Our discovery identifies a mechanism for vascular remodeling as an adaptation to increased circumferential stretch.Excitation–transcription (E-T) coupling can initiate and modulate essential physiological or pathological responses in cells, such as neurons and cardiac myocytes. Although vascular myocytes also exhibit E-T coupling in response to membrane depolarization, the underlying molecular mechanisms are unknown. Our study reveals that E-T coupling in vascular myocytes converts intracellular Ca 2+ concentration ([Ca2+]i) activates Ca2+/calmodulin-dependent kinases (CaMK) and promotes gene transcription. This signaling pathway is referred to as excitation–transcription (E-T) coupling. Although vascular myocytes can exhibit E-T coupling, the molecular mechanisms and physiological/pathological roles are unknown. Multiscale analysis spanning from single molecules to whole organisms has revealed essential steps in mouse vascular myocyte E-T coupling. Upon a depolarizing stimulus, Ca2+ influx through Cav1.2 voltage-dependent Ca2+ channels activates CaMKK2 and CaMK1a, resulting in intranuclear CREB phosphorylation. Within caveolae, the formation of a molecular complex of Cav1.2/CaMKK2/CaMK1a is promoted in vascular myocytes. Live imaging using a genetically encoded Ca2+ indicator revealed direct activation of CaMKK2 by Ca2+ influx through Cav1.2 localized to caveolae. CaMK1a is phosphorylated by CaMKK2 at caveolae and translocated to the nucleus upon membrane depolarization. In addition, sustained depolarization of a mesenteric artery preparation induced genes related to chemotaxis, leukocyte adhesion, and inflammation, and these changes were reversed by inhibitors of Cav1.2, CaMKK2, and CaMK, or disruption of caveolae. In the context of pathophysiology, when the mesenteric artery was loaded by high pressure in vivo, we observed CREB phosphorylation in myocytes, macrophage accumulation at adventitia, and an increase in thickness and cross-sectional area of the tunica media. These changes were reduced in caveolin1-knockout mice or in mice treated with the CaMKK2 inhibitor STO609. In summary, E-T coupling depends on Cav1.2/CaMKK2/CaMK1a localized to caveolae, and this complex converts [Ca2+]i changes into gene transcription. This ultimately leads to macrophage accumulation and media remodeling for adaptation to increased circumferential stretch.Elevation of intracellular Ca In contrast, local Ca2+ release from the RyRs (Ca2+ sparks) in vascular smooth muscle cells activates nearby large conductance Ca2+-activated K+ (BK) channels in the absence of global [Ca2+]i increases, and this generates spontaneous transient outward currents. Spontaneous transient outward currents lead to membrane hyperpolarization, as well as a decrease in VDCC activity and vascular tone  . CICR inlar tone . This escaveolin .2+]i turns on Ca2+-dependent enzymes, resulting in activation of transcription factors and subsequent changes in gene-transcription patterns /cyclic AMP-responsive element binding protein (CREB) pathway is activated by sustained depolarization in vascular smooth muscle  CREB phosphorylation in vascular myocytes, 2) macrophage accumulation in adventitia, and 3) vascular remodeling. In summary, E-T coupling in vascular myocytes can modulate macrophage migration and subsequent inflammation, thus altering the vascular structure.Previously we reported that caveolin-1 (cav1), an essential component of caveolae in smooth muscle, can support effective conversion between electrical and i imaging showed that neither KN93 nor STO609 had any effects on VDCC-mediated [Ca2+]i elevations . For a more quantitative analysis of effects of the drugs on CREB phosphorylation, an ELISA was utilized using arterial lysates , inhibited CREB phosphorylation in vascular myocytes D. CaMKK  lysates E. SimilaSI Appendix, Fig. S3A). As expectedly, these myocytes responded to a depolarizing stimulus (60 mM K+ solution) with a robust increase in [Ca2+]i . These findings strongly suggest that these primary vascular myocytes have Ca2+ signaling features that are common to freshly isolated myocytes. mRNA expression of CaMKK1 in these primary myocytes was not detected . As shown in Accordingly, we sought to identify the isoforms of CaMKK and CaMK involved in E-T coupling in these myocytes. Given the difficulty of knockdown or overexpression of exogenous proteins in native tissue, we utilized primary, unpassaged vascular myocytes from mouse aorta . Immunosdetected F, and thSI Appendix, Fig. S3D) decreased CREB phosphorylation in the primary vascular myocytes  mice, a 60 mM K+ solution-induced depolarization failed to result in CREB phosphorylation (v1.2 expression in cav1-KO myocytes was very similar to that of WT cells (Camk2g) mRNA . Caveola disruption using methyl-β-cyclodextrin  also inhibited CREB phosphorylation in mouse mesenteric artery . In contrast, cav1 overexpression in cav1-KO myocytes  restored CREB phosphorylation  microscope that provides the possibility of selective detection of fluorescence signals just beneath the plasma membrane .To further evaluate the functional effects of a Camembrane , 19. Sigd CaMKK2 D was det-KO mice E. PLA an2+ influx through Cav1.2 directly activates CaMKK2 within the same complexes. To address this point, primary vascular myocytes were transfected with CaMKK2 that had been labeled with the genetically encoded Ca2+ indicator GGECO1.1  in these WT vascular myocytes . These results also strongly suggest that caveolae compartmentalize Cav1.2 and CaMKK2 and promote direct signal transmission from Cav1.2 to CaMKK2 in vascular myocytes.Datasets from immunostaining experiments cannot prove that CaGGECO1.1  . Therefore, cav3 may partially support the physical interaction between Cav1.2 and CaMKK2 independently of cav1 or caveolae.We also noted that signaling from Caisrupted . PLA ana2+/CaM binding and phosphorylation at the Thr177 site by CaMKK  of cav1 resulted in reestablishment of CaMK1a phosphorylation . These results are consistent with cav1 having the ability to initiate molecular complexes containing Cav1.2, CaMKK2, and CaMK1a in arterial myocytes. As shown in Accordingly, our working hypothesis was that CaMK1a is localized within caveolae where it forms molecular complexes with Ca-KO mice A. PLA an-KO mice B and freTo gain further insight into these signaling component dynamics, changes in the number of CaMK1a puncta in the plasma membrane were counted with or without STO609, a CaMKK2 inhibitor. Normalized CaMK1a puncta density in the plasma membrane was unchanged at 5 min after depolarization, but deceased at 30 min. This decrease in the normalized CaMK1a puncta density was reversed by the treatment with STO609 H. Coloca2+ levels, we overexpressed the nuclear-localized parvalbumin-based Ca2+ buffer protein (PV-NLS-GFP) (2+ levels after depolarizing stimuli without any effects on cytosolic Ca2+ levels (To determine the importance of nuclear CaNLS-GFP) . Overexp+ levels J. Howeve+ levels K nor int+ levels L. These + (control) or 60 mM K+ (depolarizing) solutions for 1 h, after which induced genes were compared. This differential expression gene (DEG) analysis identified 138 up-regulated and 5 down-regulated genes . Up-regulated genes that had log2(fold-change) values more than 2 are listed in Cxcl1, Cxcl2, Ccl2, Ccl7), leukocyte adhesion , degradation of extracellular matrix (Adamts4 and Adamts9), or inflammation (Il6). These up-regulated genes were further analyzed using qPCR . Mesenteric artery preparations were perfused with either 5 mM King qPCR C. ImportSI Appendix, Fig. S7 A and B). The perfusion of sodium deoxycholate also reduced acetylcholine-induced vasodilation . In the endothelium denuded arteries, depolarization-induced gene induction was comparable to those seen with control arteries . These results strongly suggest that the observed gene induction in this study mainly occurs in vascular myocytes, rather than endothelial cells.Next, we sought evidence that the observed changes occurred predominantly in the smooth muscle cells. We acknowledge the possibility that endothelial cells also participate in depolarization-induced gene transcription. Accordingly, the endothelium was physically removed by perfusing 0.2% sodium deoxycholate   E-T coupling, 2) leukocyte accumulation, and 3) vascular remodeling using our ex vivo and in vivo models. First, we examined whether increased perfusion pressure can cause CREB phosphorylation. Using the ex vivo model, high pressure (80 mmHg) increased the P-CREB (+) cell ratio, and this change was blocked by Nic, KN93, or STO609 . In contrast, physiological pressure (40 mmHg) did not increase this P-CREB (+) cell ratio; that is, the ratio values were approximately equal to those of mesenteric arteries that had been rapidly fixed with ice cold 4% PFA, or incubated with no transluminal pressure gradient (0 mmHg). When the same elevated pressure maneuver was also applied to mesenteric arteries from cav1-KO and MβCD-treated tissues, the P-CREB (+) cell rate was not significantly changed by the stimulus .It has previously been reported that accumulation of leukocytes (such as neutrophils and monocyte/macrophages) can initiate vascular remodeling –18. It iSI Appendix, Fig. S9A) of the tunica media were increased 7 d after ligation in WT mice. In contrast, none of the changes in the P-CREB (+) cell ratio . These results are consistent with the hypothesis that caveolae and CaMKK2 are critical for high pressure induced CREB phosphorylation, macrophage accumulation and vascular remodeling.Finally, we examined the effects of STO609 on high pressure-induced vascular remodeling in vivo. STO609 (1 mg/kg) was administered intraperitoneally before surgery, and also three times per week after surgery A. STO609v1.2/CaMKK2/CaMK1a) that is located within caveolae of vascular myocytes. Increasing transmural pressure in the mesenteric artery by ligation in vivo significantly enhanced CREB phosphorylation, macrophage accumulation, and medial layer remodeling in cav1- and CaMKK2-dependent manner  can induce translocation of CaMKK2 into the nucleus and CaMK4 activation, resulting in alternative splicing of BK channels. This has been termed “excitation-alternative splicing coupling,” and an increase in action potential duration was also observed. Our data also suggest a slight decrease in cell surface levels of CaMKK2 30 min after 60 mM K+ stimuli . Cav1 binding motifs are well characterized , which is consistent with the predominant caveolar localization of these molecules. It is known that CaMK1a can phosphorylate CREB upon Ca2+/CaM binding and phosphorylation of Thr177 by CaMKK2 (312VVRHMRKLQL321) (263KR264)  demonstrate that maintained high pressure applied to mesenteric artery causes CREB phosphorylation in vascular myocytes, as well as macrophage accumulation in adventitia and significant medial remodeling. These important changes are induced by activation of the Cav1.2/CaMKK2/CaMK1a axis. We suggest that high pressure can activate Cav1.2 in vascular myocytes directly via membrane stretch  and cav1-KO mice were obtained from Japan SLC and the Jackson Laboratory, respectively (7BL/6 and+ or 60 mM K+ Hanks solution using a smoothened needle inserted into the first branch of the mesenteric arteries (details in SI Appendix). The perfusion rate was maintained at a 0.15 mL/min to create physiological shear force (∼5 dyn/cm2) and to avoid endothelial cell activation using a peristaltic pump. In the present study, second- and third-order mesenteric arteries were used unless otherwise stated.The mesenteric arterial beds were perfused with 5 mM K2 for 5 to 7 d to expand semiconfluent. Then, medium was switched to DMEM with 0.5% FBS, 1× ITS  supplement and 200 μM L(+)-ascorbic acid sodium for an additional 2 to 3 d. siRNA or viral vectors were applied during this period (details in SI Appendix).Single mouse mesenteric artery myocytes were freshly isolated from second- and third-order mesenteric arteries . To obtaSI Appendix).From three adjacent second-order mesenteric arteries, the first and third arteries were ligated at the distal end of the vessels using 8-0 nylon suture . This crSI Appendix).Cav1 and CaMKK2 was labeled with mCherry or GGECO1.1  at the NF/F0, where F is the sum fluorescence intensity within the regions of interest  in the TIRF area during measurements, and F0 is the baseline F value obtained as the average intensity of the ROI for 5 s before stimulation. GGECO1.1 puncta whose maximal increase in fluorescence intensity due to depolarization stimulus was higher than 5× SD of baseline values were accepted as positive GGECO1.1 puncta (details in SI Appendix).Two-dimensional single-molecular imaging were done using a TIRF imaging system (Nikon) , 19. ForSI Appendix).Confocal images were obtained using a laser-scanning confocal fluorescent microscope . For P-CREB analysis, fluorescence signals from secondary antibodies binding to P-CREB primary antibodies and nuclear indicators (Hoechst or TO-PRO3) were converted into binary images. Next, fluorescence signals of nuclei colocalized with P-CREB signals were extracted by image arithmetic operations using the NIS Elements software. The ratio of the number of nuclei colocalized with P-CREB signals to the total number of nuclei was calculated. For each artery, three image sections (215 μm × 215 μm) were acquired and the calculated ratio values were averaged . When Ca2+ responses of myocytes transfected with PV-NLS-GFP were recorded, cells were loaded with 6.7 μM CaSiR/AM and Hoechst. ROIs corresponding to whole-cell regions and nuclei were created according to the fluorescence of CaSiR and Hoechst, respectively. The sum of CaSiR intensity and area corresponding to cytosol were calculated by subtracting these values of nuclei from those of whole-cell regions.Intracellular Cat test after the application of the F test. Data from more than two groups were compared using one-way or two-way ANOVA followed by Tukey or Dunnett tests. In all cases, P < 0.05 was considered to be significant. All data were obtained from at least three independent experiments.Pooled data are shown as the mean ± SEM. The significance of differences between two groups was evaluated using the two-tailed SI Appendix. Details are also provided of sources and reference numbers for reagents, recombinant DNA, and oligonucleotides necessary for replication in SI Appendix.All details of immunostaining, ELISA, histological analysis, siRNA knockdown, real-time qPCR, RNA-seq analysis, in situ PLA, and drugs are provided in Supplementary File


Intracranial atherosclerosis (ICAS) is one of the most common causes of ischemic stroke, but there are few animal models that can recapitulate its pathological features. In this study, we examined ICAS pathological features and anatomic distributions using three types of hyperlipidemic rabbit models. We also investigated the effect of different lipoprotein profiles and hypertension on ICAS.We examined Watanabe heritable hyperlipidemic (WHHL) rabbits, apoE knockout (KO) rabbits and wild-type rabbits (WT) fed a cholesterol diet, in addition to WT rabbits fed a standard diet as a control. The whole brain was dissected and embedded in paraffin. Serial sections were stained with either hematoxylin/eosin or elastica van Gieson, or immunohistochemically stained with monoclonal antibodies against macrophages and smooth muscle cells. We investigated (1) the presence of cerebral atherosclerosis; (2) the lesion locations in the cerebral arteries; (3) the degree of lumen stenosis; (4) pathological features and cellular components of the lesions in these rabbits; and (5) whether hypertension affects ICAS.ICAS was detected in apoE and WHHL rabbits, but not in WT rabbits. Compared with apoE KO rabbits, WHHL rabbits had greater ICAS. The lesions of cerebral atherosclerosis were mainly distributed at the bifurcations of the posterior cerebral artery, basilar artery and vertebral artery, and they were basically characterized by smooth muscle cells and extracellular matrix with few macrophages. The extent of the ICAS in WHHL rabbits was significantly increased by hypertension.ICAS was detected in WHHL and apoE KO rabbits, and occurred in specific locations in the cerebral arteries. Hypertension promotes the development of ICAS in the setting of hypercholesterolemia. Cerebral atherosclerosis is widely considered the most important cause of ischemic stroke. The prevalence of cerebral atherosclerosis is as high as 50% of ischemic stroke patients in different populations –3. HowevIt is well known that cerebral atherosclerosis develops later and exhibits more variability among different populations than aortic and coronary atherosclerosis , 5. HumaAs the pathological features and pathogenesis of cerebral atherosclerosis may differ from those of extracranial atherosclerosis, it is necessary to develop appropriate animal models. The cerebral arteries unlike extracranial arteries are generally resistant to hypercholesterolemia in experimental animals –18; therIn this study, we pathologically examined cerebral atherosclerosis in rabbits. For this purpose, we used three kinds of hypercholesterolemia rabbits along with normal control rabbits. ApoE KO and WT NZW rabbits aged 7 months  were fed a cholesterol diet for 16 weeks. The cholesterol diet used for this experiment was composed of the normal standard diet  , which was supplemented with 0.3% cholesterol and 3% soybean oil, which did not affect the body weight . These tAfter fasting for 16 hours, blood was collected from rabbits, as described previously . In brievia the common carotid artery until the liquid from the internal jugular vein became clear.To map the lesion distribution in the cerebral vascular system, we first performed rabbit brain vascular casting. For this purpose, three NZW rabbits were sacrificed by intravenous injection of an overdose of sodium pentobarbital solution (5ml per rabbit). A longitudinal incision was made in the middle of the neck to expose the common carotid artery. Then, the proximal part of the carotid common artery was ligated and a “V” shape surgical incision was made 5cm away from the proximal ligation, through which one 16-gauge catheter  was inserted into the bilateral common carotid arteries and fixed stably with sutures. After cutting the internal jugular vein, rabbits were perfused with saline solution The casting solution was prepared according to the manufacturer’s protocol. Briefly, red pigment  was added in the amount of 2% to Base solution A and stirred vigorously for intensive mixing. Then, the solution was divided into two equal parts and half was prepared with 40% of Catalyst , whereas the remaining solution was mixed with Promoter C  completely. Afterward, the casting agent solution was injected at approximately 8-10ml/min until the casting agent was visualized in the vertebral arteries. The tissue was corroded in 20% KOH solution  for approximately 48h at 4°C. Vascular casting was collected, and the peripheral tissues were washed off carefully and kept at 37°C for drying.The whole brain was isolated and fixed in 10% buffered formalin solution. After fixing, the whole brain was dissected into 10 slices (approximately 10mm thick) at intervals of 3mm in the region of posterior circulation and 6mm in the region of anterior circulation to prepare paraffin specimens  and elastica van Gieson (EVG) staining. The lesions were also immunohistochemically stained with monoclonal antibodies against rabbit macrophage RAM11  and smooth muscle α-actin HHF35 . HE-stained specimens were first examined under a light microscope to find lesions along with evaluations of the lesion location and pathological features. The lumen stenostic rate was measured using EVG-stained specimens and calculated as atherosclerotic lesion area/lumen area x % as we did for coronary atherosclerosis . Lesionat-test and ANOVA were used. P-values less than 0.05 were considered significant.All data were expressed as the mean ± SEM. Data were analyzed using GraphPad Prism 7.0 . The Shapiro-Wilk test was used to verify the normal distribution of all data. The Student’s As shown in To investigate the cerebral arterial pathology, we first performed cerebrovascular casting in order to illustrate the anatomic distribution of all arteries in the brain using normal WT rabbits Figure 2We then examined apoE KO rabbits fed a cholesterol diet because apoE KO rabbits on a normal regular diet exhibited mild hyperlipidemia, which is not atherogenic . Among 1In contrast to WT and apoE KO rabbits fed a cholesterol diet, 87% (13/15) of WHHL rabbits exhibited differing degrees of cerebral atherosclerosis in 3 major branches: PCA (43%), BA (73%) and VA (26%), but not in other branches Figure 2p<0.01) due to significantly increased macrophages and smooth muscle cells in the lesions of hypertensive WHHL rabbits (p<0.05).As 87% of WHHL rabbits exhibited cerebral atherosclerosis, we further investigated the influence of renovascular hypertension on lesion formation in these rabbits. As shown in In the current study, we performed a systemic examination of cerebral atherosclerosis using three hypercholesterolemic rabbits with different lipoprotein profiles. Similar to the previous study , cholestHowever, only a few apoE KO rabbits exhibited tiny lesions in the cerebral arteries, which is consistent with other studies , 34. ThiFurthermore, we analyzed WHHL rabbits in which aortic and coronary atherosclerosis started to form as early as 3 months. Although WHHL rabbits have been extensively studied, the incidence and distribution of cerebral atherosclerosis was not clarified. Differing from cholesterol-fed WT and apoE KO rabbits, WHHL rabbits had high levels of plasma LDLs, similar to human familiar hypercholesterolemia. In the current study, WHHL rabbits aged 12 to 31 months exhibited a higher incidence of cerebral atherosclerosis 87%) and severe stenosis than apoE KO rabbits, suggesting that LDLs are more atherogenic than β-VLDLs in terms of cerebral atherosclerosis, which is consistent with the human imaging and pathological observations % and sev, 36. FurIn spite of this, the lesions of cerebral atherosclerosis in WHHL rabbits are structurally stable, and characterized by smooth muscle cells and extracellular matrix unlike the foam cell-rich lesions observed in aortic atherosclerosis in these rabbits. These features are similar to those of human intracranial atherosclerosis, which comprises more stable lesions than extracranial atherosclerosis , 6, 7. HIn conclusion, cerebral atherosclerosis can be generated in both apoE KO and WHHL rabbits, but the latter exhibited more lesions. The high incidence and severity of cerebral atherosclerosis in WHHL rabbits suggest that LDLs are more atherogenic than remnant lipoproteins. Furthermore, hypertension promotes the development of cerebral atherosclerosis in the setting of hypercholesterolemia.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Animal Care Committee of the University of Yamanashi.JF designed the experiments and provided the resources. XT, MN and HZ performed the experiments and analyzed the data. LC, YC and HY bred animals and participated in experiments. XT and JF wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported in part by JSPS KAKENHI (JP17K08783 and JP15H04718), the National Natural Science Foundation of China (No. 81941001 and 82100482), the JSPS-CAS Bilateral Joint Research Program (JPJSBP 120187204) and Innovation team program supported by Guangdong Province (2020KCXTD038). XT was a recipient of the China Scholarship Council.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In our publication, we concluded that neither cytosolic Ca2+i nor pHi were involved, and suggested a direct effect of anesthetics on gap junction channel proteins. However, while a direct effect cannot be excluded, based on the potentiating effect of caffeine and theophylline added to anesthetics and data published over the past three decades, we are now reconsidering our earlier interpretation and propose an alternative hypothesis that uncoupling by heptanol, halothane and isoflurane may actually result from a rise in cytosolic Ca2+ concentration ([Ca2+]i) and consequential activation of calmodulin linked to gap junction proteins.Cell–cell communication via gap junction channels is known to be inhibited by the anesthetics heptanol, halothane and isoflurane; however, despite numerous studies, the mechanism of gap junction channel gating by anesthetics is still poorly understood. In the early nineties, we reported that gating by anesthetics is strongly potentiated by caffeine and theophylline and inhibited by 4-Aminopyridine. Neither Ca A gap junction hemichannel is made of six radially arranged proteins named connexins in vertebrates and innexins in invertebrates. Connexins/innexins contain two extracellular lops EL and El2 2+]i) i)  in the h2+]i) ,5,6,7. Sin (CaM) ,7,8,9,10in (CaM) ,12,13 prIn the last four decades, many studies have reported that gap junction channels are sensitive to anesthetics. Channel gating induced by long-chain alcohols (heptanol and octanol) was first reported by Johnston and coworkers  in crayfIn spite of over four decades of research, the mechanism by which anesthetics cause gap junction channel gating is still poorly understood. Johnston and coworkers  proposed2+ and pH in crayfish axons uncoupled by heptanol, halothane and isoflurane i rise, and increased Ca2+ sensitivity in CaM.In 1991, our data seemed to also exclude the role of cytosolic Caoflurane . Howeversthetics , we are sthetics . As a re2, 13.5 and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 5 (pH 7.5). Either 2.8–5.6 mM 1-heptanol, 9.5–28.5 mM halothane or 23.6 mM isoflurane was added to SES in the presence and absence of either 10–20 mM caffeine or 10-20 mM theophylline. For testing the effect of low pHi i and [H+]i were measured with ion-sensitive microelectrodes based on neutral-carrier sensors. Ca2+ microelectrodes used the calcium cocktail (ETH 129) and H+ microelectrodes used the proton cocktail tri-n-dodecylamine. At the time, we thought that these ion-selective microelectrodes were insensitive to anesthetics. Later on, however, ion-selective microelectrodes were proven unreliable in the presence of anesthetics [2+]i or [H+]i caused by acetate-induced cytosolic acidification o, ranging from 7 to 27 mM, nor blockers of Ca2+ entry, such as Cd2+ (500 μM) or nisoldipine (10 μM), significantly change the heptanol-uncoupling efficiency . Wi. Wi16]. 2+]i and [H+]i. However, as mentioned in the previous section, after our publication, ion-selective electrodes have been reported not to be very reliable in the presence of anesthetics i measured with Quin-2, in exocrine pancreas treated with heptanol or octanol at concentrations that greatly increased amylase release. However, this is curious because amylase release is known to be caused by a sustained [Ca2+]i rise [2+ sensitivity of Quin-2 might have been affected by alkanols. In agreement with this report [2+ [2+]i rise. In a careful double whole-cell patch-clamp study on embryonic chick cardiac cells, Veenstra and DeHaan i rise ,27,28; ts report , some stport 2+ ,30,31,32,31,322+id DeHaan  reportedir words : “A poss et al.  indicat2+]i by 20–70% in mouse whole-brain synaptosomes [2+]i rise, monitored by the Ca2+-indicator arsenazo III, in octanol-treated squid axons [2+]i by a Ca2+ leak through both inositol -trisphosphate-(IP3)- and ryanodine-receptor channels (RyR) [2+]i increase [2+] i by releasing it from the sarcoplasmic reticulum (SR) via the RyR’s Ca2+-release channel  . In vasc+ uptake ; the haly a role . In a moy a role , and thechannels . In frog channel . 2+]i in heart i by releasing it from Ca2+ stores i by releasing it from the SR via the RyR’s Ca2+-release channel i and causes Gj to drop by 95% in N2a cells expressing human Cx43, but not in cells expressing human Cx40. Significantly, the human Cx40 does not have the CaM-binding site at the second half of the cytoplasmic loop (CL2), while Cx43 does i [2+]i. The reason that now, three decades later, we are reevaluating the earlier interpretation of the lack of Ca2+i participation in the effect of heptanol, halothane and isoflurane on gating is based on a number of facts, as follows:The ion-selective electrodes  may not 2+ sensitivity [2+]i.Anesthetics increase the CaM’s Casitivity ,80. ThisHalothane binds to CaM .2+ release via both IP3- and RyR-receptor channels [Anesthetics cause Cachannels .2+]i by releasing Ca2+ from stores i cause gap junction particle crystallization in heart i . Indeed,2+ from internal stores and activating CaM. We realize that this hypothesis needs to be further tested experimentally and it should be stressed that our data were obtained entirely on crayfish axons, which express innexins rather than connexins. Furthermore, there are mysterious data; for example, there is evidence for a strong inhibitory action of 4-aminopyridine on the heptanol effect (+ channel blocker, the effect of other K channel blockers should be tested as well. Moreover, the CaM hypothesis needs to be tested with CaM inhibitors, inhibition of CaM expression and overexpression of CaM mutants.In view of these findings, while we cannot exclude a direct effect of anesthetics on gap junction proteins and/or lipids, we propose the hypothesis that heptanol, halothane and isoflurane induce gap junction channel gating by releasing Cal effect A. Since


Differentially expressed gene analysis, pathway analysis and modelling for predicting platelet decrease in AML were performed. DEG analysis and pathway analysis revealed 157 genes and eight pathways specific for PD-AML, respectively. Most of the eight pathways were significantly involved in G-protein-coupled receptor-related pathway, cytokine-related pathway, and bone remodeling pathway. Among the key genes involved in at least one pathway, three genes including CSF1R, TNFSF15 and CLEC10A were selected as promising biomarkers for predicting PD-AML (0.847 of AUC in support vector machine model). This is the first study that identified biomarkers using RNA expression data analysis and could help understand the pathophysiology in AML with low platelet count.Acute myeloid leukemia (AML) patients are at risk of bleeding due to disease-related lack of platelets and systemic coagulopathy. Platelets play a role in hemostasis. Leukemic blasts have been shown to alter platelet activation in vitro. Here we investigated biomarkers associated with thrombocytopenia in normal karyotype AML (NK-AML). From The Cancer Genome Atlas database, case-control study was performed between normal karyotype (NK) platelet-decreased AML (PD-AML, platelet count < 100 × 10 In patit counts .Malignant cells activate platelets and the activated platelets can attach to the cancer cells, forming a layer of platelets, hiding the malignant cell from cellular components of the immune system ,4,5. ThePlatelets and AML cells are known to be mutually affected. In AML, platelets show a wide range of defects, including abnormal metabolism, lifespan and aggregation, and additional platelet dysfunctions . PlateleAlthough interactions between leukemia cells and platelets have been observed in vitro and in vivo, the factors contributing to thrombocytopenia remain unknown. The prognostic value of platelet production, such as platelet count and megakaryopoiesis, has not been clearly determined in AML ,16. Howe9/L because the normal platelet count ranges from 150 × 109/L to 450 × 109/L, but there is some dispute as to whether platelet numbers in the range 100 × 109/L to 150 × 109/L should be indicated as having true or borderline thrombocytopenia [Thus, we investigated the biomarkers associated with thrombocytopenia in this report. We studied AML patients with normal karyotypes (NK-AML) to eliminate the effects of chromosomal abnormalities on platelets. The criterion for thrombocytopenia was set at 100 × 10ytopenia .9/L, the patients were classified into PD-AML  and PND-AML  groups. The clinical features of the patients are shown in p = 0.078). Except for platelet count and hemoglobin level, there was no significant difference between the PD-AML and PND-AML groups in the other clinicopathological factors, including age, gender, ethnicity, bone marrow findings, and mutation profile.We enrolled 37 NK-AML patients. Based on a platelet count cut-off value of 100 × 10Homo sapiens ), G-protein-coupled receptors (GPCR) downstream signaling, signaling by GPCR and C-type lectin receptors ), cytokines and inflammatory response ), GPCR signaling and Janus kinase signal transducer and activator of transcription (JAK-STAT) Molecular Variation 1 ) and bone remodeling ; BioCarta, http://www.biocarta.com/ (accessed on 1 November 2021))  were upregulated while nine genes  were downregulated in the PD-AML group, compared with the PND-AML group (IFNB1 (five pathways), IL1B (five pathways), PDGFA (four pathways), and IL5RA (four pathways). These genes were mainly associated with cytokine-related and GPCR-related pathways ) . Of the ML group . To invepathways .CSF1R, TNFSF15, CLEC10A, CCR5, PDGFA, WNT10A, TNFRSF11A, MUC1, DAGLA, IL1B, RAMP1, SUCNR1, MUC16, PLXNB1, and IL5RA) were calculated . To evaluate the performance of the models, ROC curves were plotted and are shown in After the DEG analysis, pathway analysis and filtering steps, 15 genes were found to be involved in cell signaling pathways and to have statistically different expression levels between PD-AML and PND-AML. Next, the MDG of 15 genes  and is mainly expressed in macrophages. The receptor regulates the production, differentiation, and function of macrophages by CSF1. Macrophages are known to have a pro-inflammatory or anti-inflammatory phenotype [CSF1R expression decreases the overall survival of follicular lymphoma, correlates with increased invasiveness and adverse prognostic factors such as high histological grade of breast cancer, and shows an advanced clinical stage at detection of breast cancer [TNFSF15 encodes tumor necrosis factor superfamily-15, a multifaceted cytokine, is mainly produced by endothelial cell in established blood vessels, and in turn inhibits angiogenesis [CLEC10A encodes C-type lectin domain containing 10A. CLEC10A is expressed on a number of immune cells and involved in CLEC pathway and many immune system-related processes [CSF1R, which is known as a poor prognostic factor and is the most important biomarker in our study, were downregulated in PD-AML, it is possible that CSF1R may be correlated with low platelet counts in NK-AML. These findings need further research.Second, the importance of 15 genes selected through sequential filtering steps was calculated as MDG scores to determine the highly specific genes associated with PD-AML. The top three genes were n PD-AML . The assn PD-AML A–C. In t CLEC10A D–F. CSF1henotype . In earlhenotype ,34,35. Thenotype . The inthenotype . Severalt cancer ,39. Thust cancer . TNFSF15ogenesis . Increasogenesis ,43. CLECrocesses . The CLErocesses ,45. The rocesses . The assFinally, three gene models were generated using LR, RF, and SVM. Comparing these models, it was observed that the SVM and RF algorithms performed better than the LR algorithm . The sensitivities and specificities of the models were less than 70% and greater than about 80%, respectively. The relatively low sensitivities may be due to the delayed diagnosis of some cases in the PND-AML group. If this hypothesis is reasonable, future studies should set a higher sensitivity cut-off.9/L which does not reflect the actual risk of bleeding in acute leukemia because platelets are usually transfused when the platelet count is less than 10 × 109/L to 20 × 109/L. Second, RNA expression data in the TCGA downloaded from the GDAC did not contain data from normal populations. The biomarkers in this study were selected and evaluated using the significant difference in RNA expression between PD- and PND-AML groups. Hence, the models for the biomarkers showed excellent performance in the ROC analysis. However, if data from normal populations were included, more meaningful results could be obtained. Third, the size of the patients studied was small and there was a lack of validation cohort to correctly assess the power of the algorithms. To validate our models, we used LOOCV which is a special case of k-fold cross-validation with k = n, the number of observations. Although the LOOCV is feasible when the sample size is small, we have not used methods such as meta-analysis or validation in other cohorts. However, it is expected that these limitations will be resolved through further studies. Although we did not evaluate the relationship between the biomarkers and the risk of bleeding in this study, we identified a biomarker related to low platelet counts in NK-AML and further studies using this may be possible.This study has several limitations. First, we divided the two groups based on a platelet count cut-off of 100 × 10In conclusion, we identified the biomarkers related to low platelet counts in NK-AML by RNA expression data analysis. Although there have been studies about the interaction between platelets and leukemic cells in vivo and in vitro, there have been no studies that identified biomarkers using RNA expression data analysis. This can help clarify the pathophysiology of AML with low platelet count. To fully study the clinical significance of low platelet counts in NK-AML, a large-scale multi-cohort study must be done.https://gdac.broadinstitute.org/ (accessed on 1 November 2021)). Cases without RNA expression data or clinical information were excluded. NK-AML cases with no definite driver mutation were selected for analysis. Patients with NPM1 and CEBPA mutations, which are often observed in NK-AML, were also excluded. To investigate the clinicopathological difference between platelet-decreased AML (PD-AML) and platelet-not-decreased AML (PND-AML), AML cases with platelet counts <100 × 109/L were selected as case group while AML cases with platelet counts ≥100 × 109/L were considered the control group.RNA expression data and clinical information (Gene level 3) of 200 AML cases in The Cancer Genome Atlas (TCGA) were downloaded from the Genomic Data Analysis Center (GDAC) Firehose Repository ). Twenty-seven pathways with q value < 0.05 were selected. After literature review and manual curation, eight pathways previously reported to be related with platelet signaling were finally selected [To select DEGs, we performed t-test for the genes that passed the normality tests (Kolmogorov–Smirnov and Shapiro–Wilk test) using the RNA expression values of each gene between the PD-AML and PND-AML groups. Otherwise, for the genes that did not pass the normality tests, Wilcoxon rank sum test and DESeq2 package in R  were appselected ,29,30,48m is the number of classes in the target variable and if is the ratio of this class. The higher the MDG value, the higher the importance of the feature in the model. We selected the top ranked features (MDG > 1.5) that significantly decreased in the Gini impurity index.Genes with normalized read count < 1 in >50% of the cases were excluded. To select the key features discriminating between the two groups, we calculated the mean decrease Gini (MDG) for each gene using the randomForest package in R . The GinUsing selected features, logistic regression (LR), randomForest (RF), and support vector machine (SVM) were performed. The models were validated using leave-one-out cross validation (LOOCV). To evaluate the performance of the models, the receiver operating characteristic (ROC) curve was plotted using the R package, ROCR . The arep values were inferred from an independent t-test, Wilcoxon rank sum test, chi-square for trend or Fisher’s exact test, as appropriate. All statistical analyses were performed using R version 3.6.3 (https://www.r-project.org (accessed on 18 July 2021)). Principal component analysis (PCA) and visualization were performed using the factoextra R package (https://cran.r-project.org/web/packages/factoextra (accessed on 1 November 2021)). The heatmap was drawn using the R package gplots (http://cran.r-project.org/web/packages/gplots (accessed on 1 November 2021)). Network analysis and visualization were done using Cytoscape software (version 3.8.2) [p values < 0.05 were considered statistically significant.Clinical characteristics were presented as number or mean with percentage or standard deviation and n 3.8.2) . p value


Mutations in CASQ2 have been linked to the arrhythmia catecholaminergic polymorphic ventricular tachycardia (CPVT2) that occurs with acute emotional stress or exercise can result in sudden cardiac death (SCD). CASQ2G112+5X is a 16 bp (339–354) deletion CASQ2 mutation that prevents the protein expression due to premature stop codon. Understanding the subcellular mechanisms of CPVT2 is experimentally challenging because the occurrence of arrhythmia is rare. To obtain an insight into the characteristics of this rare disease, simulation studies using a local control stochastic computational model of the Guinea pig ventricular myocyte investigated how the mutant CASQ2s may be responsible for the development of an arrhythmogenic episode under the condition of β-adrenergic stimulation or in the slowing of heart rate afterward once β-adrenergic stimulation ceases. Adjustment of the computational model parameters based upon recent experiments explore the functional changes caused by the CASQ2 mutation. In the simulation studies under rapid pacing (6 Hz), electromechanically concordant cellular alternans appeared under β-adrenergic stimulation in the CPVT mutant but not in the wild-type nor in the non-β-stimulated mutant. Similarly, the simulations of accelerating pacing from slow to rapid and back to the slow pacing did not display alternans but did generate early afterdepolarizations (EADs) during the period of second slow pacing subsequent acceleration of rapid pacing.Calsequestrin Type 2 (CASQ2) is a high-capacity, low-affinity, Ca The net result in an increase in myocyte Ca2+ content .Increase in luminal dependence: CASQ2 is the major Ca. Gyorke  reported(c)cycle): Increased Ca2+ in cytosol due to an increase in L-type current as well as increased RyR2 channel activity, affects the SERCA2A cycling rates, and they pump Ca2+ back to SR rapidly increasing SR load  was significantly (p < 0.05) from the mutant myocyte, the wild-type myocyte with β-adrenergic stimulation, and the mutant myocyte with β-adrenergic stimulation. When compared to the experiments by Knollman and co-worked in transgenic mice ds in dyad in comparison to bulk myoplasm. Kubalova [2+ inactivation of L-type channels—a slow phase that depends on Ca2+ flow through the channels (Ca2+ current-dependent inactivation) and a fast one that depends on Ca2+ released from the SR, Ca2+ (Ca2+ release-dependent inactivation). Hence, SR released Ca2+ is the most effective inactivation mechanism in the inhibition of Ca2+ entry through the channel. The inactivation of the L-type Ca2+ channel shown to depend linearly on the rate and magnitude of the Ca2+ release from the RyRs [2+ serves as the feedback mechanism to the L-type channels and their amplitude decreases with higher SR Ca2+ release [L-type channels have two types of inactivation states–voltage-dependent inactivation and CaKubalova  distinguthe RyRs . Experim release .2+. The end-systolic SR volume is increased after a shorter beat which leads to a greater end-diastolic volume for the next longer beat and this process is more prominent in the rapidly paced heart [ncx is responsible for the prolongation of APD during large Ca+ transient [+ ions enter the cell for every Ca2+ ion extruded, this increase in driving force elevates the inward membrane current which prolongs the APD.Our finding agrees with Saitoh et al. , who demed heart . The APDed heart . In thisransient . Since t2+ overloading in this case. The alternate beat missing is caused by incomplete recovery of Na+ channels from inactivation (h-gate) during the relative refractory period [+ channels are available to initiate the incoming beat. Within the milliseconds of their activation, most of the Na+ channels undergo inactivate which is faster than total deactivation. The inactivated channels gradually reach the closed state and by the time the relative refractory period arrives, the depolarizing stimulus has passed. In rapid pacing, Na+ channels have the relative refractory period where they have not fully recovered from the previous inactivation. During alternans, simulations show a partially inactivated Na+ current as well as alternate action potentials and an increased Incx. The model suggests that the longer the plateau, the larger the Incx, which increases the late influx of Na+ current consistent with experiment [+ channels are still inactivated at the time of new beat Na+ channels fail to activate to depolarize the membrane and the voltage-gated L-type Ca2+ channels fail to activate resulting in a skipped beat.In the pattern of the longer and shorter beat in alternans, sometimes longer beat is prolonged and the shorter beat goes missing. This is also a form of alternans, but the model suggests different mechanisms other than SR Cay period ,52. The periment . When th2+ during diastole and/or excessive Ca2+ influx via L-type channel [2+ overload by increasing RyR2 opening probability allowing for more Ca2+ release from SR. The model simulations also indicated that under these conditions more L-type channels undergo Ca2+-dependent inactivation after a longer beat. After a long beat, there is a very short diastolic phase and might not have enough time required for reloading of SR for the upcoming beat which makes the beat a shorter beat. Although simulations had 40% L-type increases during β-adrenergic stimulation, a diastolic activation of L-type channels and the ensuing Ca2+ influx was never observed. Based on simulation findings, these studies are unable to support the DADs being the mechanism of an arrhythmia in the heart having mutation in the protein of CASQ2 expressing genes under the rapid pacing seen during β-adrenergic stimulation.Many researchers believe there are two ways DADs can occur during β-adrenergic stimulation, the aberrant leaking of SR Ca channel . Observa2+ release caused by intracellular Ca2+ loading, β-adrenergic stimulation during stress or exercise, and a resurgence of electrogenic NCX current [2+ dynamics caused by β-adrenergic receptors in the mutant myocyte caused elongated APD. Weiss et al. [Ca and the second major current is Incx but in the simulations, the primary reason causing EADs is spontaneous Ca2+ release and the secondary causes are Incx and ICa. Iyer et al. [2+ is important in reducing the probability of spontaneous Ca2+ release but the stabilization factor, CASQ2 is deleted in CASQG112+5x mutation and it provoked all the instability in the mutant myocyte. January et al. and Antoons et al. suggested that EADs were caused as a result of reactivation of L-type channels following the prolonged plateau phase however, in the simulations spontaneous Ca2+ release occurs before reactivation of L-type channels [2+ into the dyadic subspace even late in the plateau phase and the same phenomenon is observed in the simulations also but analysis of the simulations could not support it as a primary source of EADs. At the same time, it was seen the electrogenic Incx current is also higher than the prior slow phase.There are three mechanisms of EADs—spontaneous SR Ca current ,56. Hower et al.  stated tchannels ,59. Beca2+ before and after rapid pacing. 2+ level is higher (1092 M vs. 1009 M) than during the slow phase. According to Volders et al. [2+ release during systole, and inward Incx contributes to the production of EADs during β-adrenergic stimulation. The simulations suggests that spontaneous Ca2+ release from the overloaded SR and the elongated APD caused by electrogenic inward current Incx causes EADs to happen. This is consistent with the experimental finding by Priori et al. [2+ uptake of ISO exposed myocytes, Xie et al. [2+ release and EADs. Simulations accelerating pacing from slow (1 Hz) to rapid (5 Hz) and back to slow (1 Hz) did not display cellular alternans but did generate early afterdepolarizations (EADs) during the period of slow pacing that followed the period of rapid pacing. The model suggests that the rapid pacing loads the cytosol and sarcoplasmic reticulum with Ca2+, which in the CPVT mutant with increased RyR2 open probability can trigger spontaneous Ca2+ release which activates Na+-Ca2+ exchange resulting in action potential prolongation. These studies suggest that spontaneous Ca2+ release due to SR overload is the underlying cause of the arrhythmia in these patients.These simulation results are supported by the experimental literature. In experiments, the APD lasted longer due the inward current upon repolarization ,61. Simis et al. , arrhythi et al.  that bloe et al.  were ablThere are potential study limitations. The model simulations would produce consistent results across the 10 simulations for each simulation protocol . This is not unusual for computational studies because parameter variations are chosen that produce the normal and pathological behavior consistently to ease study of the underlying mechanisms. In CPVT in patients, the 4-year ad 8-year observed event rate is 31% and 58%, respectively , with a 2+ dynamics and membrane currents. The simulations show that Ca2+ overload is an important feature in producing the EAD’s seen with the reduction in pacing frequency after a period of rapid pacing. The phosphorylation of the L-type Ca2+ channel increases Ca2+ entry into the myocyte. The phosphorylation of phospholamban increases the sequestration of Ca2+ into the SR. The increase in RyR2 open probability by phosphorylation increases the number of spontaneous sparks (Ca2+ leak) that leads to triggering of the EADs. These defects align with targets of current treatment of CPVT, which includes the administration of β blockers, flecainide to reduce RyR2 opening, L-type Ca2+ channel blockers such as verapamil and use of an implantable cardioverter defibrillator (ICD) [= channel by entering the pore during the open state and getting trapped there preventing further opening [2+ entry by reducing the myoplasmic Na= content thereby enhancing NCX extrusion of Ca2+ [+, decreases intracellular Ca2 by increasing extrusion through NCX. This was shown in the previous study on alternans in a rat ventricular myocyte model by these authors [The simulation studies demonstrated the mechanisms of arrhythmia and the role of aberrant Caor (ICD) ,4,8,68.  opening . On the  of Ca2+ . In the  authors .Many experiments are conducted in mice to understand CPVT because they are mutable to transgenic expression of calsequestrin mutants. This study was performed in the guinea pig, which has a long action potential unlike the mouse, which is more similar to the human action potential. Experiments in mice show that with low pacing rates, there is an increased probability of spontaneous release events late in the action potential compared to wild type ,71. Thes2+ dynamics play a major role for the heart to beat properly. The mutation in the CASQ2 expressing genes affects the Ca2+ dynamics by extending the opening probability of RyR2 channels with the increase in luminal dependence. Based on these simulation results, these simulation studies were able to present that alternans and EADs are the main underlying mechanisms to generate arrhythmia in autosomal dominant CPVT2.Ca2+ dynamics causes a weaker/stronger beat, longer/shorter diastolic interval, smaller/larger Incx and an increase/decrease in Ca2+-dependent inactivation which all affect the SR Ca2+ alternately. When the larger release of SR Ca2+ occurs to the cytoplasm, it ends up creating a stronger beat while smaller SR Ca2+ transients produce a weaker beat developing alternans. It was also confirmed by almost double numbers of Ca2+ sparks were discharged in a stronger beat than the weaker beat. A beat-to-beat change in SR Ca2+ load give rise to Ca2+ alternans which, in turn, result in cardiac alternans and APD alternans. Due to the limitations in our model, model simulations are unable to explain this mechanism by Ca2+ waves or mini waves.A subcellular disruption in Ca2+ release from the loaded SR. The late reactivation of L-type channels and spiking in electrogenic Na+-Ca2+ exchange current also aid to generate them.Though EADs are also to be the precursor of arrhythmia both in tachycardia and bradycardia because it seems phase 2 EADs prefer low pacing rather than high pacing. Our simulation showed that the heart with mutant myocyte sprints with low pacing to high pacing back and forth, it can generate arrhythmia and the EADs are the mechanism behind it. These EADs generate due to the spontaneous Ca


In the current study, we characterized how acute myeloid leukemia (AML) promotes the generation of senescent-like CD8We analyzed NanoString, bulk RNA-Seq and single-cell RNA-Seq data from independent clinical cohorts comprising 1,896 patients treated with chemotherapy and/or immune checkpoint blockade (ICB).+ T cells were impaired in killing autologous AML blasts and that their proportion negatively correlated with overall survival (OS). We defined what we believe to be new immune effector dysfunction (IED) signatures using 2 gene expression profiling platforms and reported that IED scores correlated with adverse-risk molecular lesions, stemness, and poor outcomes; these scores were a more powerful predictor of OS than 2017-ELN risk or leukemia stem cell (LSC17) scores. IED expression signatures also identified an ICB-unresponsive tumor microenvironment and predicted significantly shorter OS.We show that senescent-like bone marrow CD8The IED scores provided improved AML-risk stratification and could facilitate the delivery of personalized immunotherapies to patients who are most likely to benefit.ClinicalTrials.gov; NCT02845297.John and Lucille van Geest Foundation, Nottingham Trent University’s Health & Wellbeing Strategic Research Theme, NIH/NCI P01CA225618, Genentech-imCORE ML40354, Qatar National Research Fund (NPRP8-2297-3-494).  Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease . We rece+ T cell infiltration has been shown to correlate inversely with OS in select tumor types, including AML, because of the establishment of highly dysfunctional T cell states , CTLA4, HAVCR2 (encoding TIM3), CD160, and 2B4 (encoding CD244), and display an impaired ability to secrete effector cytokines and to exert cytotoxic functions. Senescent T cells downregulate costimulatory molecules CD27 and CD28, express senescence-associated surface markers B3GAT1 and KLRG1, as well as MAPK p38 and γ-H2AX intracellular molecules, remain metabolically active and continue to secrete proinflammatory cytokines  in combination with immune checkpoint blockade (ICB) with pembrolizumab  (designated as AZA+Pembro). We integrated these with publicly available gene expression data from multiple cohorts of children and adults with AML to validate our RNA metric of immune effector dysfunction (IED), and we analyzed BM samples collected longitudinally at the time of AML onset and response assessment (https://doi.org/10.1172/JCI159579DS1). The derived gene signatures of IED correlated with molecular features of leukemia stemness and with distinct clinical characteristics. IED gene sets served as a reliable biomarker to stratify OS after standard-of-care therapy and ICB, both in AML and in melanoma   , 17.+ T cells in both experimental conditions. Consistent with previous observations  and nonsenescent (CD3+CD8+CD57–KLRG1–) T cells against autologous AML blasts using an anti–CD3/CD33 bispecific T cell engaging (BiTE) antibody construct (+CD8+CD57+KLRG1+) T cells in baseline BM samples was associated with significantly worse OS (P = 0.004) after treatment with standard chemotherapy (+CD8+CD57+KLRG1+ T cells = 31.9%). Senescent-like T cells measured at time of response assessment in 22 patients from the JHU1 cohort who achieved a CR also correlated with shorter OS  previously shown to be expressed by circulating CD8+ T cells from patients with AML  and correlated the expression of genes in the immunosenescence signature with markers of immune cells and leukemia blasts.Transcriptional profiling of the tumor microenvironment (TME) has been used to identify immunological signatures, characterize biological processes, and develop predictors of protective immunity , 26. We with AML  and to bwith AML  and on dwith AML . We usedCD34, CD38, IL3RA, KIT), or with markers of accessory cells of the monocyte/macrophage lineage  gene sets within the BM microenvironment might reveal core biological processes involved in antitumor immune responses and in therapeutic outcomes. To this end, both TCGA-AML and Beat-AML cases were split into quartiles based on average expression levels of the 7 T cell senescence-associated genes. Gene set enrichment analysis (GSEA) was used to identify core gene sets accounting for the enrichment signal in immunosenescencehi (highest quartile) versus immunosenescencelo cases (lowest quartile). Among the 4,872 curated gene sets from the ImmuneSigDB, only gene sets with a FDR of less than 0.05 and a normalized enrichment score of more than 2.0  were carried forward for leading-edge analysis. We reasoned that those genes contained in the leading edge would represent biologically related genes enriched for a phenotype of interest , dysfunction and/or exhaustion    , and sennd ETS1) . We herend ETS1) , solid tnd ETS1)  34, 35), 35CXCR3nd ETS1)  2). The. TheCXCRnd ETS1) . Furthernd ETS1) , as wellnd ETS1) –39 (Suppnd ETS1) , B and Cnd ETS1) –43, we fnd PD-L1 . A princnd PD-L1 .https://www.leukemia-net.org/home/) risk category, or mutation count  revealed significantly higher IED172 scores at baseline in patients with primary induction failure  compared with those achieving complete remission . We. We46). on count , A and Bon count  or in thutations . These fated AML , 48 and ated AML . The anaTP53 and RUNX1 mutational status in de novo AML  score has previously been associated with poor clinical outcomes and with novo AML , 52. Thenovo AML , A and Bnovo AML , immune novo AML  53), an, anTP53 novo AML . This finovo AML  54). Wh. WhTP53 edict OS .FLT3-ITD and NPM1-mutational status at diagnosis  statistical method to fit an L1-regularized linear model  that reviagnosis . On straividuals , 30 wereividuals . High PIAs shown in + and NK cells, characterized by the expression of inhibitory molecules, and inversely correlated with leukemia burden  and the same weights as those provided in the original publication , A and Bhi cases , C–F. SpS of 11% , were fuy of 55% . Furtherectively , G–J.2 statistics (P < 0.001) in this modeling framework than the LSC17 score (P = 0.001). In addition, the interaction between the 2 continuous variables was statistically significant (P = 0.013), indicating that a higher PI20 value will increase the association between the LSC17 score and OS. Taken together, these analyses suggest that the PI20 score and its integration with the LSC17 score could provide accurate prognostic risk stratification.We formally tested the interaction between senescence- and stemness-related pathways by a multiplication term in the Cox PH model. As shown by the Wald χatistics , the PI2n = 31 paired BM samples; P = 0.0044) and OS   . In lineE134589)  and in BE134589) . FinallyE134589) . These dP = 0.011; P = 0.0015; To further examine the effect of induction chemotherapy on IED scores, we generated nCounter gene expression data using serial BM samples from a large number of donors with newly diagnosed AML (the Studien Allianz Leukämie [SAL] and Johns Hopkins University 2 [JHU2] cohorts), totaling 90 patients and 183 BM specimens that were longitudinally collected at time of diagnosis and response assessment) . The IEDCD3G, CD8A, CD8B, CD28, GZMK, and GZMB), cosignaling molecules , myeloid  and dendritic cell differentiation , and in chemoattraction  after chemotherapy , leukemia stem cells , and candidate genes overexpressed in AML  were downregulated after chemotherapy, consistent with recent scRNA-Seq and immunohistochemistry data  in the PIlo group, prompting us to examine the ability of a published IFN signature to predict OS . The analysis of scRNA-Seq data from 8 patients with relapsed/refractory AML treated with AZA + nivolumab ; ivolumab  confirmeecursors . In lineecursors . Compare8 scores . Taken tGZMA, GZMB, PRF1, KLRD1, and NCR1), T cell and NK cell cosignaling molecules , cytokine receptors , IFN responsiveness (ISG20), and T cell signaling  . We identified 43 genes that were significantly differentially expressed in both postchemotherapy and post-AZA+Pembro BM samples (We sought to identify genes at the intersection of responses to chemotherapy and AZA+Pembro. We examined DEGs between matched posttreatment (available in 31 patients after cycle 2) and pretreatment BM samples in the immunotherapy cohort. Treatment with AZA+Pembro resulted in upregulation genes associated with immune effectors (d ZAP70) . As withd ZAP70) , AZA+Pem samples , and we  samples . This pr samples .lo cases, patients in the PI24hi group had lower numbers of lymphocyte clusters and tumor infiltrating lymphocyte (TIL) patches and higher myeloid/macrophage RNA scores  . The PI2A scores . Further subtype ; P = 0.0n = 41) or combination anti-PD-1 + anti-CTLA-4 , and had significantly higher PFS and OS rates  from pretherapy lesions of nonresponders, i.e., patients with progressive or stable disease, compared with responders  and were found to be associated with leukemia stemness and with poor response to induction chemotherapy. OS prediction afforded by validated clinical cytogenetic categories and experimental LSC17 signatures  and associated clinical data were obtained with written informed consent from the donors in accordance with the Declaration of Helsinki on research protocols approved by the Ethics Committee of TU Dresden and Studienallianz Leukämie, Germany (EK98032010), and by the Institutional Review Boards of the Children’s Hospital of Philadelphia (no. 10-007767) and Johns Hopkins University.S Rutella and LL conceptualized and designed the study. S Rutella, JV, FM, TY, RM, S Reeder, VR, and LL developed the methodology. RM, HA, MK, HAK, JFZ, AA, BW, HAA, MDM, SKT, MB, IG, and LL acquired, obtained consent from, and managed patients, as well as processing patient samples. S Rutella, JV, FM, S Reeder, TY, RM, BD, BRB, VR, BW, HAA, MDM, SKT, MB, IG, and LL analyzed and interpreted data. S Rutella wrote the manuscript. S Reeder, JV, FM, RM, BD, BRB, JFZ, SKT, MB, IG, LL reviewed and/or revised the manuscript. S Rutella and LL supervised the study.


However, there is emerging evidence that Ca2+ mishandling alone is insufficient to fully explain diabetic heart dysfunction, with focus shifting to the properties of the myofilament proteins. Our aim was to examine the effects of diabetes on myofilament Ca2+ sensitivity and Ca2+ handling in left ventricular tissues isolated from the same type 2 diabetic rat hearts. We measured the force-pCa relationship in skinned left ventricular cardiomyocytes isolated from 20-week-old type 2 diabetic and non-diabetic rats. Myofilament Ca2+ sensitivity was greater in the diabetic relative to non-diabetic cardiomyocytes, and this corresponded with lower phosphorylation of cardiac troponin I (cTnI) at ser23/24 in the diabetic left ventricular tissues. Protein expression of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), phosphorylation of phospholamban (PLB) at Ser16, and SERCA/PLB ratio were lower in the diabetic left ventricular tissues. However, the maximum SERCA Ca2+ uptake rate was not different between the diabetic and non-diabetic myocardium. Our data suggest that impaired contractility in the diabetic heart is not caused by SERCA Ca2+ mishandling. This study highlights the important role of the cardiac myofilament and provides new insight on the pathophysiology of diabetic heart dysfunction.Systolic and diastolic dysfunction in diabetes have frequently been associated with abnormal calcium (Ca Our group5 and others7 have found that contractility is impaired in type 2 diabetic hearts, in advance of heart failure development. Decades of research has suggested that abnormal calcium (Ca2+) flux impairs cardiac contractility and relaxation in the type 2 diabetic heart9. For example, cardiomyocytes from type 2 diabetic hearts with depressed cardiac function have decreased Ca2+ flux during systole and diastole, reduced Ca2+ transient decay rate, and increased sarcoplasmic reticulum Ca2+ leakage10. Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) is the major Ca2+ pump regulating cytoplasmic Ca2+ flux, and defects in SERCA have frequently been associated with impaired cardiac function in diabetes12. SERCA expression is upregulated in the right atria13, but unchanged in the left ventricular tissues14 obtained from type 2 diabetic patients with impaired diastolic function. However, protein expression does not always reflect the intrinsic activity of SERCA. Vetter et al. found that increased SERCA Ca2+ uptake improved myocardial contractility in type 1 diabetic rat hearts15, suggesting that reduced SERCA activity contributed to cardiac dysfunction in diabetes; presumably by deranging cytosolic Ca2+ flux.Diabetes impairs heart functionincreased in type 2 diabetic hearts17, and that intracellular Ca2+ transient amplitudes are unaffected by diabetes19. Misra et al. found that SERCA Ca2+ uptake rate was increased in type 2 diabetic cp/cp rat hearts, but intracellular Ca2+ concentration was not different at basal conditions17. Daniels et al. also recently found that cardiac function could be restored in diabetic rat trabeculae by kinase inhibition without any change in Ca2+ flux20. These findings suggest that SERCA alone does not regulate the intracellular Ca2+ state, and that altered intracellular Ca2+ regulation is not the only factor causing contractile dysfunction in diabetic hearts.However, others have reported that SERCA activity is 2+ sensitivity of cardiac myofilaments is altered in the type 2 diabetic heart. The Ca2+ sensitivity of the myofilament is affected by transient post-translational modifications of myofilament proteins21, particularly cardiac troponin I (cTnI)23. In both type 1 and type 2 diabetic animal models, myofilament Ca2+ sensitivity of cardiomyocytes has been reported as unaltered24, increased25 or decreased26. The obese Zucker diabetic fatty (ZDF) rat is a type 2 diabetic model with well-established cardiac inotropic and lusitropic dysfunction27. Our group recently found that myofilament Ca2+ sensitivity was increased in 20-week-old type 2 diabetic ZDF rat cardiomyocytes, and this was associated with reduced phosphorylation of cTnI at Ser23/2428. Interestingly, increased myofilament Ca2+ sensitivity would be expected to enhance cardiac contractility by increasing the affinity of thick and thin filament interaction29. Therefore, it is unclear why contractile dysfunction still existed in these hearts. One possibility is that deranged SERCA activity reduces intracellular Ca2+ concentrations. Reduced SERCA Ca2+ uptake has been associated with reduced systole Ca2+ transient amplitude and impaired cardiac contractility in type 2 diabetic mouse heart11. Reduced SERCA-mediated Ca2+ uptake activity may decrease sarcoplasmic reticulum Ca2+ content, hence less intracellular Ca2+ would be available for the subsequent contractions, and this may explain systolic dysfunction in the diabetic heart.An alternative explanation for impaired contractility is that the Ca2+ sensitivity is increased in type 2 diabetic heart28 which is not normally associated with contractile dysfunction, we therefore hypothesised that SERCA expression and activity would be reduced in 20-week-old type 2 diabetic rat heart.As our group has recently found that myofilament CaP < 0.0001), confirming their diabetic status, as previously reported27. However, body weight was not different between the diabetic and non-diabetic rats .Non-fasting blood glucose was higher in the diabetic versus non-diabetic rats  was not different between the diabetic and non-diabetic hearts  pathway36. Bockus and Humphries found that inotropic and lusitropic responses were impaired in type 1 diabetic mice following acute activation of PKA37. Thaung et al. also found that the expression of Gs protein, which increases cAMP level during β-adrenergic stimulation, was reduced by 28% in this type 2 diabetic ZDF rat model27. Therefore, our data suggest that cAMP/PKA signalling may be impaired in the diabetic heart. Interestingly, Daniels et al. found that contractility and relaxation were restored in diabetic rat trabeculae without any change in sarcoplasmic reticulum Ca2+ flux when Ca2+/calmodulin dependent kinase II (CaMKII) was inhibited20. Unfortunately, the authors did not determine whether this effect was associated with alterations in the phosphorylation of cTnI or other myofilament proteins known to alter Ca2+ sensitivity.Our finding that the diabetic ZDF rat heart had greater myofilament Ca2+ sensitivity may be involved in diabetic diastolic dysfunction. Increased baseline systole38, prolonged systolic duration39 and slower diastolic relaxation13 have been identified as early manifestations of diabetes, which precede cardiovascular disease. The greater myofilament Ca2+ sensitivity in the diabetic rat hearts in this study may suggest stronger binding of Ca2+ ions to cardiac troponin C (cTnC), which would delay or reduce the rate of Ca2+ dissociation from cTnC41. This increased affinity of Ca2+ for the troponin complex would prolong thick and thin filament interactions, thereby ‘delaying’ the onset of relaxation, leading to incomplete cardiac relaxation as often described in the diabetic heart27.Though not the focus of our study, the functional consequences of increased myofilament Ca2+-myofilament interaction. We found that skinned diabetic cardiomyocytes tended to produce less maximal force  when bathed in supra-physiological Ca2+ (pCa 4.5). In such conditions, it is assumed that all thick and thin filament cross-bridges are activated, and any difference in force is explained by differences in the quantity of force producing cross-bridges per unit area. Jweied et al. similarly found that maximal force was 29% lower in diabetic human skinned cardiomyocytes (P = 0.08 vs. non-diabetic controls)42 and van den Brom et al. found that maximal force was 68% lower in 14-week-old type 2 diabetic ZDF rats with impaired cardiac function6. Zhang et al. found that the area of the actin filament was 8% lower in streptozotocin-induced diabetic rat myofilaments, and that the sarcomeric organization of actin was disorganized compared to non-diabetic myofilaments18. A reduction in the content or quality of force generating proteins in the diabetic left ventricular cardiomyocyte would explain the consistent finding that maximal force is reduced in isolated heart experiments in this model27. It is also possible that the increase in myofilament Ca2+ sensitivity would compensate for this loss of generated force by eliciting greater contractile activation at lower intracellular Ca2+ concentrations.Our data may indicate that diabetes impacts the force generating components of the myofilament itself. It is possible that diabetes reduced cardiomyocyte force generation by interfering with the Ca27 and coincide with increased myofilament Ca2+ sensitivity28. SERCA is the primary Ca2+ pump in cardiomyocytes; however, the lack of change in SERCA activity may indicate that an alternative Ca2+ transporter is altered by diabetes. Several studies have suggested that NCX activity is increased in diabetic cardiomyocytes11, and may ‘compete’ with SERCA to remove Ca2+ from the cytosol. We did not measure NCX activity in the present study. However, NCX expression was not different between the diabetic and non-diabetic myocardium, which may indicate a normal Ca2+ efflux under basal condition. Similarly, Belke et al. found that NCX activity was not different between type 2 diabetic and non-diabetic hearts9. A more exhaustive comparison of the various Ca2+ ion channels , and the dynamic interactions between Ca2+ and the myofilaments may be necessary to better understand the mechanism of diabetic heart dysfunction.Previous studies by our group have consistently shown that type 2 diabetic ZDF rats have impaired cardiac contractility2+ sensitivity and lower phosphorylation of cTnI at Ser23/24. SERCA/PLB ratio was reduced, however, SERCA Ca2+ uptake activity was unaltered in the diabetic hearts, suggesting that impaired contractility may be associated with changes in intracellular Ca2+ concentration independent of SERCA. These findings suggest an important role for myofilaments and generate new questions regarding the pathophysiological mechanism in diabetic heart dysfunction.Our data show that the type 2 diabetic left ventricular cardiomyocytes have greater myofilament Cafa/fa; n = 5) rats and their non-diabetic  littermates. Animals were obtained from an in-house colony, with the original breeding pairs obtained from Charles River Laboratories . Animals were housed in groups of 2 per cage with a standard 12-h light–dark cycle (0700 – 1900) and stable temperature of 21 ± 1 °C. Animals were fed with rat chow Purina 5008 (LABDIET) and had access to food and water ad libitum.Experiments were conducted on 20-week-old male type 2 diabetic Zucker Diabetic Fatty  to confirm diabetic status.43. The left ventricular tissues from the different groups were blinded and randomised to eliminate bias. A unique identification number was assigned to the left ventricular tissue of each animal and kept until all the measurements were completed. Briefly, left ventricular samples were defrosted in relaxing solution containing the following (mM): 100 KCl, 1.75 EGTA, 10 imidazole, 4 ATP-Mg, and 5 MgCl2; pH 7.0. The left ventricular tissue was mechanically disrupted and incubated in relaxing solution supplemented with 1% Triton X-100 for 8 min to permeabilise the sarcolemmal membranes. The resulting isolated, skinned cardiomyocyte preparations were washed twice in relaxing solution. Thereafter, a skinned cardiomyocyte preparation (1–3 cells) was attached between a force transducer and a piezoelectric motor with silicon adhesive. Sarcomere length of the skinned cardiomyocyte preparation was adjusted to approximately 2.20 µm for isometric force measurements. The force was measured as a function of pCa (− log10[Ca2+]) in solutions ranging from pCa 9.0 (relaxing solution) to pCa 4.5 .Force measurements were performed on isolated skinned cardiomyocyte preparations at 15 °C as described by Diffee and Nagle2+ concentration, 2+ concentration that produced 50% of maximal force which is used as an index of myofilament Ca2+ sensitivity.Force was first measured in pCa 9.0 and 4.5 solutions and then in randomly ordered submaximal pCa solutions, followed by another measurement made in pCa 9.0 and 4.5 to assess any decline in cardiomyocyte performance. Measurements were continued in another three to four randomly ordered submaximal pCa solutions until a full force-pCa sigmoidal curve was obtained. This was followed by a last control measurement in pCa 9.0 and 4.5 to assess force rundown in the cardiomyocyte preparations. Skinned cardiomyocyte preparations without clear striations for sarcomere length measurement and that did not maintain more than 80% of maximal force at the end of the experimental protocol were discarded and data not used. For each activation, when steady-state force was reached, force was measured by abruptly slackened the skinned cardiomyocyte preparation by 20% of its initial length and transferred to pCa 9.0. Total force was calculated as the difference between steady-state force and the baseline force immediately after the skinned cardiomyocyte preparation was slacked. Active force was determined by subtracting passive force in pCa 9.0 from total force. Maximal force (force developed in pCa 4.5) and passive force (force developed in pCa 9.0) were normalised to the cross-sectional area (CSA) of the skinned cardiomyocyte preparation 3, 0.5 PMSF, 0.5 DTT, 0.2 g/L trypsin inhibitor, and 0.2 g/L benzamide, pH 6.90. The tissue homogenate was centrifuged at 2300g for 15 min at 4 °C. The pellet was discarded and the supernatant was centrifuged at 50,000g for 3.5 h at 4 °C. After centrifugation, the supernatant was discarded and the pellet was re-suspended in 1 mL sarcoplasmic reticulum isolation buffer supplemented with 650 mM KCl, and incubated on ice for 45 min, followed by another centrifugation at 2300g for 10 min at 4 °C. The pellet was discarded and the supernatant was centrifuged at 50,000g for 4 h at 4 °C. The supernatant was discarded and the resulting pellet containing the isolated sarcoplasmic reticulum vesicles was re-suspended in isolation buffer, snap frozen in liquid nitrogen, and stored at − 80 °C for the SERCA Ca2+ uptake assay.Left ventricular tissue (~ 0.3 g) was mechanically disrupted in sarcoplasmic reticulum isolation buffer, which contained (mM): 290 sucrose, 10 imidazole, 3 NaN2+ uptake into the cardiac sarcoplasmic reticulum vesicles was measured according to the assay described by Beca et al. with some modifications44. The assay was performed at 30 °C in a 96-well plate with 100 µg of vesicles in a final volume of 100 µL of uptake buffer per well containing (mM): 100 KCl, 4 MgCl2, 20 HEPES, and 10 sodium oxalate, pH 7.0. The starting Ca2+ concentration was adjusted to approximately 1.5 µM as assessed by the fluorescence ratio of fura-2. The reactions were initiated with a premix containing (mM): 1.5 Na2ATP, 1.5 creatine phosphate and 2 U/ml creatine phosphokinase. The fluorescence signals at 340 nm and 380 nm were recorded at 20-s intervals for 25 min with a multi-mode microplate reader . The rate of reaction for sarcoplasmic reticulum Ca2+ uptake corrected for the amount of vesicle protein was calculated using the following formula:Ca2+ ([Ca2+]free), and the maximum Ca2+ uptake rate was estimated from the plot by fitting a curve to the Michaelis–Menten equation.The data were plotted as rate of reaction against concentration of free Ca2+ buffering capacity. In a separate experiment, thapsigargin, a potent SERCA inhibitor45 completely inhibited Ca2+ uptake in isolated sarcoplasmic reticulum preparation  skimmed milk and probed with specific antibodies for total cTnI , phosphorylated cTnI Ser23/24 ,SERCA2a , Badrilla Ltd), total PLB , phosphorylated PLB Ser16 , phosphorylated PLB Thr17 , NCX1 , or GAPDH , with corresponding horseradish peroxidase-conjugated secondary antibodies . Proteins were visualised using an enhanced chemiluminescence detection system . The protein expression of total cTnI, SERCA2a, total PLB and NCX1 was normalised to GAPDH to correct for protein loading, whereas the phosphorylated proteins were normalised to the corresponding total proteins. Although some studies have suggested an alteration in GAPDH protein expression in diabetes48, others16 and our previous data14 did not find any change.The protein expression of total cTnI, phosphorylated cTnI at Ser23/24, SERCA2a, total PLB, phosphorylated PLB at Ser16 and Thr17 and NCX1 was determined as described previouslyt-tests were used to test for differences between non-diabetic and diabetic groups. Results are presented as means ± SEM, a value of P < 0.05 was considered statistically significant.Statistical analyses were performed using GraphPad Prism v.8.3.0. D’Agostino & Pearson test and Saphiro-Wilk test were used to check for a normal distribution. Student’s unpaired All experiments and the use of animals were approved (AUP-19-179) and conducted in accordance with the guidelines of the Animal Ethics Committee of the University of Otago and in adherence to the NZ Animal Welfare Act 1999. All experiments were performed in compliance with the ARRIVE guidelines.Supplementary Figures.


Confocal microscopy was used in conjunction with live cell imaging to analyze the influence of rHuOVGP1 and P4 on [Ca2+]i in the sperm tail and to examine the involvement of CatSper channels in their effect on [Ca2+]i. Western blot analysis was performed to assess the protein levels of p105, a major tyrosine-phosphorylated sperm protein.Fluorometric flow cytometry was performed to examine the effects of rHuOVGP1 on [Ca2+]i in human sperm at the beginning of capacitation and further increases and sustains the level of [Ca2+]i in the sperm tail following the addition of P4. Inhibition of CatSper channels impedes the effects of rHuOVGP1 on [Ca2+]i in the sperm tail. P4 alone can increase pY of a major human sperm protein, p105, yet yields a further increase when used in combination with rHuOVGP1.rHuOVGP1 increases [Ca2+]i at the beginning of capacitation in part through CatSper channels which, in turn, leads to the downstream event of pY of sperm proteins and enhancement of sperm capacitation.The present study revealed that rHuOVGP1 may work with P4 to upregulate [CaThe online version contains supplementary material available at 10.1007/s10815-022-02591-0. The mammalian oviductal fluid within the lumen of the oviduct is essential for gamete transport and maturation, sperm selection and capacitation, fertilization, early embryonic development, and transport of the embryo to the uterus . It was 2+ ions i with Fluo3, sperm cells at a concentration of 4 × 106 cells/mL were added to 500 mL of modified Biggers-Whitten-Wittingham medium  supplemented with 0.3% BSA to enable sperm capacitation. The indicator 7-Aminoactinomycin D (7AAD) (2 mL per 2 × 106 sperm cells) was added to all experimental reactions to assess sperm viability prior to data acquisition. Intracellular Ca2+ was then assessed using flow cytometry . Following 3 min of baseline fluorescence reading, rHuOVGP1  was added, respectively, to the cell suspension (4 × 106 cells/mL), and the data were acquired for further 10 min. As controls, the fluorescent intensity of unstained non-capacitated sperm cells (4 × 106 cells/mL) as well as individual stained cells were measured. Data were analyzed using FlowJo , and the median of Fluo3 fluorescence was recorded as a measurement of intracellular Ca2+.Following preparation for assessment of [Ca2+]i with Fluo3 as described above. Sperm cells (2 × 106 cells/mL) in non-capacitating medium were plated in 35-mm dishes  pre-coated with Concanavalin A  as previously described i were determined by measuring the fluorescence intensity of Fluo3. The assessment of [Ca2+]i using flow cytometry for each experimental sample was performed by comparing the baseline fluorescence (− 200 s) with the fluorescence at further times-point following the addition of rHuOVGP. The data were expressed as a fold change using two-way ANOVA with Dunnett’s post hoc test. The comparison of the intensity of fluorescent staining between rHuOVGP1-treated cells at different concentrations of rHuOVGP1 and the untreated cells at a specific time-point was analyzed using Student’s t tests.In all experimental groups and controls, a total of 50 to 100 sperm cells in each individual group were examined and assessed. Statistical tests were performed using Prism 9 . Changes of [Ca2+]i using live cell imaging was performed at the 16- . The intensities of the bands were normalized to those of α-tubulin. The level of tyrosine phosphorylation of sperm proteins at each time-point was compared to the 0-h time-point and expressed as a fold increase using two-way ANOVA with Dunnett’s post hoc test. The comparison between treated cells and untreated cells at a specific time-point was analyzed using Student’s 2+]i during capacitation. Flow cytometry experiments were carried out where sperm samples were incubated in capacitating medium followed by the addition of different concentrations of rHuOVGP1  at t = 0 for 10 min. Additionally, a sample incubated in non-capacitating medium was analyzed to act as a baseline for fluorescent intensity. The level of [Ca2+]i in sperm, indicated by the intracellular calcium indicator Fluo3, appeared to gradually increase when sperm cells were exposed to capacitating (BWW) medium  medium to obtain baseline measurements (1 min). Following baseline measurements in BWW, the same cells were treated in the presence or absence of rHuOVGP1 (100 mg/mL) with live cell imaging continued being recorded for 10 min and subsequently treated with P4 (1 mM) for 5 min. Representative images of the live cell imaging over time are shown in Fig. 2+]i followed by a gradual decrease . These findings imply that, during sperm transport in the female reproductive tract, the interaction of sperm with the female oviductal secretions, of which OVGP1 is a major component, is particularly important in preparing the sperm to meet the oocyte for subsequent fertilization. Without sufficient interaction with OVGP1, although P4 released from cumulus cells can induce a surge of calcium influx that leads to sperm capacitation essential for acrosome reaction and subsequent fertilization, the level of [Ca2+]i cannot be sustained and will diminish. On the contrary, sperm that come into contact with both OVGP1 and P4 maintain the increased level of [Ca2+]I and likely maintain a greater potential to fertilize the oocyte.Using recombinant human oviductin rHuOVGP1) in conjunction with live cell imaging technique, we have been able to demonstrate, for the first time, an additional role of rHuOVGP1 which works in conjunction with P4 in enhancing sperm capacitation. Previous findings in our laboratory have demonstrated that rHuOVGP1 can bind to sperm to enhance tyrosine phosphorylation of sperm proteins, a biochemical hallmark of capacitation , 28. Howcitation –42. As pcitation , 37, in GP1 Fig. . Particu in conju+]i Fig. , b, whicin sperm –42. More2+ influx in human sperm to CatSper channels [2+]i in vitro. To examine if CatSper channels play a role in the increased in influx of [Ca2+]i caused by rHuOVGP1, we used a specific inhibitor for CatSper channels, HC-056456, and performed live cell imaging to examine if rHuOVGP1 increases [Ca2+]i by way of the CatSper channels. HC-056456 was used at both concentrations of 3 and 10 mM because previous research demonstrated that the use of HC-056456 at these concentrations was found to be an effective blocker of CatSper channels i by way of CatSper channels in the sperm tail at the onset of capacitation. We are hypothesizing that OVGP1 binds to a plasma membrane receptor of the principal piece of the human sperm tail. Binding of OVGP1 to its receptor might start the initial abolition of inhibition of the CatSper channel that could prepare for the subsequent action of P4. The resulting increase of influx of [Ca2+]i, in turn, leads to the downstream event of tyrosine phosphorylation of sperm proteins and enhancement of sperm capacitation and fertilizing competence of sperm. The true nature of a putative OVGP1 receptor and its mechanism of action remain elusive and need further investigations.Collectively, our experimental results demonstrated that rHuOVGP1 and P4 function most effectively in combination to enhance human sperm capacitation through increasing i in the sperm tail, it is worth mentioning that a significant increase in [Ca2+]i was also detected in the sperm head in the presence of both rHuOVGP1 and P4. It is now known that the calcium influx is an absolute requirement for acrosome reaction to occur in all invertebrates and mammalian sperm [2+ influx channels in the sperm head plasma membrane responsible for calcium entry and intracellular calcium ion levels are voltage-activated and voltage-dependent calcium channels [2+]i in the sperm head. The mechanism of action of OVGP1 in regulating Ca2+ in the sperm head and its possible involvement in acrosome reaction need further exploration.Although the present study focused on investigating the effects of OVGP1 and P4 on i in human sperm tail is more pronounced in sperm with low motility compared to sperm with normal motility  procedure.According to the WHO laboratory manual for the examination and processing of human semen, the lower motility limit for normozoospermic samples is 40% . Due to 2+]i in human sperm at the beginning of capacitation. Furthermore, treatment of sperm with rHuOVGP1 further increases and sustains the level of [Ca2+]i in the sperm tail following the addition of P4. Inhibition of CatSper channels impedes the effects of rHuOVGP1 on [Ca2+]i in human sperm. This suggests that OVGP1 may, in part, influence Ca2+ influx through the CatSper channels. Lastly, we have demonstrated that P4 alone can increase tyrosine phosphorylation of a major human sperm protein, yet yield further increase when rHuOVGP1 and P4 are used in combination. The current findings could be of significance to the field of infertility as a better understanding of the function of OVGP1 could improve the success rates of conventional IVF procedures by supplementing the capacitating medium currently used in fertility clinics with rHuOVGP1. Improving fertility treatments for infertile couples remains a high priority in reproductive medicine. It is hoped that the positive effects on sperm capacitation resulting from OVGP1 accompanied by the secretion of P4 by cumulus cells encircling the post-ovulatory oocyte can open new avenues for further investigation into the mechanism that regulate human OVGP1in sperm function.The present investigation aimed at gaining a better understanding of the function of human OVGP1 in enhancing sperm capacitation. The present research revealed that rHuOVGP1 increases [Ca2+]i in human sperm at the beginning of capacitation. Table shows the level of Fluo3 median intensity from the flow cytometry analysis of [Ca2+]i in sperm. Following 3 min (shown as -200 to -20 s) of baseline fluorescence reading, rHuOVGP1  was added, respectively, to the cell suspension (time = 0 s), and the data were acquired for a further 10 min. The level of Fluo3 median intensity of each concentration of rHuOVGP1  over time was compared to that prior to the addition of rHuOVGP1 at -45 s. n = 7 patients; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (DOCX 492 KB)Supplementary file1 rHuOVGP1 increases [Ca2+]i in human sperm heads following treatment with P4. a: Representative images of calcium live cell imaging experiments with or without rHuOVGP1 (100 mg/mL) treatment. High magnifications of sperm cells indicated by blue (pre-P4) and red (post-P4) dashed boxes are shown on the right side of the figure with regions of interest (ROIs) of sperm heads outlined in yellow. Sperm cells were imaged for 1 min in BWW to obtain baseline measurements, followed by subsequent imaging the same cells treated with or without rHuOVGP1 (100 mg/mL) for 10 min and progesterone  for 5 min. b: Line graph of the relative mean fluorescent intensity of Fluo3 from the live cell imaging analysis of [Ca2+]i in sperm heads treated with or without rHuOVGP1 (100 mg/mL) followed by the treatment with P4 (1 mM). Data are represented as fold of change in fluorescent labeling ± SEM; n = 6 patients; * p < 0.05 (DOCX 816 KB)Supplementary file2 The effects of rHuOVGP1 on the level of i in sperm tail treated with or without rHuOVGP1 (100 mg/mL) followed by the treatment with P4 (1 mM). Data are represented as fold of change in fluorescent labeling ± SEM; control group n = 6 patients; sperm with > 40% motility n = 4 patients; sperm with ≤ 40% motility; n = 2 patients (DOCX 270 KB)Supplementary file4 Segregation of results for normal and low motility sperm treated with rHuOVGP1 followed by subsequent treatment with P4. Line graph of the relative mean fluorescent intensity of Fluo3 from the live cell imaging analysis of [CaBelow is the link to the electronic supplementary material.