CCR1, CXCR4, CCR2, CX3CR1, CCR7 and CCRL2), adhesion  and purine (including P2RY2 and P2RY13) receptor subfamilies. The downregulated receptors include adhesion GPCRs, such as LPHN1, GPR125, GPR56, CELSR3 and GPR126, protease-activated receptors (F2R and F2RL1) and the Frizzled family receptors SMO and FZD6. Interestingly, specific deregulation was observed in genetically distinct subgroups of AML, thereby identifying different potential therapeutic targets in these frequent AML subgroups.Acute myeloid leukemia (AML) is associated with poor clinical outcome and the development of more effective therapies is urgently needed. G protein-coupled receptors (GPCRs) represent attractive therapeutic targets, accounting for approximately 30% of all targets of marketed drugs. Using next-generation sequencing, we studied the expression of 772 GPCRs in 148 genetically diverse AML specimens, normal blood and bone marrow cell populations as well as cord blood-derived CD34-positive cells. Among these receptors, 30 are overexpressed and 19 are downregulated in AML samples compared with normal CD34-positive cells. Upregulated GPCRs are enriched in chemokine ( They are key transducers that bind a vast diversity of ligands allowing the cells to adapt to their environment by regulating a wide variety of physiological processes including the control of blood pressure, heart rate, digestive processes, hormone secretion, cell growth and migration as well as vision and olfaction. Binding to their ligands leads to conformational rearrangements promoting the engagement and modulation of many distinct downstream signaling effectors that are both G protein-dependent and independent.4 For example, PAR1 is overexpressed in invasive breast carcinomas5 or advanced-stage prostate cancer.7 Likewise, the chemokine receptor CXCR4 has an important role in tumor metastasis and angiogenesis.4 Moreover, the Wnt target gene, Lgr5, identified as a marker of intestinal stem cells, is implicated in mouse intestinal tumorigenesis9 and its expression is also associated with poor clinical outcome in colorectal cancer.10Several GPCRs are critical for cell proliferation and survival, and can be aberrantly expressed in cancer cells.11 only a very limited number of agonists or antagonists acting through these receptors are currently used for cancer therapy.12 Notable exceptions include peptide antagonists of gonadotropin releasing-hormone receptor in prostate cancer,12 a somatostatin receptor agonist (Octreotide) and a growth hormone receptor antagonist (Pegvisomant) for neuroendocrine tumors,12 as well as Vismodegib, an antagonist of smoothened (SMO), approved by the US Food and Drug Administration (FDA) to treat advanced basal cell carcinoma.13Although GPCRs are targets for approximately 30% of all marketed drugs,16 Moreover, in vivo studies have shown that the use of AMD3465, a small molecule antagonist of CXCR4, increases the mobilization of AML cells into the peripheral blood and improves the efficacy of chemotherapy.17 This activity has been explored in a phase 1/2 clinical study showing that the addition of CXCR4 antagonists to chemotherapy is possible in AML and might improve the remission rate.18 The role of GPCRs in mouse leukemic cells was also suggested in a transcriptome analysis of two related leukemia clones which differ in their stem cell frequency.19 This study revealed that genes encoding GPCRs were the most differentially expressed between the two clones compared with other classes of genes (~22% versus ~5%).In acute myeloid leukemia (AML), CXCR4 overexpression has been associated with poor outcome.Despite these punctual observations, an exhaustive assessment of GPCR expression in human AML is lacking. To address this issue, we sequenced the transcriptome of a large cohort of AML samples and herein report the expression pattern of GPCRs in 148 AML samples.n=12) collected with an informed consent were provided by Héma-Québec and pre-enriched for CD34+ cells before being sorted for the CD34 APC+/CD45RA PE− cell population as previously described.20 The Cancer Genome Atlas (TCGA) RNA-Seq AML dataset has been downloaded from the TCGA website (https://tcga-data.nci.nih.gov/tcga/tcgaDownload.jsp) in November 2013 and the associated clinical informations obtained from Cancer Genome Atlas Research Network.21The 148 AML samples of the Leucegene cohort used for this study (described in +), B cells , T cells , Monocytes , total white blood cells (WBC). Sorting purity was checked on aliquots after sorting, and cells were counted, resuspended in TRIzol reagent and stored at −80 °C until RNA isolation was performed according to the manufacturer's instructions. An additional purification step on RNeasy mini columns  was performed to optimize RNA quality, which was subsequently tested on an Agilent bioanalyzer 2100 . A minimum of 50 000 cells was used for RNA Sequencing (RNA-Seq), which was performed as described below.Three unsorted fresh bone marrow samples from healthy donors were purchased from Lonza . Red blood cell lysis was performed prior to resuspending the cells in phosphate-buffered saline, 0.1% bovine serum albumin and DNase 10 μg/ml. Subpopulations were sorted on a BD Aria II sorter  based on published surface marker combinations . Periphe22RNA-Seq was performed using an Illumina HiSeq 2000 instrument . Libraries were prepared according to the manufacturer's recommendations (Illumina). RefSeq annotations were based on the UCSC January 27th 2011 version. The alignment to reference genome (hg19) was carried out using the CASAVA 1.8.2 package and Eland v2 mapping software and bioinformatic analyses were performed as described earlier.2(RPKM+1)), where +1 was added to avoid excessive variations due to very small values. Log transformation was performed to avoid overrepresentation of extreme values. Highly expressed GPCRs were selected using a threshold of 3.5 lRPKM (or 10.35 RPKM) as shown in + cells greater than 1 and less than −1, respectively.RNA-Seq data in Reads Per Kilobase per Million mapped reads (RPKM) were transformed to lRPKM (loghttp://www.guidetopharmacology.org/). To complete the classification and subdivide the class A group into further subgroups, the GRAFS phylogenetic classification of GPCRs was also used.23 Taste receptors have been added along with vomeronasal receptors, opsins and three orphan GPCRs . Overall, GPCRs were classified into 18 subfamilies  database classification downloaded from the website in July 2014  was calculated using the function FET of the add-in Fisherexact downloaded from http://www.obertfamily.com/software/fisherexact.html. The receptors associated with a specific AML genetic subgroup were identified by calculating a difference in mean expression level (lRPKM) between samples with and without the genetic abnormality. An arbitrary difference of 1.5 lRPKM and a significant Student's t test  were used as cutoff levels to identify differentially expressed GPCRs in the different genetically defined subgroups of the studied AML cohort.The GPCR subfamily enrichment analysis in the upregulated or downregulated groups was performed using a Fisher's exact test. Significance  and in normal bone marrow and peripheral blood cell populations. The 772 GPCRs analyzed in this study comprise all GPCRs included in the IUPHAR database, as well as 370 olfactory, 24 taste and 4 vomeronasal receptors. Information about the receptor subfamilies and the 18 subgroups based on their ligands is provided in Using RNA-Seq, we have evaluated the expression of GPCRs in 148 AML samples and compared it with that observed in normal cord blood-derived CD34+ cells , inv(16)(p13.1q22) or utations .MLL translocations  as well as the adhesion GPCRs, EMR1 and GPR114 and the oxysterol-binding receptor, GPR183 (downregulated). Overexpression of eight other GPCRs occurs in the inv(16) AML subgroup. These are C5AR1, CCR2, CXCR7/ACKR3, FPR1, GPR183, RXFP1, PTGIR and LPAR6 , NPM1 (n=43) or DNMT3A mutations (n=30) , inv(16) and ocations . For exand LPAR6 . AML wit fusions . FLT3-ITnd GPR56 . These rs (n=30) . DNMT3A-ADRA2C, GPR126, CYSLTR2, RXFP1, GPR153 and CXCR7/ACKR3 maintained their significant overexpression in specific AML genetic subgroups when compared with normal cell populations , adhesion  and purine (including P2RY2 and P2RY13) receptor subfamilies. This list includes GPCRs previously described as important for AML cell biology, such as CXCR4,24 as well as GPCRs that have a role in hematopoietic stem cell engraftment, such as C3AR1 and PTGER2.26 In addition, 19 receptors are downregulated in AML cells including adhesion GPCRs like LPHN1, GPR125, GPR56, CELSR3 and GPR126, protease-activated receptors (F2R and F2RL1) and the Frizzled family members SMO and FZD6.Our results revealed that 30 GPCRs are overexpressed in AML samples compared with normal CD34PTGER4 and PTGER2 are among the most highly expressed GPCRs differentiating AML from normal CD34+ cells. Preliminary results have shown that PGE2 can increase cyclic AMP production by human leukemic cells through EP2 (encoded by PTGER2) but not EP4 receptors. Further functional studies are needed to confirm these observations.27 Interestingly, highly selective EP2 antagonists (TG4-155 and TG6-10-1) have recently been developed and might therefore be available for pre-clinical studies.28 It is worth noting that different studies have highlighted the importance of PGE2, acting through four GPCRs, PTGER1–4, in the progression of many cancers including colorectal, gastric, lung or breast cancers.29Among these deregulated GPCRs, prostaglandin receptors are of particular interest. Indeed, both CXCR4, CCR7, CCR1, CCR2, CX3CR1 and CCRL2 are also of potential interest. Except for CXCR4,24 their role in AML has never been invoked. In other hematological cancers, CX3CR1 and its ligand, CX3CL1, have been proposed to be involved in the interaction between chronic lymphocytic leukemia cells and their microenvironment,30 and CCR1 has a crucial role in the pathogenesis of myeloma-associated bone disease. Interestingly, CCX721, a selective CCR1 inhibitor, improves osteolytic bone lesions in a preclinical mouse model of this disease.31 This compound is analogous to CCX354-C, an oral CCR1 antagonist that has been evaluated in clinical trial for human rheumatoid arthritis.32Chemokine receptors found to be overexpressed in AML specimens such as GPR109A, GPR109B, SUCNR1, P2RY2, P2RY13 and GPR65. To our knowledge, none of these receptors have been involved previously in hematologic malignancies. However, GPR109A is silenced in colon cancer and primary breast tumor tissues where it acts as a tumor suppressor.33 In contrast to these findings, the receptor is overexpressed in squamous cell cancers compared with normal keratinocytes.34P2RY2 mediates prostate cancer cell migration and metastasis,35 and its activation leads to increased proliferation of melanoma36 and lung tumor cells.37 This observation is context-dependent because P2RY2 activation induces apoptosis in human colorectal carcinoma cell lines.38 The pH-sensing receptor GPR65 has also been reported to be overexpressed in a significant proportion of kidney, ovarian, colon and breast tumors.39 Recent findings have also highlighted that a member of this GPCR family, P2Y14, is critical for stress hematopoiesis.40The purine receptor family members overexpressed in AML include 41 They represent another group of GPCRs for which we observed a strong expression in AML. Notably, CD97 is expressed at high levels in 100% of AML studied to date at a much higher level than found in normal CD34+ cells. CD97 is upregulated in a variety of other malignancies, including glioblastoma,42 digestive and thyroid cancers.43GPR56 and GPR114 are specifically overexpressed in FLT3-ITD-mutated samples and GPR126 is most specific to AML with MLL translocations. Notably, GPR56 has recently been identified as a leukemia stem cell marker, and its expression correlates with poor prognosis in AML.45Adhesion GPCRs are a family of receptors characterized by a long extracellular domain and an autoproteolytic site leading to an extracellular N-terminal fragment and a transmembrane C-terminal fragment that remain noncovalently associated after cleavage. These receptors have been shown to have a crucial role in development, cell migration and are increasingly being invoked as deregulated in numerous cancers.F2R or PAR1 and F2RL1 or PAR2 are downregulated in AML. PAR1 has a well-established role in thrombosis, hemostasis and inflammatory diseases. It is also involved in promoting growth and in the angiogenesis and metastasis processes of several malignancies.4 Unlike AML samples, most solid tumors show an upregulation of PAR1 expression. Our findings are in accordance with the results of a recent study that demonstrated that PAR1 expression is downregulated in primary AML cells.46 Furthermore, Par1 homozygous null mutant mice develop leukemia with a shorter latency than control animals in secondary transplantation experiments.46PAR2 is involved in migration and/or proliferation of many cancer cells, including breast,47 pancreatic48 or colon49 cancer and seems to have a role in tumor angiogenesis through vascular endothelial growth factor production in cancer cells.50Two protease-activated GPCRs SMO and FZD6 are also downregulated in all AML samples except AML with EVI1 rearrangements, which have a FZD6 median expression close to CD34+ cells. SMO and Patched are two receptors mediating Hedgehog signaling. This pathway is aberrantly activated in many solid tumors including basal cell carcinoma.51 This feature has been exploited for the development of Vismodegib, an antagonist of SMO.13 However, in contrast to several other cancers, SMO expression in AML cells is five times lower than in CD34+ cells in our study. Vismodegib is currently being investigated in a phase II clinical trial  in AML patients in combination with ribavirin, because it could potentially overcome the resistance of leukemic cells to ribavirin.52Two frizzled class receptors ADRA2C, which is upregulated in AML with t, could be an interesting target to explore, because many agonists and antagonists of this receptor are available, including several FDA-approved drugs such as the antihypertensive drug clonidine and the antidepressant Mirtazapine. CYSLTR2, a receptor for the inflammatory mediators cysteinyl leukotrienes, is also overexpressed in FLT3-ITD- and NPM1-mutated samples. In aggressive breast tumors, this gene has been described as an independent prognostic factor in combination with CYSLTR1.53 Selective agonist (NMLTC4)54 and antagonists, such as HAMI3379ref.  or BayCysLT2,56 have been developed for CYSLTR2. Other interesting targets are the GPCR members that are overexpressed in AML with inv(16) or AML M4  might be explored for anti-leukemic activity.57It is tempting to speculate that the GPCR members that are differently expressed in frequent AML genetic subgroups and more specifically expressed in AML cells compared with normal hematopoietic cells could be exploited for the development of novel therapeutic approaches. In this context, r AML M4 . For exaIn conclusion, we provide the first comprehensive transcriptome analysis of GPCRs in AML, which reveals that these surface receptors are potential novel therapeutic targets in AML. Using available agonists/antagonists to leukemia-enriched GPCRs, specific drugs can now be tested in preclinical models and, when active, in clinical trials for AML therapeutics.


Ageing is associated with an increased risk of cardiovascular disease and arrhythmias, with the most common arrhythmia being found in the atria of the heart.Little is known about how the normal atria of the heart remodel with age and thus why dysfunction might occur.2+ transient that have implications for the function of the atrial in the elderly.We report alterations to the atrial systolic Ca2+ in the old atria.We describe a novel mechanism by which increased Ca buffering can account for changes to systolic CaThe present study helps us to understand how the processes regulating atrial contraction are remodelled during ageing and provides a basis for future work aiming to understand why dysfunction develops.2+ homeostasis. In the atria, age‐associated changes in the action potential have been documented. However, little is known about remodelling of intracellular Ca2+ homeostasis in the healthy aged atria. Using single atrial myocytes from young and old Welsh Mountain sheep, we show the free Ca2+ transient amplitude and rate of decay of systolic Ca2+ decrease with age, whereas sarcoplasmic reticulum (SR) Ca content increases. An increase in intracellular Ca buffering explains both the decrease in Ca2+ transient amplitude and decay kinetics in the absence of any change in sarcoendoplasmic reticulum calcium transport ATPase function. Ageing maintained the integrated Ca2+ influx via ICa‐L but decreased peak ICa‐L. Decreased peak ICa‐L was found to be responsible for the age‐associated increase in SR Ca content but not the decrease in Ca2+ transient amplitude. Instead, decreased peak ICa‐L offsets increased SR load such that Ca2+ release from the SR was maintained during ageing. The results of the present study highlight a novel mechanism by which increased Ca buffering decreases systolic Ca2+ in old atria. Furthermore, for the first time, we have shown that SR Ca content is increased in old atrial myocytes.Many cardiovascular diseases, including those affecting the atria, are associated with advancing age. Arrhythmias, including those in the atria, can arise as a result of electrical remodelling or alterations in Ca Ageing is associated with an increased risk of cardiovascular disease and arrhythmias, with the most common arrhythmia being found in the atria of the heart.Little is known about how the normal atria of the heart remodel with age and thus why dysfunction might occur.2+ transient that have implications for the function of the atrial in the elderly.We report alterations to the atrial systolic Ca2+ in the old atria.We describe a novel mechanism by which increased Ca buffering can account for changes to systolic CaThe present study helps us to understand how the processes regulating atrial contraction are remodelled during ageing and provides a basis for future work aiming to understand why dysfunction develops. AFatrial fibrillationNCX+/Ca2+ exchangeNaPLBphospholambanSERCAsarcoendoplasmic reticulum calcium transport ATPasePPprotein phosphataseRyRryanodine receptorSRsarcoplasmic reticulumAge is a major risk factor for cardiovascular disease and, as life expectancy continues to increase, so too does the prevalence of cardiovascular disease  is the most commonly seen. AF has a greater probability of occurring when atrial myocytes have remodelled in response to diseases such as heart failure and previous episodes of AF, or as a consequence of the ageing process itself. Disease‐associated remodelling at the level of the cardiac myocyte includes alterations to excitation–contraction coupling contributing to contractile dysfunction  Ca content are decreased in aged patients  to define how Ca2+ handling is altered and (ii) to determine the mechanisms that are responsible for altered Ca2+ homeostasis in the old atria.Several facets of Caet al. et al. et al. et al. 2+ handling changes with age.The sheep atrium was chosen for the present study because of its similarity to human atria   and old (>8 years) female Welsh Mountain sheep deemed to be in their last quintile of life by clinical examination Pope,  without All procedures comply with The UK Animals (Scientific Procedures) Act 1986 and EU directive 2010/63. The ARRIVE guidelines were followed for reporting the use of animals in scientific experiments. Local ethical approval was granted by The University of Manchester Animal Welfare Ethical and Review Board.et al. et al. i.v. injection of pentobarbitone (200 mg kg−1) mixed with heparin  and then the heart was rapidly removed and rinsed in Ca2+ free solution containing (mm): 134 NaCl, 11 glucose, 10 Hepes, 10 2,3‐butanedione monoxime, 4 KCl, 1.2 MgSO4, 1.2 NaH2PO4 and 0.5 mg ml−1 bovine serum albumin (pH 7.34 with NaOH). The atria were separated from the ventricles, cannulated via the left superior atrial artery and perfused at 37°C with the Ca2+ free solution for 10 min. Collagenase type II  and protease type XIV  were added to the superfusate and the heart digested for ∼12 min. The perfusate was switched to a taurine containing solution (in mm): 113 NaCl, 50 taurine, 11 glucose, 10 Hepes, 10 2,3‐butanedione monoxime, 4 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 0.1 CaCl2 and 0.5 mg ml−1 BSA (pH 7.34 with NaOH) for a further 20 min. The left auricle was excised and myocytes were dispersed by chopping, followed by trituration. Prior to experimentation, cells were stored in a solution containing (mm): 140 NaCl, 10 glucose, 10 Hepes, 4 KCl, 1.8 CaCl2, 1 MgCl2 and 2 probenecid (pH 7.34 with NaOH) at room temperature.Single myocytes were isolated using an enzymatic digestion technique, as described previously . Cells were loaded at 5 μmol l−1 for 8 min and allowed to de‐esterify for at least 45 min prior to experimentation. Fluorescence was excited at 488 nm, emitted light captured >515 nm and intracellular Ca2+ calculated using:Kd is the dissociation constant of Fluo‐5F at 37°C (1035 nmol l−1)   in the pipette solution : 5 4‐aminopyridine, 0.1 BaCl2 and 0.1 DIDS. Micropipettes (2–3 MΩ) were filled with (in mm): 125 KCH3O3S, 20 KCl, 10 NaCl, 10 Hepes and 5 MgCl2 and then titrated to pH 7.2 with KOH. ICa‐L was elicited by a 100 ms voltage step from a holding potential of −40 mV. We predict a liquid junction potential of −8.2 mV for all data, which has not been corrected for. To determine whether increasing Ca buffering in young atrial myocytes could produce an ‘old’ systolic Ca2+ transient, young atrial myocytes were incubated with 20 μm EGTA for 10 min. All experiments were performed at 37°C within 12 h of myocyte isolation.Electrophysiological experiments were performed simultaneously with [CaICa‐L and Na+/Ca2+ exchange (NCX)] were quantitatively measured, as described previously . The integral was also corrected for Ca2+ removal by non‐electrogenic mechanisms .Sarcolemmal fluxes i below the resting level, Bmax is the maximum buffering capacity for Ca2+, [Ca2+]i represents the free intracellular Ca2+ and Kd is the dissociation constant of the buffers. Ca buffer power was calculated from the Bmax and Kd values obtained above using the equation  was calculated from the resting and peak systolic [Ca2+]i and the buffer properties. The amount of Ca2+ released from the SR was then calculated by subtracting the integral of the L‐type Ca2+ current from this value. The interaction between sarcolemmal Ca2+ fluxes and SR Ca2+ release were assessed by measuring both the gain of CICR and the fractional release of Ca2+ from the SR. Gain was calculated using the equation:The increase of Ca2+ from the SR was calculated using eqn The fractional release of Caet al. et al. −1 for 20 min at room temperature). Cells were excited at a wavelength of 488 nm and emitted light collected >500 nm. Imaging was performed at an xy resolution of 310 nm and 310 nm separation between z stacks. Cells were subsequently patch clamped and capacitance measured to generate a capacitance–volume relationship. This relationship was not altered by age and we therefore used a combined value of 4.80 ± 0.18 pF pl–1.The capacitance to volume relationship was quantified using an SP2 confocal microscope , as described previously  and the caffeine‐evoked Ca2+ transient in the presence of 10 mmol l−1 Ni+ (kNi+caff) were calculated by fitting single exponentials. Correction factors were calculated for each age group using eqn Correction factors were calculated in a further series of experiments as described previously . Differences between young and old animals were determined using linear mixed modelling  to account for multiple cellular data points from a single sheep. Thus, for statistical purposes, in the present study, n is the number of animals. The Kolmogorov–Smirnov or the Shapiro–Wilk statistic  was used to test for normality of data distribution. Data were transformed appropriately (e.g. log10 transform) to achieve a normal distribution. In a small number of cases where data could not be normalized with an appropriate transformation, linear mixed modelling was performed if the frequency distribution of the data was symmetrical or n was sufficiently large  increased with age by 17 ± 7%  and capacitance (patch clamp) were performed in young and old cells. Volume increased by 45% with age . Increased volume was accompanied by an equivalent increase in cellular capacitance such that the ratio of capacitance to volume was unchanged during ageing . A mean value of capacitance for a given cell volume was therefore generated (4.80 ± 0.18 pF pl–1) and used to assess cell volume from measured capacitance in subsequent experiments.Typical calcein‐AM stained atrial myocytes from young and old hearts are shown in Fig. 2+ transients recorded from single atrial myocytes under voltage clamp control are shown in Fig. C. Ageing resulted in a 25 ± 8% decrease in Ca2+ transient amplitude . Normalizing typical Ca2+ transients from experiments similar to Fig. C reveals a decrease in the rate constant of decay of the systolic Ca2+ transient with age . kSYS was calculated by fitting a single exponential to the entire decay phase of the Ca2+ transient and was reduced by 13 ± 6% during ageing (P < 0.05) .Typical Ca2+ transient decays as Ca2+ is removed from the cytosol by SERCA (dominant pathway), NCX and minor pathways . During the systolic Ca2+ transient, all Ca2+ removal pathways contribute; in contrast, during the caffeine‐evoked Ca2+ transient , SERCA is negated as a result of the continued presence of caffeine. Thus, the canonical method to calculate the rate of Ca2+ removal by SERCA (kSR) is to fit the caffeine‐evoked Ca2+ transient with a single exponential and the rate constant of decay (kCAFF) is then subtracted from that of the systolic Ca2+ transient (kSYS). Normalizing caffeine‐evoked Ca2+ transients  suggested that there was no effect of age on kCAFF, which is consistent with the data in Fig. I. kSR, however, decreased by 19 ± 7% with age . Subsequent experiments were designed to determine the mechanism responsible for decreases in (i) kSR and (ii) Ca2+ transient amplitude.The Caent Fig. E, SERCAnts Fig. H suggeskSR is often accounted for by decreased SERCA function, we first explored expression levels and phosphorylation status of relevant proteins. Surprisingly, protein levels of SERCA and PLB (including the SERCA:PLB ratio) remained unchanged with age . Furthermore, we were unable to detect any change in PLB phosphorylation at Ser16 or Thr17, which is consistent with no change in the levels of the protein phosphatases PP1 and PP2A . Taken together, these data suggest that SERCA function does not alter with age. An alternative explanation for the age‐associated decrease in kSR would be an increase in leak of Ca2+ from the SR . Thus, our data provide no evidence to support the hypothesis that either decreased SERCA function or increased SR leak can account for the decrease in the rate of decay of the Ca2+ transient. Because increased Ca buffering can slow the Ca2+ transient decay  records the change in free Ca2+ . SERCA is effectively disabled in the continued presence of caffeine and Ca2+ is removed from the cell largely by NCX . Because, upon caffeine application, a small amount of Ca2+ is removed by pathways other than NCX, this must be accounted for to accurately calculate the change of (Δ) total Ca. Correction factors were determined in a separate series of experiments for young and old atrial myocytes (see Methods) . The corrected integral of INCX thus gives a measure of the change in total cellular Ca (free Ca2+ plus bound Ca) .Experiments such as that shown in Fig. a2+ Fig. A, upperds) Fig. G and H.Ca) Fig. A, lower2+ over the falling phase of the caffeine‐evoked Ca2+ transient  and the data fitted with a hyperbolic function  with no change in the maximum buffering capacity . The buffering power (eqn P < 0.05), suggesting that increased Ca buffering contributes to the slowed decay of the Ca2+ transient.Total Ca was plotted against free Ca2+ was converted to total Ca using eqn Bmax and Kd values from the cells' own Ca buffer curve calculations were used. The falling phase of total Ca for both the systolic Ca2+ and caffeine‐evoked Ca2+ transients were differentiated with respect to time (d[CaT]/dt) to give a rate of Ca2+ removal independent of Ca buffering and then plotted against free Ca2+ . Data in Fig. C (where the effects of Ca buffering were removed) were fit with linear regressions and the gradient of fits to the caffeine‐evoked Ca2+ transient subtracted from that of the systolic Ca2+ transient. The resultant value describes the SERCA‐dependent slope (independent of Ca buffering) and this was unchanged with age . Thus, decreased kSR is not the result of a decrease in SERCA function but rather increased Ca buffering in the old atria slows the decay of the Ca2+ transient. The mechanism underlying the decrease in Ca2+ transient amplitude was investigated next.Although the protein data shown in Fig. a2+ Fig. C. Data age Fig. D. Thus,INCX  gives a measure of the amount of Ca2+ that was stored within the SR. Thus, we aimed to determine whether decreased SR load underlies the decrease in Ca2+ transient amplitude with age. We found, however, that SR Ca content was actually increased with age by 29.6 ± 8%  . EGTA decreased both the amplitude of the systolic Ca2+ transient  and the rate of decay of systolic Ca2+, as shown by the normalized records  and mean data in Fig. D. It is important to note that, as shown previously, increasing Ca buffering with EGTA results in fast and slow Ca2+ decay kinetics  which presumably resulted from the addition of a buffer of higher affinity than the cells endogenous buffers. Our data suggest that increasing Ca buffering recapitulates the effects of ageing on the systolic Ca2+ transient.To confirm whether increased Ca buffering mimics the effects of ageing on the systolic Cants Fig. A. EGTA ent Fig. B and thrds Fig. C and meKd Fig. E which ICa‐L can alter SR function, we next set out to determine any role for ICa‐L in age‐associated atrial remodelling.However, increased buffering cannot explain (i) the increase in SR Ca load or (ii) the lack of change in the amplitude of the total Ca transient despite an increase in SR Ca load. Because changes in ICa‐L was reduced by 18.6 ± 7% in old atrial myocytes . Integrated Ca2+ entry, however, was unaltered  suggesting slowed ICa‐L inactivation in the old atria. It was not possible to fit ICa‐L decay with a single exponential, making any comparison between young and old data sets complicated. Slowed ICa‐L inactivation was not resolved by calculating the absolute rate of inactivation of exponents in Fig. E. However, the decreased amplitude of the fast component of ICa‐L inactivation with age  is consistent with slower ICa‐L inactivation. Example traces in Fig. C were chosen to highlight slowed kinetics of the fast component of decay in the old cell, which, together with the decrease in peak, maintains the current integral.Peak ICa‐L results in an increase in SR Ca content  is sufficient to explain the increase in SR Ca content with age. Here, the age‐associated decrease in peak ICa‐L was mimicked in young atrial cells by pharmacological inhibition of ICa‐L with nicardipine to the levels observed in old animals . SR Ca content was assessed from the integral of the caffeine‐evoked NCX current in young atrial cells in the absence and presence of nicardipine. Reducing ICa‐L with nicardipine increased SR Ca content by 22 ± 2%  compared to 29.6 ± 8% during ageing. The discrepancy between these values is probably a result of the nicardipine‐induced decrease in integrated Ca2+ entry , which was maintained during ageing. Thus, we suggest that the age‐associated increase in SR Ca load is explained by decreased peak and maintained integral of ICa‐L.Previously, we have shown that decreasing atrial ICa‐L could explain why the total Ca transient amplitude was maintained during ageing despite an increase in SR Ca load. First, we calculated fractional SR Ca release using /SR Ca content  when assessed using /peak ICa‐L despite increased SR Ca load. Thus, our data suggest that SR Ca release is maintained during ageing because decreased fractional release opposes increased SR Ca load, maintaining the total Ca transient.Accordingly, we investigated whether decreased peak tent eqn . Fractiotent eqn ; we nextling eqn . Gain, o2+ entry via ICa‐L does not change with age, Ca2+ efflux must also be maintained to preserve flux balance. Although a decrease in the amplitude of the free Ca2+ transient would be expected to decrease Ca2+ efflux, a slowing of decay would be expected to increase efflux. To assess the net effect, the integrated area underneath the free Ca2+ transient was calculated and found not to change with age . Thus, during ageing, the effects of increased buffering (decreased amplitude and slowed decay) act in concert to maintain Ca2+ efflux. A summary of the age‐associated changes influencing systolic Ca2+ is provided in Fig. Finally, because integrated Caage Fig. G and H.2+ handling in old sheep. We show that ageing resulted in: (i) cellular hypertrophy; (ii) decreased amplitude and rate of decay of the systolic Ca2+ transient brought about by an increase in intracellular Ca buffering; (iii) decreased peak ICa‐L with maintained integrated Ca2+ entry; and (iv) increased SR Ca load arising as a consequence of decreased peak ICa‐L.In the present study, we have described and elucidated mechanisms for alterations in atrial intracellular Caet al. et al. et al. et al. et al. et al. et al. et al. et al. Healthy ageing in sheep results in an increase in atrial cell dimensions and cellular capacitance. Increasing cellular capacitance with age has been reported previously in the dog right atrium  and in the ventricle, as reported previously .Less than 1% of Ca2+ efflux from the cell must equal Ca2+ influx. The integrated Ca2+ influx is unaffected by ageing , suggesting that any increase in the affinity of troponin for Ca2+ is not sufficient to maintain atrial contraction during ageing.It is unclear what is responsible for the increase in buffering with age. We report no change in ICa‐L might appear to be an obvious candidate accounting for the decrease of Ca2+ transient amplitude with age. However, during ageing, the decrease of peak Ca2+ current is not accompanied by any change of integrated influx. The fact that the integrated influx is constant means that, in the steady‐state, the efflux must also be unchanged. This requires (irrespective of what happens to the peak current) that the Ca2+ transient, or the amount of Ca2+ seen by NCX, must be unaltered. In older atria, efflux is maintained by slowed decay of systolic Ca2+ as discussed above.Initially, decreased peak ICa‐L does, however, result in an increase in SR Ca content in old atrial cells. Peak ICa‐L acts as the trigger for SR Ca release, whereas the integral acts to load the cell, and thus the SR, with Ca2+. Decreased peak ICa‐L decreases the fractional SR Ca release, resulting in less Ca2+ being available for extrusion from the cell, which presumably acts to increase SR Ca content. Because the integral of ICa‐L is unaltered by age, this effect is unopposed in the old atria.The decrease in peak ICa‐L increases SR Ca content to a level where the combination of decreased peak ICa‐L and increased SR Ca content results in the same release of Ca2+ from the SR in older as in younger atria  was decreased in the old atria, as was the rate constant following correction for Ca2+ removal by pathways other than SERCA (kSR). Initially, this might suggest an age‐associated decrease in SERCA function. However, when SERCA activity was calculated directly from changes of total Ca, SERCA was actually unaltered.It is important to emphasize that, had we not considered Ca buffering in the present study, we would also have calculated a decrease in SERCA function. We found the rate of decay of systolic Ca2+ handling that we report in myocytes from old sheep could promote AF by several mechanisms. First, the increase in SR Ca content could increase the propensity to spontaneous diastolic Ca2+ release, leading to delayed after depolarisations  slows the Ca2+ transient decay more than when loading with the AM ester, suggesting that, under our experimental conditions, the AM ester buffers Ca2+ less than the pentopotassium salt. We have estimated passive Ca2+ buffering of Fluo‐5F and we expect ∼9% of total Ca to be bound to the indicator in our experiments. We suggest this will have a negligible effect on our data and, because buffering by Fluo‐5F is unaltered by age, this will have no effect on our conclusions.Furthermore, in our experience, loading with the pentopotassium salt of Fluo‐5F  voltage clamp technique. Given the differences in capacitance and known access resistance, even in the uncompensated state, any difference in voltage errors would be less than 1 mV in these experiments. In the present study, a holding potential of −40 mV was used to isolate ICa‐L has been reported in studies of dogs , a University of Manchester Stepping Stone Award (KMD), British Heart Foundation Project Grants (PG/09/062 and PG/12/89/29970), a British Heart Foundation Clinical Research Training Fellowship , The BHF Michael Frazer PhD Studentship (FS/07/003) and a European Union 6th Framework STREP Project ‘Normacor’.


Developing effective and humane on-farm euthanasia methods is essential for all livestock industries to ensure that animals do not suffer and are killed humanely. Approved methods are lacking for commercial meat rabbits, potentially leading to poor welfare. We assessed several methods of on-farm killing of cull rabbits of different ages to determine which methods were most effective and humane. These included blunt force trauma (the most commonly used method on rabbit farms), a novel mechanical cervical dislocation device, and a non-penetrating captive bolt device. We evaluated method effectiveness by examining animal reflexes and behaviours after applying the method as well as by examining radiographs of rabbit heads for signs of skull damage, and by assessing the degree of trauma to the brain through dissection and microscopy, because more trauma is generally correlated with enhanced method effectiveness and irreversibility. We found that blunt force trauma resulted in an unacceptably high failure rate, particularly in mature rabbits, whereas the mechanical cervical dislocation and non-penetrating captive bolt devices were both highly effective for killing rabbits humanely and irreversibly. The non-penetrating captive bolt device was the most effective with a 100% success rate and could be used on all rabbits weighing more than 150 g.n = 12) confirmed tissue damage prior to gross dissection and microscopic evaluation. All 63 rabbits euthanized by the NPCB device were rendered immediately and irreversibly insensible. The MCD device was effective in 46 of 49 (94%) rabbits. Method failure was highest for BFT with euthanasia failures in 13 of 58 (22%) rabbits. Microscopically, brain sections from rabbits killed with the NPCB device had significantly more damage than those from rabbits killed with BFT (p = 0.001). We conclude that BFT is neither consistently humane nor effective as a euthanasia method. MCD is an accurate and reliable euthanasia method generally causing clean dislocation and immediate and irreversible insensibility, and the NPCB device was 100% effective and reliable in rabbits >150 g.The commercial meat rabbit industry is without validated on-farm euthanasia methods, potentially resulting in inadequate euthanasia protocols. We evaluated blunt force trauma (BFT), a mechanical cervical dislocation device (MCD), and a non-penetrating captive bolt device (NPCB) for euthanasia of pre-weaned kits, growers, and adult rabbits. Trials were conducted on three commercial meat rabbit farms using 170 cull rabbits. Insensibility was assessed by evaluating absence of brainstem and spinal reflexes, rhythmic breathing, and vocalizations. Survey radiographs on a subsample of rabbits ( Livestock producers must have effective methods in place to euthanize sick, injured, and cull animals ,2,3. FewCervical dislocation (CD) is another method commonly used for rabbit euthanasia ; howeverOur objective was to evaluate the effectiveness of several physical methods for euthanizing adult and juvenile rabbits, including BFT, a novel mechanical cervical dislocation device (MCD) device and a NPCB device, as determined by onset of rapid and irreversible insensibility and degree of induced brain damage. Based on previous studies examining the efficacy of the NPCB device in poultry, we hypothesized that this device would be most effective and humane for farmed rabbits.n = 58), MCD (n = 49), and NPCB device (n = 63). Minimum sample size requirements to detect differences with an error rate of 5% between techniques and age groups (using 10% variability) was estimated from previous euthanasia studies [2]. All methods were tested on New Zealand white-like rabbits across three age groups: pre-weaned kits (2 to 5 weeks), growers (6 to 12 weeks), and adults (>12 weeks). The procedures and protocol for this research were reviewed and approved by the University of Guelph Animal Care Committee (AUP3366). The University of Guelph holds a Good Animal Practice certificate issued by the Canadian Council on Animal Care and is registered under the Ontario Animals for Research Act. All rabbits used in this study were ones targeted for euthanasia by producers and procedures were conducted on-farm, necessitating convenience sampling. The majority of rabbits that are culled on-farm are young animals  with lesser numbers of does and very few mature bucks, given their very small relative population on any given farm. Necessity for euthanasia of individual rabbits in this study was determined by operators and reasons included sick or injured rabbits, unthrifty rabbits that were unlikely to survive to market weight, and rabbits reaching the end of reproductive utility. Three commercial meat rabbit farms  from across southwestern Ontario were recruited to participate in this study. Producers were experienced in rabbit production and had raised meat rabbits commercially full-time for at least eight years. Between May and August 2015, a total of 170 rabbits were euthanized over 28 trial days by the following methods: BFT  used BFT as a routine euthanasia method. Blunt force trauma was the primary method for all age groups of rabbits on Farms 2 and 3 , and was used on pre-weaned kits only on Farm 1 . The BFT method used by producers varied, but in all cases, the rabbit was suspended by its back legs prior to striking the animal on the head with a heavy object or striking the rabbit against a hard surface. http://www.rabbitwringer.com, West Grove, PA, USA)  . The rabThe NPCB device  is a commercially available modified pneumatic nail gun weighing 750 g, with a mushroom-shaped nylon bolt head that extends 2 cm from the end of the barrel . The devAn initial pilot study was conducted at the University of Guelph on 22 donated rabbit cadavers of varying sizes and ages, weighing between 0.9 kg to 4.7 kg, to determine the appropriate placement and required pressure for the NPCB device. The correct location and pressure were determined by dissection of the head following application of the device, looking for maximum brain damage without breaking the skin or injuring other anatomical sites, such as the eye or frontal sinuses, which might contribute to poor esthetic outcome as well as increased biosecurity risk. From this initial cadaver work, and based on preliminary observations and discussions with abattoir personnel who use this device routinely by discharging twice in rapid succession for killing rabbits prior to slaughter, it was determined that the NPCB device should be positioned to rest lightly on the frontal and parietal bones, in the center of the forehead, with the barrel placed in front of the ears and behind the eyes . The reqwww.random.org) to the operator to use their preferred euthanasia technique for the size and age of rabbit or to research personnel (JLW) to use the NPCB device. Blind scoring techniques could not be used because it was obvious at the time of examination which method had been applied. Immediately after each euthanasia method was applied, an observer assessed the rabbit for insensibility by evaluating rabbit reflexes, postures, behaviours, and palpable heartbeat. Reflexes assessed included pupillary, palpebral, toe and ear pinch withdrawal, inner nostril poke, and corneal. Reflexes were selected based on use in other studies examining insensibility in rabbits . A post hoc Tukey’s test indicated that total time convulsing following BFT was shorter than for the NPCB device (p = 0.01) and MCD (p = 0.03). Overall, clonic and tonic convulsions in rabbits did not follow a clear pattern or transition and occurred variably. Convulsion occurrence and strength seemed to depend on the health status of the animal, with healthier animals having more aggressive convulsions, although this was not specifically scored in this study. A pattern predictive of a return to sensibility was convulsions suddenly stopping versus slowly fading out. No convulsions occurred when rabbits were sensible. Cardiac arrest occurred at 160 s ± 5 after the method was applied with a range across all successful euthanasia of 70 s to 388 s. Heartbeat was challenging to palpate and auscultate for young pre-weaned rabbits, emaciated rabbits, and for rabbits euthanized by BFT.The average total convulsion time for BFT, the NPCB device, and MCD was 50 s ± 7, 62 s ± 4, and 58 s ± 7, respectively. There was a difference between groups as determined by one-way ANOVA [F = 5.03, After application of the NPCB device, subcutaneous swelling was immediately evident at the site of application. No distal vertebral fractures or hip luxations were noted after application of MCD, and dislocations were only detectable with palpation. Rabbits were difficult to restrain for BFT and this difficulty in safely restraining them may have attributed to method failure. Rabbits were easily restrained for application of the NPCB device and the relatively large bolt and barrel of the device to the size of the head of the rabbit allowed for easy alignment.Survey radiographs of the four growers euthanized by the NPCB device demonstrated a depressed cranium, some with skull fragments embedded in the brain and all rabbits had noticeable swelling from subcutaneous hemorrhage . Those e2  = 120.82, p < 0.001 for macroscopic subcutaneous and χ2  = 82.36, p < 0.001 for subdural hemorrhages, respectively. There was a statistically significant difference between all three methods for skull fractures, χ2  = 93.10, p < 0.001, with the NPCB device scoring the highest, followed by BFT then MCD.Macroscopic evaluations consistently demonstrated marked brain hemorrhage for rabbits euthanized with the NPCB device. Swelling of the tissue above the skull (subcutaneous hematoma) resulted in high macroscopic subcutaneous hemorrhage scores . Rabbits2  = 9.60, p = 0.008, whereas no differences were noted between the other age groups. Subcutaneous and subdural macroscopic hemorrhages were not significantly different between age groups across all methods. Comparisons for age within treatment groups indicated that within the BFT group, pre-weaned kits scored significantly higher than adults for macroscopic subcutaneous hemorrhage, χ2  = 8.19, p = 0.02, and for skull fractures, χ2  = 9.81, p = 0.006. Within the NPCB group, adults had a significantly lower skull fracture score than pre-weaned kits and growers, χ2  = 9.81, p = 0.001. Within the MCD group there was less macroscopic subdural hemorrhage in the growers than the adults, χ2  = 8.32, p = 0.02. These were the only statistically significant differences among age groups.Comparing between age groups, skull fracture scores were higher for pre-weaned kits compared to adult rabbits across all methods, χ2  =9.06, p = 0.01. There were no significant differences in hindbrain parenchymal hemorrhage scores between methods. Comparing brain sections from the mid-brain, the NPCB device scored significantly higher than other methods for mid-brain subdural hemorrhage, χ2  = 15.03, p = 0.001, and scored significantly higher than BFT for mid-brain parenchymal hemorrhage, χ2  = 9.22, p = 0.010. Comparing brain sections from the cortex, there was no significant difference in cortex subdural hemorrhage scores between methods, but NPCB cortex parenchymal hemorrhage scores were significantly higher than other methods, χ2  = 19.957, p < 0.001.Histologic analysis of the three sections from each brain indicated that rabbits euthanized with the NPCB device had the highest average subdural and parenchymal hemorrhage scores across brain sections, except for the parenchymal score in the hindbrain . Compari2  = 12.39, p = 0.002  = 9.584, p = 0.008. There were no other significant differences within groups for microscopic brain hemorrhage scores. Within the MCD group, comparing all brain section scores, there was significantly more microscopic parenchymal hemorrhage in the hindbrain than in the cortex, χ = 0.002 . Within 2  = 12.31, p = 0.001, χ2  = 10.94, p = 0.005 respectively  = 6.72, p = 0.04.The highest overall microscopic subdural and parenchymal hemorrhage score for each brain, regardless of brain section origin, indicated that all brains had some form of hemorrhage. Average highest overall subdural and parenchymal hemorrhage scores were significantly higher for the NPCB device than BFT, χectively . There wOur results demonstrated that the NPCB device was 100% effective for euthanizing all age groups of commercial meat rabbits tested  and that the MCD device was also a highly effective technique with an overall 6% failure rate, largely attributable to inexperience with one specific weight range. Blunt force trauma had the highest failure rate with 13 of 63 animals (22%) unsuccessfully euthanized, including a 43% failure rate (8 of 14) in adult rabbits. Based on these findings we cannot recommend blunt force trauma as a euthanasia method for commercial meat rabbits. Several reasons may have accounted for BFT failures, one of which is insufficient force. Li et al.  found thThe device used for MCD also resulted in a high degree of accuracy with clean dislocation at the base of the skull. Dislocations were only slightly palpable, limiting the ability to check for correct application other than through assessment of sensibility, although subcutaneous cervical swelling and hemorrhage was often marked after application. Gap distances in poultry after application of manual cervical dislocation are significantly larger (~5 cm) than those found in this study making them easier to palpate . A smallThe immediate insensibility noted following MCD in rabbits in this study is very different from findings in poultry, in which brainstem reflexes are reported to persist for several seconds after application ,15. ErasThere was a significant difference between all three euthanasia methods in the number of skull fractures. No skull fractures were predicted to occur during MCD in accordance with proper technique. Although no vertebral fractures were seen, some rabbits had minor occipital bone fractures caused by pressure of the device during downward application force. The mild skull fractures noted rarely following application of MCD are unlikely to contribute to poor animal welfare since insensibility was immediate after application, which occurs <2 s after sliding the head into the V-shaped opening. The difference in skull damage between BFT and NPCB rabbits is in line with minimal microscopic brain damage with BFT, even when euthanasia was successful. Across all euthanasia methods, pre-weaned kits had more skull fractures than adults. Skull thickness correlates with age and this finding likely occurred because of the thinner skulls of younger rabbits, which fractured more readily. This supports our recommendation for altering pressure settings for the NPCB device according to animal age and degree of skull thickness. Finnie et al.  used a dThe NPCB device caused significantly more focal macroscopic subcutaneous hemorrhage than the other two methods. The strength of subcutaneous hemorrhage findings for evaluating euthanasia efficacy is minimal compared to the other measurements, based on research evaluating cervical crushing in turkeys . SensiblEsthetics are an important consideration when evaluating euthanasia methods as euthanasia can have detrimental mental effects on the operator and observers . IncreasAmong the three euthanasia methods evaluated in this study, there were differences in the training and experience operators had with their preferred methods. None of the producers had ever been formally trained in BFT, but all had been using the technique for eight or more years. The operator for the MCD device had been using this method for six months prior to this study and was self-taught based on an online video. The operator of the NPCB device had received training from an experienced NPCB device operator and had practiced the technique on rabbit cadavers during the pilot study. With any physical method of euthanasia there can be a learning curve  and thishttp://www.rabbitwringer.com, West Grove, PA, USA). This device accommodates different sizes of rabbits with an adjustable neck plate. The device does not pose a physical safety or biosecurity risk for the operator. The NPCB device used was the Zephyr-E, an expensive device at ~$900 USD . Restraint of pre-weaned kits increases the proximity of the device to the hand leading to a potential safety risk. The manufacturer of the device indicates that accidental misfire of the device to the hand would result in bruising. Our results are specific to this particular instrument design (including the modified end of the bolt) and the size of the rabbit. Re-validation would be required if modifications to the device were attempted or if the device was applied to rabbits of a breed other than the New Zealand white-like rabbits and outside the weight range of 0.15 to 3.5 kg evaluated in this study.Cost and operator safety are important considerations to ensure that validated euthanasia methods are practical. Blunt force trauma does not require a specific tool, but safety is a significant concern, as difficulty in restraining a rabbit could result in self injury. External bleeding and tissue discharge are also biosecurity safety concerns for BFT. The MCD device can be made on-farm for ~$35 USD or purchased for ~$90 USD (Rabbit Wringer, Our findings indicate that blunt force trauma is neither a humane nor an esthetically acceptable method for killing meat rabbits of any age and it is not recommended for on-farm euthanasia of rabbits. Both the non-penetrating captive bolt device and the mechanical cervical dislocation device used in this study provided effective and humane single step methods for euthanasia of rabbits >150 g.


In darkness, the continuous current entering the cone outer segment through cGMP-gated (CNG) channels is carried in part by Ca2+, which is then extruded back to the extracellular space. The mechanism of Ca2+ extrusion from mammalian cones is not understood. The dominant view has been that the cone-specific isoform of the Na+/Ca2+, K+ exchanger, NCKX2, is responsible for removing Ca2+ from their outer segments. However, indirect evaluation of cone function in NCKX2-deficient (Nckx2−/−) mice by electroretinogram recordings revealed normal photopic b-wave responses. This unexpected result suggested that NCKX2 may not be involved in the Ca2+ homeostasis of mammalian cones. To address this controversy, we examined the expression of NCKX2 in mouse cones and performed transretinal recordings from Nckx2−/− mice to determine the effect of NCKX2 deletion on cone function directly. We found that Nckx2−/− cones exhibit compromised phototransduction inactivation, slower response recovery and delayed background adaptation. We conclude that NCKX2 is required for the maintenance of efficient Ca2+ extrusion from mouse cones. However, surprisingly, Nckx2−/− cones adapted normally in steady background light, indicating the existence of additional Ca2+-extruding mechanisms in mammalian cones.Calcium ions (Ca Upon light-induced closure of CNG channels, the Ca2+ inflow is reduced but its continuous extrusion out of the outer segment results in a decline in cytoplasmic Ca2+ in the outer segment2+ regulates several steps of the cone phototransduction cascade, which in turn modulate the recovery of the cone light response as well as background light adaptation32+ modulation, response recovery is greatly delayed and the photoreceptor cell saturates under low background light6In vitro evidence indicates that the only mechanism of extrusion of Ca2+ out of the outer segments of vertebrate photoreceptors is by cell-specific Na+/Ca2+, K+ exchangers (NCKX)910Nckx2−/−) mice found no alterations in cone number or in cone-driven bipolar cell responses assayed by photopic b-wave measurements from in vivo electroretinogram (ERG) recordings2+ homeostasis of mammalian cones, leaving the mechanism of its extrusion from cone outer segments unknown. Here, we addressed this question by directly analyzing the physiological properties of NCKX2-deficient mouse cones in dark- and light-adapted conditions.The detection of light in the vertebrate visual system and its transduction into an electric signal take place in the outer segments of rod and cone photoreceptors. Light triggers a phototransduction cascade that ultimately causes the closure of cyclic nucleotide-gated (CNG) channels in the photoreceptor outer segments1+/Ca2+, K+ exchanger, NCKX2, has been shown to be expressed in chicken and human cones by in situ hybridizationNckx2−/− . To investigate whether ablation of NCKX2 expression led to compensatory changes in expression of other known retinal exchangers in cones, we examined expression of NCKX1 in dissociated retinal cells. NCKX1 is known to be specifically expressed in rodsGnat1−/− sample indicating a lack of compensatory changes in the expression of NCKX1. Having established that NCKX2 is expressed in mouse cone outer segments and that its deletion does not affect cone survival, we next investigated the functional role of this exchanger in cones.The presumptive cone-specific Na retinas , as were retinas  and the  retinas . Quantif2+ extrusion from the outer segments of cone photoreceptors would be expected to delay the Ca2+-mediated negative feedback on the cone phototransduction cascade and result in abnormally slow response recovery. However, a recent study found that the deletion of the putative cone-specific Na+/Ca2+, K+ exchanger, NCKX2, did not alter the b-wave recorded in photopic (cone-driven) light conditions14in vivo ERG recordings, particularly at subsaturating flash intensities16ex vivo ERG recordings which allowed us to obtain flash responses from an isolated retina (see Methods for details). This methodology provides excellent signal-to-noise ratio which allows quantitative analysis of dim flash responses16ex vivo recordings allow pharmacological block of the b-wave to unmask the full time course of the photoreceptor-driven a-wave. Thus, we began our analysis of the role of NCKX2 by comparing the ex vivo a-wave photoresponse in Nckx2−/− mice and their littermate controls. To isolate the a-wave from the downstream components of the retinal response, including the b-wave, we perfused the retina with a cocktail of synaptic blockers (see Methods for details). The cone component of the mouse retina response is typically overwhelmed by the much larger rod-driven response . The deletion of Gnat1 prevents the rods from generating photoresponses while preserving normal retinal morphology1921Gnat1−/−) mice (Gnat1−/−Nckx2−/−) mice (2+ via NCKX2 plays a key role in the timely inactivation of the cone phototransduction cascade within the time course of the cone flash response (~1 s). Notably, the deletion of NCKX2 led to a subtle but statistically significant decrease of the maximal response amplitude (Rmax) but did not change the sensitivity of cones to light flashes (I0)  mice  and NCKX/−) mice  confirme/−) mice ; Table 1hes (I0) , Table 1hes (I0) . Howeverhes (I0)  or in rehes (I0) , indicathes (I0) .2+ from the cone outer segments would also be expected to delay sensitivity adjustment during light adaptation. To examine this issue, we exposed control (Gnat1−/−) retinas (Gnat1−/−Nckx2−/−) retinas  results in several-fold larger cone single photon response and dramatically delayed response recovery2+ from cone outer segments, it would be expected that its deletion would largely delay the onset of the Ca2+-mediated feedback on GCAPs so that the physiological phenotype in Nckx2−/− cones would be comparable to that in GCAPs knockout cones. However, we found that the deletion of NCKX2 in mouse cones produces only a slight delay in the response recovery without affecting substantially the amplitude of the single photon response , the background light response amplitude of Nckx2−/− cones reaches steady state comparable to that of control cones  for the maintenance of Ca2+ homeostasis in cones, because complete block of Ca2+ extrusion would be expected to lead to abnormally high and toxic Ca2+ levels23Block of the major Caresponse . Thus, tol cones . TogetheNckx1−/− rods2+ entry when Ca2+ extrusion is disrupted. In contrast, the maximum light response is comparable between control and NCKX2-deficient cones, indicating a normal complement and function of cone CNG channels  in NCKX2-deficient cones.Interestingly, we recently found that disruption of NCKX1, the rod-specific exchanger isoform, leads to down-regulation of the rod CNG channel and greatly diminished maximal light responses in channels . This di2+ still appears to proceed despite the removal of NCKX2. Thus, our results reveal the existence of a novel, yet to be identified NCKX2-independent cone Ca2+ extrusion pathway. One possibility is that another exchanger is also expressed in the outer segments of cones. We show that the rod-specific exchanger NCKX1 is not expressed in mouse cones2+ in the outer segment could move down the concentration gradient into the inner segment through the connecting cilium and then be taken up by intracellular stores, such as mitochondria+/Ca2+ exchanger NCX1 has been reported in the cone inner segments2+ ATPase (PMCA) expressed in cones could also be involved in the regulation their Ca2+ homeostasisBased on these results, the emerging model is that NCKX2 plays an important role in the rapid modulation of the phototransduction cascade at the sub-second time scale of the mouse cone response. Consistent with this concept, the rapid onset of cone light adaptation is delayed in the absence of NCKX2 . HoweverNckx2−/− mice were kindly provided by Jonathan Lytton  which produce no rod photoresponses. Experiments were done with young adult mice (2–3 months-old) from either sex.The use of mice in these experiments was in accordance with the guidelines established by the animal care and use guidelines of our respective universities and all experimental protocols were approved by the Animal Studies Committees of Washington University and the University of Southern California. Li et al.. To faciEyecups were marked at the superior pole by cauterization. Eyes were placed in fixative for 5 min . The lens and cornea were dissected out, and the remaining eyecup further fixed overnight. Fixed tissues were rinsed in 0.1M cacodylate buffer, dehydrated by graded ethanol washes and embedded into epoxy resin. Tissues were sectioned at 1 μm thickness along the vertical meridian and images were acquired on a Zeiss Axioplan2 microscope  using a 63X objective.Nckx2−/− retinas. For the dissociated cells, retinas were chopped in PBS. The cell suspension was placed on gelatin-coated glass slides and centrifuged for 3 min at 500 rpm. The cells were fixed for 5 min in 4% formaldehyde in PBS, washed, and processed for immunofluorescence as described for frozen sections. The primary antibodies used were LUMI-J that binds cone arrestinEyes were enucleated and fixed in 4% formaldehyde in phosphate buffered saline (PBS) for 1 hour. The cornea and lens were then removed and the remaining eyecup was placed back in 4% formaldehyde in PBS for another 2 hours. After this second fixation step the retinal pigmented epithelium was carefully peeled away from the neuroretina. The remaining tissue was further fixed in 4% formaldehyde in PBS for 1 hour. The fixed tissue was then washed 3 times in PBS and blocked with 2% bovine serum albumin, 2% goat serum, 0.3% Triton X-100 in PBS for 1 hour. Neuroretinas were then incubated overnight at 4 °C with the following primary antibodies: rabbit NCKX2 N2F . First the cones were highlighted using the threshold function. This was followed by processing the image into binary form. The cones were then counted using the “analyze particles” function. A minimum of 7 fields from two retinas was used to quantify the number of cones.Confocal images of whole-mount retinas with PNA stained cones were acquired using a 40X objective. Each image was analyzed using the ImageJ freeware . Half of the dorsal retina was transferred to the recording chamber on filter paper with the photoreceptor side up. To remove the electrical components of higher-order neurons, 2 mM L-glutamate was added to the perfusion solution . The electrode solution under the retina also contained 10 mM BaCl2 to suppress the glial component of the response. All solution chemicals were purchased from Sigma-Aldrich, Saint Louis, Missouri, USA). Responses were amplified by a differential amplifier , low-pass filtered at 30 Hz, and digitized at 1 kHz. For light stimulus, 500-nm flash light or white light (>410 nm) was delivered after attenuation with neutral density filters. Sensitivity of flash response was estimated by fitting the following equation:Transretinal recordings from R is response amplitude, Rmax is maximal response amplitude, I is flash intensity, and Io is the flash intensity required to produce half-maximal response. Time to peak was estimated as the time from the onset of the flash to the peak of a response from the linear range (<30% Rmax). From the same responses, integration time was estimated as the integral of the dim flash response normalized to its peak amplitude, and recovery time constant was estimated as the single exponential fit to the late phase (past 50%) of the response recovery. The residual response in background illumination, Rres, was measured from a saturating flash response at the end of the 2 s step light exposure and Rres/Rmax was fit to the Hill equation:where Ist is step light intensity, Ib is the step light intensity to produce half-maximal response amplitude, and k is the Hill coefficient.where Nckx2−/− littermate control mice expressing rod transducin α were isolated by using double flash technique as described previously2 carbogen at 37 °C  supplemented with 40 μM DL-AP4 , 2 mM L-Aspartate and 100 μM BaCl2 to isolate the photoreceptor component of transretinal ERG signal. Data was sampled at 10 kHz and low-pass filtered at 300 Hz (8-pole Bessel filter). We used pre-flash (flash length 1 ms) of 12,000 photons (530 nm) μm−2 to saturate rods and test flashes from 565 to 732,000 photons μm−2 (flash length 1 ms except for the brightest flashes up to 4 ms) with inter-stimulus interval of 200 ms to elicit pure cone responses without contribution from rod photoreceptors that remained saturated at that time.Cone responses in WT and their By selecting the dorsal, M-opsin-rich part of the retina for recordings and by using longer wavelength test flashes (500 nm and 565 nm), we focused our studies on cones expressing M-opsin. Considering that in the mouse retina most cones express M-opsin or co-express M-opsin and S-opsinTwo-tailed unpaired Student’s t-test was used to test for the significance of difference in the mean values of two sample groups. P-values of <0.05 were considered to be statistically significant.How to cite this article: Sakurai, K. et al. The Na+/Ca2+, K+ exchanger 2 modulates mammalian cone phototransduction. Sci. Rep.6, 32521; doi: 10.1038/srep32521 (2016).


Ananas comosus) stem demonstrates several therapeutics properties, including acceleration of wound healing. Tacorin is a water crude extract derived from the stem of A. comosus with high protein content. The effect of tacorin on wound healing in vivo was examined using rats with an induced injury. Wound closure was faster with tacorin treatment than in the untreated group. An in vitro study was conducted on mammalian cells (3T3‐L1) to observe the effect of tacorin on cell proliferation. Tacorin was first heated to inactivate its proteolytic activity. It increased the viability of 3T3‐L1 cells in a dose‐dependent manner. Excessive inflammation was suppressed by tacorin as shown by decreased tumor necrosis factor α expression. Treatment with tacorin increased the expression of transforming growth factor β, a major player in tissue remodeling. Moreover, tacorin also reduced the expression of MMP‐2 to accelerate the recovery of the wound. Taken together, tacorin is able to accelerate the wound‐healing process by increasing cell proliferation, suppressing inflammation and accelerating tissue remodeling.Wound healing is a complex biological process that involves integration of hemostasis, inflammation, proliferation and tissue remodeling. An extract of pineapple ( DLBSDexa Laboratories of Biomolecular SciencesILinterleukinMMPmatrix metalloproteinaseTGFtransforming growth factorTNFtumor necrosis factore cytokines such as tumor necrosis factor (TNF), transforming growth factor (TGF) and interleukin (IL). These cytokines will stimulate cell migration and proliferation and formation of the tissue matrix A complex series of interactions between different cell types, cytokine mediators and the extracellular matrix are required in the wound healing process. The normal phase of wound healing involves hemostasis, inflammation, proliferation and remodeling. Each phase is distinct, continuous and overlapping There are many types of wounds that can damage skin. Deeper wounds require medical attention to prevent infection and loss of function. However, most wounds are superficial and can be cared for at home. This usually requires cleaning, application of antibacterial ointment and covering with an adhesive bandage. The purpose of medical care for wounds is to prevent infection and loss of function. In several cases, cosmetic treatment is used, but it is not the primary consideration for wound care.p‐nitroanilide (pNa) substrate. On the other hand, bromelain preferentially hydrolyzes Bz‐Arg‐Arg‐pNa Crude pineapple stem preparations have previously been shown to contain two cysteine proteases: bromelain and ananain Ananas comosus, a medicinal plant with highest proteolytic activity in other parts such as fruit and fruit core extract. Tacorin has been developed by Dexa Laboratories of Biomolecular Science (DLBS), one of the biggest pharmaceutical companies in Indonesia, which is exploring various natural compounds for medicinal applications in vivo and in vitro studies were conducted. The in vitro study used 3T3‐L1 cells, while the in vivo study was conducted with rats with an induced injury. The expression of several cytokines and growth factors was quantified with enzyme linked immunosorbent assay (ELISA) using specific antibodies.Tacorin is a crude extract from the stem of A. comosus  were collected and ground using a blender . The protein fraction from each plant part was extracted using a customized press machine , followed by separation at 8930 g at 4 °C for 15 min using a Kubota 7780 centrifuge , and filtration through 0.1 μm and 5 kDa membrane with the QuixStand benchstop system . The filtrate was subsequently dried using a Mini‐Lab fluid bed dryer . Tacorin was obtained from the plant part of A. comosus with highest concentration of protein content and protease activity. Protease in tacorin must be inactivated for cell treatment, and inactivation was carried out by heat exposure at various temperatures (60 and 80 °C) and time incubations .Plant parts of Characterization of tacorin was carried out by analyzing the protease activity, protein and amino acid contents. The protein profile of tacorin was also obtained by tricine SDS/PAGE.et al. m potassium phosphate buffer (pH 7.5) was added to a 0.25 mL sample. The mixture was incubated for 10 min at 37 °C. The reaction was stopped by adding 250 μL of 110 mm trichloroacetic acid (Sigma‐Aldrich). A blank was prepared by adding trichloroacetic acid to the crude enzyme, followed by the substrate. After vortexing for 5 s, 2 mL of reaction mixture was placed in an Eppendorf tube and centrifuged at 9200 g for 10 min. A half‐volume of supernatant was added to 1.25 mL Na2CO3 (Sigma‐Aldrich) and 0.25 mL Folin  reagent. The absorbance was measured by UV/Vis spectroscopy at 660 nm. One unit of tacorin is the amount of enzyme that hydrolyzes casein to produce color equivalent to 0.5 nmol of tyrosine per minute at 37 °C and pH 7.5 .The proteolytic activity assay was performed according to Rowan Protein concentration was quantified using the Bradford method The protein profile of tacorin was performed by tricine SDS/PAGE in 10% and 16% gel concentration using ultra low molecular mass markers (1.02–26.6 kDa). Proteins were visualized by Coomassie Brilliant Blue R‐250 The amino acid profile was analyzed using high performance liquid chromatography (HPLC). Tacorin was injected into a Waters AccQ‐Tag amino acid analysis column  and eluted with a mixture of AccQ‐Tag eluent A, acetonitrile and HPLC‐grade water, based on the manufacturer's instructions.Rattus novergicus), weight 170–220 g, were used in this study, seven rats used for the negative control group and the rest for the tacorin group. Rats were caged individually in polysulfone cages and housed under standard conditions . All procedures in this study have been reviewed and approved by Dexa Laboratories of Biomolecular Sciences Animal Care and Use Committee with protocol number DOC‐DLBS‐PROC‐APC‐027 and carried out in accordance with Association for Assessment and Accreditation of Laboratory Animal Care International .A total of 14 female Wistar rats (−1 bw) and xylazine (10 mg·kg−1 bw) intraperitoneally prior to wound induction. The abdomen was opened by making a vertical incision. A 2.5 cm midline abdominal excision was made from the midpoint of the abdomen to the anterior of the urethra, and then surgery was performed for 2 cm on each side right and left of the uterine area. The wound was then closed by the one‐layer closure technique with continuous lock stiches of 4.0 chromic catgut sutures.After 1 week of acclimatization, the rats were anesthetized with ketamine (75 mg·kg−1 bw) and a treated group (tacorin 80 mg·kg−1 bw – human dose). Purified water and tacorin were daily administered by the oral route. Then a 1.7% (v/w) blood sample of each rat was taken daily up to 5 days (D0–D5). Blood samples were further analyzed for the expression of TNF‐α and TGF‐β. Tissue was collected from the left and right of the rats' uterine area at days 0, 3, 5 and 7 and were used for analysis of expression of matrix metalloproteinase 2 (MMP‐2). Table Wounded rats were divided into an untreated group  as a comparator. Cells were treated at various concentrations of both proteins for 24 h. Cell viability was quantified with a 3‐‐2,5‐diphenyltetrazolium bromide tetrazolium  assay.The 3T3‐L1 cell line  was maintained in DMEM supplemented with 10% BSA and 1% penicillin/streptomycin . Cells were seeded at a density of 4 × 10−1 in phosphate‐buffered saline; PBS) was immobilized in a 96‐well microtiter plate  overnight at 4 °C. After incubation, the plates were washed three times in washing buffer . One hundred microliters of blocking solution [5% skim milk (Sigma‐Aldrich) in PBS] was added to each well. Afterwards, 100 μL of primary antibody  in blocking solution was added, followed by incubation for 2 h at room temperature on a shaker. Plates were washed as before. One hundred microliters of secondary antibody‐tagged horseradish peroxidase  in blocking solution was added to each well. The plates were incubated for 1 h at room temperature. Finally, the plates were washed four times and 50 μL of tetramethylbenzidine (Sigma‐Aldrich) was added. The color intensity of the solution was read in a microtiter reader at 650 nm.The expression levels of TNF‐α, IL‐6 and TGF‐β were measured with ELISA using specific antibody . One hundred microliters of diluted antigen  for 30 min. The suspension was frozen and thawed twice in a deep freezer, and then centrifuged at 2500 g for 30 min. The supernatant was collected and dialyzed on ice‐cold dialysis buffer  for 48 h. The extracted protein was then diluted in 10 μL of zymogram buffer  and analyzed by electrophoresis (SDS/PAGE) using 10% acrylamide. After electrophoresis, the electrophoretic gel was incubated in 2.5% (v/v) Tween 20 for 30 min, in 50 mm potassium phosphate buffer (pH 7.0) for 3 h, and then stained with Coommassie Brilliant Blue R‐250 for 30 min and destained using destaining solution .The expression of MMP‐2 was detected using zymogram analysis. Approximately 25 mg of uterine tissue was extracted with 1 mL of isolation buffer  are a cell‐derived proteolytic enzyme family with 26 identified members Tacorin is a water extract containing proteolytic activity even toward 3T3 cell surface protein that contributes to cell adhesion. Protease‐treated cells had reduced contact with neighboring cells and the plate surface caused by digestion of the cell surface proteins that are involved in cell adhesion. The proteolytic digestion of membrane proteins by protease also showed complete inhibition of fibroblast adhesion on fibronectin‐coated plastic dishes. Therefore, the proteolytic enzyme in tacorin must be inactivated prior to use to avoid false‐negative result. This inactivation was conducted through a physical (heat) method. The proteolytic activity assay of tacorin was conducted using casein Bromelia balansae Mez had no activity when exposed at 37 °C for 120 min. Complete activity loss of B. balansae Mez is observed when incubated at 75 °C. The activity of bromelain from A. comosus was decreased by 20% when incubated at 50 °C and 100% when incubation was continued at 80 °C for 8 min.The influence of temperature and incubation time on bromelain activity was reported in several studies We examined the effect of temperature on tacorin proteolytic activity. Bromelain was also studied as a control. Upon exposure at 60 °C for 30 min, the activity of tacorin was reduced by 60% Fig. . For lonSeveral parameters relevant to the wound healing process are cell regeneration, including proliferation, cell growth and maturation. To observe the effect of tacorin on the proliferation phase, we measured the viability of 3T3 cells in the presence and absence of tacorin and control protein. Tacorin treatment increased cell viability in a dose‐dependent manner Fig. . At the A. comosus stem. The bioactive protein fraction in tacorin contains glycine, proline, glutamine and alanine, which are amino acids that are important in the wound healing process. The effects of protein and amino acid in the wound healing process are through remodeling the expression of cytokines and growth factors. In addition, those proteins and amino acids are involved in inflammation and tissue regeneration as well. Thus, tacorin is a promising wound healing therapeutic agent.Tacorin is a crude protein extracted from in vivo study and carried out data analysis. LA contributed to writing the paper, planned and performed the in vitro study and analyzed data. RRT participated in supervising the whole experiment, and reviewed the entire data and the manuscript.PR designed the experiments, prepared the paper, provided and characterized tacorin, performed the The authors are responsible for the content and writing of the paper and declare no conflict of interest in this work. The corresponding author is fully responsible for the submission of this publication.