This modification has the potential to modulate SNARE complex formation required for neurotransmitter release in an activity-dependent manner. To determine the extent to which T14 phosphorylation occurs in vivo in the mammalian retina and characterize the pathway responsible for the in vivo phosphorylation of T14, we utilized quantitative immunofluorescence to measure the levels of STX3 and STX3 phosphorylated at T14 (pSTX3) in the synaptic terminals of mouse retinal photoreceptors and rod bipolar cells (RBCs). Results demonstrate that STX3B phosphorylation at T14 is light-regulated and dependent upon the elevation of intraterminal Ca2+. In rod photoreceptor terminals, the ratio of pSTX3 to STX3 was significantly higher in dark-adapted mice, when rods are active, than in light-exposed mice. By contrast, in RBC terminals, the ratio of pSTX3 to STX3 was higher in light-exposed mice, when these terminals are active, than in dark-adapted mice. These results were recapitulated in the isolated eyecup preparation, but only when Ca2+ was included in the external medium. In the absence of external Ca2+, pSTX3 levels remained low regardless of light/dark exposure. Using the isolated RBC preparation, we next showed that elevation of intraterminal Ca2+ alone was sufficient to increase STX3 phosphorylation at T14. Furthermore, both the non-specific kinase inhibitor staurosporine and the selective CaMKII inhibitor AIP inhibited the Ca2+-dependent increase in the pSTX3/STX3 ratio in isolated RBC terminals, while in parallel experiments, AIP suppressed RBC depolarization-evoked exocytosis, measured using membrane capacitance measurements. Our data support a novel, illumination-regulated modulation of retinal ribbon-style synapse function in which activity-dependent Ca2+ entry drives the phosphorylation of STX3B at T14 by CaMKII, which in turn, modulates the ability to form SNARE complexes required for exocytosis.Neurotransmitter release at retinal ribbon-style synapses utilizes a specialized t-SNARE protein called syntaxin3B (STX3B). In contrast to other syntaxins, STX3 proteins can be phosphorylated  Retinal ribbon-style synapses, elaborated by the first two neurons in the visual throughput pathway, continuously convey visual information over many log units of dynamic range. These remarkable signaling abilities are supported by a number of unique features, one of which is the expression of specialized presynaptic proteins such as syntaxin3B (STX3B). STX3B is a t-SNARE protein that is expressed almost exclusively in retinal photoreceptors and bipolar cells, where it is highly enriched in the synaptic terminals . Similarin vitro phosphorylated by Ca2+/calmodulin-dependent protein kinase II  is regulated by light in a CaAll of the animal procedures were approved by the Animal Welfare Committee of the University of Texas Health Science Center at Houston. Prior to use, adult male and female mice, strain C57BL/6J  were maintained on a standard 12 h light/dark cycle. Sacrifice was performed by cervical dislocation followed by decapitation. To minimize potential effects of circadian rhythm on syntaxin 3 (STX3) phosphorylation, animals for each set of experiments were sacrificed at the same time of day, typically between 12 and 1 pm.in vivo experiments, mice were unrestrained and free to move about their cage until the time of sacrifice. Two groups of mice were dark-adapted for 4 h, beginning 1 h after morning light onset. Immediately following dark-adaptation, one group of mice was sacrificed and retinal eyecups dissected free under infrared illumination and fixed, while under maintained dark conditions. The second group of mice was exposed to 15 min of a bright flashing light stimulus  superimposed upon a dim background light (20 lux). Animals were then sacrificed, retinal eyecups dissected free and fixed.For the ex vivo experiments, two groups of mice were dark-adapted for 4 h, beginning 1 h after morning light onset. Animals were sacrificed and retinal eyecups prepared under infrared illumination. Eyecups were bathed in an oxygenated Hanks solution that contained (in mM): 128 NaCl, 5 KCl, 0.34 Na2HPO4, 0.44 KH2PO4, 10 HEPES, and 10 glucose, supplemented with either 2 CaCl2 and 1 MgCl2 (“2Ca”) or 0 CaCl2 and 2 MgCl (“0Ca”) pH ≈7.4, ≈315–320 mOsm. For the dark/light experiments, the left eyecup from each animal was maintained under dark-adapted conditions and then fixed. The right eyecup was exposed to 15 min of a bright flashing light stimulus  superimposed on a dim light background (20 lux) and then fixed. Similarly, for the 2Ca/0Ca experiments, one eyecup from each animal was placed in the 2Ca external solution and the other in the 0Ca solution.For the 2, 0.5 CaCl2, and 10 HEPES, or 153 NaCl, 2.6 KCl, 3.0 MgCl2, 0 CaCl2, and 10 HEPES (“low Ca”). Following the settling phase, coverslips were either incubated for an additional 30 min in the low Ca solution or incubated in a “high Ca” solution that was designed to elevate the intraterminal free Ca2+ concentration. The latter had a reduced Na+ concentration, a perturbation that we have previously shown activates the reverse mode of the Na+/Ca2+ exchanger in the RBC synaptic terminal to produce a modest, sustained elevation in the spatially-averaged intraterminal free Ca2+ concentration (2+” solution contained (in mM): 30 NaCl, 123 choline chloride, 2.6 KCl, 1 MgCl2, 2.0 CaCl2, and 10 HEPES. Kinase inhibitors were applied during the settling and incubation phases. For all, the solution pH was adjusted to ≈7.4 and the osmolarity was 290–300 mOsm. Following treatment, cells were fixed for 10 min in 4% formaldehyde.Rod bipolar cells were dissociated from the retinae of light-adapted mice by enzymatic digestion followed by mechanical trituration  and platntration . The “hiRetinal eyecups were fixed for 10 min at room temperature in freshly prepared 4% formaldehyde, rinsed with PBS, cryoprotected in 30% sucrose and stored at 4°C overnight. Processing of retinal eyecups was similar to that described previously , 2013. IFixed cells were blocked for 2 h at room temperature in PBS supplemented with 5% donkey serum and 0.4% Triton X-100 and then incubated overnight at 4° in PBS supplemented with 2% donkey serum, 0.4% Triton X-100 and primary antibodies (see below). After washing in PBS, cells were then incubated for 2 h in PBS containing the secondary antibodies (see below). Cover slips were washed and mounted with Prolong Gold anti-fade with DAPI mounting medium . Slides were stored in a light protective holder at 4°C before imaging.Syntaxin 3 was immunolabeled with a mouse-monoclonal antibody initially received as a generous gift from Dr. Muna Naash  and thenin vivo and ex vivo experiments, quantification of the pSTX3 and STX3 signals was obtained from the analysis of five images from two different histological sections from each eyecup at 0.5 μm intervals from randomly chosen locations 50 to 80% from the distance of the optic nerve head to the periphery.Image acquisition and data analysis were conducted in similar manner to that described previously . Rod sph2+) using independent sample t-tests. Tissue from multiple (2–4) animals was sampled per experimental condition. Figure images have been cropped and are displayed as maximal intensity projections.Tissue sections were analyzed using the ROI function of SimplePCI software  that allowed for the manual selection of ROIs based on STX3 immunolabeling. ROIs were defined as having a STX3 immunoreactivity that was greater than 20% threshold of the total intensity range. ROIs representative of rod spherules were selected based upon STX3 immunolabeling, location in the upper OPL, round shape and smaller size relative to cone pedicles, and the lack of arrestin-labeling. ROIs representative of cone pedicles were chosen based on the large, STX3-positive plaques associated with cone-arrestin and a location at the bottom of OPL. ROI’s representative of RBC terminals were identified based upon STX3 immunoreactivity, size and location at the bottom of the IPL. For each OPL image, data from ≈30–60 rod terminals and ≈5–15 cone terminals were obtained. For each IPL image, data from ≈25 RBC terminals were obtained. The mean pixel intensity values from each ROI in an image were collected and the pSTX3/STX3 ratio per ROI calculated. The individual ratios from a given image were not normally-distributed, and therefore the median value rather than the mean was used to denote the pSTX3/STX3 ratio representative of each image. The value per animal or eyecup was calculated as the mean of the representative image values, and the value across animals or eyecups per condition was calculated as the average of these values and expressed as the mean ± s.e.m. As such, the value “n” denotes the number of animals or eyecups per condition rather than the number of terminals. Results were compared across the two conditions . Normality of the data was verified using column statistics. Two-tailed independent sample t-tests and One-Way ANOVA followed by Tukey’s posthoc multiple comparisons test were used as appropriate. Data are expressed as mean ± s.e.m, where n = the number of mice per condition. For immunocytochemistry on isolated RBCs, tissue from multiple (3–7) animals was sampled per experimental condition. Figure images are displayed as maximal intensity projections.Methods for adjustment of confocal settings and image acquisition were similar to those described above for retinal sections. Isolated RBC were identified by their characteristic morphology and PKC immunoreactivity . A minim2 and 5%CO2) contained (in mM): 125 NaCl; 1.2 CaCl2; 0.5 MgCl2; 1.5 KCl; 1.3 NaH2PO4; 10 D-Glucose; 23 NaHCO3, with pH 7.3, 294–296 mOsm. Retinal slices were allowed to rest in the dark for ≥1 h under continuous perfusion prior to use. Slices were transferred under dim light to the recording chamber. The external bath solution used for recordings was identical to that above except that it additionally contained (in μM): 100 picrotoxin, 50 μM 1,2,5,6-Tetrahydropyridin-4-yl methylphosphinic acid (TPMPA), 10 strychnine to block GABAA, GABAC, and glycine receptors, respectively. To minimize artifacts in the capacitance record associated with perfusion, bath perfusion was halted during capacitance measurements.To measure exocytosis from RBCs, retinal slices (≈200 μm thick) were prepared under dim red light illumination using a handmade, semiautomatic chopper. The external bath solution  was applied approximately 1 min prior to the depolarizing pulse train stimulus and the current–voltage relationship evaluated for efficacy of K+ channel blockade and isolation of the presynaptic, L-type Ca2+ current : 100 Cs-Methanesulfonate; 20 TEA-Cl; 0.2 MgClof 25 μM . Measure current . Exocyto current .m, and membrane conductances Gm and Gs. For each 100 ms sine wave segment, the mean values for Cm, Gm, and Gs were recorded. Measurement of Cm was temporally halted during a depolarizing voltage step and for the first 25 ms thereafter to minimize the confounding contributions of current flow and channel gating to Cm  and the mean resting membrane conductance, Gm, . In addition, there was no statistical difference in the resting Cm  or in the standard deviation of the resting Cm signal (baseline noise) . Data were exported to IGOR Pro  for analysis of membrane current and Cm, Gm, and Gs. Two-tailed independent sample t-tests with or without Welch’s correction were used, as appropriate. Statistical analyses were performed in Excel  and Prizm 7 .Membrane capacitance measurements were made with the use of an EPC-10 patch-clamp amplifier and Patchmaster software . In brief, a 500-Hz, 20-mV peak-to-peak sinusoidal voltage stimulus was superimposed on a holding potential of −65 mV . The resng to Cm , 2010. RStaurosporine  was made as a 2.15 mM stock solution in DMSO and used at a final concentration of 100 nM. KN93  was made up as a 3.35 mM stock solution in MQ water and used at a final concentration of 1 μM. KN92  was made up as a 3.6 mM stock in DMSO and used at a final concentration of 1 μM. Myristoylated-AIP , a membrane permeant analog of the CaMKII inhibitor AIP (Autocamtide-2-related inhibitory peptide), was made as a 293 μM stock solution in MQ water and used at a final bath concentration of 100 nM. In the isolated cell experiments, drugs were added to the external bath solution. For drugs made up in DMSO, addition of DMSO to the bath solution at the same concentration as in the test condition had no effect on pSTX3B or pCaMKII levels (not shown). For electrophysiological measurement of exocytosis, a 1 mM stock solution of AIP  was made in MQ water and added to the internal pipette solution at a final concentration of 25 μM . All othin vitro biochemical studies have shown that T14 of STX3 is phosphorylated by Ca2+/CaMKII  and total STX3 levels in the synaptic terminals of rod and cone photoreceptors and RBCs were measured using quantitative immunofluorescence. To this end, retinal sections were double-labeled with a phospho-specific antibody that recognizes pSTX3, which we previously developed and extensively characterized  and a STp = 0.2673, t = 1.182, and n = 6,5). By contrast, pSTX3 levels were approximately two times higher in the dark-adapted mice than in light-exposed mice . These data suggest a selective, light-driven decrease in the phosphorylation of STX3 at T14. To quantify the change in STX3 phosphorylation, we calculated the pSTX3/STX3 ratio for each condition. Results show that the pSTX3 to STX3 ratio in rod terminals was nearly two times higher in the dark-adapted mouse, when rods are depolarized and releasing neurotransmitter, as compared to the light-exposed mouse, when rods are expected to be quiescent . Cone terminals, on the other hand, showed little evidence of a light-regulated change in the pSTX3 to STX3 ratio . Furthermore, the light-dependent increase in pSTX3 labeling does not arise from a light-regulated change in total synaptic STX3  but rather a specific light-dependent increase in pSTX3B  Given that RBCs are depolarized by light, these findings suggest that pSTX3 levels are higher in depolarized terminals relative to quiescent ones. Taken together, results obtained rod photoreceptors and rod bipolar cells indicate a correlation between periods of neuronal activity and the phosphorylation of STX3 at T14.2+, which enters nerve terminals during synaptic activity, was required for phosphorylation of STX3 at T14. To this end, eyecups were isolated from dark-adapted mice and bathed in our standard, Ca2+-containing bath solution or in a nominally Ca2+-free bath solution. Isolated eyecups were then subjected to either an additional 15 min of darkness or a light stimulus for 15 min.We next asked whether external Ca2+ conditions, we saw a significantly higher pSTX3 to STX3 ratio in the dark than in the light , recapitulating the results observed in the live mouse experiments. This dark-driven phosphorylation of STX3B at T14 in rod terminals . In the light-exposed eyecup, removal of external Ca2+ did not further reduce the pSTX3 to STX3 ratio .As shown in erminals , top wassolution , bottom.2+ conditions approached significance , possibly reflecting the greater ease with which isolated eyecups could be oriented to face the light stimulus relative to live mice. Importantly, removal of external Ca2+ significantly reduced the dark-adapted levels of STX3 phosphorylation at T14 , indicating that cone terminals also exhibit Ca2+-dependent STX3 phosphorylation at T14. There was no further decrease in the pSTX3 to STX3 ratio in photoreceptor terminals in light-exposed eyecups in the absence of external Ca2+ .In cone terminals, the ability of the light stimulus to reduce the pSTX3 to STX3 ratio attained in the dark-adapted state under normal Ca2+ entry, then in RBC terminals, phosphorylation levels should be highest in light-exposed eyecups when external Ca2+ is present and remain at or near baseline levels when in the dark or when bathed in a Ca2+-free solution in the light. The results depicted in 2+, RBC terminals in light-stimulated eyecups exhibited an approximately two-fold higher pSTX3B to STX3B ratio relative to those of dark-adapted eyecups . That external Ca2+ was required for this light-driven doubling of the pSTX3 to STX3 ratio was evidenced by the virtual abolition of the light-driven increase in Ca2+-free external solution  and maintenance of the pSTX3 to STX3 ratio at a level indistinguishable from that observed in the dark-adapted eyecup in the absence of Ca2+ . Taken together, the results of this set of experiments demonstrate that the light-regulated phosphorylation of STX3 on T14 in synaptic terminals of retinal photoreceptor and rod bipolar cells is dependent upon external Ca2+, which presumably enters the synaptic terminals via voltage-gated Ca2+ channels during synaptic activity , confirming this interpretation.To probe the signaling pathway downstream of Caparation , 2010. Tractions , 2012. Tntration . As show3 levels , suggestin vitro (2+ (“High”) produced an increase in pCaMKII labeling in RBC synaptic terminals relative to RBC terminals bathed in nominally Ca2+-free solution (“Low”) .Given that STX3 is phosphorylated at T14 by CaMKII in vitro , we addiin vitro . As show2+ and pSTX3 levels in the synaptic terminals of acutely isolated RBCs to test for a role for CaMKII. To validate this approach, we first ascertained whether inhibition of phosphorylation, in general, would prevent the Ca2+-mediated increase in pSTX3. To this end, acutely isolated RBCs were treated with the non-specific kinase inhibitor staurosporine . Furthermore, staurosporine lowered the residual pSTX3 to STX3 ratio of RBC synaptic terminals bathed in Ca2+-free solution . Staurosporine treatment was also found to suppress the Ca2+-dependent increase in pCaMKII immunolabeling , holding the ratio at a level indistinguishable from that of unstimulated RBC terminals . It also suppressed the Ca2+-induced increase in pCaMKII levels  blocked the Ca2+-evoked increase in pSTX3. Pooled data from multiple experiments show that the Ca2+-triggered elevation of the pSTX3 to STX3 ratio was reduced in the presence of mAIP  to a ratio that was not dramatically different from that observed when external Ca2+ was nominally zero . Interestingly, mAIP did not suppress the Ca2+-evoked elevation of pCaMKII in these experiments  or the mean Ca2+ current amplitude measured at −20 mV .The above data are consistent with a model in which STX3 is phosphorylated at T14 in a Cacle pool . To avoicle pool ] was delcle pool . To allo current . Evaluatp = 0.0059, t = 4.309, and n = 6,5). These results indicate a major role for CaMKII in the regulation of exocytosis in RBC synaptic terminals. Because changes in exocytosis could also result from changes in Ca2+ entry, we additionally evaluated the effects of AIP on the train-evoked Ca2+ current. To correlate the total train evoked exocytosis with total train-evoked Ca2+ entry, we calculated the mean total Ca2+ current for the pulse train by taking the sum of each Ca2+ current evoked by an individual depolarizing voltage step within a stimulus train. As shown in 2+ current for the pulse train failed to reveal a significant difference between control and AIP-treated rod bipolar cells . This stimulation protocol and the levels of total Ca2+ current produced are sufficient to trigger fusion of the rapidly-releasable and releasable vesicle pools  of STX3 T14 phosphorylation/dephosphorylation in the regulation of exocytosis.How might STX3 phosphorylation at T14 modulate release? We have shown previously that a phosphomimetic T14 mutation of STX3 interacts more readily with its SNARE binding partner SNAP-25 than wild-type STX3 , suggest2+-dependent modulation of the secretory pathway is particularly relevant to the visual, auditory and vestibular systems, where information about the outside world, covering many log units of dynamic range, is encoded in a largely analog fashion by ribbon-style synapses. To maintain this ability, these synapses must efficiently replace vesicles lost to stimulus-evoked fusion with new, fusion-competent (primed) vesicles. Studies in retinal bipolar cells going back more than a decade have shown that the delivery of vesicles to the fusion-competent state is accelerated by the elevation of presynaptic Ca2+ (2+-dependent processes regulate the availability of vesicles for release. The first involves a vesicle-associated phosphoprotein called synapsin that tethers synaptic vesicles in the cytosol to the actin cytoskeleton (2+ and calmodulin and has been implicated in the activity-dependent preparation of vesicles for fusion and in Ca2+-dependent short-term plasticity at conventional synapses (2+-accelerated progression to the fusion competent state in a retinal ribbon-style synapse. Intriguingly, both Ca2+ and calmodulin have been suggested to speed the delivery and attachment of synaptic vesicles to the synaptic ribbons (2+-dependent phosphorylation of STX3B at T14 by CaMKII may subserve this function.Important for all neurons, Catic Ca2+ , howeverskeleton . In respskeleton . The secskeleton . Munc13 synapses . In thissynapses , an imposynapses . Secondlsynapses . Togethe ribbons . However2+-dependent phosphorylation of STX3B. STX3A, on the other hand, is widely expressed throughout the body, where it plays roles in exocytosis and apical trafficking, particularly within the immune, renal and digestive systems (2+-dependent phosphorylation of STX3 at T14. Finally, we note that neither STX3 isoform has been reported at the ribbon-style synapses of cochlear hair cells (STX3 has two major splice forms, STX3A and STX3B. STX3A and STX3B are virtually identical from the N-terminus through approximately the middle of the SNARE binding domain, differing only in the second half of the SNARE domain and the transmembrane domain due to the use of different exons in the 3’ regions of the two mRNAs . Thus, t systems . Given tir cells . This suThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Animal Welfare Committee of The University of Texas Health Science Center at Houston.RH, HL, and RJ conceived the presented idea and designed the experiments. JC, HL, YW, XL, MK, and AH conducted experiments. All authors analyzed data and contributed to interpretation. JC, RH, and AH prepared the manuscript with input from all authors.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.


Ca2+/calmodulin-dependent protein kinase II-δ (CaMKIIδ) is a well-recognized key molecule in calcium dysregulation in cardiomyocytes. Elevated cellular stress is known as a common feature during pathological remodeling, and c-jun N-terminal kinase (JNK) is an important stress kinase that is activated in response to intrinsic and extrinsic stress stimuli. Our lab recently identified specific actions of JNK isoform 2 (JNK2) in CaMKIIδ expression, activation, and CaMKIIδ-dependent SR Ca2+ mishandling in the stressed heart. This review focuses on the current understanding of cardiac SR calcium handling under physiological and pathological conditions as well as the newly identified contribution of the stress kinase JNK2 in CaMKIIδ-dependent SR Ca2+ abnormal mishandling. The new findings identifying dual roles of JNK2 in CaMKIIδ expression and activation are also discussed in this review.Calcium homeostasis in the cardiomyocyte is critical to the regulation of normal cardiac function. Abnormal calcium dynamics such as altered uptake by the sarcoplasmic reticulum (SR) Ca Thux (ICa) , 130. Thhannels) , 84, 93.to LTCCs , 77. CICto LTCCs . When anlic Ca2+ , 76. CICtraction , 89.2+ and cytosolic Ca2+ is removed through the sodium–calcium exchanger (NCX) to the extracellular space and pumped back to the SR through the cardiac sarcoplasmic reticulum Ca2+-ATPase (SERCA2), while another small portion of Ca2+ is taken up by mitochondria via mitochondrial Ca2+ uniporters as well as a small Ca2+ efflux via the plasma membrane Ca2+-ATPase (PMCA) [2+ levels in systole and low levels in diastole [2+ release via RyR2 channels and reuptake via the predominant Ca2+ pump SERCA2a isoform (SERCA2a), and to a much lesser extent SERCA2b isoform, critically mediate the cytoplasmic Ca2+ concentration, which is essential in cardiac contraction and relaxation of each heartbeat [During cardiac muscle relaxation, LTCCs close and terminate the influx of Cae (PMCA) , 50, 107diastole , 69. Theeartbeat .2+ in ECC, even a small amount of aberrant Ca2+ release resulting from slowly developing pathological changes in the intracellular Ca2+ homeostasis can potentially have escalating negative consequences for the myocyte and ultimately the whole heart. Under pathological conditions including HF, ischemia–reperfusion (IR) injury, post-MI, atrial fibrillation (AF), and ventricular arrhythmias, abnormal SR Ca2+ dynamics result in electrical and mechanical dysfunctions and myocardial maladaptive function . However, increased diastolic RyR2 channel activity could be responsible for increased diastolic SR Ca2+ leak and reduced systolic fractional Ca2+ release for a given L-type voltage-gated Ca2+ current (Ica) as the release trigger [2+ leakage along with an impaired SR Ca2+ uptake in HF slows down the intracellular Ca2+ decline and then elevates the amount of diastolic intracellular Ca2+ concentration, which leads to increased sodium (Na+) influx via NCX for removing the elevated intracellular Ca2+ outside of the cell membrane. As a result, increased diastolic SR Ca2+ leak promotes aberrant Ca2+ events (Ca2+ sparks and waves) and the inward NCX current produces abnormal triggered activities such as delayed after-depolarizations (DADs) and initiates atrial arrhythmias such as atrial fibrillation  and ventricular arrhythmias including ventricular tachycardia and ventricular fibrillation  [2+ handling also occurs in AF pathogenesis as discussed in detail below.For instance, HF is a common disorder in which the cardiac output does not meet the needs of the body resulting from dysfunctional contractility, impaired electrical conduction, and abnormal energy metabolism , 30. Durng heart , 142. In trigger , 19, 121hythmia) , 81. Thi2+ dynamics. CaMKII is a well-recognized pro-arrhythmic kinase, promoting abnormal SR Ca2+ dynamics via phosphorylation of Ca2+ handling proteins in the heart. There are four highly conserved isoforms of CaMKII  widely expressing in the body, while the δ isoform is predominantly expressed in the heart. Extensive studies demonstrate that activated CaMKIIδ is critically involved in phosphorylation of RyR2 at the site of Ser2815, resulting in sensitized RyR2 channels and profoundly increased diastolic SR Ca2+ leak that in turn promotes triggered activities and arrhythmia initiation in pathologically altered ventricles in HF [2+ mishandling and triggered activities (delayed afterdepolarizations (DADs)) in the atria of chronic AF patients and post-operative AF patients [2+ activities and profibrotic activity-caused structural remodeling in chronic AF patients associated with HF [2+ activities [2+ mishandling was recently found to underlie AF pathogenesis in rabbit and mouse models of aging and holiday heart syndrome, in a tachy-pacing canine AF model, and in a spontaneous AF CREM mouse model [2+ binding affinity of SERCA2a by phosphorylating phospholamban (PLB) at the threonine-17 site (PLB17) to release the inhibitory PLB from SERCA2a, which enhances SERCA2a Ca2+ affinity and stimulates SR Ca2+ uptake [2+ uptake in failing hearts [2+ handling, CaMKIIδ is also known to regulate other ion channels such as Ca2+ [+ [+ [V1.5) enhances a late depolarizing current (INa. Late), which leads to prolonged action potentials (APs) and disrupted Ca2+ handling and promotes arrhythmogenic DADs [+ channels, it has been suggested that CaMKIIδ activation regulates potassium Kv channels and reduces Ito and IK1 currents, which prolong APs and increase Ca2+-triggered repolarization, and ultimately enhance arrhythmogenicity [2+ homeostasis and ECC in pathologically remodeled hearts , and how stress JNK2 signaling and/or CaMKIIδ interact with other pathological signaling pathways during the process of disease development. All of these questions merit further investigation.Over the years, our understanding of the underlying mechanisms of physiological CaAccumulating evidence suggests that suppression of CaMKIIδ function can mitigate arrhythmias and various heart diseases in animal models provoking a great deal of interest in the development of CaMKIIδ inhibitors as possible anti-arrhythmic therapeutic agents , 158. Al


Calcium homeostasis plays an essential role in maintaining excitation–contraction coupling (ECC) in cardiomyocytes, including calcium release, recapture, and storage. Disruption of calcium homeostasis may affect heart function, leading to the development of various heart diseases. Myocardial ischemia/reperfusion (MI/R) injury may occur after revascularization, which is a treatment used in coronary heart disease. MI/R injury is a complex pathological process, and the main cause of increased mortality and disability after treatment of coronary heart disease. However, current methods and drugs for treating MI/R injury are very scarce, not ideal, and have limitations. Studies have shown that MI/R injury can cause calcium overload that can further aggravate MI/R injury. Therefore, we reviewed the effects of critical calcium pathway regulators on MI/R injury and drew an intuitive diagram of the calcium homeostasis pathway. We also summarized and analyzed calcium pathway-related or MI/R drugs under research or marketing by searching Therapeutic Target and PubMed Databases. The data analysis showed that six drugs and corresponding targets are used to treat MI/R injury and involved in calcium signaling pathways. We emphasize the relevance of further detailed investigation of MI/R injury and calcium homeostasis and the therapeutic role of calcium homeostasis in MI/R injury, which bridges basic research and clinical applications of MI/R injury. Myocardial ischemia/reperfusion (MI/R) is an inevitable process in the treatment of cardiovascular diseases such as acute myocardial infarction, thrombolysis, coronary angioplasty, and cardiac arrest . MI/R in2+ is an important cytoplasmic signaling molecule in most cellular responses and is mainly distributed in extracellular and intracellular organelles, such as sarcoplasmic reticulum (SR) and mitochondria. Calcium plays a vital role in excitation–contraction coupling (ECC) of heart tissue. When cardiac action potential occurs, extracellular Ca2+ enters the cell through L-type Ca2+ channels (LTCC), and intracellular Ca2+ activates ryanodine receptor 2 (RyR2) and more Ca2+ is released from the sarcoplasmic reticulum releases  plasmic reticulum Ca2+-ATPase (SERCA) recaptures intracellular Ca2+ back to SR. On the other hand, sodium/calcium exchanger (NCX) expels Ca2+ from the cells and the dissociation of Ca2+ from myofilament protein relaxes the myocardial cells  interacts directly with LTCC (Cav1.2); LTCC is regulated by G protein, adenylate cyclase, PKA, PP1, and PP2A, which affects channel activity. Some studies have shown that loss or deficiency of LTCC function can lead to short QT intervals and sudden cardiac death , and Cav2 and Cav3, which are non-LTCCs. Cav1.2 and Cav1.3 are co-expressed in multiple tissues  and play similar regulatory roles. Cav1.1 and Cav1.4 are expressed only in skeletal muscle and retina, respectively . LTCC chribution . At myocac death . Cav1.2 ac death . Therefo2+ sensitivity, and SR Ca2+ content , and phosphatases are involved in RyR2 phosphorylation. PKA-induced RyR2 phosphorylation (Ser-2808) promotes FKBP12.6 and RyR2 dissociation, thereby opening RyR2 and promoting calcium-activated calcium release (The FK506 binding proteins (FKBP) are expressed in cardiomyocytes and act as essential RyR2 regulators. FKBP12 and FKBP12.6 subtypes correspond to low and high RyR2 affinity, respectively. Reportedly, FKBP12.6 and not FKBP12 can inhibit RyR2 activity. FKBP12.6 and RyR2 association keeps RyR2 closed, preventing calcium release from the SR, thereby, ensuring heart relaxation . PKA, Ca release . This prRyR2 phosphorylation caused by CaMKII (Ser-2814) is more critical than that by PKA. Studies have shown that atrial fibrillation induced in FKBP12.6 KO mice can be achieved by inhibiting CaMKII phosphorylation RyR2 instead of PKA, thereby inhibiting SR calcium leakage and delayed depolarization . Decreas2+ is returned to SR through SERCA; excessive Ca2+ can also be expelled from cells through the NCX, resulting in myocardial cell relaxation , SLC8A2 (NCX2), SLC8A3 (NCX3) . In mammNa+:Ca2+ . The dird f-loop .NCX participates intracellular calcium homeostasis regulation and is implicated in the occurrence and development of various diseases, such as heart failure, arrhythmia, diabetes, hypertension, and cerebral ischemia. The role of NCX in cardiovascular diseases is debatable. Some studies have shown that heart-specific NCX knockout mice can resist MI/R injury, which may be associated with calcium overload inhibition . Other s2+ back to the SR, causing myocardial relaxation , ATP2A2 , and ATP2A3 (SERCA3a-f), each of containing multiple subunits. SERCA2a is the most important, mainly expressed in the heart and slow-twitch skeletal muscle, and mainly involved in cardiomyocyte ECC. SERCA1 is mainly expressed in fast-twitch skeletal muscle, SERCA2b mainly in smooth muscle and non-muscle tissue, SERCA2c mainly in epithelial and hematopoietic cells and SERCA3 primarily in hematopoietic cells . SERCA hlaxation . SERCA cal loops .2+ uptake by increasing SERCA activity, and reduce reperfusion-induced calcium overload. SERCA is an essential calcium homeostasis and is associated with the pathological mechanisms of various diseases; SERCA is expected to become an important target for cardiovascular disease.A variety of molecules regulate SERCA; SERCA also affects intracellular calcium homeostasis. Phospholamban (PLB) phosphorylation can release the inhibitory effect on SERCA; PKA affects SERCA sensitivity to calcium . SERCA oPLB located in SR contains 52 amino acids (6.1 kD), easily forms a polymer (22 kD), which interacts with SERCA and inhibits its activity . PLB con2+-releasing proteins such as RyR2, LTCC, IP3R, and regulate calcium recalculation by PLB and SERCA phosphorylation  stores and releases Caunctions .2+ release other than that in the normal calcium cycle is called SR calcium leakage, which is mainly mediated by RyR2. Calcium leakage can cause cardiac systolic dysfunction by reducing effective SR Ca2+ release, cause diastolic dysfunction by increasing diastolic cytosolic Ca2+, and induce arrhythmias  formation. Additionally, mitochondria are also involved in processes such as apoptosis, signal transduction, cell proliferation, and metabolism . The carchondria . CalciumMitochondria are not involved in myocardial ECC regulation under physiological conditions. However, due to the large calcium capacity of mitochondria, which accounts for about 30% of the calcium capacity in plasma and tissue cells, they compensate by alleviating intracellular calcium accumulation during calcium overload in myocardial cells . Studies2+ is driven by a large electrochemical gradient across the plasma membrane into the cells. Cells use this external source of signal Ca2+ by activating various entry channels, such as voltage-operated channels . Excessive intracellular Na+ will promote Na+ excretion and Ca2+ intake by NCX, which significantly increases intracellular calcium levels, hence, leading to calcium overload. When blood flow and oxygen supply to the cardiac tissue returns to normal, extracellular pH level is further increased, HNX and NCX activities are enhanced, and intracellular calcium overload is further aggravated , in the myocardial cells. OFR can decompose cell membrane phospholipid components and damage membrane structure, which leads to increased membrane permeability and excessive extracellular Ca+ influx . OFR als+ influx .2+ plays a vital role in cardiac ECC, so calcium overload will further exacerbate the degree of MI/R injury in cardiac muscle cells. Excessive intracellular calcium will enter the mitochondria, resulting in mitochondrial calcium overload; this inhibits ATP production, exacerbates energy metabolism disorders, and eventually leads to myocardial cell apoptosis  pathway is a database that integrates genomic, chemical, and system function information, linking gene catalogs from genomes that have been fully sequenced to higher-level system functions at cell, species, and ecosystem levels , such as nifedipine, nisoldipine, diltiazem, verapamil, nimodipine, and amlodipine . Therefoecretion . In a wo2+ plays an essential role in maintaining cell proliferation, division, and energy metabolism. The calcium signal pathway-related protein expression and ion channel regulation mechanism connect arrhythmia, myocardial hypertrophy, and heart failure in cardiovascular diseases, making the development of these diseases a cause and effect  is complex, and the targets are not single. For cardiovascular diseases, which involve multiple signaling pathways, TCM has its unique advantages. For example, the past . BesidesIn fact, nearly half of the drugs listed in 2+ movement into and out of the plasma membrane and sarcoplasmic reticulum; calcium circulation disorders can cause various heart diseases. Changes in the Ca2+ steady-state, including decreased SR Ca2+ reuptake, abnormal calcium channels, SR Ca2+ leakage, or significantly decreased SERCA2a expression can cause various heart diseases, including ischemic heart disease, reperfusion injury, hypertrophic cardiomyopathy, diastolic cardiomyopathy, and heart failure , National Key Research and Development Project (grant number 2017YFC1702504 and 2018YFC1707408), and the National Natural Sciences Foundation of China (grant number 81891012).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The mutation slows Ca2+-binding kinetics, and the structural differences between wild type and E340A indicate that the mutation disrupts a central interdomain “communication hub” governing Ca2+ binding/dissociation. MD simulations reveal altered dynamics in regions mediating Ca2+ occlusion, a critical step in SERCA’s alternating access mechanism. The mutation stabilizes a more occluded state of the Ca2+ sites. The strict conservation of Glu340 among P-type ATPases is the result of its critical role in interdomain communication between the cytosolic headpiece and the transmembrane domain, ensuring a delicate balance between dynamics of ion binding, occlusion, and release—key steps in the transport process.We present a crystal structure, functional data, and molecular dynamics (MD) simulations of the sarco(endo)plasmic reticulum Ca 2+-ATPase (SERCA) is a P-type ATPase that transports Ca2+ from the cytosol into the sarco(endo)plasmic reticulum (SR/ER) lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca2+-binding sites and the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A. The mutated Glu340 residue is strictly conserved among the P-type ATPase family of membrane transporters and is located at a seemingly strategic position at the interface between the phosphorylation domain and the cytosolic ends of 5 of SERCA’s 10 transmembrane helices. The mutant displays a marked slowing of the Ca2+-binding kinetics, and its crystal structure in the presence of Ca2+ and ATP analog reveals a rotated headpiece, altered connectivity between the cytosolic domains, and an altered hydrogen bonding pattern around residue 340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilization of the Ca2+ sites in a more occluded state, hence displaying slowed dynamics. This finding underpins a crucial role of Glu340 in interdomain communication between the headpiece and the Ca2+-binding transmembrane region.The sarco(endo)plasmic reticulum Ca It pumps cytosolic Ca2+ ions across the sarco- or endoplasmic reticulum (SR/ER) membrane at the expense of ATP, a vital function in all living cells, particularly in the context of muscle contraction, Ca2+ signaling, and cell survival , phosphorylation (P), and actuator (A) domains, while the Ca2+-binding sites are located in the transmembrane (TM) part of the protein, which consists of 10 α-helices (M1 through M10) , and luminal release of these ions. This is associated with conformational transitions between so-called E1, E1P, E2P, and E2 forms (The molecular Caugh M10) A. ATP bi2+ transport  that are thought to relay the movements of the A domain to the M1–M3 helices. The P domain, however, is structurally very tightly integrated into the cytosolic ends of M4 and M5. M4 is linked to the so-called P1 helix (Pro337-Cys344), a short α-helix that runs roughly parallel to the membrane surface, at the membrane-facing side of the P domain. In such a location, P1 may be a key element of interdomain communication: it connects directly to a β-strand in the P domain ending with the phosphorylated aspartate residue, and it makes contact with the cytosolic end of M3 and to the loop between M6 and M7 (L6-7), which has been shown to play an important role in SERCA catalysis –17 Fig.A.2+ transport mechanism it is mandatory to obtain information on the structural relations of residues critical in mediating the communication between the membranous Ca2+-binding sites and the cytosolic phosphorylation site. Glu340 is a centrally positioned residue in the P1 helix at the interface between the phosphorylation domain and the cytosolic ends of M3 through M7  (To understand the Carough M7 A. It is able S1) .2+ interactions , and we examined its functional properties, including ATPase activity and Ca2+-binding kinetics. Notably, unlike other structurally characterized mutants, SERCA E340A is catalytically active, i.e., capable of completing a full catalytic cycle, albeit with altered kinetics. Furthermore, molecular dynamics (MD) simulations of both wild-type (WT) and E340A structures embedded in a lipid bilayer supported our conclusions derived from structural and functional studies. Our data link the structure to Glu340’s functional importance and provide insight into the interdomain communication that guides Ca2+ transport by SERCA.In this study, we have determined the crystal structure of yeast-expressed rabbit SERCA1a mutant E340A at 3.2-Å resolution in the Can = 9)  C, i.e., n = 9) , 17. Theved here –24.2+ binding to SERCA E340A, we measured the time course of the Ca2+-binding transition, by recording changes in intrinsic tryptophan fluorescence upon addition of Ca2+ to the E2 state of the protein. The E2 to Ca2E1 transition of E340A is significantly slower than that of the WT, with an increase of t1/2 of about threefold . This suggests that the effect of the E340A mutation on the overall turnover of SERCA may be caused by a delay in the conformational change associated with Ca2+ binding, i.e., the HnE2 → E1 → Ca2E1 transition (see further discussion below).In order to assess whether this overall deceleration is due to a reduced rate of CaSI Appendix, Table S2) allows for a detailed structural comparison with the WT structure. When superposed, the WT and E340A structures deviate by a substantial rmsd of 2.3 Å over all main chain atoms. Nevertheless, when superposed on the C-terminal transmembrane helix bundle M5 to M10 , the E340A mutant and WT have an almost identical arrangement of the remaining transmembrane helices M1 to M4 (main chain rmsd = 0.56 Å), with a small 1.6-Å bend at the cytosolic end of M3 in E340A . At the membrane-facing side of the P domain, the distance between Pro337 at the distal end of the P1 helix and Pro312 in M4 (the nearest residue to Pro337 at the approximate level of the membrane surface) decreases from 15.6 Å in WT to 12.3 Å in E340A. The pivot points of this headpiece movement are at the cytosolic ends of M2, M4, and M5. Whereas M2 and M4 stay very rigid throughout their entire lengths, M5 undergoes a slight bend in the region between its N-terminal end (at Phe740), which is embedded in the P domain, up till residue Gly750, which sits at the “rear side” of P1, exactly opposite residue 340 (Inset and Movie S1).In contrast to the almost identical arrangement in the TM domain, there is a pronounced shift (approximately 10 Å) in the position of the cytosolic headpiece, which is rotated further “downward” toward the membrane surface in E340A , their concerted movement relative to the rest of the structure in E340A is reflected by an overall main chain rmsd for the entire headpiece of 1.16 Å. The P and N domains appear to move together as one rigid body (main chain rmsd = 0.47 Å for the P/N body), whereas the A domain is displaced slightly further, with the distance between the N and the A domain increasing by approximately 3 Å in the final position.E1 to the Ca2+-bound E1 form (Ca2E1): when Ca2+ binds to WT SERCA, the entire P1 to M3 network moves upwards relative to L6-7 and M5, by about 4.5 Å, while the overall headpiece rotates down toward the membrane surface (E1  and thePDB 3N8G ] structu2+-binding movement in E340A led us to ask whether this mutation might lead to a stabilization of a Ca2+ occluded state, the state that follows Ca2+ (and nucleotide) binding in the catalytic cycle. Ca2+ occlusion is normally stabilized by the initiation of phosphorylation by ATP and not by Ca2+ binding alone , leaving only two contacts intact between these two domains (Arg134 to Asp426 and Thr171 to Glu486), whereas the A domain’s contacts to the P domain remain intact  C and D. 2E1 form of WT SERCA ) was recovered from pMT2 vector initially used for expression in COS cells  and clonescribed . TransfoS. cerevisiae, light membranes were prepared and solubilized by DDM for subsequent purification by streptavidin affinity chromatography , and 0.5 mg/mL DDM, together with some thrombin remaining from the elution procedure. The protein concentration in the purified fraction was typically in the 0.05- to 0.15-mg/mL range, depending on the batch. Further purification for crystallization trials was carried out as described previously for the SERCA E309Q mutant .Expression and purification of the E340A mutant SERCA1a was done as previously described for the WT enzyme . Brieflytography , 33, 35.Q mutant , involvi12E8 and 50 µM free Ca2+ to limit time-dependent inactivation of the SERCA1a (Steady-state ATPase activity measurements were performed using an enzyme-coupled assay by measuring the rate of NADH oxidation (followed by absorbance at 340 nm) in the presence of 0.02 mg/mL lactate dehydrogenase, 0.04 mg/mL pyruvate kinase, 1 mM phosphoenolpyruvate, 0.3 to 0.4 mM NADH, 5 mM MgATP  and in t SERCA1a , at 30 °2, 20% glycerol (vol/vol) and 2 mg/mL DDM, at 20 °C. Initial Ca2+ concentration was adjusted to 105 µM on top of the contaminating Ca2+ (3 to 5 µM) already present in the buffer. This was followed by the addition of 5 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EG), reducing [Ca2+]free to about 100 nM. An extra addition of 12.5 mM CaCl2 (Ca) allowed to reach a final [Ca2+]free of about 7.6 mM to recover the initial level of fluorescence. Data were fitted with the GraphPad Prism program with an exponential two-phase association law on the first 60 s of the raw data following Ca2+ addition.Intrinsic fluorescence was measured with a Fluorolog spectrofluorimeter (Horiba), in a temperature-regulated and continuously stirred 2-mL quartz cuvette. Excitation and emission wavelengths were set at 295 and 320 nm, with bandwith of 2 and 5 nm, respectively. Integration time for recording of the signal was 2 s. SERCA intrinsic fluorescence changes were measured with purified WT or purified E340A mutant suspended at a protein concentration of about 10 µg/mL in a buffer containing 50 mM Mes-Tris pH 6.5, 5 mM MgCl2+, Mg2+, and AMPPCP to 10, 3, and 1 mM, respectively, and crystallization trials were carried out using the vapor diffusion technique. The crystallization drops were prepared as 1-µL sitting drops, with each drop containing a 1:1 mixture of the concentrated, DOPC-relipidated, and C12E8-solubilized Ca2+-ATPase solution and the reservoir solution. Diffraction-quality crystals were obtained with reservoir solutions containing 200 mM lithium acetate, 18 to 22% PEG6K, 6 to 9% glycerol, and 3% tert-butanol. The E340A crystals varied markedly in appearance from crystals obtained with WT under similar conditions. Hence, the E340A crystals displayed a rectangular stick morphology, whereas WT crystals were diamond shaped , and E340A in space group P21212 with unit cell parameters , which are hitherto unknown parameters for SERCA. Phasing was done by molecular replacement with the Ca2E1-AMPPCP form [1T5S PHENIX  and PDB 6RB2 E340A). The protein atoms were described using the CHARMM36 force field , and buiA. The prARMM-GUI . GeneratThe systems were energy minimized using the steepest descent method, then equilibrated with positional restraints on heavy atoms for 100 ps in the NPT ensemble at 310 K with the V-rescale thermostat and semiisotropic Parrinello-Rahman pressure coupling , 43. ProAll simulations were run using GROMACS 2019 . SimulatSupplementary FileSupplementary FileSupplementary File


Mineralocorticoid receptors (MRs) in the brain play a role in learning and memory, neuronal differentiation, and regulation of the stress response. Within the hippocampus, the highest expression of MRs is in area CA2. CA2 pyramidal neurons have a distinct molecular makeup resulting in a plasticity-resistant phenotype, distinguishing them from neurons in CA1 and CA3. Thus, we asked whether MRs regulate CA2 neuron properties and CA2-related behaviors. Using three conditional knockout methods at different stages of development, we found a striking decrease in multiple molecular markers for CA2, an effect mimicked by chronic antagonism of MRs. Furthermore, embryonic deletion of MRs disrupted afferent inputs to CA2 and enabled synaptic potentiation of the normally LTP-resistant synaptic currents in CA2. We also found that CA2-targeted MR knockout was sufficient to disrupt social behavior and alter behavioral responses to novelty. Altogether, these results demonstrate an unappreciated role for MRs in controlling CA2 pyramidal cell identity and in facilitating CA2-dependent behaviors. In the brain, glucocorticoid and mineralocorticoid receptors  mediate learning and memory, emotional states, and behavioral and physiological responses to stress. In most cells, including neurons, GRs and MRs act as transcription factors that control vast downstream networks of other transcriptional regulators, so activation of either receptor by the endogenous ligands cortisol or corticosterone (CORT) initiates complex programs of gene expression and repression –3. AlthoIn contrast with GRs, which are expressed in virtually all cell types in the rodent brain, MRs are expressed primarily in neurons of limbic regions such as the hippocampus, lateral septum, and amygdala , 12, 14.Cornu Ammonis (CA) fields, MR protein and mRNA (Nr3c2) are both strongly expressed by pyramidal cells (PCs) during embryonic development [In the hippocampal elopment , 23 and elopment . Althougelopment , 26, theelopment  and confelopment , 29, whielopment –32. Receelopment , 32–36. Nr3c2, using single-molecule fluorescent in situ hybridization, confirmed that Nr3c2 was co-expressed with known CA2 pyramidal cell markers, aggrecan (Acan) and purkinje cell protein 4 (Pcp4) 16.5 and is highly concentrated in CA2 by E18.5 , lastingp4) Fig. . RNA-Seqp4) Fig. . This cop4) Fig. , suggestNestin promoter-driven cre recombinase (NesCre) to delete Nr3c2 embryonically in the whole-brain (see Methods). MRs are already expressed in the hippocampus by embryonic day 16.5 [+ mice  is a robust marker of CA2, starting by PN14 [+ mice, demonstrated here in mice older than PN45 , and these axon terminals can be labeled with antibodies raised against VGLUT2 . TherefoAmigo2 promoter-driven cre recombinase (Am2Cre) (see Methods). This line primarily targets cre to CA2 pyramidal neurons [+ animals  , spironolactone (MR-specific antagonist), or vehicle  under the cle Fig. . After 7cle Fig.  and decrcle Fig. , consistcle Fig. . As woulcle Fig. . These d+ ), embryonic neuronal loss of MRs enabled LTP in CA2 from P14-P18 mice . Synaptic responses recorded from CA2 neurons from NesCre– mice showed the typical resistance to the LTP pairing protocol (99.0 + /– 0.02% baseline). Intrinsic properties did not change in a way that could explain this enhanced plasticity  is normally not observed in stratum radiatum of CA2 pyramidal neurons , and preice Fig. . ExcitatNr3c2 in forebrain neurons, mice were shown to have deficits in social recognition memory and hyper-reactivity to novel objects [– and Am2Cre– mice spent more time investigating the novel mouse than the familiar mouse, suggesting they had a social memory of the familiar mouse, or at least had some preference for investigating novelty  exhibited hyper-reactivity, as measured by an increase in total distance traveled and a higher speed of movement when compared with their NesCre– littermates , and differ depending on whether the deletion is embryonic, affecting all neurons (NesCre+), or postnatal, targeting CA2 pyramidal neurons (Am2Cre+). Altogether, however, these studies provide evidence that MR deletion targeted to CA2 pyramidal neurons, is sufficient to produce some of the same behavioral deficits as the larger MR deletions using Nestin or CaMKII to drive expression of cre recombinase.To further assess for the possibility of an anxiety-like phenotype in the MR knockout mice, we studied the mice in an elevated plus maze. In this task, we observed no difference in the amount of time spent in or the number of entries into the open arms between NesCre+ mice, suggesting that these inputs may also be disrupted with a targeted postnatal deletion of MRs  of MR expression in mouse hippocampal area CA2 as assessed by conditional deletion of the receptor. Our findings demonstrate that MR expression is required for the acquisition of CA2-specific pyramidal neuron gene and protein expression, as well as for the maintenance of these expression patterns throughout development and into adulthood. Although the neurons comprising the anatomical CA2 were grossly intact following MR deletion, as observed with NeuN and DAPI stains, these cells failed to express the molecular markers that normally distinguish CA2 neurons from neighboring CA1 and CA3. In fact, both protein and mRNA analyses indicated that genes normally highly expressed in CA1 and CA3 increased in expression in MR knockout animals, suggesting that MRs are not only critical for the development and maintenance of CA2’s unique molecular profile, but also in suppressing CA1- and CA3-specific gene expression. Surprisingly, embryonic deletion of MRs also disrupted inputs from the SuM into both CA2 and DG, further defining their role in hippocampal development. Some of the CA2 proteins disrupted by MR deletion are also highly expressed in DG e.g., PCP4), and a loss of these proteins in both regions may be suggestive of their role in facilitating the development of hippocampal inputs. While not reaching significance, VGLUT2 staining to visualize SuM inputs into CA2 was also trending down in Am2Cre, and a lThe dramatic changes in CA2’s defining SuM input and in its molecular profile were accompanied by the physiologically abnormal enabling of Schaffer collateral LTP in CA2 pyramidal cells in mice with embryonic deletion of MRs. This phenomenon has also been observed in mice deficient for the plasticity-restricting protein RGS14 and in hippocampal slices treated with an enzyme that degrades CA2 PNNs , 46. We As expected because many hormone receptors become downregulated under constant activation , 43, 44,+) or postnatal, CA2-targeted deletion (Am2Cre+) of the gene encoding MRs, display many of these same deficits in behaviors as previously described for mice with postnatal forebrain deletion (using the CaMKII promoter). Specifically, we observed increased reactivity to novelty and a loss of social preference for a novel mouse. These findings emphasize a role for MRs in CA2 in regulating behavior, with deficits from loss of MR being similar to previous observations of cognitive and social deficits in mice with silenced, or lesioned CA2 neurons [Some clues concerning the function of MRs in the hippocampus come from pharmacological studies using specific agonists and antagonists of MRs, as well as studies using genetic techniques to manipulate MR expression. Pharmacological manipulations of MR signaling or conditional deletion or overexpression of MRs in the forebrain have pronounced effects on hippocampus-dependent spatial tasks , 53, anx neurons , 42, 59. neurons , 60 and  neurons , 36, 52 + mice traveled more, at a faster speed, and spent significantly less time in the center of a novel open field than did their cre-negative littermates. Within this group, males traveled more and faster than did females. These data suggest an increase in anxiety-like behavior in NesCre+ mice, with a potentially stronger effect in males. The same anxiogenic effects were not observed in the Am2Cre+ mice, indicating that these effects in NesCre+ mice are likely due to loss of MRs outside of the hippocampus or, alternatively, due to the loss of SuM inputs into CA2 in the NesCre+ mice, which might rely on a secreted factor from CA2 for their development. In fact, Am2Cre+ mice displayed reduced anxiety-like behavior in the elevated plus maze, spending more time in the open arms than did their cre-negative littermates. This is consistent with the finding that MR blockade with MR-specific antagonists can produce anxiolytic-like behavioral responses [The role of MRs and/or CA2 in anxiety are likely to be complex and are possibly sex-dependent. NesCreesponses , 61; thuThe results of this study show that many of genes that make CA2 unique in the hippocampus are under the transcriptional control of MRs, and that by deleting MRs embryonically, postnatally, or in adulthood, CA2 pyramidal neurons are drastically altered. This transformation is evident by the changes in protein and mRNA expression, disruption of SuM inputs into CA2, the presence of LTP in CA2, and behavioral changes observed in both social and non-social contexts. The requirement of a single, autoregulatory transcription factor for both the embryonic acquisition and adult maintenance of neuronal identity has been demonstrated in other model organisms and may in fact be a fundamental organizational principle for the nervous system –64. Such+/fl+) mice, with loxP sites flanking exons 5–6 [Nestin-cre line (B6.Cg-Tg(Nes-cre)1Kln/J, Jax 003771), the Amigo2-cre line (B6.-Nr3c2 < tm2Gsc > Tg(Amigo2-cre)8Ehs) [Emx1-cre line (B6.129S2-Emx1tm1(cre)Krj/J, Jax 005628). All offspring were genotyped using Transnetyx . All cre-positive mice used in each experiment were homozygous for the MR-floxed gene. Both males and females were used for all experiments and were not analyzed separately, unless otherwise reported. For each experiment, a power analysis was completed to determine the number of mice needed per group for statistical comparisons. Groups were determined based on genotypes (cre– or cre+). Exact animal numbers for each immunofluorescence experiment are shown in Supplemental Figs. All animal protocols were approved by the National Institute of Environmental Health Sciences Animal Care and Use Committee and are in accordance with the National Institutes of Health guidelines for care and use of animals. Mice were housed on a 12 h:12 h light-dark cycle with same-sex littermates, with up to five animals per cage. Mice were provided water and food ad libitum. All animals were crossed on a c57BL/6J background Jax 000664). Homozygous floxed mineralocorticoid receptor 8Ehs) , or to t. HomozygMice were sacrificed at several developmental time points to assess molecular changes as a result of MR deletion over the course of the lifespan. An early postnatal PN4) time point was chosen to determine the specific effect of embryonic deletion of MR (NesCre) at a time before the postnatal deletion (Am2Cre) occurred. Another subsest of animals was sacrificed at PN14 because this is a critical window during which many CA2 markers, including PNNs, begin to express , 46. Lat time poit-test. If the standard deviations of the two groups were not equal, Welch’s correction was implemented . All quantifications are shown as mean ± SEM, with dots representing each individual animal. All post-processing of images was performed in ImageJ after quantification and applied equally across compared genotypes in accordance with ethical policies on image integrity and standards.All images for quantification were taken on a Zeiss Epifluorescence microscope. Quantifications were made using original image files with ImageJ (1.49 v) and analyzed by an investigator blinded to genotype. Background fluorescence was subtracted using an area measured from outside of the hippocampal pyramidal layer. A region of interest (ROI) was highlighted in area CA2 using molecular and anatomical markers with the same size ROI used for every image. A minimum of two hippocampal measurements were averaged for each animal. Fluorescence intensities were normalized to the control group  and analyzed with a two-sided, unpaired 12) mice were anaesthetized with ketamine  and xylazine , then placed in a stereotaxic apparatus. Before surgery, animals were administered buprenorphine (0.1 mg/kg subcutaneous) for pain relief. An incision was made in the scalp, a hole drilled over the target region for infusion, and a 27-ga cannula connected to a Hamilton syringe by a length of tube was lowered into the CA2 region of the left hippocampus . For each infusion, mice were infused unilaterally with 0.5 µl of rAA5V-hSyn-GFP-cre over a 5-min period. The cannula was left in place for an additional 10 min before removing. The scalp was sutured and the animals were returned to their home cage for recovery. Animals were group housed for 3 weeks then sacrificed for immunofluorescence following the protocol outlined above.For infusion of rAAV5/hSyn-GFP-cre virus  were deeply anesthetized with Fatal Plus as described above and brains were quickly harvested on ice in an RNase-free environment. A sagittal cut was made to separate the two hemispheres. Each hippocampus was dissected free and the ventral one-third (approximate) was removed. Hippocampal tissue was immediately frozen on dry ice and stored at –80 °C for subsequent processing. RNA was extracted using the Qiagen RNeasy Mini Kit , and the concentration of each sample was measured using a nanodrop spectrophotometer. mRNA levels were determined using the NanoString (www.nanostring.com) platform utilizing a custom Code Set that measured 109 endogenous RNAs and 11 housekeeping genes . All but one sample passed nSolver’s initial QA/QC controls , and this sample was omitted from analysis. All replicates were well correlated (R > 0.95). Normalized data (log2 of counts) were imported into Partek  and quantile normalized for further QA/QC and statistical analyses. To identify significant differences in RNA expression, a t-test on treatment groups was performed with post-hoc Benhamini-Hochberg false-discovery rate for each group comparison. Six low-expressing genes from the custom code set did not quantify above noise and therefore were not included in further analysis   of pH 7.4 . Brain slices were cut coronally at 300 μm using a vibrating microtome (Leica VT 1000 S) and allowed to recover at room temperature in a submersion holding chamber with ACSF (in mM): 124 NaCl, 2.5 KCl, 2 MgCl2, 2 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, and 17 d-glucose bubbled with 95% O2 with 5% CO2. Whole-cell recordings were made from pyramidal neurons in area CA2, which were identified visually using differential interference contrast (DIC) optics . Glass borosilicate pipettes were filled with a potassium gluconate internal solution , with a tip resistance between 3–4.5 MOhms. Data were collected using Clampex 10.4 and analyzed using Clampfit software (Axon Instruments). Series and input resistances were monitored by measuring the response to a 10 mV step at each sweep and cells were included for analysis if series and input resistance changed by < 30%. Recordings were not compensated for series resistance. To assess excitatory transmission in P14-18 slices, whole-cell recordings were performed in voltage-clamp mode, and postsynaptic currents (PSCs) were evoked with a bipolar cluster electrode  placed in stratum radiatum. For long-term potentiation (LTP) experiments, baseline excitatory PSCs (EPSCs) were collected every 15 s for at least 5 min after which a pairing protocol was used, consisting of 1.5 min of 3 Hz presynaptic stimulation (270 pulses) paired with postsynaptic depolarization to 0 mV in voltage-clamp mode. Data were averaged and normalized to baseline. Biocytin was used to fill CA2 neurons to show representative neurons targeted in all LTP experiments , Pcp4 (Cat#402311), and Acan (Cat#439101). The slides were imaged on a Zeiss LSM 880 inverted confocal microscope with a 40x oil immersion lens. Acquisition parameters were set using 3plexed negative controls  in each of the three channels  so that any signal above the level of background was acquired. Area CA2 borders were defined using Pcp4 as molecular marker.Animals were deeply anaesthetized with Fatal Plus as described above, followed by rapid decapitation. Brains were harvested and frozen in Tissue Plus® O.C.T Compound  over dry ice. The brains were cryosectioned at 20 μm and mounted on SuperFrost® Plus slides (Fisher Scientific). The slides were then processed for single-molecule FISH according to the RNAscope Fluorescent Multiplex kit instructions . The following probes were used: GSE116343). Briefly, laser capture microdissected samples (n = 3 per region) targeting the cell body region of CA1, CA2, CA3, and dentate gyrus were obtained and sequenced using an Illumina NextSeq 500 instrument, acquiring 100 bp paired-ends reads to a depth of 50 million reads per sample ( + /– 10 mil). Raw reads were pseudoaligned to GENCODE (vM12) gene models and the mm10 mouse genome and quantitated per gene using Salmon  [2 expression from six donors was collected for MR and GR expression from each of the hippocampal subregions, CA1, CA2, CA3, and DG. The expression values for each region were transformed into an MR:GR ratio for each donor. A one-way ANOVA was conducted to compare the ratios, and Bonferroni post-hoc analysis was used to determine significant differences between CA2 and each of the other regions.RNA-Seq data were obtained from previously published work , 67 (Gensize 31) –68. The size 31) . Log2 exn = 5 males and 5 females per group) were briefly anesthetized with 2.5% isoflurane and subcutaneously implanted with a 21-day release pellet  containing either 15 mg corticosterone (Cat#G-111), 25 mg spironolactone , or vehicle (Cat#C-111). After 7 days, animals were perfused and brains were processed for immunofluorescence, as described above. One control male was removed from the study because he removed his wound clips and pellet shortly after surgery. No sex differences were detected and therefore males and females were collapsed for analyses. Comparisons were made using a one-way ANOVA, with a priori LSD post hoc comparisons between the control group and each of the two drug groups.Mice  were brought to the behavior room and allowed to habituate for a minimum of 20 min before each behavioral task. All behavioral experiments were performed during the light cycle. For each task, all subjects were run within the same 3 h circadian window. All mice were tested in each behavioral task with a minimum of 24 h between each task to minimize interference from previous HPA axis activation on subsequent tasks. Social assays were completed a minimum of 72 h after the last non-social task. The experimenter was blinded to genotype during all behavioral testing. Four animals were excluded from analysis for the following reasons: one Am2Cre+ male was inadvertently left out of the elevated plus maze, and therefore not included in any analyses; one Am2Cre– male fell off the elevated plus maze and was removed from the study; one Am2Cre+ female jumped out of the arena during novel object testing and was removed from the study; one NesCre– female was removed from the study as an outlier (z-score = 3.44 for both distance traveled and speed during sociability task). Final animal number per group were as follows: NesCre–n = 18 , NesCre+ n = 13 , Am2Cre–n = 20 , Am2Cre+ n = 17 . Post hoc comparisons between sexes were made within each group, and, unless otherwise reported, no sex differences were observed during any behavioral task.Animals . Each animal was placed on a clean elevated plus maze in the center and left to investigate for 5 min. The animal was removed after 5 min and returned to its home cage. Movement about the maze, including distance traveled, speed, and entries into and duration spent in the open arms were recorded using Noldus Ethovision software. Comparisons between cre-negative and cre-positive mice were completed using a two-tailed t-test. Each arena was thoroughly cleaned with an ammonia solution between each animal. The following day , each animal was again placed in the same clean, empty arena and allowed to investigate for 10 min in order to habituate to the environment. On the fourth day, two identical novel objects were placed on the edges of the arena , animals were placed in a clean, empty novel arena and allowed to investigate for 10 min, after which they were returned to their home cage. The animals’ reactivity to the novel environment, including distance traveled, speed, and duration spent in the center of the arena, was recorded using Noldus Ethovision and compared between cre-negative and cre-positive mice using a two-tailed ena Fig. . The aniThe following week, animals were tested for social investigation and social discrimination. Animals were placed in an arena with three chambers (with a door between the chambers) and a small perforated barrier in one corner of each of the top and bottom chambers. They were allowed to investigate the entire arena for 10 min. After 10 min, the mice were gently moved into the center chamber and the doors were closed. A novel sex- and age-matched conspecific was added behind one of the barriers Fig. . The dooSupplemental Information


Metabolic derangements, including depressed high-energy phosphate stores due to insulin deficiency or insulin resistance, as well as hormone imbalance and ultrastructural alterations, are also known to occur in the diabetic heart. It is pointed out that the activation of the sympathetic nervous system and renin–angiotensin system generates oxidative stress, which produces defects in subcellular organelles including sarcolemma, sarcoplasmic reticulum and myofibrils. Such subcellular remodeling plays a critical role in the pathogenesis of diabetic cardiomyopathy. In fact, blockade of the effects of neurohormonal systems has been observed to attenuate oxidative stress and occurrence of subcellular remodeling as well as metabolic abnormalities in the diabetic heart. This review is intended to describe some of the subcellular and metabolic changes that result in cardiac dysfunction in chronic diabetes. In addition, the therapeutic values of some pharmacological, metabolic and antioxidant interventions will be discussed. It is proposed that a combination therapy employing some metabolic agents or antioxidants with insulin may constitute an efficacious approach for the prevention of diabetic cardiomyopathy.Although the presence of cardiac dysfunction and cardiomyopathy in chronic diabetes has been recognized, the pathophysiology of diabetes-induced metabolic and subcellular changes as well as the therapeutic approaches for the prevention of diabetic cardiomyopathy are not fully understood. Cardiac dysfunction in chronic diabetes has been shown to be associated with Ca Type 1 diabetes occurs as a result of insulin deficiency due to a defect in insulin-producing β-cells in the islets of Langerhans of the pancreas, while type 2 diabetes occurs as a consequence of insulin resistance/insensitivity of insulin receptors . In factDiabetes has also been shown to be associated with the activation of sympathetic nervous system (SNS) and increased outflow of catecholamines, and the activation of the renin–angiotensin system (RAS) and increased formation of angiotensin II. The elevated levels of these vasoactive hormones due to insulin-deficiency are considered to produce marked defects in myocardial metabolism and remodeling of sarcolemma (SL), sarcoplasmic reticulum (SR), mitochondria (MT) and myofibril (MF) ,11,12,13In order to demonstrate that cardiac dysfunction in diabetes occurs progressively, and to examine the relationship between heart dysfunction and subcellular abnormalities, we have previously reported the time-course of changes in subcellular remodeling following the induction of diabetes in two well-established experimental models of type 1 diabetes using streptozotocin (STZ) and alloxan ,30,31. T+-K+-ATPase, SL Na+-Ca2+-exchanger, SR Ca2+-stimulated ATPase, SR Ca2+-uptake and MF Ca2+-stimulated ATPase was evident at 2 wks, whereas changes in cardiac function (LVEDP and LVSP), heart rate and mean arterial pressure were apparent at 4 wks after alloxan-induced diabetes [+-K+-ATPase, SL Na+-Ca2+-exchanger, SR Ca2+-stimulated ATPase, SR Ca2+-uptake and MF Ca2+-stimulated ATPase were seen at 3 wks after the induction of diabetes with STZ. Taken together, cardiac dysfunction may be either associated with or due to subcellular abnormalities as a consequence of increase in plasma glucose and decrease in plasma insulin levels. Therefore this article aims to discuss the significance of subcellular defects with respect to Ca2+-handling in cardiomyocytes in diabetic heart. It also intends to describe hormonal imbalance that is associated with elevated plasma levels of catecholamines, angiotensin II and 5-hydroxytryptamine (5-HT) as well as increased fatty acid oxidation in diabetes and that leads to the generation of oxidative stress. It is the oxidative stress that is considered to be the main cause of subcellular abnormalities and altered Ca2+-handling in the cardiomyocytes and subsequent cardiac dysfunction in diabetes (2+ concentration (verapamil), catecholamine action (propranolol), angiotensin II action , 5-HT (sarpogrelate), metabolic defects (propionyl-L-carnitine) and oxidative stress (vitamin E) should be indicated in attenuating subcellular remodeling for improvement of cardiac function and therapy of diabetic cardiomyopathy [diabetes ,30. Furtdiabetes  show thadiabetes . Accordimyopathy ,26. Metabolic and ultrastructural derangements associated with insulin deficiency and/or insulin resistance can be seen to play an important role in the development of cardiac dysfunction in chronic diabetes ,7,8,32. In view of the involvement of mitochondria in the metabolism of glucose and FFA and their main function to produce ATP, a defect in energy production due to an impairment of the electron transport system in these organelles can be seen to be associated with increased formation of oxyradicals and occurrence of oxidative stress. It should be mentioned that although plasma levels of both glucose and FFA are elevated in diabetic subjects, glucose metabolism is markedly reduced due to impaired glucose transport in cardiomyocytes ,38,39. FSeveral studies have investigated the morphological appearance of hearts from control and experimental diabetic animals ,26,43. T2+ has been shown to produce cell damage and contractile failure [2+-handling abnormalities have been associated with cardiac dysfunction in diabetic cardiomyopathy. A depression in the activity of SL Na+-K+ ATPase in the diabetic heart has been suggested to promote the entry of Ca2+ [+- Ca2+ exchanger activity can be seen to raise the intracellular concentration of Ca2+ [2+-pump activity has been reported to depress the extrusion of Ca2+ from the diabetic cardiomyocytes [2+/Mg2+-ecto ATPase in diabetic heart has been considered to promote the entry of Ca2+ [+-H+ exchanger would favor the accumulation of H+ for the release of Ca2+ from the intracellular Ca2+-stores [2+-binding and L-type Ca2+-channel density were suggested to reduce Ca2+-influx into the diabetic heart [2+-channels are biphasic in nature, where at initial stages of diabetes, these changes promote Ca2+-entry, but at later stages, the reduction in SL Ca2+-channel density could lead to depressed cardiac contractile activity [An excessive amount of intracellular Ca failure ,26,44, a of Ca2+ ,46 where of Ca2+ ,47,48. Tmyocytes ,48,49. T of Ca2+  whereas +-stores ,51,52. Tic heart . It is pactivity . 2+-uptake and Ca2+-pump ATPase activities [2+-release activity was also depressed in hearts from diabetic animals [2+-transport activities in the SR membranes, were impaired in the diabetic heart [2+-uptake and -release activities in the diabetic heart seems to occur due to changes in the phosphorylation/dephosphorylation status of both SR and SL Ca2+-cycling proteins [2+-handling abnormalities in SR would tend to favor the occurrence of intracellular Ca2+-overload [2+-antagonist, verapamil, attenuated defects in subcellular activities [2+- uptake and SR Ca2+-stimulated ATPase activities were decreased in the diabetic heart as compared to control values. Chronic treatment of diabetic rats with verapamil resulted in improvement of cardiac function as well as altered myofibrillar ATPase activity and SR Ca2+-pump activities without affecting the plasma glucose/insulin levels. Accordingly, it was suggested that normalization of subcellular organelle function in diabetic cardiomyopathy by verapamil may be related to its effects in controlling the entry of Ca2+ into the cardiac cell [Several investigators have observed defects in SR Cativities ,26; SR C animals ,56,57. Vic heart ,26,28,54proteins ,58,59,60overload ,62,63,64tivities ,65. Tabliac cell . +-K+ ATPase, Na+-dependent Ca2+-uptake and ATP dependent Ca2+-uptake, as well as SR Ca2+-uptake and Ca2+-release activities and MF Ca2+-stimulated ATPase activity in diabetic hearts, were attenuated by propranolol treatment [In view of the activation of SNS and increased level of plasma catecholamines in early stages of diabetes ,34, it hreatment . These o+-K+-ATPase, Na+-dependent Ca2+-uptake, and Ca2+-pump, as well as reduced SR Ca2+-release and Ca2+-pump activities and decreased MF Ca2+-ATPase activity in the diabetic heart, were also improved  is activated during the development of cardiac dysfunction in diabetes ,67. The improved . These r2+-handling, it was suggested that the improvement in cardiac function in response to sarpogrelate in diabetes is due to a facilitation of glucose transporter expression levels and possibly insulin production [Since there is an increase in platelet aggregation and the circulating levels of 5-HT have been reported to be elevated in diabetes ,68, the 2+-pump and SL Na+-K+ ATPase activities were improved by treatment of animals with etomoxir, an inhibitor of carnitine palmitoyltransferase [2+-pump, SL Na+-K+ ATPase and SL Na+-Ca2+ exchange as well as cardiac function without any changes in SL Ca2+-pump or MF Ca2+-stimulated ATPase activities . Interestingly, the acute effects of β-endorphin infusion in patients with mild to moderate CHF have been observed to improve LVEF, reduce systemic vascular resistance and diminish neurohormonal activation; these changes were associated with stimulation of the GH/IGF-1 pathway . It is t2+-handling abnormalities, defects of the MF would be more associated with insensitivity of MF to Ca2+. It is likely that Ca2+-handling defects, metabolic derangements and ultrastructural abnormalities may be a consequence of hormonal imbalance as well as occurrence of oxidative stress and intracellular Ca2+-overload. Pharmacological interventions that inhibit the SNS, RAS, 5-HT receptors and Ca2+-channels, as well as agents that can modulate fatty acid metabolism and those that can exert antioxidant actions, have been shown to partially prevent or normalize subcellular remodeling and cardiac dysfunction. Accordingly, it is suggested that a combination therapy with different interventions (as summarized in The complexity of diabetic cardiomyopathy is confounded by the diverse range of mechanisms that are involved in the pathogenesis of cardiac dysfunction in diabetes. Increases in plasma levels of norepinephrine, angiotensin II and 5-HT have been observed at different stages of diabetes, which may contribute to the metabolic changes as well as functional alterations of the heart. In addition, depressed cardiac contractility in diabetes may also be due to subcellular remodeling in cardiomyocytes at the level of the SL, SR and MF. While the SL and SR defects in diabetic heart appear to result in Ca


AML is a heterogeneous disease both in genomic and proteomic backgrounds, and variable outcomes may appear in the same cytogenetic risk group. Therefore, it is still necessary to identify new antigens that contribute to diagnostic information and to refine the current risk stratification.  The expression of C-type lectin-like molecule-1 (CLL-1) in AML blasts was examined in 52 patients with newly diagnosed or relapsed/refractory AML and was compared with two other classic markers CD33 and CD34 in AML, in order to assess the value of CLL-1 as an independent biomarker or in combination with other markers for diagnosis in AML. Subsequently, the value of CLL-1 as a biomarker for prognosis was assessed in this malignant tumor. high and CLL-1low groups according to cutoff 59.0%, we discovered that event-free survival and overall survival (OS) of the CLL-1low group were significantly lower than that of the CLL-1high group, and low CLL-1 expression seems to be independently associated with shorter OS.  The results showed that CLL-1 was expressed on the cell surface of the majority of AML blasts (78.8%) and also expressed on leukemic stem cells in varying degree but absent on normal hematopoietic stem cells. Notably, CLL-1 was able to complement the classic markers CD33 or CD34. After dividing the cases into CLL-1 These preliminary observations identified CLL-1 as a biomarker for diagnosis and prognosis of AML. Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults, which is characterized by the accumulation of immature myeloid cells in the bone marrow (BM) leading to hematopoietic dysfunction . AML is +/CD38– compartment. In the setting of AML, CLL-1 is detected on the majority of AML blasts varying from 77.5% to 92% at diagnosis. More importantly, CLL-1 is also present on leukemic stem cells (LSCs), which is the main cause of treatment failure and leukemia relapse [Human C-type lectin-like molecule-1 , dendritic cell-associated lectin 2 (DCAL2), or killer cell lectin like receptor-1 (KLRL1)) is emerging as a surface marker of blasts in AML these years. It is a type II transmembrane glycoprotein which plays a role in immune regulation as an inhibitory receptor –10, thou relapse –15. Acco relapse , 16–23. In this study, we first examined the expression of CLL-1 in samples of newly diagnosed or relapsed/refractory AML patients and compared it with other classic markers in AML, in order to assess the value of CLL-1 as an independent biomarker or in combination with other markers for diagnosis in AML and subsequently assessed the value of CLL-1 as a biomarker for prognosis in this malignant tumor.9/L; platelet count > 100 × 109/L. This study was approved by the Ethics Committee of the Guangdong General Hospital and Sun Yat-sen University Cancer Center.From April 2015 to March 2020, a total of 52 prospectively accrued diagnostic patients with de novo or relapsed AML from the Department of Hematology, Guangdong General Hospital, and Sun Yat-sen University Cancer Center were enrolled in this study. The patients who simultaneously suffered from other malignant tumor were excluded from this study. All samples and medical information were collected with informed patient consent and in accordance with the Declaration of Helsinki. Diagnosis of patients was based on morphology using the French-American-British (FAB) classification, immunophenotyping, cytogenetics, and molecular genetics. Relapse risk for acute promyelocytic leukemia (APL) was classified according to Sanz score . Genetic4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4), and washed with phosphate-buffered saline (PBS) containing 0.1% fetal calf serum (FBS). The frozen samples were prepared using red blood cell lysis buffer, then frozen in RPMI 1640 media  containing 20% FBS and 10% dimethylsulfoxide and subsequently stored in liquid nitrogen. When analysis was needed, the cells were thawed and suspended in prewarmed RPMI with 10% FBS at 37°C and enabled to recover for 45 min. The prepared samples were then used to perform multiparameter flow cytometry (FCM) subsequently.BM samples were collected at diagnosis after informed consent from 52 AML patients. In two cases, BM was not available, and PB was used. Five control normal BM samples were obtained from healthy volunteers in the setting of BM harvest for allogeneic BM transplantation after informed consent. The majority of the samples were analyzed freshly. Red blood cells were lysed afterward by 10 minutes of incubation on ice, using 10 mL lysis buffer . PBS was used as a negative control, because for these specific antibodies, isotype controls provided the same results. The gating strategy has been described in detail before . In short test (two tailed) was used for continuous variables. Survival analysis was performed using the Kaplan-Meier method, and survival between groups was compared using log-rank tests. Univariate and multivariate analyses were used to estimate the prognostic impact of different variables in overall survival (OS) and event-free survival (EFS) using the Cox regression model. OS was measured from the date of diagnosis to the date of death from any cause or the last follow-up. EFS was measured from the date of diagnosis to the date of disease progression or relapse or death from any cause or the last follow-up. Analyses were performed using SPSS version 19 statistical software, and two-tailed values of p < 0.05 were considered significant.We evaluated the predictive value of CLL-1 expression level using the area under the receiver-operating characteristic (ROC) curve and determined the cutoff point to maximize the sum of the sensitivity and specificity. Categorical variables were compared using the chi-square test. The Student's To analyze the expression profile of CLL-1 on AML blasts, we defined positive antigen expression as the expression of the antigen in more than 20% of the sample cells. In the prospectively collected test set, 41 of 52 (78.8%) patients stained CLL-1 positive with different intensities, consistent with our previous report . Represep < 0.05), but no difference with CD33  and 28 of 47 (59.6%) AML samples, respectively. Interestingly, of the 7 CD33-negative samples, 3 were positive for CLL-1 antigen. Furthermore, of the 19 CD34-negative samples, 15 were positive for CLL-1 antigen. When we compared the antigen expression levels, we found that CLL-1 was more frequently expressed than CD34 (ith CD33 .+CD38− stem cells from part AML patients (n = 17) and healthy donors (n = 5). We observed that CLL-1 expression on LSCs showed interindividual variability from 2.5% to 52%. And most importantly, the expression level on LSCs was higher than on HSCs   and 19 CLL-1low cases (<59.0%). As seen in low group was significantly related to lower BM blast percentage (p < 0.05). More importantly, the CLL-1low group was closely related to CR rate which was significantly lower than the CLL-1high group . However, we did not find any significant correlation between CLL-1 expression level and other clinicopathological features including gender, age, disease status, WBC count at diagnosis, FAB subtype, and risk group (data not shown).We established the cutoff value of CLL-1 expression at 59.0% with an area under curve (AUC) of 0.694  and OS (p = 0.012) compared to the higher group. In view of the prevalence of risk-adapted therapy is common in AML nowadays, it was also important to check the possible additional value of CLL-1 in different risk status of AML patients. We found that the CLL-1high patients (n = 9) had better EFS than the CLL-1low patients (n = 7) in the poor-risk group  and OS (p < 0.001) were significantly better among patients who attained a CR after one cycle of chemotherapy than those without a CR , the median EFS and OS were 15 months  11.9-18.0) and 23 months (95% CI 16.2-29.8), respectively. The estimated 2-year EFS was 43.4% (95% CI 25.6%-61.2%) while the estimated 2-year OS was 42.9% (95% CI 25.5%-60.3%). During the follow-up period, 23 patients died due to disease progression. After having established the definition of CLL-1 Figures .p = 0.002), and the significant factors affecting OS included treatment response (p < 0.001) and CLL-1 expression (p = 0.019). Multivariate analysis showed that the treatment response and CLL-1 expression remained significantly independent factors for OS . However, the differences in gender , age , WBC counts , BM blasts counts , and risk stratification  were not statistically significant.Univariate and multivariate analyses of potential prognostic factors for EFS an OS are summarized in AML is a highly heterogeneous disease with different pathophysiological characteristics, therapeutic response, and prognosis . The leu+/CD38− LSCs with heterogeneous expression pattern [NCT03038230) [Upon analysis of 52 de novo or relapsed AML samples, we confirmed that CLL-1 was expressed on the cell surface of the majority of AML blasts (78.8%), clearly indicating that the CLL-1 could be used to be the routine FCM in AML. Consistent with previously published work, we found that CLL-1 was also present on the CD34 pattern , while a pattern –15, 28,  pattern , antibod pattern , 17, bis pattern –20, and  pattern , 22, 23 3038230) . We have3038230) .− subgroup. Notably, we are the first to compare the antigen expression levels of CLL-1, CD33, and CD34 simultaneously on AML bulk cells.How would CLL-1 perform compared with the classic markers CD33 and CD34? In the present study, we found that CLL-1, CD33, and CD34 were positive in 78.8%, 86.0%, and 59.6% of AML samples, respectively. Interestingly, CLL-1 was complemented with CD33 or CD34 as diagnostic markers and  therapeutic targets, because 42.9% of the CD33 negative AML samples expressed CLL-1, and 78.9% of the CD34 negative samples expressed CLL-1. Moreover, when the CLL-1 and CD33 were combined, 92% (46 of 50) of the AML samples could be identified. Consequently, our coexpression analysis suggested that combinatorial detecting approaches might enhance diagnosing efficiency in AML, and combinatorial targeting approaches might enhance therapeutic efficacy in AML that should be validated in the future. Besides, CD34, a stem cell-specific marker in AML, is widely used to monitor minimal residual disease (MRD). The addition of CLL-1 maybe valuable in increasing the likelihood of upfront MRD marker identification by FCM in the CD34high and CLL-1low according to cutoff 59.0%, we found that the low expression level of CLL-1 was significantly correlated with lower BM blast percentage, and what is more, the CR rate after cycle 1 of the CLL-1low group was significantly lower than the CLL-1high group (p < 0.05). We know that low remission rates often indicate poor prognosis, and we did demonstrate that both the EFS and OS were significantly worse among patients who did not reach CR than those did. Meanwhile, no CR was demonstrated to be an independent factor associated with EFS and OS in multivariate Cox regression model. However, a statement on the possible correlation with other clinicopathological features, such as gender, age, disease status, WBC count at diagnosis, FAB subtype, and risk group, cannot be presently made probably due to the relatively small number of patients.We next analyzed the relationship between CLL-1 expression level and clinicopathological features. We found that CLL-1 was nonrandomly expressed on AML samples throughout the different FAB subtypes and risk groups. After having established the definition of CLL-1high and CLL-1low groups, we discovered that EFS and OS of the CLL-1low group were significantly lower than the CLL-1high group (p < 0.05). Furthermore, in univariate Cox regression model analysis, we found that the CLL-1low group was an independent prognostic value associated with OS, and multivariate Cox regression model analysis showed that CLL-1low was still independent from other well-established factors. However, the low expression level of CLL-1 did not maintain its value in predicting EFS. Taking into consideration that risk stratification is critical in AML, we analyzed the impact of CLL-1 expression in each risk subgroup. The results showed that the CLL-1high patients had better EFS and OS than the CLL-1low patients (p < 0.05) in the poor-risk group. This implied that CLL-1high patients in the poor-risk group had a more intermediate prognosis comparing to poor-risk CLL-1low patients, who actually have a worse prognosis. As a result, the CLL-1 expression should be incorporated into future risk-adapted therapy and prognosticating relapse risk in this subset of poor-risk AML patients. We did not find similar results in other risk subgroups probably due to the small numbers. This study agreed with Wang et al., who also demonstrated that CLL-1low indicated poor prognosis (p < 0.001 for EFS and OS) in patients with AML. Nevertheless, they detected the CLL-1 expression only in de novo CD34+ Non-M3 AML, while we evaluated the CLL-1 expression in nonselected patients including de novo and relapsed AML, further supporting its high value in prognosis. Besides, some studies have compared the expression of CLL-1 on AML samples at diagnosis, treatment, and relapse and found no significant difference [When we compared the survival between CLL-1fference , 29–31. Concerning the mechanism of low CLL-1 expression in leukemic blasts as a predictor of poor prognosis, it remains unclear. It was previously reported that CLL-1 might play a role in the control of cell maturation , so we cTaken together, we have demonstrated that CLL-1 is one of the promising surface molecules for AML diagnosis; meanwhile, CLL-1 is easy to measure in clinical practice and thus can be incorporated into the routine practice of most clinical laboratories. Furthermore, we have proved that CLL-1 is an effective tool to predict the survival of AML patients, so it can be used as a supplement to the current AML prognostic risk stratification system and may optimize the clinical management of AML. However, the relatively small number of patients and the short follow-up time limited us to draw a robust conclusion in our study, and cytogenetic and molecular genetic profiles were not complete for all patients. Further and larger-scale studies are required to more clearly define the significance of CLL-1 expression in AML and also to elucidate the underlying mechanisms in the future.low group were significantly lower than the CLL-1high group, and low CLL-1 expression seems to be independently associated with shorter OS. These results suggested that CLL-1 may serve as a biomarker for diagnosis and prognosis of AML.In summary, we report that CLL-1 was expressed on the cell surface of the majority of AML blasts (78.8%) and also expressed on leukemic stem cells in varying degree but absent on normal hematopoietic stem cells. Notably, CLL-1 was able to complement the classic markers CD33 or CD34. In addition, we discovered that EFS and OS of the CLL-1