptake and release  was significantly elevated in MLP-deficient hESC-CMs, leading to higher levels of extracellular Ca2+ entering the cytoplasm during systole, and triggering the SR to release more Ca2+. Higher amplitude of calcium transients in MLP-deficient hESC-CMs was subsequently observed as early as day 15 of cardiac differentiation as compared with WT hESC-CMs. Similarly, SERCA expression was observed to be decreased at both the mRNA and protein levels in MLP-deficient hESC-CMs, resulting in reduced cytoplasmic Ca2+ transport back into the SR, and resulting in the elevation of diastolic [Ca2+]. As a result, SR Ca2+ load in MLP-deficient hESC-CMs was observed to be decreased because of increased RyR Ca2+ leak and diminished SR uptake. As a consequence of defective SR Ca2+ cycling, Ca2+ transients in MLP-deficient CMs show distinct alterations such as decreased amplitude, prolonged time to peak, reduced the rate of Ca2+ removal, and elevated resting [Ca2+], consistent with previously reported findings in failing CMs41. Prolonged calcium uptake and increased cytoplasmic calcium concentration in MLP-deficient hESC-CMs obstruct sarcomeric relaxation, leading to diastolic dysfunction.Given our findings in MLP KO hESC-CMs, we propose a model to illustrate the pathogenesis underlying MLP-associated HCM and HF Fig. . Based o2+-dependent pathways including calmodulin, CaMKII, and calcineurin have been shown to be key regulators of cardiomyopathy23. Cytoplasmic Ca2+ binds to calmodulin, which induces expression of ANF by calmodulin-binding transcriptional activator 2 (CAMTA2). In addition, CAMTA2 participates in hypertrophic response by binding to NKX2-542. Ca2+ binding to calmodulin also activates Ca2+-sensitive enzymes such as calcineurin and CaMKII, which have been shown to be overactivated in HCM and HF28. In line with these findings, MLP-deficient hESC-CMs were observed to have increased expression of calmodulin and phosphorylated CAMKII. In our cell models, overactivation of Ca2+-Calmodulin-CaMKII signaling in MLP-deficient CMs is most likely due to elevated cytoplasmic Ca2+. However, we found calcineurin was not increased at the protein level in MLP-deficient hESC-CMs. A possible explanation is that MLP, calcineurin, and calsarcin-1 can form a trimeric complex at the Z-disk and MLP is essential for calcineurin anchorage to the Z-disk43. Compartmentalization of calcineurin at the Z-disk is essential for coordinated dephosphorylation of NFAT transcription factors, which also are localized at the Z-disk45. Calcineurin was observed to be dislocated from the Z-disk in MLP-deficient hESC-CMs, which could inhibit the activation of Ca2+-calcineurin–NFAT signaling.Several Ca2+ can also be transported into the mitochondria by mitochondrial Ca2+ uniporters46. Elevated resting [Ca2+] in MLP-deficient CMs leads to even higher Ca2+ transport into the mitochondria, which has been shown to damage mitochondria34. MLP-deficient hESC-CMs demonstrate hallmarks of mitochondrial injury such as decreased mtDNA/nDNA ratio, Mitotracker green intensity, and reduced mitochondria density. Mitochondrial fragmentation was observed in MLP-deficient hESC-CMs, mimicking findings observed in patient HF47. Increased ROS were also observed in MLP-deficient hESC-CMs. Elevated mitochondrial ROS levels and ROS mediated damage has been demonstrated both in patients with HF and in preclinical models of the disease49. Increased ROS in MLP-deficient CMs can be explained by the several hypotheses. Frist, abnormal mitochondria observed in MLP-deficient CMs are a major source of ROS production. Second, increased energy demand due to inefficient sarcomeric ATP utilization in HCM31 require higher output of mitochondria, which could lead to further generation of ROS. It is known that Ca2+ uptake by SERCA, NCX, and Ca2+ ATPases in diastole are a process requiring significant energy consumption50. However, less ATP production due to reduction in mitochondrial density and injury in MLP-deficient hESC-CMs could hinder this process causing a further increase in cytoplasmic Ca2+, further forming a positive-feedback loop and thereby aggravating the progression of HCM and HF. Reduced ATP production and increased energy consumption in MLP-deficient CMs result in an increased AMP/ATP ratio, which can activate AMPK as evidenced by increased levels of phosphorylated AMPK.Resting Ca21 and long QT syndrome37. However, genetically modified PSC models are associated with several challenges in accurately modeling human heart disease. First, hESC-CMs are developmentally immature, and are characterized by transcriptional profiles more similar to fetal human CMs than adult CMs13. Second, hESC-CMs are not at the stage where they can model disease phenotypes at the tissue and organ levels such as interstitial fibrosis and endothelial cell dysfunction. Lastly, mutations in over 100 causal genes have been identified to engender cardiomyopathy in human patients, and our findings may only be specific to CSRP3 defects resulting in MLP deficiency. In spite of these limitations, our findings are the first to provide evidence of elevated cytoplasmic [Ca2+] as an initiating factor in the development of HCM and HF phenotypes caused by MLP deficiency in human CMs. These findings further demonstrate a possible mechanism to prevent development of this disease by early intervention via normalization of Ca2+ dysfunction.Genetically modified PSC-derived CMs circumvent the limitations associated with transgenic animal models of human heart disease and have been used to model numerous genetic cardiovascular disorders in vitro, including HCM2 or CaCl2 . The cells were cultured at 37 °C with 5% CO2. hESCs were differentiated into ESC-CMs using a small molecule-based method as previously described13. hESC-CMs were purified with the lactate metabolic-selection method51.H9 and derivative lines were maintained on feeder-free Matrigel  and fed daily with E8 medium . Cells were routinely passaged every 3 days at 70–80% confluency using 0.5 mM EDTA in PBS without MgClCSRP3 were designed using online tools (http://crispr.mit.edu). Two sgRNAs (TGGGGCGGAGGCGCAAAATG and ATGGTAGACGGTCTTTTCAC) were tested for editing efficiency, and the first one was selected for genome editing in H9 cells. sgRNA was cloned into the epiCRISPR plasmid as previously reported11. Briefly, 2 × 106 H9 cells were disassociated using 0.5 mmol/L EDTA, then these cells were electroporated with 2.5 μg epiCRISPR plasmid in 82 μL stem cell P3 solution + 18 μL supplement  using 4D nucleofector system with the CA137 program (Lonza). Transfected cells were seeded onto Matrigel-coated 10 cm petri dish and cultured in E8 medium with 10 µM Rho kinase inhibitor Y-27632 for the first 24 h. At day 3, cells were selected by puromycin (0.3 μg/mL). The surviving colonies were picked into a 24-well plate and expanded for PCR screening.Single-guide RNAs targeting CSRP3−/−-GCaMP: 2.5 μg AAVS1_sgRNA plasmid  and 2.5 μg pAAVS1-PC-GCaMP6f plasmid  were electroporated together into H9 and CSRP3−/− ESCs. Methods for puromycin screening, selection, and identification of clones are as described above.Generation of H9-GCaMP and The ESC cells and ESC-CMs were fixed with 4% PFA, permeabilized in PBS containing 0.5% Triton X-100  for 15 min, and blocked with 3% BSA (Sigma) for 60 min at room temperature. After that cells were incubated with primary antibodies overnight at 4 °C. Cells were then washed three times using PBS for 5 min each. Then cells were incubated with secondary antibodies diluted in 3% BSA for 1 h at room temperature. After three washes with PBS for 5 min each, cells were counterstained with DAPI  for 5 min. Images were taken under Leica DMI 4000B. The full list of primary and secondary antibodies used and their appropriate dilution are provided in Table ESC-CMs were singularized with CardioEasy CM dissociation buffer (Cellapy). Cells were washed three times with PBS and fixed with chilled (4 °C) fixation buffer (BD Biosciences) for 10 min at room temperature. Next, the samples were incubated with the primary antibody (cTnT) for 30 min. After three washes with PBS, cells were incubated with the secondary antibody (Alexa Fluor 488) for 30 min. Then the samples were washed in PBS and assessed using FACS analysis .2. After washed by PBS, cells were resuspended after centrifugation in PBS. Samples were stored on ice until flow cytometry was run on. The results were analyzed with FlowJo X program. Mean signal fluorescence intensity was used to compare mitochondrial content and ROS production in MLP KO CMs relative to WT CMs.Quantification of mitochondria and reactive oxygen species (ROS): Dissociated hESC-CMs were stained using 50 nM Mitotracker green , 10 μM Cell ROS green , and 2.5 μM MitoSOX Red . Live cells were incubated with these dyes  for 20 min at 37 °C and 5% COCSRP3−/−-GCaMP derived cardiomyocytes were seeded onto confocal dishes. Intracellular calcium flux was imaged using a confocal microscope  at 40×. Spontaneous Ca2+ transients were acquired at 37 °C and 5% CO2 using line-scan mode at a sampling rate of 1 ms/line. A total of 8192 lines were acquired for 8.192 s recoding. For detection of caffeine-evoked Ca2+ release, 10 mM caffeine in Ca2+ free solution was used to evoke sarcoplasmic reticulum (SR) Ca2+ transients in hESC-GCaMP-CMs. The results were analyzed with Image J and Igor.H9-GCaMP and 5 cells with TRIzol  and treated with DNase I (Life Technologies) for about 30 min at 37 °C to eliminate DNA contamination. For qRT-PCR, 1 μg of RNA was reverse transcribed into cDNAs using the PrimeScriptTM reverse transcription system , following the manufacturer’s instructions. QRT-PCR was performed on iCycler iQ5 (Bio-Rad) using 2× SYBR Master Mix (Takara). The relative quantification of gene expression was carried out according to the ▵CT method. Primer sequences are listed in Table Total RNA was extracted from about 2 × 10Cell pellets were washed with cold PBS and resuspended in Mammalian Protein Extraction Reagent  with 5 mM EDTA , containing a protease inhibitor cocktail  and phosphatase inhibitor cocktail . Samples were incubated on ice for 30 min and vibrated every 10 min and then centrifuged at 12,000 rpm for 15 min. The protein concentration of the supernatant was measured by BCA method. Equal amount of denatured protein were resolved by electrophoresis 8–12% (depending on protein size) sodium dodecyl sulfate polyacrylamide gels. The gel was transferred onto a PVDF membrane at 300 mA for 90 min using gel transfer device (Bio-Rad). Then the membrane was blocked with 5% nonfat milk powder at room temperature for 1 h. Membranes were incubated with primary and secondary antibodies of appropriate concentrations listed in Table t test for two groups. Significant differences were considered when P < 0.05.All data are presented as means ± standard errors of the means (S.E.M.). Statistical significance was evaluated using two-sided Student’s Supplemental FiguresTable S1Table S2Supplementary figure legends.