Synapses differed in the voltage dependence of release: Those residing at the IHC' pillar side activated at more hyperpolarized potentials and typically showed tight control of release by few Ca2+ channels. We conclude that heterogeneity of voltage dependence and release site coupling of Ca2+ channels among the synapses varies synaptic transfer within individual IHCs and, thereby, likely contributes to the functional diversity of SGNs. The mechanism reported here might serve sensory cells and neurons more generally to diversify signaling even in close‐by synapses.The cochlea encodes sound pressures varying over six orders of magnitude by collective operation of functionally diverse spiral ganglion neurons (SGNs). The mechanisms enabling this functional diversity remain elusive. Here, we asked whether the sound intensity information, contained in the receptor potential of the presynaptic inner hair cell (IHC), is fractionated via heterogeneous synapses. We studied the transfer function of individual IHC synapses by combining patch‐clamp recordings with dual‐color Rhod‐FF and iGluSnFR imaging of presynaptic Ca 2+ channel‐mediated glutamate release.Analysis of individual synapses reveal that the ability of auditory inner hair cells to translate a wide range of sound intensities is linked to presynaptic heterogeneity in Ca Diversity is implemented at several levels, i.e., circuits, neurons, and subcellular functional units such as synapses. Sensory systems employ such multiscale diversity as well as adaptation to deal with the challenge of encoding a wide range of stimulus intensities   in mature mouse IHCs relates near‐linearly to Ca2+ influx when varying the number of open Ca2+ channels , we patch‐clamped IHCs and simultaneously imaged postsynaptic iGluSnFR fluorescence by spinning disk confocal microscopy . We observed a mild rundown of the ΔF‐iGluSnFR over repetitive stimulation: The mean peak ΔF amplitude of the last three points was 36.75 ± 7.03% smaller than the mean of the first three points (14 step depolarizations over 5 min).Next, we probed the effect of the imaging plane on the iGluSnFR signal. We applied 50‐ms‐long step depolarizations in seven different planes each separated from the next one by 0.5 µm  ; Fig P < 0.0001), while ΔCm were detectable only at 5 ms  and in the whole recorded voltage range . These findings corroborated our observation in Fig As a complementary approach to validate ΔF‐iGluSnFR as a readout of exocytosis, we probed it by applying brief (10‐ms) step depolarizations from the holding potential (−87 mV) to −57 mV in 10 mV increments up to 23 mV  per SV. The sustained component of exocytosis amounted to 42.7 a.u./s (~ 185 SV/s per AZ) for the recorded synapses compared to 242 fF/s , we applied 10‐ms‐long step depolarizations from the holding potential (−87 mV) to −62 mV in 5 mV increments up to −22 mV  for glutamate release on average was −47.11 mV with considerable variance     close to 1) when varying the number of open Ca2+ channels. Here, we found a near‐linear dependence  of glutamate release at single AZs on the whole‐cell Ca2+ influx when depolarizing IHCs within the range of physiological receptor potentials . Varying the voltage in this hyperpolarized range changes the open‐channel number, and for Ca2+ nanodomain control, this is more relevant than the change in single‐channel current as once the channel opens the ensuing Ca2+ signal tends to saturate the Ca2+ sensor of release . Taken together, imaging of glutamate release as a function of whole‐cell Ca2+ influx corroborates the notion of a Ca2+ nanodomain‐like control of release in mature IHCs . We combined ruptured patch‐clamp of IHCs with sequential dual‐color imaging of first synaptic Ca2+ signals and then glutamate release  chemical Ca2+‐indicator Rhod‐FF. We isolated Ca2+ signals at individual AZs using strong cytosolic buffering (10 mM EGTA in the patch pipette) and increased [Ca2+]e (5 mM) . The hot spots of Rhod‐FF fluorescence elicited by depolarizations localized to the plasma membrane , while applying 50‐ms‐long step depolarizations (ranging from −57 to −17 mV) in a pseudo‐randomized fashion  of Ca2+‐influx . In conclusion, the sequential dual‐color imaging approach allowed us to study Ca2+ dependence of release at individual AZs.Next, we studied the voltage dependence of synaptic Caence Fig  while aprane Fig  and to trane Fig , indicathion Fig . We usedflux Fig  and glutflux Fig  were −42tion Fig . We rest2+ influx (QCa), synaptic Ca2+ influx (∆F/F0 of Rhod‐FF), and glutamate release (iGluSnFR‐AUC), we observed fundamental heterogeneity of AZs within and across IHCs. The threshold for glutamate release was −48.27 ± 6.47 mV  and showed a broader distribution than that of the whole‐cell Ca2+ influx . As previously reported  as well as between synaptic and whole‐cell Ca2+ influx . The V1/2 values of synaptic Ca2+ influx and glutamate release correlated . The observed discrepancy of the average m estimates from single‐synapse analysis and the m estimated based on the whole‐cell QCa might at least partially be explained by the exclusion of synapses at the densely innervated basal IHC cap. We suspect that there are a fair number of synapses with m < 2 in the basal cap. The m estimate based on the whole‐cell QCa was lower on average than the m estimate obtained in the perforated‐patch configuration on P15–19 IHCs at 1.3 mM [Ca2+]e using a similar stimulus protocol . This difference is compatible with the hypothesis that strong Ca2+ buffering by 10 mM EGTA favors the operation of release sites under Ca2+ nanodomain control. Since the data of Fig 2+ channel–exocytosis coupling  and with high EGTA buffering , supporting the lack of obvious glutamate release quench in these high buffering conditions. Nevertheless, probing the synaptic Ca2+ dependence of release in more physiological buffering conditions remains important task for future studies. When operationally defining m < 2 (“near‐linear”) as indicative of Ca2+ nanodomain‐like control of release, we found approximately half of the synapses (29 out of 55 synapses) to operate in this scenario. The other half showed a broad spread of m values reaching up to 8, compatible with Ca2+ microdomain‐like control of release despite the presence of 10 mM EGTA. Taken together, single‐synapse imaging of Ca2+ influx and glutamate release revealed a heterogeneity of the apparent Ca2+ dependence of release that is likely due to differences in the coupling of Ca2+ channels to release sites among the IHC AZs. This heterogeneous coupling of Ca2+ channels to release sites likely contributes to the heterogeneous output of IHC AZs.Next, we estimated the apparent Caflux Fig  and to tflux Fig  for the flux Fig , and EV3evel Fig  was high2+ influx and glutamate release as a function of position along the pillar–modiolar axis. Figure 1/2 and a wider dynamic range compared to the modiolar synapses in the given cell  that we previously reported for an earlier postnatal stage (P14–20) , V1/2 , and dynamic range  of release. Ca2+ cooperativity did not show a significant correlation with the position along the pillar–modiolar axis  on the 11th‐dimensional space of single‐synapse properties  and obtained three synapse clusters . To visualize the clusters in two or three dimensions, we performed principal component analysis (PCA) and used the first three principal components that explained 79% of the variance  indicating a Ca2+ nanodomain‐like control of release as well as a wide dynamic range (27.52 ± 9.37 mV) and a hyperpolarized activation threshold (−53 ± 5.07 mV) of release. In contrast, the third synapse subtype showed a high m  with smaller dynamic range (15.53 ± 3.95 mV) and a more depolarized threshold  during primary manipulation of the open‐channel number. Hence, different from what was proposed based on an AZ summation model  developmental competition of synapses, (ii) transsynaptic signaling from SGNs, and (iii) planar polarity signaling. One interesting idea is that, during development, pioneer SGN axons  glutamate spill‐over from neighboring synapses, (ii) saturation of iGluSnFR and other non‐linear effects resulting from the glutamate binding to endogenous receptors in the postsynapse, and (iii) contamination of synaptic Cabbon Fig .2+ influx . Subsequently the wound was sutured. The whole procedure was performed under general isoflurane anesthesia. For analgesia, buprenorphine (0.1 mg/kg) was injected subcutaneously prior the surgery, additional local analgesia (xylocaine) was applied to the skin and post‐surgery pain was covered by metamizol (1.33 mg/kg) provided in the drinking water for 5 days. The recovery of the animals was monitored on a daily basis. All animals were kept in a 12‐h light/dark cycle, with access to food and water ad libitum and together with the mother until the end of the weaning period (∼ P21). Injected WT mice were used for experiments either 1 week (P15–19) or 2 weeks (P21–26) after the injection.All experiments were done in compliance with national animal care guidelines and were approved by the University of Göttingen Board for Animal Welfare and the Animal Welfare Office of the State of Lower Saxony (permit number: 17‐2394). Postnatal AAV injections were made into scala tympani of the right ear through the round window  or clicks of 0.03 ms were presented at 40 Hz (tone bursts) or 20 Hz (clicks) in the free field ipsilaterally using a JBL 2402 speaker.Recordings of auditory brainstem responses (ABR) were performed on P29 mice as previously described . For immunostaining of the whole‐mount organs of Corti, the apical turns of organs of Corti were dissected out and washed three times with PBS. For immunostaining of the cryosections, the cochlea was decalcified in 0.5 M EDTA overnight. After a PBS washing step, the cochleae were incubated in 25% sucrose in PBS at 4°C. The cochlea was frozen in Tissue‐Tek and cryosectioned with section thickness of 16 µm.The samples  were blocked with a goat serum dilution buffer  for 1 h at room temperature in a wet chamber. The blocking was followed by an overnight incubation with the primary antibodies at 4°C. After 3 × 5 min PBS washing steps, the samples were incubated with the secondary antibodies for 1 h at room temperature. Following the final 4 × 5 min PBS washing steps, the samples were mounted in mounting medium . The primary antibodies used were the following: mouse anti‐CtBP2 . The IHC basolateral membranes were exposed by cleaning of nearby cells with a suction pipette by approaching from either pillar or modiolar side. All experiments were conducted at room temperature (20–25°C). Patch pipettes were made from GB150‐8P or GB150F‐8P borosilicate glass capillaries  for perforated and ruptured patch‐clamp recordings, respectively. To decrease the capacitive noise, pipettes were coated with Sylgard and their tips were polished with a custom‐made microforge. All patch‐clamp recordings were done simultaneously with fluorescent imaging of iGluSnFR or of iGluSnFR and Ca2+.The apical 2/3 turn of organs of Corti was acutely dissected from P15 to P26 animals in HEPES Hanks' solution containing (in mM): 5.36 KCl, 141.7 NaCl, 10 HEPES, 0.5 MgSO2+ current and membrane capacitance (Cm) measurements, the extracellular solution contained the following (in mM): 110 NaCl, 35 TEA‐Cl, 2.8 KCl, 1 MgCl2, 1 CsCl, 10 HEPES, 1.3 CaCl2, and 11.1 d‐glucose  and was introduced into the recording chamber via a perfusion system. The pipette solution contained (in mM): 130 Cs‐gluconate, 10 TEA‐Cl, 10 4‐AP, 10 HEPES, 1 MgCl2, as well as 300 mg/ml amphotericin B . The intracellular solution also contained the TAMRA‐conjugated dimeric CtBP2/RIBEYE‐binding dimer peptide . The patch pipette solution contained (in mM): 111 l‐glutamate, 1 MgCl2, 1 CaCl2, 10 EGTA, 13 TEA‐Cl, 20 HEPES, 4 Mg‐ATP, 0.3 Na‐GTP and 1 l‐Glutathione . For fluorescent imaging, 800 μM of the low‐affinity chemical Ca2+ indicator Rhod‐FF tripotassium salt  was applied to evoke Ca2+ influx : 2.8 KCl, 102 NaCl, 10 HEPES, 1 CsCl2+ current rundown exceeded 25%.For all IV recordings, the IHCs were step depolarized for 20 ms from −87 to +63 mV in 5 mV increments. The IV recordings were used to assess the fitness of the cell, and recordings were discarded when the CaImaging experiments were performed with a spinning disk confocal scanner  mounted on an upright microscope  with 63×, 1.0 NA objective . The spinning disk speed was set to 2,000 rpm to avoid uneven illumination. A scientific CMOS camera  with a pixel size of 103 nm was used to acquire images. iGluSnFR and Rhod‐FF or TAMRA peptide were excited by diode‐pumped solid‐state lasers with 491 nm and 561 nm wavelength, respectively (Cobolt AB).To avoid photobleaching, iGluSnFR‐expressing SGN boutons were detected via low intensity 491 nm excitation. The imaging plane for the target IHC was selected when several transduced boutons were visible, in the mid‐basal section of the cell, avoiding the high synapse density at the basal pole. A brief step depolarization was applied to the cell to check for the functional signal in the given plane. iGluSnFR fluorescence was acquired at 50 Hz simultaneously with patch‐clamp recordings. The iGluSnFR signal was evoked by step depolarizations of different durations to different voltage values, as it is specified in every dataset.2+ and glutamate release, as described above for iGluSnFR imaging, the imaging plane was selected based on the baseline fluorescence of iGluSnFR. Once the middle plane was set, the fluorescence of Rhod‐FF was imaged at 100 Hz while Ca2+ currents were triggered by applying five voltage ramps  in five alternating planes separated by 0.5 μm. To precisely control the Z‐plane, a piezo positioner for the objective  was used. After the Ca2+ imaging, the iGluSnFR signal was acquired at 50 Hz by applying 50‐ms‐long step depolarizations from the holding potential of −87 to different voltage values. Depolarizations  were applied in a pseudo‐randomized manner and covered the dynamic range of IHC glutamate release.For the sequential dual‐color imaging of Ca2+ charge (QCa) was calculated by the time integral of the leak‐subtracted current during the depolarization step. ∆Cm was calculated as the difference between the average Cm 400 ms before and after the depolarization, skipping the initial 100 ms after the depolarization.Electrophysiological recordings were analyzed using custom‐written programs in Igor Pro 6.3. Whole‐cell CaImage and further data analysis and visualization were done in Python (Python software foundation) with custom written code using the following Python libraries: NumPy, Pandas, Matplotlib, Skimage, SciPy, Sklearn, statmodels, and Seaborn.0, an average of 15 frames before stimulus) from the fluorescence images acquired during/after stimulation . The ∆F image was median‐filtered with a two‐dimensional pixel array size of 4–6 depending on the signal amplitude. To create a mask for ROI detection, maximum entropy thresholding was applied to the median‐filtered ∆F image. To label and separate individual ROIs, a watershed segmentation algorithm was used. A single mask was generated per cell, using the recording with strongest stimulation, and applied for all images. Individual ROIs, corresponding to postsynaptic SGN boutons, were further confirmed by the nearby presence of presynaptic ribbon peptide (TAMRA‐conjugated dimeric CtBP2‐binding peptide). The fluorescence of every pixel in the defined ROI was averaged over time for further analysis. The background fluorescence was calculated by averaging 60 × 60 pixels in the pillar region of the image, where no iGluSnFR fluorescence is expected: By their anatomy, SGNs innervate IHCs and leave the cochlea toward the modiolus.The ∆F image was created by subtracting baseline fluorescence (F0). ∆F was normalized to F0 to create ∆F/F0 traces. For peak detection, ∆F/F0 traces were smoothened using a Hanning window function with a window size of 7. To correct for photobleaching, we fitted a single exponential to ∆F/F0 traces. The area under the curve (AUC) was estimated by calculating the area between ∆F/F0 and the fit in an interval of 40 frames from the beginning of the stimulus.The average background value was subtracted from the raw fluorescence traces (F). Following background subtraction, ∆F traces were generated by subtraction of mean baseline fluorescence , the following function was fitted to the 30 points of photobleaching corrected ∆F/F0 traces after stimulation.To obtain decay constant . A mean mask was generated per cell using all the recordings. ROIs were confirmed by the presence of a corresponding Ca2+ “hot spot”.The ROIs were picked as described above. Differently, a Gaussian filter with sigma of 1–3 was applied consistent with the detection of Ca2+ “hot spot”.Similarly, a ∆F image was created from the mean time series, in this case, by averaging all the trials from five recorded planes. This ∆F image was treated the same way as described for iGluSnFR‐ROI detection. The created mask was applied to all recording planes and the plane with the maximum ∆F for a given ROI was used for further analysis. This way we used the plane with the highest signal for each Ca0 as described above. Fluorescence–voltage (FV) relations for iGluSnFR were estimated from the step depolarizations to different voltage values. iGluSnFR‐AUC for each depolarization was calculated as described above.To remove the noise caused by the spinning disk speed at 2,000 rpm (33.3 Hz), obvious in the Fourier amplitude spectrum, the raw traces were filtered with a 33.3 Hz band‐stop filter. The obtained traces were background‐subtracted and normalized to F1/2) and slope factor (k) of glutamate release.A Boltzmann fit was used to estimate the two fitting parameters: voltage of half‐maximum activation  was obtained with this equation. The resulting fit was used to estimate V1/2 by minimizing the scalar at the mid‐point. The reversal potential (Vr) was fixed to +47.6 mV after LJ potential (17 mV) correction. In addition, this fit was used to calculate the fractional activation curve, dividing it by the extrapolated linear fit to the decay of fluorescence. To estimate fractional V1/2,Pactivation and k1/2,Pactivation  were comparable to the ones from the raw fluorescence traces.The peak of the Rhod‐FF signal was obtained by averaging three frames corresponding to the voltage values between −17 and 3 mV during ramp depolarization. Dynamic ranges were calculated from the fits as the voltage range of 10–90% of the maximal activation. Threshold was calculated as the voltage value where there is 10% of the maximal activation. Note that VCa and iGluSnFR‐AUC were plotted against each other in the voltage range of −57 to −17 mV. To obtain single‐synapse glutamate release–Ca2+ signal/whole‐cell Ca2+ charge relationship, a power function was fitted:FV fits for Rhod‐FF or Q2+ "hot spot" to the normalized major axis. A number was assigned to a given synapse on the normalized scale from 0 (pillar side) to 1 (modiolar side).To estimate the cell boundary, an ellipse was fitted to the baseline fluorescence of Rhod‐FF. We defined the pillar–modiolar axis of the cell as the major axis of the ellipse. We calculated the shortest distance from the center of a given CaK‐means clustering algorithm (K = 3) was applied to the whole dataset of 11 synaptic properties for three clusters  or Wilcoxon's signed rank test . Comparison of dispersion was performed by Levene's test. We used one‐way ANOVA for multiple comparisons followed by post hoc Tukey's test. The Pearson correlation coefficient was used to test for linear correlation. Significant differences are reported as *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.All the statistical tests were performed in Python (Python Software Foundation). Averages are expressed as mean ± SD or as mean ± SEM (specified in the figure legends), and box plots indicate 25–75 quartile with whiskers reaching from 10 to 90%. Datasets were checked for normal distribution by D'Agostino & Pearson omnibus normality test and for equality of variances. For normally distributed data, unpaired two‐tailed student's TM and ÖDÖ designed the study. ÖDÖ performed the experiments and the analysis. TM and ÖDÖ prepared the manuscript.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Review Process FileClick here for additional data file.


Background and Methods: Acute myeloid leukemia (AML), which starts in the bone marrow, is a group of hematopoietic stem cell disorders. Chloride intracellular channel 4 (CLIC4) is regulated by p53, c-Myc, and TGF-β. It induces the NF-κB-dependent activation of HIF (hypoxia-inducible factor) and participates in tumor growth through its microenvironmental function. However, its prognostic value in AML remains unclear, as well as its co-expression biomarkers. In this study, we evaluated the prognostic significance of CLIC4 expression using two independent large cohorts of cytogenetically normal AML (CN-AML) patients. Multivariable analysis and multi-omics analysis with weighted correlation network analysis (WGCNA) in the CN-AML group were also presented. Based on CLIC4 and its related genes, microRNA–target gene interaction network analysis and downstream gene ontology analysis were performed to unveil the complex functions behind CLIC4.Results: We demonstrated that the overexpression of CLIC4 was notably associated with unfavorable outcome in the two independent cohorts of CN-AML patients , as well as in the European LeukemiaNet (ELN) Intermediate-I group , the National Comprehensive Cancer Network Intermediate Risk AML group , and the non-M3 AML group . Multivariable analysis further validated CLIC4 as a high-risk factor in the CN-AML group. Multi-omics analysis presented the overexpression of CLIC4 as associated with the co-expression of the different gene sets in leukemia, up/downregulation of the immune-related pathways, dysregulation of microRNAs, and hypermethylation around the CpG islands, in open sea regions, and in different gene structural fragments including TSS1500, gene body, 5′UTR region, 3′UTR region, and the first exon. By further performing WGCNA on multi-omics data, certain biomarkers that are co-expressed with CLIC4 were also unveiled.Conclusion: We demonstrated that CLIC4 is a novel, potential unfavorable prognosticator and therapeutic target for CN-AML. As having a key role in CN-AML, the interactions between CLIC4 and other genomics and transcriptomics data were confirmed by performing microRNA–target gene interaction network analysis and gene ontology enrichment analysis. The experimental result provides evidence for the clinical strategy selection of CN-AML patients. NPM1, ASXL1, TP53, RUNX1, FLT3-ITD, double mutation of CEBPA, etc. (Acute myeloid leukemia (AML) is a genetically and clinically heterogeneous hematopoietic malignancy. The outcome of AML patients is generally poor and is strongly influenced by gene mutations and chromosomal alterations . CurrentOver the past decade, the biological and physiological role for ion channels in tumor development has been widely studied. Ion channels are integral membrane proteins preserving the normal physiology of cells and regulating many cellular, organellar, and physiological processes. Evidence reveals the multitask possibility of the participation of ion channel proteins in the tumorigenesis and leukemogenesis process . The abnCLIC4 is involved in p53- and c-myc-mediated apoptosis and transforming growth factor beta (TGF-β) signaling modulation in multiple cell types  and participated in tumor growth through its microenvironmental function , a novel class of intracellular organelle ion channels, are now being widely surveyed to explore their role in tumor biology. When overexpressed heterologously, the CLIC protein family is able to generate an outwardly rectifying chloride channel activity . Among tll types . CLIC4 hpoptosis . Studiesfunction , 12. Subarcinoma , 14. Spe pathway . HoweverCLIC4 as a novel, potential unfavorable prognosticator and therapeutic target for CN-AML. We also explored CLIC4-associated genomic and epigenomic patterns to further elucidate its key role in CN-AML. Our study represented direct evidences for CLIC4 expression as a prognostic biomarker in multiple CN-AML datasets. We also brought insights into CN-AML risk stratification and potential therapeutic targets in leukemia prognosis and treatment.Here, we presented This study was approved by local institutional review boards and written informed consent was provided for all patients according to the Declaration of Helsinki.The first cohort was derived from the whole AML cohort (GSE6891) with 185 primarily untreated CN-AML patients  diagnosed and collected at Erasmus University Medical Center from 1990 to 2008. Conventional cytogenetics examined more than 20 metaphases from bone marrow (BM) samples to determine normal karyotype.The second independent cohort was derived from the multicenter AMLCG-1999 trial of the German AML Cooperative Group (GSE12417) with 163 primarily untreated CN-AML patients . This cohort was used to validate the results of the first cohort.1. Overall survival (OS) and event-free survival (EFS) data were obtained from the GEO database and their publications.All the clinical, cytogenetic, molecular, and microarray data of these CN-AML patients were obtained from the Gene Expression Omnibus (GEO) databaseCLIC4 represented a normal distribution. A significant distinction was shown along the median value  as well as Student’s t-test were used to validate differences in the mRNA, microRNA, and DNA methylation profiles between the CLIC4high and CLIC4low groups , relapse, or death. Between-group comparisons of OS and EFS were performed by the Kaplan–Meier and log-rank tests. The association of w groups . All ana2 , one of the popular gene co-expression network identification tools , was adob portal . The tarCILC4 expression in the BM, peripheral blood (PB), and CD34+ cell samples from AML patients and healthy donors. The results showed that CLIC4 was significantly overexpressed in AML BM than in normal BM  (P = 0.0005)  (Two public GEO microarray datasets (GSE9476 and GSE30029) were used to compare = 0.045) , which w 0.0005) . CD34+ c = 0.04) . All resCLIC4high group tends to have fewer NPM1 mutations (P = 0.0209) and more FLT3-ITD patients (P < 0.0001), while no significant differences between the CLIC4low and CLIC4high groups were observed in the CEBPA and N-ras/K-ras mutation groups . Furthermore, the CLIC4high group was more likely to overexpress BAALC, MAPKBP1, RUNX1, and TCF4, which were proven to contribute to adverse outcomes in CN-AML. Interestingly, patients in the CLIC4high group were more likely to relapse than those in the CLIC4low group    and 1.36 higher risk of OS (P < 0.01). Other adverse factors included the presence of FLT3-ITD (P < 0.01 for both EFS and OS), the presence of FLT3-TKD (P < 0.01 for OS), and mutated N-ras/K-ras (P < 0.05 for EFS). For the cohort of 115 ELN Intermediate-I patients, the CLIC4high group had 1.23 times higher risk of EFS (P < 0.05) and 1.31 higher risk of OS (P < 0.01). Other adverse factors included the presence of FLT3-TKD (P < 0.01 for OS) and mutated IDH1/IDH2 (P < 0.05 for OS). These results indicated that the overexpression of CLIC4 was an independent risk factor for both OS and EFS in CN-AML patients and in the ELN Intermediate-I category.In order to further validate the prognostic role of  0.0001)  as well = 0.012) . To furt cohorts . For theCLIC4high group both in the NCCN Intermediate Risk category   (de novo CN-AML patients (GSE12417) was further studied, which showed that CLIC4high was significantly associated with shorter OS    and in n 0.0001) . Another 0.0032) .CLIC4 in leukemogenesis, we derived the CLIC4-associated gene expression profiles from high-throughput sequencing in the 73 CN-AML patients from the TCGA dataset. A total of 1,185 upregulated 538 downregulated genes were found to be significantly associated with the CLIC4high group (FDR-adjusted P < 0.01) (P = 6.461e-32) (+ cells isolated from the BM of patients with chronic myeloid leukemia (CML) compared to those from normal donors  (P = 3.249e-11) (P = 1.406e-11) (P = 4.282e-5). Five hundred and thirty-eight downregulated genes were analyzed as previously described. A total of 23 genes were among the top 100 probe sets for pediatric AML subtypes with chimeric MLL fusions  (P = 1.563e-40) and myeloid leukocyte-mediated immunity .To gain a better understanding of the biological value of  < 0.01) . The 1,1461e-32)  22). An. AnCLIC4400e-25)  23). Be. BeCLIC4249e-11)  (24). In406e-11)  16). Th. ThCLIC4033e-19) . The topCLIC4, analyzed from the MSigDB data, further provided new insights into the leukemogenesis of CN-AML. A total of five upregulated and 17 downregulated pathways showed significant associations with the CLIC4high group | > 1]  and in open sea regions    were further derived to investigate the different methylation patterns between the C)| > 1] . Secondlheatmaps .via the TSUNAMI web portal. We ended up with 13,082 mRNA genes, 681 microRNA genes, and 15,843 methylation profiles. We considered them as the combined multi-omics biomarkers  and performed biomarker-wise normalization  before performing the WGCNA.As the genomics and transcriptomics data remain noises, it is necessary to filter out some genes before the multi-omics co-expression analysis by WGCNA. We filtered out mRNA genes with the lowest 20% means and variances of expression values, filtered out microRNA genes with the lowest 20% means and variances of expression values, and filtered out methylation profiles with the lowest 80% means and variances of expression values CLIC4 is located at the module with a dark turquoise color which contained 25 mRNA genes, five microRNAs , and 238 methylation profiles. The complete co-expression module list is provided in a By selecting the soft threshold (power) = 6 , minimumCLIC4). We found that those genes were enriched in the chromosome location 19p13.3 , in agreement with several existing literatures  and more FLT3-ITD patients (P < 0.0001) and remarkably high expressions of BAALC (P = 0.0153), MAPKBP1 (P = 0.0153), RUNX1 (P = 0.0026), and TCF4 (P < 0.0001), which were proven to contribute to unfavorable outcomes in CN-AML. The CLIC4high group also had more relapse patients than the CLIC4low group . Other synchronized changes of microRNA–target gene (such as GPR132 targeted by miR-146b and PYCARD targeted by miR-125a) might promote leukemogenesis in CN-AML  and The Cancer Genome Atlas.SH, ZH, and PC designed and performed the study and wrote the manuscript. ZH and CF analyzed the data and revised the manuscript. All authors reviewed the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Accordingly, epicardial mapping revealed a markedly enhanced homogeneity in ventricular conduction. NO2-OA treatment of isolated cardiomyocytes lowered the number of spontaneous contractions upon adrenergic isoproterenol stimulation and nearly abolished ryanodine receptor type 2 (RyR2)-dependent sarcoplasmic Ca2+ leak. NO2-OA also significantly reduced RyR2-phosphorylation by inhibition of increased CaMKII activity. Thus, NO2-OA might be a novel pharmacological option for the prevention of VT development.Nitro-fatty acids are electrophilic anti-inflammatory mediators which are generated during myocardial ischemic injury. Whether these species exert anti-arrhythmic effects in the acute phase of myocardial ischemia has not been investigated so far. Herein, we demonstrate that pretreatment of mice with 9- and 10-nitro-octadec-9-enoic acid (nitro-oleic acid, NO However, patients suffering from AMI with ventricular tachycardia (VT) or -fibrillation (VF), still have a fourfold increased risk of in-hospital mortality2.Due to significant advances in the therapy of acute myocardial ischemia (AMI), e.g. more aggressive approaches to coronary revascularization and secondary prevention, AMI mortality has declined drastically over the last decades2+ signaling within cardiomyocytes of the ischemic border zone, thus leading to ventricular conduction blocks and enhanced VT vulnerability4. In particular, oxidation and subsequent autophosphorylation of Ca2+/calmodulin dependent kinase II (CaMKII) cause malfunctioning of ryanodine receptor type 2 (RyR2) channels and dysregulation of phospholamban (PLN)-dependent cytosolic Ca2+ clearance leading to an intracellular Ca2+ leak from the sarcoplasmic reticulum (SR)6. Consequently, depolarization of the membrane potential is enhanced thereby causing inactivation of Na+ channels with further slowing of ventricular conduction7.The mechanisms accounting for the development of acute VT are diverse: in the acute ischemic phase, 15–30 min after AMI, the development of so called 1B arrhythmias has been linked to increased catecholamine release and disturbances in CaREDUCE-IT which demonstrates that prophylactic treatment with icosapentaenoic acid-ethyl (VASCEPA) significantly reduces cardiovascular events, the rate of cardiac arrests, and the number of sudden cardiac deaths (SCD)8.As the identification of AMI patients at risk for VT remains difficult, a therapeutic strategy would have to be applied to a broad patient population. Therefore, dietary intervention with endogenously produced anti-arrhythmic modulators appears as an attractive therapeutic approach. This concept has been recently supported by the randomized double blind trial 2-OA) is an endogenously generated unsaturated fatty acid nitroalkene derivative that is formed by oleic acid reaction with nitric oxide and nitrite-derived nitrogen dioxide (•NO2)10. NO2-OA is conferred with an electrophilic β-carbon that rapidly and reversibly reacts with nucleophilic cysteine-, and to a lesser extent histidine, residues via a Michael addition11. The covalent nitro adduct is essential for the metabolic effects of NO2-OA since native oleic acid is not electrophilic and ineffective in various studies14. Nitro-fatty acids have been identified as potent endogenous anti-inflammatory mediators that affect a number of signaling mediators, transcriptional regulatory proteins and ion channels, including nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NF-κB), kelch-like ECH-associated protein 1 (Keap1/Nrf2), mitogen-activated protein kinases (MAPK), peroxisome proliferator-activated receptor gamma (PPARγ) and transient receptor potential channels (TRP) channels17. Preclinical studies have demonstrated therapeutic benefit in a variety of disease models such as pulmonary hypertension, nephritis and, of relevance to herein, reperfusion damage after myocardial ischemia (I/R) and a subsequent attenuation of cardiac fibrosis, reduced infarct size and preserved left ventricular function20. Since NO2-OA has passed safety clinical phase I trials21 these results have been translated into two, currently recruiting, clinical phase II trials targeting pulmonary hypertension and primary focal segmental glomerular sclerosis23.Nitro-oleic acid  was drastically reduced in animals pretreated with NOent Fig. D indicat2-OA pretreated- compared to vehicle treated animals  is shown in Fig. 2-OA on action potential (AP) development, we performed patch-clamp analysis of isolated adult cardiomyocytes under baseline conditions. The treatment with NO2-OA revealed no differences in the AP morphology regarding resting potential 28. The number of arrhythmic events which appeared aside from a basic 1 Hz pacing stimulus (e.g. caused by early after depolarizations (EAD) and delayed after depolarizations (DAD)), was increased after beta-adrenergic stimulation with isoproterenol (Iso)3. Crucially, this increase was prevented by NO2-OA treatment  and at the end of the RyR2 inhibitor tetracaine treatment (red cross)  Fig. C. Final ent Fig. D.Figure 2+ levels have been closely correlated with increased SR Ca2+ leak by RyR2-phosphorylation30. Of interest, caffeine treatment of short-time paced cardiomyocytes (1 Hz) demonstrated equal SR Ca2+ loads in vehicle- and NO2-OA treated cells. SR Ca2+ load was significantly elevated after Iso stimulation, an effect which was slightly, but not significantly, attenuated upon additional NO2-OA treatment  activity33, was increased upon Iso treatment and not further affected by NO2-OA treatment 34. Although the absolute rates of VT and VF have declined during the era of percutaneous coronary intervention, patients suffering from VT or VF prior to revascularization still have a significantly impaired outcome and are at a higher risk for stent thrombosis. These data may underestimate the true incidence, since prehospital SCD before fibrinolysis or any other interventions were not included in this analysis35. In the present study, we modeled a clinical scenario of myocardial ischemia by LAD ligation and demonstrated that intraperitoneal injection of NO2-OA 20 min before he ischemic episode sustainably reduced ventricular arrhythmic events specifically in the acute phase after AMI.Herein, we show that the electrophilic fatty acid nitroalkene NO2-OA as a therapeutic option for the treatment of acute VT after AMI is underlined by further beneficial effects of this fatty acid nitroalkenes in various cardiovascular diseases. As we and others have demonstrated, the application of NO2-OA preserves left ventricular ejection fraction and reduces infarct size in a mouse model of AMI36. Moreover, NO2-OA also protects from angiotensin II induced atrial fibrillation12.The prospect of using NO3. The concomitantly enhanced sympathetic tone leads to cellular Ca2+ overload, resulting in a disturbed Ca2+ homeostasis, accompanied by diastolic Ca2+ leak further increasing the vulnerability for VT37. On a cellular level, alterations in Ca2+ transients have been linked to the emergence of alternans and afterdepolarizations which may trigger VTs4, as well as to reduced sodium channel availability resulting in ventricular conduction slowing. This increases the wavelength of reentry circuits thereby increasing the vulnerability to VTs40.Mechanistically, the occurrence of ventricular arrhythmic events during the acute phase of AMI is attributed to development of proarrhythmic substrates with subsequent formation of reentry circuits2-OA on cellular Ca2+ transients and sarcoplasmic Ca2+ load, both important in arrhythmia development in AMI, we performed analyses of electrically stimulated isolated cardiomyocytes. We did not detect major changes in both Ca2+ transient characteristics and in SR Ca2+ load upon NO2-OA treatment.To investigate the effects of NO2+ clearance into the SR by SERCA2a5, at its CaMKII specific phosphorylation site Thr17 under Iso stimulation. Iso increased PLN phosphorylation but no further change was detectable upon treatment with NO2-OA. This indicates that NO2-OA does not influence cytosolic Ca2+ clearance by CaMKII mediated phosphorylation of PLN on this specific site, although we could not fully exclude potential other target sites or phosphorylation mechanisms being modified by NO2-OA.We quantified PLN phosphorylation, a protein regulating cytosolic Ca2-OA-mediated VT prevention in detail, we measured RyR2-dependent Ca2+ leak from the SR in isolated adult cardiomyocytes treated with Iso. We observed an increased number of arrhythmic events (e.g. EADs and DADs) upon Iso treatment that was markedly reduced by NO2-OA. Therefore, we hypothesized that NO2-OA beneficially influences CaMKII-dependent modulation of Ca2+-homeostasis. Indeed, CaMKII activity and subsequent RyR2 phosphorylation on the critical serine residue (Ser2814), which has been linked to CaMKII-mediated Ca2+ leak in cardiomyopathy and catecholamine treatment41, was significantly attenuated by NO2-OA in Iso-stressed cardiomyocytes 3.As this study was performed in a small animal model, a number of limitations may apply when trying to translate the present findings to human pathology. The mechanisms underlying VT in the setting of AMI are the result of multiple alterations, e.g. loss of viable myocardium with loss of cell-to-cell contact, changes in cell membrane potential, ion composition, and other factors predisposing for sudden cardiac death which are only partly mirrored by our experimental setting2-OA. However, if other mechanisms, e.g., those responsive to acidosis and cell hypoxia, are altered by NO2-OA remains to be investigated.Excessive catecholamine stimulation may be just one of multiple pathways leading to VT development during AMI modulated by NO2-OA modulates a critical pathway of electrical remodeling following AMI and thus has potential as a pre-emptive anti-arrhythmic strategy for patients at risk for developing AMI.In summary, we show for the first time that, by inhibiting CaMKII, NO2-OA rapidly stabilizes myocellular Ca2+ homeostasis by inhibition of CaMKII activity in the setting of AMI  were used for all animal studies. All animal studies were approved by the local Animal Care and Use Committees , NRW, Germany) and follow ARRIVE  guidelines.26.LAD ligation to induce myocardial ischemia was performed as described by Mollenhauer et al.26.A detailed stimulation protocol can be found within the Supplemental Material. In short, right ventricular stimulation was performed 20 min after induction of myocardial ischemia, while the mouse was kept under anesthesia, according to a standardized protocol that initially included a programmed ventricular stimulation with a fixed S1S1 interval to whose last S1-impulse a short-coupled additional stimulus was applied. After 10 s of recovery, automated burst stimulation was performed according to the protocol. VT were defined as a series of repetitive ventricular ectopic beats lasting for > 200 ms26. Field potentials were recorded using a 128-channel, computer-assisted recording system (Multichannel Systems). Data was evaluated according to Lammers et al.46. In short, inter-electrode conduction latencies  were calculated for all neighboring electrodes. Of each neighboring quadruplet of electrodes, the largest latency was taken and plotted as a phase map. From this phase map the mean latency of conduction was calculated. The variation coefficient of the phase map was calculated (Percentile (P): P5–95/P50) to receive the index of inhomogeneity as velocity independent factor of conduction inhomogeneity. Phase maps were calculated using custom-programmed software (Excel).In brief, directly after electrophysiological investigation the heart was exposed by thoracotomy. A 32-electrode microelectrode array  was positioned on the epicardial surface of the left ventricle apico-septally of the peri-ischemic region as described previously2-OA), administered at 20 nmol/g body weight, was solvated in polyethylene-glycol/ethanol , or 100 ml of vehicle . These preparations were injected i.p. 20 min prior to LAD ligation. NO2-OA was synthesized as previously described47.Nitro-oleic acid  as described previously2+ imaging with Fura-2 dye (IonOptix)28.All experiments were performed at 37 °C within 6 h after cell isolation, as described previously using ratiometric Ca2+transient analyses the cells were incubated with either 0.1% EtOH as control, 10 µM Iso in 0.1% EtOH or 10 µM Iso and 5 µM NO2-OA in 0.1% EtOH for 10 min. Calcium transients were recorded under electrically stimulated biphasic field pulses  at a frequency of 1 Hz for 1 min.For CaSR Ca2+leak and load were measured according to a modified protocol48. Fura-2 loaded ventricular cardiomyocytes were incubated with 10 µM Iso for 7 min and stimulated for 3 min at 1 Hz, 20 V, 4 ms until cellular Ca2+ transients reached a steady state followed by a burst stimulation with 4 Hz for 30 s. Directly after the last pulse the pacing was stopped and the normal Tyrode solution was substituted by a 0 Na+ /0 Ca2+ Tyrode supplemented with 10 mmol/l EGTA and 1 mmol/l of the RyR2 inhibitor tetracaine in which Na+ was replaced by Li+. This condition allowed measuring intracellular Ca2+ levels in a closed system without trans-sarcolemmal Ca2+ fluxes and prevents SR Ca2+ leak into the cytoplasm. After 40 s recording time the solution was switched back to 0 Na+ /0 Ca2+ Tyrode without tetracaine for another 40 s to unblock the ryanodine receptors and allow potential Ca2+ leak. SR Ca2+ leak was estimated as the difference between the Fura-2 ratio recorded at the end of the 0 Na+ /0 Ca2+ Tyrode perfusion with and without tetracaine. At the end of the protocol, 10 mM caffeine was applied to evaluate the total SR Ca2+ content.Ca2+sensitivity: Fura-2 loaded ventricular cardiomyocytes were incubated with Tyrode solution containing 0.25 mM, 0.5 mM, 1 mM or 2 mM of Ca2+ together with either 10 µM Iso in 0.1% EtOH or 10 µM Iso and 5 µM NO2-OA in 0.1% EtOH. For each calcium concentration cells were paced at 1 Hz for 30 s following a 20 s non-pacing period in which pro-arrhythmic calcium release events was counted. Ca2+ transient height was determined within the last paced Ca2+ transient before the non-paced period.2+ transient characteristics were calculated by the IonWizard (IonOptix) software.Ca26. Briefly, membranes were incubated with the following antibodies overnight at 4 °C: anti-GAPDH , anti-P-PLN-Thr17, anti-PLN, anti-P-RyR2-Ser2814 , anti-RyR2 . Full blots are shown in the Supplemental Material section.Protein extraction and immunoblotting were performed as previously described31. Bound active CaMKII, CaMKII input, and GST-HDAC4 419-670 input were resolved by SDS-PAGE and detected by immunoblot using HDAC4  and CaMKII antibodies . Protein loading was normalized to GAPDH .CaMKII activity was assessed by detecting the amount of endogenous CaMKII that binds to GST-HDAC4 amino acids 419 to 670 (containing the CaMKII activity–dependent binding domain) as described previouslyP < 0.05 was considered statistically significant. All statistical calculations were carried out using GraphPad Prism 8.2.1 (GraphPad). *P < 0.05, **P < 0.01, ***P < 0.001.Results are expressed as mean ± standard error of the mean. Gaussian normality was tested via Shapiro–Wilk normality test. Unpaired Student’s t-test or one-way ANOVA were used for gaussian distributed data, whereas non-gaussian distributed data were analyzed using Kruskal Wallis test, each followed by appropriate post-hoc test. Results of Fig. Supplementary information.


Venetoclax, an FDA-approved Bcl-2 selective inhibitor for the treatment of chronic lymphocytic leukemia and acute myeloid leukemia (AML), is tolerated well in elderly patients with AML and has good overall response rates; however, resistance remains a concern. In this study, we show that targeting CDK9 with voruciclib in combination with venetoclax results in synergistic antileukemic activity against AML cell lines and primary patient samples. CDK9 inhibition enhances venetoclax activity through downregulation of Mcl-1 and c-Myc. However, downregulation of Mcl-1 is transient, which necessitates an intermittent treatment schedule to allow for repeated downregulation of Mcl-1. Accordingly, an every other day schedule of the CDK9 inhibitor is effective in vitro and in vivo in enhancing the efficacy of venetoclax. Our preclinical data provide a rationale for an intermittent drug administration schedule for the clinical evaluation of the combination treatment for AML. For patients who are treated with intensive standard chemotherapy, relapse often occurs. Thus, better-tolerated and more effective therapies are needed to improve treatment outcomes of patients with AML.Although the biology of acute myeloid leukemia (AML) has been well studied, the prognosis of this deadly disease remains frustratingly low, with 5-year survival rates of 66% in children and 24% in adults.4 Bcl-2, Bcl-xL, and Mcl-1 have been found to be overexpressed in AML, and their overexpression is associated with resistance to chemotherapy and poor clinical outcomes.10 In particular, Bcl-2 is overexpressed in AML cells, making Bcl-2 a promising therapeutic target.11 Venetoclax  is a BH3 mimetic and Bcl-2 selective inhibitor developed by AbbVie.13 Venetoclax improved upon prior Bcl-2 family inhibitors by not targeting Bcl-xL, whose inhibition leads to thrombocytopenia and clinical toxicity.12 Its excellent antileukemic activity against chronic lymphocytic leukemia (CLL) led to FDA approval of venetoclax in April 2016. For AML patients who cannot tolerate standard intensive chemotherapy, venetoclax in combination with azacitidine, decitabine, or low-dose cytarabine (ara-C) generated encouraging response rates and survival outcomes, which led to FDA approval in November 2018. While these early results are promising, the median overall survival was 14.2–15.2 months,15 leaving substantial room for improvement.The antiapoptotic Bcl-2 family proteins Bcl-2, Bcl-xL, and Mcl-1 are key apoptosis regulators, and their dysregulation causes resistance to chemotherapy and promotes survival of cancer cells.16 However, our lab and others have found that venetoclax treatment increases Mcl-1 protein level and its binding to Bim in venetoclax-resistant AML cell lines and primary patient samples, representing an intrinsic mechanism of resistance to venetoclax in AML.17 The small-molecule Mcl-1 inhibitor A-1210477 was able to sensitize AML cells to venetoclax by disrupting compensatory Bim-Mcl-1 binding,18 representing a promising direct approach to overcome intrinsic resistance to venetoclax in AML. Alternatively, downregulation of Mcl-1 might indirectly overcome this intrinsic mechanism of resistance to improve treatment of AML with venetoclax.Antiapoptotic proteins, such as Bcl-2, function by sequestering proapoptotic proteins, such as Bim, preventing proapoptotic proteins from binding to proapoptotic activators Bax and/or Bak and inducing apoptosis. Venetoclax is able to disrupt the interaction between Bim and Bcl-2, freeing Bim to induce apoptosis.20 Inhibition of CDK9 with flavopiridol  can downregulate Mcl-1 and cooperate with venetoclax in AML.23 There, however, remain concerns about the off-target toxicity of flavopiridol. Thus, the second-generation CDK9 selective inhibitor voruciclib was developed.24 Voruciclib has greater specificity for CDK9 than flavopiridol, downregulates Mcl-1, and synergizes with venetoclax in diffuse large B cell lymphoma in vitro and in vivo. However, the molecular mechanisms underlying the synergistic interaction between venetoclax and CDK9 inhibition are not fully understood.CDK9, a key protein in the positive transcription elongation factor complex b, is a known regulator of transiently expressed prosurvival genes, including but not limited to Mcl-1, Cyclin D1, and c-Myc.In this study, we found that voruciclib synergizes with venetoclax to induce apoptosis in both AML cell lines and primary patient samples. Further, voruciclib transiently downregulates Mcl-1, which plays a role in the synergy between voruciclib and venetoclax. Interestingly, downregulation of c-Myc by voruciclib also contributes to its synergy with venetoclax. Simultaneous inhibition of c-Myc and knockdown of Mcl-1 show further enhancement of apoptosis induced by venetoclax than inhibition of c-Myc or knockdown of Mcl-1 alone. Furthermore, an every other day schedule seems optimal for the combination of venetoclax and voruciclib in an AML cell line-derived xenograft model.25 in five AML cell lines and three primary AML patient samples  was used to determine the extent and direction of antileukemic interactions.17 venetoclax-resistant cells (THP-1) but not venetoclax-sensitive cells (MV4–11) showed an increase in Mcl-1 protein in response to venetoclax treatment. Interestingly, CDK9 inhibition by voruciclib resulted in a transient downregulation of Mcl-1 protein, which rebounded 12 h after drug treatment in both MV4–11 and THP-1 cell lines ) moderately but significantly enhanced single drug treatment but did not significantly affect the highly efficacious combination treatment Fig. . Convers29 Thus, CDK9 inhibition may downregulate c-Myc, leading to enhancement of apoptosis induced by venetoclax. CDK9 inhibition for 6 h with voruciclib, alone or in combination with venetoclax, caused major downregulation of c-Myc protein levels in both MV4–11 and THP-1 cells . A total of 15 injections were given for each single agent as monotherapy or in combination with mice receiving either 25 mg/kg/inj venetoclax p.o., 200 mg/kg/inj voruciclib p.o., or in combination, as shown in Fig. p < 0.05, Mantel–Cox; 20.9% increase in lifespan (ILS); Fig. Based on our in vitro data of the CDK9 inhibitors, a Q2D dosing schedule was chosen for our in vivo studies. 1 × 1024 reported downregulation of Mcl-1 in diffuse large B cell lymphoma cell lines after treatment with voruciclib for 6 h. In primary AML cells, Bogenberger et al.21 reported variable changes in Mcl-1 protein levels following flavopiridol treatment, similar to our results after 24 h treatment of the AML cell lines. They also observed synergistic antileukemic activity of combined venetoclax and flavopiridol in AML cells. Our results are in agreement and additionally show that voruciclib synergizes with venetoclax in AML cells. While the main target of voruciclib is CDK9, inhibition of CDK4 and CDK6 cannot be ruled out. However, Bogenburger et al.21 showed that selective inhibition of CDK4 and CDK6 does not potentiate venetoclax activity against AML cells. We also found that Bax/Bak knockdown partially rescued the cells from combination treatment , flavopiridol , Z-VAD-FMK (a pan-caspase inhibitor), and 10058-F4 (a c-Myc inhibitor) were purchased from Selleck Chemicals . Voruciclib was provided by MEI Pharma .l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cells were cultured in a 37 °C humidified atmosphere containing 5% CO2/95% air. The cell lines were authenticated in August 2017 at the Genomics Core at Karmanos Cancer Institute using the PowerPlex® 16 System from Promega . Cell lines were tested for the presence of mycoplasma by PCR on a monthly basis.32The MV4–11, U937, and THP-1 cell lines were purchased from the American Type Culture Collection . OCI-AML3 was purchased from the German Collection of Microorganisms and Cell Cultures . MOLM-13 was purchased from AddexBio . The cell lines were cultured in RPMI 1640  with 10–20% fetal bovine serum , 2 mM 35).Diagnostic AML blast samples derived from patients were purified by standard Ficoll-Hypaque density centrifugation, and the isolated mononuclear cells were then cultured in RPMI 1640 with 20% fetal bovine serum, ITS solution , and 20% supernatant of the 5637 bladder cancer cell line , as described by the manufacturer. Western blots were repeated at least three times, and one representative blot is shown. Densitometry measurements were made using Odyssey V3.0 (Li-Cor), normalized to β-actin, and calculated as the fold change compared to the corresponding no drug treatment control.Cells were lysed in the presence of protease and phosphatase inhibitors . Whole-cell lysates were subjected to SDS-polyacrylamide gel electrophoresis, electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes , and immunoblotted with anti-Mcl-1 (4572), -PARP (9542), -Bim (2819), -Bak (3814), -Bax (2774), -c-Myc (5605s), -cleaved caspase-3 , or -β-actin  antibody, as previously described.40 Results are expressed as percent Annexin V-positive (Annexin V+) cells. For the AML cell lines, experiments were performed three independent times in triplicate, and the data presented are from one representative experiment, while the experiments with the patient samples were performed once in triplicate due to limited sample. Patient samples were chosen based on availability of adequate sample for the assay. The extent and direction of the antileukemic interaction was determined by calculating the combination index (CI) values using CompuSyn software . CI < 1, CI = 1, and CI > 1 indicate synergistic, additive, and antagonistic effects, respectively.39AML cells were treated with venetoclax and voruciclib, alone or in combination, and subjected to flow cytometry analysis using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Kit , as previously described.41 Briefly, TLA-HEK293T cells were transfected with pMD-VSV-G, delta 8.2, and lentiviral shRNA or LentiORF constructs using Lipofectamine and Plus reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions. Virus-containing culture medium was harvested 48 h post transfection. Cells were transduced overnight using 1 mL of virus supernatant and 4 μg of polybrene and then cultured for an additional 48 h prior to selection with puromycin or blasticidin.The pMD-VSV-G and delta 8.2 plasmids were gifts from Dr. Dong at Tulane University. Bax, Bak, and non-target control (NTC) shRNA lentiviral vectors were purchased from Sigma-Aldrich. Precision LentiORF Mcl‐1 and RFP (red fluorescent protein) lentivirus vectors were purchased from Dharmacon . Lentivirus production and transduction were carried out as previously described.http://crispr.mit.edu). The NTC  and Mcl-1 (5′-GCTTCCGCCAATCACCGCGC-3′) vectors were generated using Feng Zhang’s protocol, which is available on Addgene’s website (www.addgene.org). Lentivirus production and transduction were carried out as described above in “shRNA Knockdown,” but psPAX2  was used instead of delta 8.2.The lentiCRISPRv2 plasmid was a gift from Feng Zhang at the Broad Institute of MIT and Harvard (Addgene plasmid 52961). Guide RNAs were designed using the CRISPR design tool (40 Mcl-1 mRNA (Hx01050896_m1) and 18s rRNA (Hs03928985_g1) were quantitated using TaqMan probes (Thermo Fisher Scientific) and a LightCycler 480 real-time PCR machine (Roche Diagnostics), based on the manufacturer’s instructions. The real-time PCR results are expressed as the mean from three independent experiments and were normalized to 18S transcripts. c-Myc transcripts were quantified using forward (5′-GTGGTCTTCCCCTACCCTCT-3′) and reverse (5′-CGAGGAGAGCAGAGAATCCG-3′) primers. These real-time PCR results are expressed as the mean from three independent experiments and were normalized to GAPDH transcripts measured by forward (5′-AGCCACATCGCTCAGACA-3′) and reverse (5′-GCCCAATACGACCAAATCC-3′) primers and SYBR Green. Fold changes were calculated using the comparative Ct method.42Total RNA was extracted using TRIzol (Thermo Fisher Scientific), cDNAs were prepared from 2 µg of total RNA using random hexamer primers and an RT-PCR Kit (Thermo Fisher Scientific), and then purified using the QIAquick PCR Purification Kit , as described previously.scid Il2rgtm1Wjl Tg1Eav/MloySzJ; Jackson Laboratory, Bar Harbor ME, USA)) were injected intravenously (IV) with 1 × 106 MV4–11 cells/mouse (day 0) and treated Q2D (every other day) from day 3 through day 31 (first day of leukemia symptoms detected in control mice) for a total of 15 injections. Treatment cohorts were administered 25 mg/kg/inj venetoclax p.o. and/or 200 mg/kg/inj voruciclib p.o. A group of vehicle-treated mice was included as a control, and five mice were included in all arms. The average mouse body weight for the study was 22.4 ± 1 g at the start of the treatment, with body weight and condition monitored 1–2 times daily for the duration of the study.NSG-SGM3 mice  and treated from day 3 through day 27 (first day of leukemia symptoms detected in control mice). Treatment cohorts were administered 25 mg/kg/inj ABT-199 p.o.  and/or 16 mg/kg/inj Palmo-ara-C i.p. Q3D . A group of vehicle-treated mice was included as a control, and five mice were included in all arms. The average mouse body weight for the study was 24.2 ± 1 g at the start of the treatment, with body weight and condition monitored 1–2 times daily for the duration of the study.In a separate trial, NSGS mice were injected (IV) with 1 × 10The experimental endpoint and efficacy response for both trials were determined for each group based on the median day for development of leukemic symptoms . The %ILS was calculated: % ILS = [T-C/C] × 100, where “T” = treated and “C” = control median day of death. All mice were provided food and water ad libitum, given supportive fluids and supplements as needed, and housed within an AAALAC accredited animal facility with 24/7 veterinary care. All animal experiments were approved by the Institutional Animal Care and Use Committee of Wayne State University.t-test. Error bars represent ± standard error of the mean (s.e.m.); significance level was set at p < 0.05 and adjusted for multiple comparisons with the Bonferroni correction. One-way ANOVA was used to compare differences between three or more groups with Dunnett correction when compared to the control or Bonferroni correction when compared to single drug treatments. Overall survival probability was estimated using the Kaplan–Meier method, and statistical analysis was performed using the Mantel–Cox test. All statistical analyses were performed utilizing GraphPad Prism 5.0.Differences were compared utilizing a two-sample Supplementary Figures S1 and S2


We here aimed to study if it might be due to a dysregulation of either the whole Ca2+ homeostasis, the reticulum–mitochondrial Ca2+ coupling, and/or the mitochondrial Ca2+ entry through the uniporter. Following a 16-week high-fat high-sucrose diet (HFHSD), mice developed cardiac insulin resistance, fibrosis, hypertrophy, lipid accumulation, and diastolic dysfunction when compared to standard diet. Ultrastructural and proteomic analyses of cardiac reticulum–mitochondria interface revealed tighter interactions not compatible with Ca2+ transport in HFHSD cardiomyocytes. Intramyocardial adenoviral injections of Ca2+ sensors were performed to measure Ca2+ fluxes in freshly isolated adult cardiomyocytes and to analyze the direct effects of in vivo type 2 diabetes on cardiomyocyte function. HFHSD resulted in a decreased IP3R–VDAC interaction and a reduced IP3-stimulated Ca2+ transfer to mitochondria, with no changes in reticular Ca2+ level, cytosolic Ca2+ transients, and mitochondrial Ca2+ uniporter function. Disruption of organelle Ca2+ exchange was associated with decreased mitochondrial bioenergetics and reduced cell contraction, which was rescued by an adenovirus-mediated expression of a reticulum-mitochondria linker. An 8-week diet reversal was able to restore cardiac insulin signaling, Ca2+ transfer, and cardiac function in HFHSD mice. Therefore, our study demonstrates that the reticulum–mitochondria Ca2+ miscoupling may play an early and reversible role in the development of diabetic cardiomyopathy by disrupting primarily the mitochondrial bioenergetics. A diet reversal, by counteracting the MAM-induced mitochondrial Ca2+ dysfunction, might contribute to restore normal cardiac function and prevent the exacerbation of diabetic cardiomyopathy.Type 2 diabetic cardiomyopathy features CaThe online version of this article (10.1007/s00395-020-00835-7) contains supplementary material, which is available to authorized users. Purified mitochondria and MAMs, localized at the bottom of the tube as a dense band and in the middle as a round layer, respectively, were collected and washed by MRB at a centrifugation of 6300g /10 min/4 °C, and then, MAMs were re-centrifuged in polycarbonate tubes using the ultracentrifuge at 100,000 g/1 h/4 °C. A ring at the bottom of the tubes was collected, being the MAMs. For ER and cytosolic fractions, the cytosolic fractions were centrifuged at 20,000 g/30 min/4 °C to remove potential remaining mitochondria. Next, the supernatants were collected and re-centrifuged at 100,000 g/1 h/4 °C: pellets were saved as ER proteins and the supernatants as cytosolic ones.Mice were euthanized by cervical dislocation, and then, hearts were extracted and placed in Starting Buffer . Atria were removed and heart tissues were minced by scissors, followed by a homogenization using a teflon–glass grinder (15–20 strokes) with 4 ml of IB1 buffer  per heart. The homogenates were centrifuged twice at 740 Proteins were stacked in a single band in the top of an SDS-PAGE gel  and stained with Coomassie blue R-250 before in-gel digestion using modified trypsin  as previously described . ResultiBiological processes were determined with the Panther software (pantherdb.org).2+ imaging experiments, mice were anesthetized with 2% isoflurane and supplemented subcutaneously with buprenorphine (0.075 mg/kg). A left thoracotomy was performed in the fourth left intercostal space and the pericardium was opened to expose the heart. 5 × 108 PFU of adenovirus in 20 µl NaCl was injected into the left ventricular wall at seven different points. The chest cavity was closed and the mice were allowed to recover for 1 week prior to cardiomyocyte isolation.The 4mtD3cpv plasmid was obtained from Dr Roger Tsien’s lab , while tProteins were extracted from cardiomyocytes or frozen hearts using complete RIPA lysis buffer  and supplemented with anti-protease (Sigma P8340) and anti-phosphatase (Sigma P5726) cocktails. Proteins were quantified with the Lowry DC Protein Assay (Bio-Rad). For IP3R immunoprecipitation, as previously described , 500 µg Phospho-AKT, AKT, CHOP, and Grp78 proteins were detected in total heart homogenates (50 µg), phospho-PDH, and PDH proteins were assessed in cardiomyocyte lysates (30 µg), while MICU1 and MCU proteins were detected in pure mitochondria samples (15 µg). Mfn2 and VDAC assessments were done on isolated cardiac MAM samples (15 µg). After sample loading, proteins were transferred onto a nitrocellulose membrane, which was blocked for 1 h with 5% bovine serum albumin (BSA)-PBS. Immunoblotting was performed by incubating the membranes overnight at 4 °C with primary antibodies: rabbit anti-IP3R1 , mouse anti-VDAC , mouse anti-Grp75 , rabbit anti-phospho-AKT , rabbit anti-AKT , mouse anti-CHOP , goat anti-Grp78 , rabbit anti-MICU1 , rabbit anti-MCU , rabbit anti-MFN2 , mouse anti-phospho-PDH , and rabbit anti-PDH  prepared in 5% BSA-PBS. Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies  were added for 1 h incubation at RT. Immunoblot development was done using the reaction substrate ECL prime reagent  with Bio-Rad Molecular Imager Gel Doc XR + (Bio-Rad). Image Lab software (Bio-Rad) was used for quantification.2+ concentrations were, respectively, followed by FRET-based Ca2+ sensors, 4mtD3cpv and D4ER, expressed in adult mouse cardiomyocytes by in vivo adenoviral delivery and monitored with excitation at 430 nm (CFP) and emission at 485 nm (CFP) and 540 nm (YFP). Live measurements were performed at 37 °C in 1 ml CCB  or 1 ml CFB (similar to CCB without 2 mM CaCl2 for D4ER measurements) using a wide-field Leica DMI6000B microscope equipped with a 40 × objective and an Orca-Flash4.0 digital camera (HAMAMATSU). Following stabilization of the resting Ca2+ level (from 1 to 2 min), mitochondrial Ca2+ uptake following IP3R Ca2+ release was induced by 10 µM IP3-AM (SiChem). Mitochondrial Ca2+ concentration was recorded similarly in a field stimulation buffer  under successive electrical stimulations at 0.5, 1 Hz, and 2 Hz for 1 min each, using the MyoPacer Field Stimulator (IonOptix). Reticular Ca2+ release through the ryanodine receptors was induced by 5 mM caffeine (Sigma C0750). YFP/CFP fluorescence ratio was calculated with MetaFluor 6.3  after removing background fluorescence. For resting cytosolic Ca2+ level measurement, cardiomyocytes were loaded with 5 µM Fura2-AM (Thermo Fisher F1221) in CCB for 30 min at 37 °C, and then washed with CCB, and fluorescence was recorded using 340/380 nm excitation and 510 nm emission.Mitochondrial and reticular CaNADH autofluorescence was recorded at 25 °C in freshly isolated cardiomyocytes (1 mg) in CCB supplemented with 2 mM pyruvate, using a Hitachi F2500 spectrofluorometer with excitation at 360 nm and emission at 450 nm. NADH calibration was achieved by 2 µM rotenone and then 10 µM FCCP for each run.Oxygen consumption rates (OCR) were monitored in freshly isolated cardiomyocytes (500 µg), in CCB supplemented with 2 mM pyruvate, using an Oroboros respirometer at 25 °C. Maximal respiration level was induced by 10 µM FCCP.Total ATP content was detected using an ATP Bioluminescence Assay Kit  on freshly isolated adult cardiomyocytes at 16 weeks of diet.2+ transients were recorded measuring the Fluo-4 signal (Thermo Fisher F14201: ex/em 490/510) with a laser scanning confocal microscope (Nikon A1R) on a 40 × objective at 37 °C. Cardiomyocytes loaded with 1 µM Fluo-4 for 30 min at 37 °C, then washed with CCB, were placed in the field stimulation buffer, and electrically stimulated with the MyoPacer Field Stimulator (IonOptix) successively at 0.5 and 1 Hz for 1 min each with a rest of 1 min in between . Images were acquired every 68 ms, and then, normalization was done to the average resting fluorescence intensity (F/F0). Ca2+ transients were analyzed using a homemade program developed with Matlab and Statistics toolbox . Transient peaks and minima were detected with a standard algorithm for finding local extrema based on the derivative of the signal and the corresponding amplitude and time of each extremum were recorded. From these values, different parameters were measured: time to peak, half-time, and time peak to basal. Cardiomyocyte shortening was evaluated in Fluo-4 loaded cells by line scanning along the long axis. Cell length was measured, using ImageJ software, in the resting state and in the maximally contracted state to express the shortening as a percentage of the resting cell length.Cytosolic CaEchocardiography was performed under a light anesthesia (ketamine 80 mg/kg ip), with a digital ultrasound system  and a 13-MHz linear-array transducer as previously described . LV end-N = 9, p = ns). Acquisitions were performed on the Vevo 3100 imaging system using a 40-MHz linear probe . E and A peak velocity was measured from the mitral flow profiles obtained by Pulsed Wave Doppler in the apical four-chamber view as well as isovolumic relaxation time (IVRT), while Tissue Doppler imaging at the septal corner of the mitral valve annulus was performed to assess E’ peak velocity. Analyses were performed offline blindly on the VevoLab software, as previously described c in HFHSD  in ventricular myocytes from SD and HFHSD cells . Supplementary Fig. 4b shows the current–voltage relationships of normalized ICa,L peak to membrane capacitance from SD and HFHSD cells. No significant difference in the density of ICa,L was observed between the two groups, nor in the density of the Na+/Ca2+ exchange current measured as the lithium-sensitive slow tail current . Monitoring of [Ca2+]c by Fluo4-AM in cardiomyocytes under field stimulation at both 0.5 and 1 Hz did not reveal any significant differences in the cytosolic Ca2+ transients, including the peak amplitude, the time to peak, the half-time, and the time peak to basal (Supplementary Table 3). However, the examination of [Ca2+]m in paced cardiomyocytes . These results support that the diet-induced functional alterations of MAM coupling precede any dysfunction of the excitation–contraction coupling at both plasma membrane and cytosolic levels while altering the field-stimulated physiological mitochondrial Ca2+ transfer.To determine if the excitation–contraction coupling was altered following 16-week HFHSD, we performed electrophysiological recordings of inward L-type Ca2+ uniporter, which is responsible for the Ca2+ entry into the mitochondrial matrix. First, we analyzed the protein level of the pore-forming protein MCU and its main regulator MICU1, since the stoichiometry of the MICU1 to MCU ratio directly controls the function of the mitochondrial Ca2+ uniporter c, a rapid mitochondrial Ca2+ clearance was recorded in both SD and HFHSD cardiac mitochondria  were significantly reduced by this extended diet reversal. Altogether, these data strongly demonstrate that the T2D-induced alterations of the reticulum–mitochondrial Ca2+ coupling and the ensuing mitochondrial and heart dysfunctions are reversible.Because of this incomplete reversal, we extended the study for 4 weeks . Enriched MAM isolation showed a significant reversal of the protein content ratio of total MAM over pure mitochondria in the HFHSD/SD compared to HFHSD hearts Fig. d. IP3R1 ine Fig. f-g. The tes Fig. h. Follow2+ coupling, resulting in mitochondrial Ca2+ mishandling and energy disruption, is an early feature of the cardiomyopathy induced by a 16-week HFHSD in the mouse model. This HFHSD-induced cardiomyopathy is characterized by a reduced mitochondrial Ca2+ uptake secondary to a reduced formation of the IP3R1/Grp75/VDAC1 Ca2+ channeling complex in MAM but without any alteration of the mitochondrial Ca2+ uniporter. Moreover, the observed HFHSD-induced MAM functional Ca2+ uncoupling contributes to the cardiomyopathy with diastolic dysfunction and mild systolic dysfunction in the absence of any detectable change in reticular Ca2+ stocks, cytosolic Ca2+ transients, and Ca2+ currents at the plasma membrane. Importantly, both the abnormalities in Ca2+ fluxes and the structural and functional alterations of the heart are reversed following a switch back to a standard diet. Altogether, our study demonstrates that the alteration of the MAM thickness towards tighter interactions not compatible with the Ca2+ transport process is an early trigger of the mitochondrial dysfunctions during diabetic cardiomyopathy.We here report that the structural and functional alteration of the reticulum–mitochondria Ca2+ sensors to evaluate the early mechanisms of DCM in adult cardiomyocytes freshly isolated from HFHSD mice.Knowing that diabetes is a metabolic syndrome that impairs many vital organs, both structurally and functionally, performing the study in vivo with a representative model of T2D, was a very crucial requisite to understand better these impairments and the mechanisms behind the altered phenotype. One major asset of this mouse model is that it recapitulates the early phase of a T2D-like cardiomyopathy, including diastolic dysfunction, LV remodeling with cardiac fibrosis, lipid accumulation, and concentric hypertrophy, together with impaired mitochondrial functions at the molecular level . One mus2+ uptake with a decreased mitochondrial ATP production in the diabetic cardiomyocyte m. Future studies will be required to decipher between these differences. Nonetheless, our results of disrupted MAMs Ca2+ coupling in the heart are in line with these studies and suggest a systemic effect of T2D. Additional work is needed to clarify the impact of cardiac insulin resistance on the reticulum–mitochondria interface structure and function. Our proteomic analysis of the cardiac MAM proteome clearly highlights a change in the protein composition by the HFHSD. The protein recruitment at the MAM might involve post-translational modifications, such as glucotoxicity-associated O-N-acetylglucosamine acylation as previously suggested , pointing out the mitochondrial Ca2+ dysregulation following the reticulum–mitochondria miscoupling as a main contributor to the development of DCM. Additionally, recent studies also demonstrated that mitochondrial bioenergetics is not only regulated by mitochondrial Ca2+ but also by cytosolic Ca2+ via the malate-aspartate shuttle  is a central mechanism of the T2D cardiomyopathy and that these initial modifications of the heart are reversible. Furthermore, this strongly supports the idea that optimization of the diet might be a very important component of the therapy aimed to attenuate the early progression of cardiomyopathy in T2D patients.A major observation of our study is that an 8-week switch back to a standard diet reestablishes the functional Ca2+ through MAMs is an early and crucial player of the mitochondrial Ca2+ mishandling and dysfunction in T2D hearts. Our work further suggests that adaptation of patients’ nutrition might prevent these early modifications and subsequently counteract the progression or even reverse the associated cardiomyopathy.In summary, we propose that the transfer of Ca2+, due to its Kd of 0.6 µM, is not the most adequate to assess both changes in the resting mitochondrial Ca2+ and in the IP3-induced peak amplitude. Higher magnitude of changes are assessed in the peak level; nevertheless, small magnitude changes in resting levels are still of biological meaningful difference.While being an invaluable asset for the study of in vivo T2D alterations, analysis in freshly isolated adult mouse cardiomyocytes knows some limitations. Intramyocardial adenoviral infection is leading to a maximum of 30% of infected cardiomyocytes. Therefore, this point precluded any further in vivo analysis of cardiac function and non-single cells targeted measurements, such as electron microscopy, OCR, and immunoblotting, since we could not select the infected cardiomyocytes. Moreover, our diet-induced T2D mouse model also triggers obesity, which prevents us from deciphering between diabetes and obesity-related effects. Finally, the 4mtD3cpv FRET-based sensor of mitochondrial Ca2+ coupling impairs mitochondrial Ca2+ stocks and bioenergetics in the diabetic heart, further altering the myocardial function, which could be reversed by diet reversal. Interestingly, the control of nutritional intake showed its direct contribution in the improvement of cardiac function in the early stage of diabetic cardiomyopathy, specifying the underlying mechanisms behind the beneficial effect of diet reversal.Diabetic cardiomyopathy is a very common complication in type 2 diabetes, notably as a result of mitochondrial dysfunction ending by a compromised cardiac function. Our study shows that alteration of the reticulum–mitochondria CaSupplementary file1 (PDF 532 kb)Supplementary file2 (PDF 1890 kb)Below is the link to the electronic supplementary material.


PKD2. PKD2 encodes polycystin-2, which forms an ion channel in primary cilia and endoplasmic reticulum (ER) membranes of renal collecting duct cells. Elevated internal Ca2+ modulates polycystin-2 voltage-dependent gating and subsequent desensitization – two biophysical regulatory mechanisms that control its function at physiological membrane potentials. Here, we refute the hypothesis that Ca2+ occupancy of the polycystin-2 intracellular EF hand is responsible for these forms of channel regulation, and, if disrupted, results in ADPKD. We identify and introduce mutations that attenuate Ca2+-EF hand affinity but find channel function is unaltered in the primary cilia and ER membranes. We generated two new mouse strains that harbor distinct mutations that abolish Ca2+-EF hand association but do not result in a PKD phenotype. Our findings suggest that additional Ca2+-binding sites within polycystin-2 or Ca2+-dependent modifiers are responsible for regulating channel activity.Approximately 15% of autosomal dominant polycystic kidney disease (ADPKD) is caused by variants in  Summary: A refutation of a common hypothesis regarding Ca2+ control mechanisms of polycystin-2 – a primary cilia ion channel, the dysregulation of which causes autosomal dominant polycystic kidney disease. PKD2 encodes for polycystin-2, a member of the polycystin subfamily of transient receptor potential ion channels (TRPP)   . Previou tissues . Primary tissues . Primary tissues . Dysreguorbidity . Variant (ADPKD) , a commo (ADPKD) . Mouse mhenotype , whereasin utero . These s2+ (2+ is elevated to micromolar concentrations (2+-dependent modulation’ (CDM) to describe this form of polycystin-2 gating regulation. Importantly, this mechanism gives polycystin-2 gating its physiological relevance, allowing Ca2+ and monovalent ions to flow into the primary cilium at its resting membrane potential. Subsequently, prolonged and elevated intraciliary Ca2+ causes polycystin-2 channels to enter an irreversible non-conducting state, through a process we have termed ‘Ca2+-dependent desensitization’ (CDD). This form of polycystin regulation was postulated to protect the cell from Ca2+ overload, by turning off the flow of Ca2+ at the source within the ciliary compartment  ion channels, so its contribution is difficult to distinguish from inositol trisphosphate (IP3R)- and ryanodine receptor channel-mediated effects , or alter the EF hand, abolish the entire function of the channel . We determined that the affinity of Ca2+ for the wild-type human CTD EF hand was 19±5 µM – a finding which approximates the value previously reported (Kd=22 µM) for this peptide . We then individually mutated the EF hand vertices to alanine to determine which positions contribute to Ca2+ binding. We found that all five vertices are involved in Ca2+ affinity for the EF hand, as all alanine substitutions increased Kd 6–68× (Table S1). Based on these results, if Ca2+ occupancy of the EF hand regulates polycystin-2 function, disrupting its affinity for this motif should translate into measurable changes in the Ca2+-dependent properties of the channel.Two groups have independently reported that human and mouse orthologs of polycystin-2 are activated by internal Caysiology . Polycyschannels . Althougchannels . The EF ractions A,B. We i peptide . Importa2+ occupancy of the EF hand is responsible for CDM and CDD of human polycystin-2, we generated double alanine mutations at the -X (T771A) and -Z vertices (E774A), which abolished Ca2+ affinity for the CTD fragment . As described previously, we established inside-out cilia patch configurations so that Ca2+ within the cilia (intraciliary) can be adjusted by the superfusate (2+ was elevated from 100 nM to 100 µM for both wild-type and -X-Z channels (2+ potency (EC50) for opening between wild-type and mutant channels was observed after fitting the integrated single channel activity to the Hill equation (Table S2). Previous work has established that CDM is a product of a hypopolarizing-shift in the voltage dependence of polycystin-2, which increases channel open probability (Po) at negative membrane potentials (2+ concentrations (V1/2) from low to high Ca2+ was −47 mV for wild-type channels, and was not different from the shift observed in -X-Z channels (Table S2). As reported previously, we observed that polycystin-2 enters an irreversible desensitized state (CDD) after prolonged exposure to high internal Ca2+ . The time course of CDD was not different between wild-type and -X-Z mutant channels, as the magnitude of cilia current was completely abolished for both channels after 3.5 min of recording. Taken together, these results demonstrate that abolishing Ca2+ occupancy of the human polycystin-2 EF hand does not alter CDM and CDD forms of channel regulation.To test whether Cafragment B. We theeviously . The staerfusate A,B  and -Z (E772A) vertices, and simultaneously insert a V5 epitope tag immediately before the termination codon in murine Pkd2 . Thus, we challenged the robustness of this interpretation by generating a second mouse strain (del-ZPkd2), in which the EF hand -Z vertex (E772), along with the preceding arginine (R771) were genetically deleted. Using isothermal titration calorimetry measurements, we confirmed that the del-Z mutation abolished Ca2+ occupancy of the EF hand . +/del-ZPkd2, del-Z/del-ZPkd2 and del-Z/-Pkd2 mice were viable to at least 12 months of age, and the kidney morphology of these mice was periodically imaged using MRI , +/del-ZPkd2 (0/19) and del-Z/del-ZPkd2 (0/18) animals developed PKD-type cysts. In our preliminary observations, a minority of the del-Z/del-ZPkd2 (4/35) mice developed bilateral cystic disease (non-PKD-type), but because of the infrequency of this phenotype and because these cysts were not observed after outbreeding, we conclude that this bilateral cyst phenotype is not likely caused by the del-Z mutation. Owing to the limited resolution of MRI, we conducted histology experiments on sectioned kidneys from six mice from each genotype. Consistent with the MRI results, we did not observe any obvious cysts in the kidney sections from the other mice showing homozygous expression for del-ZPkd2 alleles . The half-maximal voltage-dependence of channel opening (V1/2) was not different from the three genotypes at low Ca2+. Under elevated intraciliary Ca2+, we observed a small but insignificant difference (−4 mV) in V1/2 when comparing the cilia channels measured from +/+Pkd2 and del-Z/del-ZPkd2 mice (Table S2). The potency of Ca2+ opening polycystin-2 channels was not different from +/+Pkd2 (EC50=1.2±0.4 µM), +/del-ZPkd2 (EC50=1.4±0.4 µM) and del-Z/del-ZPkd2 (EC50=1.4±0.3 µM) mice . In addition, we observed no difference in the onset of CDD between cilia currents recorded from +/+Pkd2 and del-Z/del-ZPkd2 mice . As discussed previously, polycystin-2 was reported to function as an intracellular Ca2+ release channel (2+ release was attenuated by heterologous overexpression of a truncating mutation (L703X) in a porcine kidney-derived cell line (LLC-PK1). As L703X removes the EF hand along with the entire CTD in polycystin-2, it is conceivable that removing the EF hand motif was responsible for the attenuated Ca2+ response to vasopressin  and Ca2+ channels (Cavs), and Ca2+-activated potassium channels (KCa) are modulated by internal Ca2+  within the channel, or that an unknown Ca2+-binding protein associates with polycystin-2 to regulate its gating. Results from calorimetry and NMR spectra methods suggest that the EF hand is the only Ca2+ binding site in the CTD of polycystin-2  and large-conductance Ca2+-activated K+ (BK) channels, in which separate Ca2+-sensor proteins bind to motifs within the channel to control gating , in agreement with previous calorimetry studies (2+ (∼90 nM) is regulated by cytoplasmic buffering proteins. However, as the primary cilia retains a high concentration of Ca2+ (390-580 nM) at rest, and the EF hand motifs are near the ion-conducting pore, they are likely to experience elevated Ca2+ in the micromolar range  in the coding region of the PKD2 EF hand . However, there are several reported frameshift variants, which result in premature truncations of the CTD , 5% glycerol, 0.25 mM EDTA (pH 8.0), 1 mM PMSF (RPI), 25 µg/ml lysozyme and DNase I (GoldBio). The suspension was then lysed by sonication and clarified by centrifugation . Filtered supernatant was loaded into a HisPur cobalt Superflow agarose column (Thermo Scientific). The column was washed with 10 column volumes (CV) of buffer A, 5 CV of buffer A plus 5 mM imidazole, and finally eluted with 3 CV of buffer A+100 mM imidazole. The eluate was desalted by dialysis in 150 mM NaCl, 20 mM Tris/HCl, 20 mM imidazole (pH 7.4). SDS-PAGE confirmed the expression, solubility and purity of the peptide. All of our solutions were formulated with ultrapure water (Milli-Q IQ 7005 water purification system), which has less than 0.29 ng/l Ca2+ ions present. The binding affinity was recorded based on the heat difference between the sample cell and reference cell measured by MicroCal iTC200. Forty successive additions of 1 µl CaCl2 (2 mM) were added into the sample cell containing 400 µM of the respective purified peptides at 1 min intervals. The cells were insulated by an adiabatic jacket held at 25°C. The heat of dilution of Ca2+ was not subtracted from data sets as the signal was prohibitively small for several of the mutant channels. The exothermic energy of the bimolecular interaction between Ca2+ and peptide during each injection was analyzed by integrating the change in heat to generate the binding isotherm. The resulting relationship was fit (Origin 7.0) with a one-site independent binding model to determine the binding affinity (Kd).The C-terminal fragment of human polycystin-2 (I704-P797) was cloned into the PET19b vector and transformed into BL21 (DE3) competent cells (New England Biolabs) for bacterial expression. The translated peptide has a 10× His tag on its N-terminus. Additionally, the alanine mutants of the individual Cahttp://crispr.mit.edu/). To edit the mouse genome, single-stranded oligodeoxynucleotide (ssODN) donor templates for T769A and E772A point mutations, and another with a V5 tag in-frame at the C-terminus, were synthesized (IDT). A sgRNA plasmid was constructed and linearized, followed by in vitro transcription of sgRNAs using MEGAshortscript Kit (Invitrogen). The yield and quality of sgRNA were assessed by absorbance ratio and gel electrophoresis  and two donor ssODN templates were microinjected into zygotes, followed by culture and transfer of blastocysts into the uterus of pseudopregnant ICR female mice  for the T769A and E772A mutations (5′-GCTCACGCTCGGTCAGTTCC-3′) and the inserted V5 tag (5′-CACGTGTGGATTATTAGGCA-3′) were designed using the CRISPR Design tool , +/del-ZPkd2  and del-Z/del-ZPkd2  were MRI scanned at 9 and 12 months of age. Nine-month-old cPkd2 mice  were fed doxycycline through drinking water for 1 week to genetically attenuate Pkd2 expression , as described previously  were fixed in 40% paraformaldehyde for 24 h, sectioned on a cryostat, mounted on glass coverslips and stained with hematoxylin and eosin (H&E). Images of medial sections from both kidneys were analyzed using FIJI (ImageJ) to identify cystoid foramen. Images were processed as black and white, and reverse-negative. The images were then analyzed using the particle analysis search protocol, in which the lower limit threshold for circular foramen was set to 20 µm to identify cystoid foramen in the tissue samples. del-ZPkd2 mice were crossed with our previously established ARL13B-EGFP mouse, so that primary cilia could be visualized from living cells during our cilia electrophysiology recordings described in the next section  were isolated from wild-type or eviously . Brieflyeviously . To gene2+ ions present. The internal solution (bath) contained (in mM): 120 NaMES, 10 NaCl and 10 HEPES. Ca2+ was buffered with 5 EGTA, 5 Na4-BAPTA {Glycine, N,N′-bis[N-(carboxymethyl)]-,tetrasodium} and 0.5 EDTA; free Ca2+ was calculated using Maxchelator and titration of 1 M CaCl2 solution  mOsm with d-mannitol. Whole-cell currents used to measure CDD were also recorded in symmetrical sodium concentration, placing the internal recording solution in the pipette electrode and the external recording solution in the bath. Data were collected using an Axopatch 200B patch-clamp amplifier, Digidata 1550B and pClamp 10 software. Single channel currents were digitized at 50 kHz and low-pass filtered at 10 kHz. Intraciliary conditions were controlled using an Octaflow II rapid perfusion system (ALA systems) in which the patched cilia and electrode were held in the perfusate stream. Data were analyzed by Igor Pro 7.00 (Wavemetrics). The polycystin-2 open probability (Po) current-voltage relationships were fitted to a Boltzman equation, f(x)=1/(1+exp[V-V1/2]/k), to estimate the half-maximal voltage (V1/2) required to open the channels. The potency of Ca2+ opening polycystin-2 channels was estimated by integrating the single channel current measured in response to elevating the internal Ca2+ concentration ([Cain]). The average integrated current was fitted to the Hill equation, f(x)=base+(max-base)/{1+(EC50/[Cain]), to estimate the effective concentration of Ca2+ (EC50) required to half maximally stimulate the polycystin-2 response.The electrophysiologist was blinded by a third party in the laboratory, whereby test groups were assigned a letter to conceal the genetic identity of the cells being evaluated. The identity of the cells remained unknown by the electrophysiologist until the analysis was complete. Ciliary ion currents were recorded using borosilicate glass electrodes polished to resistances of 14-23 MΩ using the cilium patch method described previously . Single solution ; pH was 2+ responses were measured from pIMCD cells 24 to 72 h after cells were isolated. Before seeding the cells, glass-bottom dishes were pre-coated with poly-L-lysine and laminin (Sigma-Aldrich). The cells were incubated for 1 h at 37°C in complete medium after loading with 2 µg of Fura-2/AM (Invitrogen). The cells were then washed with and stored for 15 min in Tyrode's solution (in mM): 140 NaCl, 4 KCl, 2 MgCl2, 2 CaCl2, 10 Glucose and 10 HEPES. Cells were adjusted in pH to 7.4 with NaOH and osmolarity to 300 mOsm with d-mannitol. Cells were then placed under an inverted wide-field microscope equipped with a 20× objective lens (Olympus IX81), and the stage temperature was held at 37°C (Tokai Hit). Images of Fura-2 fluorescence remission at 520 nm were captured every 2 s during excitation at 380 nm (Ca2+ free) and 340 nm (Ca2+ bound). Images were acquired and analyzed using SlideBook (Intelligent Imaging Solutions), which synchronizes the filter wheel changer  and the camera . In each trial or replicate (n), the emission fluorescence (340:380 nm) ratio of 15 to 30 cells was recorded after subtracting the background fluorescence levels. This ratio was averaged at the start (for resting level) and the response was reported after extracellular vasopressin or carbachol treatment . At least three replicates (n) were performed with cells isolated from five animals of the same genotype.Vasopressin-mediated intracellular Cat-test power analysis of the recorded variance from pilot study results from each assay, where target power=0.9 and α=0.05. Statistical comparisons were made using one-way ANOVA or two-tailed unpaired Student's t-tests using OriginPro software (OriginLab) or Excel (Microsoft). Experimental values are reported as the mean±s.d., unless otherwise stated. Differences in mean values were considered significant at P<0.05. All of our results are normally distributed as per a Shapiro–Wilk Test.Sample sizes were determined based on 2-sample