Ca2+ signals must be tightly controlled for a healthy heart, and the impairment of Ca2+ handling proteins is a key hallmark of heart disease. The discovery of microRNA (miRNAs) as a new class of gene regulators has greatly expanded our understanding of the controlling module of cardiac Ca2+ cycling. Furthermore, many studies have explored the involvement of miRNAs in heart diseases. In this review, we aim to summarize cardiac Ca2+ signaling and Ca2+-related miRNAs in pathological conditions, including cardiac hypertrophy, heart failure, myocardial infarction, and atrial fibrillation. We also discuss the therapeutic potential of Ca2+-related miRNAs as a new target for the treatment of heart diseases.In hearts, calcium (Ca Heart disease (HD) is a major concern in global health. The number of patients with heart disease worldwide has nearly doubled in the last 10 years . HD is aThe coronavirus disease 2019 (COVID-19) is spreading worldwide, with over 170 million infected and 3.7 million deaths across nearly 200 countries. People with HD or those who have had a stroke are associated with an increased risk of severe complications from COVID-19 . There i2+) are critical for regulating muscle contraction, and abnormal Ca2+ homeostasis in cardiac cells is critical in the pathogenesis of common HD. In this review, we will describe the role of the main Ca2+ handling proteins in cardiac function and recent findings related to the mechanism of regulation of Ca2+ homeostasis via microRNAs (miRNAs) in diseased hearts and their potential for HD therapy.In the heart, calcium ions  increases above 1 µM, allowing interaction between the contractile elements during systole and diastole, and [Ca2+]i decreases to approximately 100 nM, causing dissociation of the contractile elements [2+]i, extracellular Ca2+ enters the cytoplasm across the plasma membrane (sarcolemma), or intracellular Ca2+ is released from Ca2+ storage organelles, including the sarcoplasmic reticulum (SR). In cardiomyocytes, the sarcolemma Na+/Ca2+ exchanger (NCX) is the primary mechanism by which Ca2+ is released from cells. Activation of the sarco/endoplasmic reticulum Ca2+-adenosine triphosphatase 2 (SERCA2) pump promotes the reuptake of cytosolic Ca2+ into the SR. Numerous other molecules are involved in Ca2+ homeostasis in cardiomyocytes i during ECC, and in cardiomyocytes, most Ca2+ is stored in the SR within the milli-molar range. Ca2+-buffering proteins  within the SR lumen are important Ca2+ sensors and contribute to deciphering Ca2+ transients and signals during ECC.The contraction movement of hearts is carried out by excitation–contraction coupling (ECC), which occurs in cardiomyocytes through Cagulation . Ca2+ re2+ plays two pivotal roles in the ECC. Ca2+ drives myofilament activation and regulates sarcolemma ionic currents that are responsible for normal electrical rhythms. A series of ECC events in cardiomyocytes are as follows: (1) initiation and propagation of an AP along with the plasma membrane, (2) rapid spread of the potential along with the transverse tubule system (T-tubule system), (3) dihydropyridine receptors -mediated detection of changes in membrane potential, (4) ryanodine receptors (RyRs) stimulation due to allosteric interaction of the LTCC with the SR RyRs, (5) release of Ca2+ through stimulated RyRs in SR and transient increase in [Ca2+]i, (6) transient activation of the cytosolic Ca2+ buffering system and the contractile apparatus, followed by (7) disappearance of Ca2+ from the cytosol mediated by its movement to the mitochondria via the mitochondrial Ca2+ uniporter (MCU), its expulsion by the NCX and plasma membrane Ca2+–ATPase (PMCA) at the sarcolemma, and its final reuptake by the SR through the SERCA2. Both the duration and intensity of cardiac AP affect the regulation of Ca2+ fluxes and contractility in cardiomyocytes.Cardiac ECC is the process whereby an electrical stimulation  of the surface membrane with an action potential (AP) triggers a cardiomyocyte to depolarize and contract. Ca2+-mediated calmodulin (CaM)-dependent kinase (CaMK) signaling, which is activated by increased cytosolic Ca2+content, thereby inducing regulation of ECC. More recent studies have reported a process called excitation–transcription coupling (ETC), which reveals a similar role of Ca2+ in controlling gene transcription in cardiomyocytes to that of Ca2+-dependent signaling of excitation–contraction coupling i, decreased SR Ca2+ uptake, increased SR Ca2+ leak, and decreased SR Ca2+ content. SERCA2a and RyR2 are primarily responsible for the sequestration and release of SR Ca2+. Both SERCA2a and RyR2 are regulated by post-translational modifications (PTMs) and interacting partner proteins. Therefore, strategies to restore impaired SR Ca2+ homeostasis caused by an abnormality in these two Ca2+ handling proteins have been extensively studied for the treatment of HF. The major SR Ca2+-handling proteins reported to be associated with HD are summarized in ECC defects that cause contractile dysfunction and cardiac arrhythmia are typical features of HF. In failing cardiomyocytes, a major defect in Ca2+]i and prolongation of diastolic Ca2+ decay. SERCA2a is the dominant SERCA isoform in hearts and determines the clearance of more than 70% of cytosolic Ca2+ in humans and 90% of cytosolic Ca2+ in rodents i and reactive oxygen species (ROS). Initially, the elevation of [Ca2+]i triggers Ca2+binding to CaM, activating CaMKII. Activation of CaMKII by Ca2+-CaM depends on the local Ca2+ level and the frequency of Ca2+ release. When [Ca2+]i increases briefly, CaMKII returns to its inactive form after Ca2+-CaM dissociation. However, the continuous presence of Ca2+-CaM allows for autophosphorylation of CaMKII, which causes “CaM trapping” to maintain CaMKII activity even at low [Ca2+]i conditions. For example, CaMKII can be activated through PTMs induced by ROS independent of Ca2+-CaM but also activated in response to βAR/PKA stimulation i [SERCA2a is also a target of  [Ca2+]i . In addi [Ca2+]i .miR-212/miR-132 cluster were upregulated in cardiomyocytes under hypertrophic conditions. The miR-212/miR-132 family is a calcineurin/NFAT signaling cascade regulator that directly downregulates the anti-hypertrophic Forkhead box protein O3 (Foxo3) transcription factor. Ucar et al. reported that miR-212/132 knockout mice protect cardiac function from pressure-overload-induced HF, whereas cardiac miR-212/132 overexpressing mice displayed pathological HT and HF phenotypes [Serca2a gene [Expression levels of the enotypes . Recent a2a gene . Preclina2a gene . CDR132La2a gene .2+-dependent signaling during cardiac HT and HF through the inhibition of CaMKII.Additionally, it was reported that dysfunction of miR-30, miR-148/152, and miR-625 is likely to be associated with CaIschemic heart disease or myocardial infarction (MI) is the most common underlying cause of HF. In pathological hypertrophy, adverse cardiac remodeling after myocardial ischemia eventually leads to cardiac dysfunction and decreased performance. Among the complex signaling networks that characterize myocardial remodeling, the distinct processes are cardiomyocyte loss, cardiac hypertrophy, alteration of extracellular matrix homeostasis, fibrosis, autophagy defects, metabolic abnormalities, and mitochondrial dysfunction.2+ transport system with cytosolic Ca2+ overload are significant contributors to myocardial I/R injury [2+ uptake have been reported in most myocardial I/R studies. Consistently, the rate of SR Ca2+ reuptake significantly decreased in the human myocardium after reverse I/R. Several studies have reported that I/R-mediated cytosolic Ca2+ overload can be reduced by pharmacological inhibition of reverse-mode NCX activity. In addition, altered expression or activity of LTCCs and CaMKII, and oxidative modifications of Ca2+ handling proteins after I/R injury have been reported in experimental animals.Although reperfusion strategies to reduce ischemic injury are widely used in clinics for MI, reperfusion following ischemia (I/R) injury is an important complication of this therapy. Cardiomyocyte death, arrhythmias, and contractile dysfunction are the main signs of myocardial I/R injury. Defects in the myocardial CaR injury . ReductiMany studies have suggested that miRNAs are associated with the progression of myocardial I/R injury and MI ,96. For miR-214 is transcribed together with miR-199a-2 and is upregulated in response to various cardiac stresses, including pressure overload, MI, and excessive calcineurin, a calcium/calmodulin-sensitive phosphatase. Aurora et al. reported that miR-214 null mice exacerbated myocardial I/R injury due to loss of cardiac contractility, increased apoptosis, and excessive fibrosis [2+ handling was confirmed in miR-214 knockout mouse hearts. miR-214 directly inhibited Ncx1, increasing NCX protein expression, and reverse mode activity miR-214 knockout mice. Furthermore, CaMKII, a key regulator of Ca2+ signaling, also contains a miR-214 binding site, and mRNA levels of CaMKII increased in miR-214 deficient hearts upon I/R compared to wildtype-controls.fibrosis . ThroughmiRNA-145 is enriched in hearts and is dysregulated in animal models after I/R injury. miR-145 is downregulated in MI rats, whereas upregulation of miR-145 expression promotes the repair of infarcted myocardium. In cardiomyocytes, miR-145 inhibits ROS-induced Ca2+ overload and Ca2+-related signals by directly targeting CaMKII [g CaMKII .miR-1 is also known to be involved in the I/R injury process [ process . While mMicroRNA dysregulation due to I/R injury involves different reactions depending on the damage process. Acute inhibition or overexpression of miRNAs after MI might be beneficial in limiting tissue damage and preventing long-term adverse remodeling and HF.2+ handling abnormalities, triggering spontaneous diastolic SR Ca2+ release. However, the SR Ca2+ load was not increased significantly, suggesting that SR Ca2+ leaks occur due to altered RyR2 function. Excess diastolic Ca2+ is removed by NCX, creating a net depolarizing current called the transient inward current that produces DADs. Several studies have demonstrated that the level of RyR2 protein is elevated in the atria of patients with paroxysmal AF. In addition, increased NCX expression and function are commonly observed in AF. Altered Ca2+ signaling may also contribute to structural and electrical remodeling. For example, Ca2+ overload can activate the calcineurin-NFAT pathway, leading to HT and fibrosis.AF is a persistent cardiac arrhythmia, especially in the elderly, which can cause or exacerbate HF. Delayed afterdepolarizations (DADs) are the most important mechanisms underlying focal atrial activity . AF atrimiR-1 and the onset of cardiac arrhythmia has been observed in animal models and humans. miR-1 expression is significantly upregulated in ventricular arrhythmias. The possible mechanisms of hyperactivated miR-1-mediated arrhythmia include enhanced Ca2+ release, dissociation of phosphatase activity from the RyR2 complex, and altered expression of K+ channels that impairs the kinetics or membrane trafficking systems. Several studies have shown that miR-1 and miR-133 may indirectly increase calcium release by targeting B56α, a regulatory subunit of PP2A. Yang et al. found that miR-1 promotes ischemic cardiac arrhythmias by targeting the KCNJ2 gene, which encodes the Kir2.1 inward rectifier K+ channel protein subunit, and the GJA1 gene, encoding connexin-43 gap junction channel protein subunit [+ current density. These results suggest different effects of miR-1 under other arrhythmia conditions; therefore, the exact mechanism of miR-1 mediated arrhythmia generation requires further study.The link between  subunit . In addi subunit . In cont subunit . This inmiR-106b~25 cluster consists of the highly conserved miR-106b, miR-93, and miR-25 and is downregulated in patients with paroxysmal AF, which is associated with elevated RyR2 expression. miR-106b and miR-93 could negatively regulate RyR2-3′UTR [2+ release, resulting in increased AF sensitivity in mice.The R2-3′UTR . MoreovemiR-208(miR-208a/b) family is a cardiomyocyte-specific miRNA encoded within introns of the cardiac myosin heavy chain genes . Of the two isoforms, increased miR-208b was consistently observed in tissues of damaged hearts, especially MI and dilated cardiomyopathy. Canon et al. observed upregulated miR-208b expression in AF cardiomyocytes [2+ handling impairment during atrial remodeling.The myocytes . AberranmiR-21, miR208b, and miR-328 contributed to adverse electrical remodeling in AF by targeting LTCC. In particular, miR-21 has been extensively studied for its role in atrial fibrosis during AF. Several studies have reported that circulating miR-208b and miR-328 are related to AF prevalence [In addition, upregulated evalence , suggestThe modified miRNAs of hearts due to AF contribute to cardiac electrical and structural remodeling. A recent meta-analysis conducted by Shen et al. reported that 283 miRNAs changed compared to AF and non-AF controls . Of the As our knowledge of the biosynthesis and functionality of miRNAs in hearts has increased over the past two decades, attempts have been made to identify HD-related miRNAs and understand mechanisms of action and downstream effectors for potential clinical applications. Inconsistent findings in the expression profiling of several miRNAs and miRNA-target relationships may be due to differences in HD models (types and stages of disease), sample sources, subjects of study, and technical issues. However, these discrepancies also indicate that miRNAs mediate gene regulation in a tissue-cell-specific manner for a particular disease state.Various strategies are being used to modify miRNA levels through restoration/replacement or suppression as a potential treatment method . A speciGene therapy has often been suggested as a technique to limit local approaches rather than systemic administration, including limited vector size, inefficient delivery to target tissues, and nuclear localization requirements for protein synthesis. In this context, miR-regulated treatment based on gene therapy may be a better choice. miR regulators are much smaller, so there are no limitations on the size of the genes being delivered . As miR To date, only a few miRNA-based drugs are still in the early phases of clinical trials, but there are clinical research efforts to investigate novel miRNA drug candidates for HD along with the miR-132 study (described above) . For exa2+ homeostasis in cardiac cells and the mechanisms of Ca2+ regulation through miRNAs, whose functions are altered during HD. A potential benefit of miRNA-based therapies is their ability to simultaneously target many genes in a given pathway and ease of application. Given the critical role of Ca2+ metabolism in cardiac pathophysiology, Ca2+ regulating miRNAs have significant potential as therapeutic agents in HD.In this review, we explored critical molecules involved in Ca


The current lack of understanding of the mechanisms leading to neurovascular deficits in glaucoma is a major knowledge gap in the field. Retinal pericytes regulate microcirculatory blood flow and coordinate neurovascular coupling through interpericyte tunneling nanotubes (IP-TNTs). We demonstrate that pericytes constrict capillaries in a calcium-dependent manner during glaucomatous stress, decreasing blood supply and compromising neuronal function. Moreover, ocular hypertension damages IP-TNTs and impairs light-evoked neurovascular responses. The reestablishment of calcium homeostasis in pericytes restores vascular and neuronal function, and prevents retinal ganglion cell death in glaucomatous eyes. This study provides important insights into the therapeutic potential of pericytes to counter vascular dysregulation in glaucoma. 2+ influx rescued hemodynamic responses, protected IP-TNTs and neurovascular coupling, and enhanced retinal neuronal function as well as survival in glaucomatous retinas. Our study identifies pericytes and IP-TNTs as potential therapeutic targets to counter ocular pressure-related microvascular deficits, and provides preclinical proof of concept that strategies aimed to restore intrapericyte calcium homeostasis rescue autoregulatory blood flow and prevent neuronal dysfunction.Reduced blood flow and impaired neurovascular coupling are recognized features of glaucoma, the leading cause of irreversible blindness worldwide, but the mechanisms underlying these defects are unknown. Retinal pericytes regulate microcirculatory blood flow and coordinate neurovascular coupling through interpericyte tunneling nanotubes (IP-TNTs). Using two-photon microscope live imaging of the mouse retina, we found reduced capillary diameter and impaired blood flow at pericyte locations in eyes with high intraocular pressure, the most important risk factor to develop glaucoma. We show that IP-TNTs are structurally and functionally damaged by ocular hypertension, a response that disrupted light-evoked neurovascular coupling. Pericyte-specific inhibition of excessive Ca Glaucoma is the leading cause of irreversible blindness worldwide, affecting 80 million people globally in 2020 . There i49Acta2 gene, independent of whether they are located on superficial or deep vascular plexuses   . The diameter of larger vessels did not change, indicating that the observed responses were not caused by upstream arterioles , and there was no reduction in retinal capillary density .Unilateral OHT was induced by injection of magnetic microbeads into the anterior chamber of the mouse eye. Microbeads were attracted to the iridocorneal angle with a magnet to block aqueous humor outflow and increase intraocular pressure (able S1) . We seleA and B) . CapillaA and B) C. PericyA and B) D–F. A siMovie S1). We observed a significant reduction of capillary blood flow in glaucomatous eyes compared with sham controls . Live TPLSM visualization showed that reduced blood flow correlated with pericyte-induced capillary constriction (Movie S4). Furthermore, the probability of capillary blood flow interruption at pericyte locations increased by fivefold in glaucoma relative to sham controls . No vascular deficits were observed in sham-operated controls using identical imaging conditions  per second , 24 , thus better reflecting early pathology onset. We recently identified IP-TNTs, nanotube-like processes mediating pericyte-to-pericyte communication in the retina, which control local neurovascular coupling and coordinate light-evoked responses between adjacent capillaries . Similar to the retina, IP-TNTs within the optic nerve head were damaged in glaucoma . IP-TNTs mediate pericyte-to-pericyte communication through (Ca2+) transients that are essential for the coordination of capillary responses (2+ transients were measured as spontaneous Ca2+ increases in IP-TNT–coupled pericytes imaged in mice expressing the Ca2+ indicator GCaMP6f downstream of the NG2 promoter (NG2-GCaMP6f) both in live retinas by TPLSM and retinal explants . A marked decrease in the frequency of these Ca2+ transients was observed following induction of OHT . Immunohistochemical analysis of retinas from Cacna1c-null mice (−/−Cacna1c) confirmed the selective absence of Cav1.2 in pericytes, whereas adjacent neurons were positive for Cav1.2, thus serving as internal controls (+/+Cacna1c) expressed Cav1.2 in both pericytes and neurons . We found a slight increase in basal capillary diameter in noninjured −/−Cacna1c retinas relative to +/+Cacna1c controls, which likely reflects the effect of reduced pericyte contractility on capillary tone, but basal blood flow, capillary blockade probability, and total number of IP-TNTs were similar, thus confirming the absence of major vascular alterations .In addition to playing a key role in pericyte-to-pericyte communication, cytosolic Caericytes . To inveexplants A–C. OHT controls A–C. A machannels , of whicchannels . To testcontrols D and E.  neurons F. Analys retinas G and H. −/−Cacna1c mice and wild-type littermate controls following induction of OHT or sham surgery. Conditional pericyte-specific Cacna1c deletion did not alter microbead-induced pressure elevation . Our data demonstrate that restoration of Ca2+ homeostasis in pericytes lacking Cacna1c preserved capillary diameter and blood flow in glaucoma, in addition to reducing the likelihood of capillary blockage, to levels found in sham controls , and that light-evoked hemodynamic responses in −/−Cacna1c mice with OHT were similar to sham controls . Collectively, these results demonstrate that 1) excessive intrapericyte Ca2+ promotes vascular dysregulation, and 2) pericyte-specific Cav1.2 deletion restores intrapericyte Ca2+ homeostasis, capillary dynamics, blood flow, and neurovascular coupling in glaucoma.Next, we examined capillary and blood flow changes in controls I–K. We acontrols M–O. In c−/−Cacna1c mice affected RGC activity, we recorded light-evoked single-RGC Ca2+ responses by TPLSM using an adeno-associated virus (AAV) encoding GCaMP6f driven by the synapsin promoter (AAV-GCaMP6f). Selective GCaMP6f expression in RGC was confirmed on retinal flat mounts and cross-sections using the RGC-specific marker RBPMS (RNA-binding protein with multiple splicing) . We focused on alpha ON-sustained (αON-S) RGCs, a major cell class characterized by maintained firing during the bright phase   . In wild-type control retinas, GCaMP6f-positive αON-S RGCs elicited a brief burst of light-evoked Ca2+ signal followed by a rapid signal decay or recovery , Ca2+ signals were reduced and recovery was significantly delayed (Movie S8). Quantitative analysis of Ca2+ signal parameters confirmed a significant reduction in the peak amplitude response and increased decay time in neurons fed by capillaries with reduced blood flow . Next, we investigated whether preventing vascular abnormalities rescued RGC function in ocular hypertensive eyes by measuring light-evoked Ca2+ responses in RGCs from −/−Cacna1c mice. Our results demonstrate that, contrary to wild-type mice showing significant alterations in αON-S RGC Ca2+ dynamics after glaucoma induction  . Cav1.2 deletion promoted RGC survival and, strikingly, preserved neuronal density at a level similar to that found in noninjured sham-operated control eyes. In contrast, substantial RGC death was observed in wild-type littermate controls    , 31. The S5 C–G) –32. To crecovery B and C. ive eyes D–G. To eood flow J and K. ecovery) D–G, Cacnesponses L–Q. Lastcontrols R–U. We c2+ influx to pericytes preserved capillary dynamics, improved blood flow, protected IP-TNTs, and rescued neurovascular coupling during ocular hypertensive stress. Importantly, the restoration of microvascular function by selectively reducing intrapericyte Ca2+ restored light-evoked RGC responses and promoted neuronal survival. Together, these findings reveal that pathological changes in pericytes caused by loss of Ca2+ homeostasis play a critical role in microcirculatory blood flow deficits that contribute to RGC dysfunction and damage in glaucoma.Glaucoma is a complex and multifactorial neurodegenerative disease. There are two main forms of the adult-onset pathology based on the mechanism of aqueous humor outflow obstruction: open-angle glaucoma (OAG) and angle-closure glaucoma. OAG, the most common type, is characterized by slow and asymptomatic damage to the optic nerve that correlates with elevated intraocular pressure . OAG incSI Appendix, Fig. S1G and Movie S4). Optogenetic activation of brain capillary pericytes was recently shown to constrict capillaries and reduce blood flow  promoter (NG2-DsRed) for selective visualization of retinal pericytes ; 2) Ca2+ indicator GCaMP6f (fast variant) downstream of the NG2 promoter (NG2-GCaMP6f), generated by crossing NG2-Cre mice  with Ai95(RCL-GCaMP6f)-D mice  which harbor the Rosa-CAG-LSL-GCaMP6f::deltaNeo conditional allele with a floxed-STOP cassette; and 3) pericyte-specific conditional deletion of the gene encoding Cav1.2 (Cacna1c) generated by crossing NG2-Cre mice  with floxed Cacna1c mice . For two-photon live imaging, we generated albino mice by backcrossing each of these lines with CD-1 mice. Animals were housed in 12-h light–12-h dark cyclic light conditions, with an average in-cage illumination level of 10 lx, and fed ad libitum. All procedures were performed under general anesthesia .Animal procedures were approved by the University of Montreal Hospital Research Center and followed Canadian Council on Animal Care guidelines. Experiments included adult female and male mice  from the following strains: 1) red fluorescent protein under the control of the NG2 . Using a micromanipulator, we used the tip of the microneedle to gently puncture the cornea, and injected the microbeads into the anterior chamber avoiding injury to ocular structures such as the lens and iris. A hand-held magnet was used to immediately attract the magnetic microbeads to the iridocorneal angle. Sham controls received an injection of phosphate-buffered saline (PBS). We applied an antibiotic eye drop to the operated eye  and allowed the animal to recover on a heating pad. We measured the intraocular pressure before and after the procedure, and biweekly thereafter, in awake animals using a calibrated TonoLab rebound tonometer (Icare). For intraocular pressure measurements, a drop of proparacaine hydrochloride  was applied to the cornea and, holding the tonometer perpendicular to the eye surface, we took and averaged 10 consecutive intraocular pressure readings per eye.Unilateral elevation of intraocular pressure was performed by a single injection of magnetic microbeads into the anterior chamber of the mouse eye as described . Briefly2 cd/m2, 6 ms) with a PowerLab unit (ADInstruments) presented using a white light–emitting diode centered relative to the pupil and located 5 mm away from the corneal apex. Stimulus onset (t = 0) and TPLSM imaging recording were synchronized offline by identifying the frame at which the light stimulus was registered. We carried out image acquisition using a wavelength of 820 nm to excite TRITC/DsRed or FITC-dextran and a mean laser power at the sample plane of 15 to 50 mW. Imaging was performed throughout the entire thickness of the retina below the sclera (depth 50 to 200 μm) and multiple fields were scanned  at 40 Hz and acquired during light stimulation.TPLSM live retinal imaging was performed as previously described . Mice we13 particles per milliliter; Addgene). The tip of a custom-made glass micropipette was inserted into the superior quadrant of the eye at an ∼45° angle, through the sclera into the vitreous body, avoiding injury to eye structures or retinal detachment.We administered the following fluorescent probes or reagents by intravitreal injection : TRITC-lectin , Fluo-4-AM , or AAV serotype 9 carrying GCaMP6f under the control of the synapsin promoter  and the vascular diameter was computed. Recordings with large-amplitude motion, which led to loss of focus during live imaging, were discarded (<5%). To rule out artifacts from movements in the z axis, we imaged the volume of capillaries by capturing five-slice stack series during light stimulation and compared it with single-plane measurements. No vessels were eliminated from our analysis and we recorded thousands of capillaries from all vascular plexuses throughout the retina using an unbiased stereological sampling approach. We normalized diameter changes after light stimulation relative to changes prior to stimulus presentation and classified them as positive or negative based on their dilation or constriction response, respectively. We calculated maximum responses by averaging the global diameter change after a light stimulus. IP-TNT z projections were done with Imaris software (Bitplane).Immediately prior to TPLSM imaging, we performed tail-vein injection of FITC-coupled dextran  or intraperitoneal injection of fluorescein  to label vessels. For live imaging of IP-TNTs, we injected fluorescently tagged lectin (Thermo Fisher Scientific) into the vitreous chamber 1 h prior to imaging. We acquired 40-Hz recordings at a resolution of 512 × 512 pixels (90 × 90 pixels at the region of interest), which were automatically corrected for residual movements with ImageJ (NIH) and the TurboReg plugin . Diameter measurements were then performed by placing a linear probe at the desired location, perpendicular to the fluorescent plane of the filled vessel, using ImageJ (NIH). After projecting the signal every five frames, the fluorescent pattern was exported to a custom R routine freely available  disector frame were acquired with an Axio Imager M2 optical sectioning microscope  and analyzed using ImageJ (NIH). The vessel diameter was measured at locations where the circular probe touched the vessel. The number of capillaries within each disector was quantified and capillary density was calculated.FITC- or Texas red–coupled dextran  was administered by tail-vein injection and blood flow was assessed by quantification of RBCs that crossed a defined location per second before and after light stimulation. RBCs do not take up dextran, and hence they were identified as shadows against the fluorescent background . CapillaBandeiraea simplicifolia) . We incubated retinal sections with the following primary antibodies: laminin 2 , Cav1.2 , RBPMS , choline acetyltransferase , and SMI-32 . We incubated antibodies in blocking solution  at 4 °C overnight for 3 d, followed by fluorophore-conjugated secondary antibodies . Flat-mounted retinas and retinal cross-sections were rinsed and mounted in antifade solution with DAPI  for visualization using an epifluorescence microscope  or a confocal microscope . Eight retinal cross-sections per eye were analyzed.Animals were deeply anesthetized and transcardially perfused with 4% paraformaldehyde (PFA) or methanol. Eyes were immediately collected and processed to generate cryosections as described , and the2+ transients in regions of interest were longitudinally recorded by TPLSM (excitation 920 nm) and signals were calculated as ΔF/F = (F − F0)*100/F0, where F0 is the fluorescence baseline and F is the fluorescence at time t. We quantified Ca2+ transients automatically using a custom R routine freely available (https://www.r-project.org) when the maximum peak was ≥3 times the SEM over baseline fluorescence, and Ca2+ transient frequency was calculated at each region of interest. We defined Ca2+ transients as rapid intracellular Ca2+ increases in individual pericytes. A subset of Ca2+ transients between IP-TNT–connected pericytes was identified and recorded as synchronous Ca2+ peaks in linked pericytes within a window of 3 s around each peak, as described , obtained the intensity value of each pixel in every frame using ImageJ (NIH), and color-coded their values over time with a custom R routine  freely available (https://www.r-project.org). Fluo-4-AM allowed us to visualize Ca2+ levels by combining the signal from 50 frames, but it did not enable the measurement of Ca2+ dynamics in pericytes and IP-TNTs. For quantification of Ca2+ signals in retinal explants, we superfused the explants from NG2-GCaMP6f mice with oxygenated bicarbonate-buffered artificial cerebrospinal fluid solution  bubbled with 95% O2, 5% CO2 at 34 °C. Time-lapse images were acquired using a Quorum Technologies confocal microscope with a CSU10B (Yokogawa) spinning head mounted on a BX61W1 fluorescence microscope (Olympus) and connected to an ORCA-ER camera (Hamamatsu Photonics). Images were captured using Volocity software (Improvision) and analyzed by ImageJ (NIH). For ex vivo quantification of Ca2+ signals, we removed the eyes, fixed them in 4% PFA, and flat-mounted the retinas. Using an unbiased stereological sampling approach, we acquired images over the entire retina with identical exposure times and gain settings for all experimental and control groups . Raw fluorescence intensity in each cell, acquired from images obtained using the same exposure time for all cohorts, was measured manually with ImageJ (NIH). Background fluorescence obtained from three surrounding square areas was subtracted to yield the final value for each region of interest. High-Ca2+ pericytes were defined as pericytes with an intensity ≥2 times the SEM over sham pericyte fluorescence. Ca2+ signals in −/−Cacna1c and wild-type littermate mice were visualized by intravitreal injection of the Ca2+ indicator Fluo-4-AM (Invitrogen) 1 h prior to imaging.We identified IP-TNTs and their associated pericytes by TPLSM in NG2-GCaMP6f mice after intravitreal injection of TRITC-lectin. Caescribed . To incr2+ responses in RGCs, we administered AAV-GCaMP6f by intravitreal injection 3 wk prior to TPLSM imaging. RGCs expressing GCaMP6f were scanned at 12 Hz and Ca2+ signals were analyzed in circular regions of interest encompassing the entire soma. We examined light-evoked Ca2+ responses by averaging the fluorescence intensity of all pixels within the region of interest using ImageJ (NIH) after background subtraction. We calculated the ΔF/F peak and decay time, defined as the time to fall to one-third of the ΔF/F peak, with a custom R routine freely available (https://www.r-project.org), and light-responsive neurons were defined as cells with ΔF/F >2.To evaluate Cax-, y-, and z-axis movement, a color camera , and image analysis software . Using the stereological random sampling method described above, RBPMS-labeled RGCs and lectin-positive IP-TNTs were counted using 3D disectors (stacks) throughout the entire retina, and the number of RGCs or IP-TNTs was calculated . IP-TNTs were visualized in flat-mounted retinas labeled with fluorescently tagged lectin (Thermo Fisher Scientific). Retinal images were obtained using an Axio Imager M2 optical sectioning microscope  equipped with an automatically controlled specimen stage for lculated , 59.F) tests. We compared values corresponding to vessel diameter, blood flow, probability of capillary blockage, Ca2+ signal amplitude, Ca2+ decay, number of IP-TNTs, RGC density, and Ca2+ transients. Stereological quantifications were compared by means of two-tailed Student’s t or Mann–Whitney U tests, where appropriate. For multiple comparisons, we used ANOVA followed by Dunnett’s, Tukey’s, or Kruskal–Wallis tests, where appropriate. A P value ≤ 0.05 was considered significant. All regression lines of diameter-change graphs were fitted with the same order between experimental and control cohorts.We always carried out data analysis blinded by third-party concealment of treatment using uniquely coded samples. The number of animals used in each experiment as well as the number of cells or structures analyzed are indicated in the figure legends. All values are provided as the mean ± SEM, and individual values are presented in each graph. Statistical analysis was performed with Prism 9 (GraphPad). We evaluated all cohorts with normality (Shapiro–Wilk) and variance (Supplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary File


Dear Editor,9/L or > 100 ×109/L . Gen. Gen7]. We evaluated the prognostic impact of the genetic classifications as well as that of each individual gene according to the treatment group. In all multivariate survival analyses, no significant interaction between dexamethasone treatment and classifications or gene mutations was found, indicating that the effect of dexamethasone did not differ significantly between the various genetic subsets.P = 0.020).When the genomic classification was tested in multivariate analysis, AML with inv(16)/CBFB–MYH11 was the only subgroup with a significant impact on overall survival (OS)  or NPM1mut/DNMT3Amut .The co-occurrence of  poor OS. This mutNPM1mut: HR, 0.56; 95% CI: 0.33-0.97; P = 0.039 and myeloid transcription factor gene fusions or mutations: HR, 0.34; 95% CI: 0.19–0.60; P < 0.001) were significantly and independently associated with better OS whereas the chromatin-modifying gene subset was associated with poorer OS .Regarding functional gene categories, 2 subsets (P = 0.001). However, there was no significant difference between intermediate and favorable prognostic groups .The ELN 2017 adverse group was independently associated with poor OS . On the contrary, CBFB-MYH11 , CEBPA , NPM1  and surprisingly, RUNX1 mutations  were significantly and independently associated with better OS. The different types of RUNX1 mutations, co-mutations, response to treatment with or without dexamethasone and outcome are shown in Table RUNX1mut AML, 13 (86.7%) achieved a complete response.Finally, we assessed the prognostic impact of each individual gene using the least absolute shrinkage and selection operator (LASSO) statistical method Table . DNMT3A Multivariate analyses for event-free survival, relapse-free survival and cumulative incidence of relapse yielded similar results (data not shown). Of note, clinical or treatment parameters including infection at diagnosis, secondary AML, hydroxyurea, albumin, LDH, fibrinogen, CD56 expression, admission to intensive care unit (ICU) or allogeneic stem-cell transplantation retained an independent prognostic value in most multivariate analyses Table .FLT3 or RAS pathways, mutations in DNA methylation genes and NPM1 were over-represented whereas other subgroups, such as RUNX1-RUNX1T1 or TP53 mutations had a very low frequency. Compared to the Taiwanese study, we found far fewer CEBPA biallelic mutations (2.6% vs. 16%) and more NPM1 (53.2% vs. 30%) or FLT3 mutations (62.3% vs. 44.5%). Apart from the difference in median age between the two cohorts (60 vs. 50 y), we have no clear explanation for this difference [This study shows that the genomics of hyperleukocytic AML differs substantially from nonhyperleukocytic AML. Signalling mutations in either ations 2.% vs. 16%FTL3 mutations (~60% of patients in our series) including ELN 2017 adverse risk patients [FLT3 inhibitor. Thus, further studies are needed to assess the impact of midostaurin in hyperleukocytic AML. Inhibition of the RAS pathway could also be a valuable avenue in this setting [A large proportion of hyperleukocytic AML patients present at diagnosis with therapeutically targetable mutations (>70%). This brings hope that the use of targeted therapies may improve their prognosis, which is still poor compared to nonhyperleukocytic AML. Midostaurin combined with intensive chemotherapy has improved the outcome of AML patients with patients . Our pat setting .RUNX1 mutations (RUNX1mut AML) is a provisional entity of the WHO 2016 classification that accounts for 10% of newly diagnosed AML. In our series of hyperleukocytic patients, the frequency of RUNX1 mutations was similar . RUNX1mut AML are classified in the ELN 2017 adverse risk group. However, RUNX1 mutations were independently associated with better OS in our cohort. A recent study using a chemogenomic approach revealed that AML cells with inactivating RUNX1 mutations were particularly sensitive to very low concentrations of glucocorticoids [RUNX1mut AML has yet to be determined.AML with rticoids . HoweverRUNX1, NPM1, or SRSF2 modulate gene expression in a manner that primes AML cells for glucocorticoid sensitivity [Studies on the mechanisms of resistance to chemotherapy or tyrosine kinase inhibitors together with high-throughput drug screening have underpinned the potential role of glucocorticoids in AML. Cytarabine resistance is associated with the acquisition of sensitivity to glucocorticoids and mutated sitivity . These rHyperleukocytic AML remains a very challenging clinical management issue. This is evidenced by the high number of multivariate analysis parameters that reflect the patients’ general condition, inflammation, metabolism or therapeutic management. Acting on these parameters could be key to limiting early complications and induction deaths. Leukoreduction with hydroxyurea, anti-inflammatory treatments, such as dexamethasone and early or direct admission to ICU have recently been proposed to this end whereas the benefit of leukapheresis has not been demonstrated . New tarSupplemental Material


In HNSCC patients, PD-1 blockade increases KCa3.1 and Kv1.3 activity along with Ca2+ signaling and mobility in CD8+ peripheral blood T cells (PBTs). The mechanism by which PD-L1/PD-1 interaction regulates ion channel function is not known. We investigated the effects of blocking PD-1 and PD-L1 on ion channel functions and intracellular Ca2+ signaling in CD8+ PBTs of HNSCC patients and healthy donors (HDs) using single-cell electrophysiology and live microscopy. Anti-PD-1 and anti-PD-L1 antibodies increase KCa3.1 and Kv1.3 function in CD8+ PBTs of HNSCC patients. Anti-PD-1 treatment increases Ca2+ fluxes in a subset of HSNCC patients. In CD8+ PBTs of HDs, exposure to PD-L1 reduces KCa3.1 activity and Ca2+ signaling, which were restored by anti-PD-1 treatment. The PD-L1-induced inhibition of KCa3.1 channels was rescued by the intracellular application of the PI3 kinase modulator phosphatidylinositol 3-phosphate (PI3P) in patch-clamp experiments. In HNSCC CD8+ PBTs, anti-PD-1 treatment did not affect the expression of KCa3.1, Kv1.3, Ca2+ release activated Ca2+ (CRAC) channels, and markers of cell activation (CD69) and exhaustion (LAG-3 and TIM-3). Our data show that immune checkpoint blockade improves T cell function by increasing KCa3.1 and Kv1.3 channel activity in HNSCC patients.Programmed death receptor-1 (PD-1) and its ligand (PD-L1) interaction negatively regulates T cell function in head and neck squamous cell carcinoma (HNSCC). Overexpression of PD-1 reduces intracellular Ca Head and neck squamous cell cancer (HNSCC) is the seventh most common cancer worldwide . Convent2+ required for downstream effector functions, such as migration and proliferation. Calcium release activated Ca2+ (CRAC), voltage-gated K+ (Kv1.3) and Ca2+ activated K+ (KCa3.1) channels are involved in the early phase of T cell activation and regulate the Ca2+ influx necessary for their effector functions. Ca2+ influx is triggered by the T cell receptor (TCR) mediated-depletion of Ca2+ stores in the endoplasmic reticulum and opening of the CRAC channels, and it is aided by Kv1.3 and KCa3.1 channels, which allow maintenance of the negative membrane potential necessary for Ca2+ influx through the CRAC channels  of HNSCC patients causes these cells to be hypersensitive to adenosine found in the tumor microenvironment, ultimately reducing their migratory abilities and restricting their infiltration into tumors  were obtained from the University of Cincinnati Medical Center. HNSCC patients included in this study were treatment-naïve and had a positive diagnosis of HNSCC by tissue biopsy , tetraethylammonium-chloride (TEA-Cl), 1,2-Bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), MgCl2 were purchased from Millipore Sigma. Sterile, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), RPMI-1640 medium, fetal bovine serum, penicillin, streptomycin, Fura-2 and phosphate buffered saline (PBS) were obtained from ThermoFisher. Phosphatidylinositol 3-phosphate diC16 (PI3P) was purchased from Echelon Biosciences. Pembrolizumab (Merck Sharpe and Dohme Corp) samples used in this study were received from the leftover pharmacy supply at Cincinnati Children’s Hospital. Atezolizumab was purchased from Biovision life sciences. PD-L1-Fc chimera was obtained from R&D systems. Stock solutions of LY394002 and TG were prepared in dimethyl sulfoxide and used at 0.1% dilution. Stock solutions of PD-L1-Fc, pembrolizumab and atezolizumab were prepared in sterile PBS.Human serum, + PBTs were isolated by negative selection using EasySep Human CD8+ T cell Enrichment kit (StemCell Technologies). Post isolation, CD8+ PBTs were maintained in RPMI-1640 medium supplemented with 10% human serum, 200 U/ml penicillin, 200 mg/ml streptomycin, 1 mM l-glutamine, and 10 mM HEPES. Activation of cells was performed either by adding 40.5 nM of phorbol-12-myristate-13-acetate  and 1.5 µM of ionomycin (Millipore Sigma) or in cell culture plates coated with 10 μg/ml anti-CD3 and anti-CD28 antibodies (BioLegend) for 72–96 h (h) as previously described  as previously described . CD8+ PBescribed . After i+ PBTs from HNSCC patients were activated for 72–96 h using PMA and ionomycin followed by treatment with the αPD-1 antibody pembrolizumab (10 μg/ml) and/or the αPD-L1 antibody atezolizumab (1 and 10 μg/ml) for 6 h prior to performing functional studies. CD8+ PBTs from HDs were plated on cell culture plates coated with PD-L1  (10 μg/ml) and activated with either PMA and ionomycin or (with anti-CD3/CD28 antibodies for 72–96 h followed by treatment with the αPD-1 antibody pembrolizumab (10 μg/ml) for 6 h before performing functional studies. For prolonged treatment with PD-L1, cells were activated for 120 h with plate-bound anti-CD3/CD8 antibodies along with PD-L1-Fc in T cell medium supplemented with 20 IU/ml of IL-2.CD8+ PBTs cells of HDs and HNSCC patients as described previously : 145 K-Aspartate, 2 MgCl2, 8.5 CaCl2, 10 EGTA, 10 HEPES, pH 7.2 TRIS buffer (1 μM free Ca2+ concentration). Borosilicate glass (World Precision Instruments) pipettes (4–5 MΩ resistance) were fabricated using a P-97 horizontal puller (Sutter Instruments). A voltage-ramp pulse protocol  was used to elicit the currents from CD8+ PBTs. Currents were recorded, amplified and digitalized  through a 16-bit A-D/D-A interface. Data acquisition was performed using pClamp 8.0 software (Molecular Devices) and signals were low pass filtered at 2 kHz and digitalized at 100 kHz. The conductance (G) of KCa3.1 channels was calculated as, the ratio of linear fraction of the currents to the slope of ramp voltage stimulus (measured in the voltage range between −100 and −80 mV) after subtraction of leak current. : 130 NaCl, 4.5 KCl, 20 CaCl2, 10 D-glucose and 5 HEPES (pH 7.4 with NaOH). 10 mM TEA-Cl was added to all extracellular solutions to block voltage-gated K+ channels. The pipette solution contained (in mM): 135 Cs aspartate, 8 MgCl2, 8 BAPTA and 1 HEPES (pH 7.2 with CsOH). A ramp  protocol was used for 100 m every 1.5 s to record DVF currents. Analysis of DVF current was performed by measuring the peak current value at the voltage of −100 mV during the ramp step protocol.For divalent free (DVF) currents, cells patched in the whole-cell configuration were pre-incubated with TG (1 µM) in external solution without Caor 2 min . DVF cur2+ fluxes were measured in activated CD8+ PBTs using the ratiometric Ca2+ sensitive dye Fura-2. Perfusion with the sarco-endoplasmic pump inhibitor TG allowed us to measure Ca2+ fluxes that are independent of TCR stimulation and only rely on downstream signaling events initiated by the release of Ca2+ from the endoplasmic reticulum (l-lysine coated coverslips followed by treatment with 1 µM Fura-2 AM (ThermoFisher) at 37°C for ∼30 min. Cells were then washed with RPMI-1640 and maintained at 37°C prior to recordings using InCyt-Im2 Ca2+ imaging system (Intracellular Imaging). Coverslips were mounted on the microscope and perfused with a Ca2+ free solution for 5 min, followed by perfusion with 1 μMTG in Ca2+ free solution for other 5 min to deplete the Ca2+ from the intracellular Ca2+ stores and open the CRAC channels. Finally, 0.5 mM Ca2+ solution was perfused for 10–15 min to allow Ca2+ influx through CRAC channels. Ca2+ free solution had the following composition (in mM): 155 NaCl, 4.5 KCl, 1 MgCl2, 10 HEPES, 10 glucose, 2 EGTA, pH 7.4. The 0.5 mM Ca2+ solution had the following composition (in mM): 155 NaCl, 4.5 KCl, 2.5 MgCl2, 10 HEPES, 10 glucose, 0.5 CaCl2, pH 7.4. A standard calibration curve was used to correlate ratiometric Fura-2 values (340/380 nm ratio) with known Ca2+ concentrations as per the protocol provided by the manufacturer. Changes in Ca2+ values [Delta (Δ) Ca2+], a measure of Ca2+ fluxing ability, were determined as the difference between the peak of Ca2+ reached after 0.5 mM Ca2+ and the baseline Ca2+ after the perfusion with 1 μMTG in Ca2+ free solution, immediately before the addition of 0.5 mM Ca2+. Statistical analysis was performed to detect significant difference in ΔCa2+ values post treatment for an individual patient or healthy donor. Only those donors who showed a statistically significant increase in the Ca2+ values before and after αPD-1 were included in the positive response (PR) group while those who did not show any statistically significant increase were included in the no-response (NR) group. Individual single cell ΔCa2+ values from donors in the PR group and the NR group were then combined to detect significance differences between the two groups. This is because of low sample quantity and variable cell count per patient.Intracellular Caeticulum . T cells+ PBT cells were maintained at a density of 1 × 106 cells/mL and stimulated with PMA/ionomycin for 72 h. Cells were then rinsed with 1x PBS, followed by staining for flow cytometry. The proportion of dead cells were determined using the Zombie UV fixable viability kit (Biolegend). The cells were then fixed with 1% paraformaldehyde (ThermoFisher), washed with 1x PBS and stained overnight with mouse anti-human anti-KCa3.1 biotinylated antibodies . Then, the cells were washed with 1x PBS and incubated for 30 min at room temperature with the following anti-human antibodies : anti-CD8-PacificBlue (cloneHIT8A), anti-CD69-APCFire750 (clone FN50), anti-LAG3-BV510 (clone 11C3C56), anti-PD1-BV605 (clone EH12. 2H7), anti-TIM3-BV786 (clone F38-2F2), and anti-streptavidin-PECy7 (clone). Next, the cells were washed with 1x PBS, and permeabilized with BD Cytofix/Cytoperm kit (BD Biosciences) as per the manufacturer’s instructions, and incubated for 30 min at 4°C with mouse anti-human anti-Calmodulin-PerCP , and anti-human anti-Ki67-BV711 (Biolegend).CD8For determination of ion channel expression, cells were fixed with 4% paraformaldehyde, washed with PBS followed by overnight incubation at 4°C with the following primary antibodies, mouse anti-human KCa3.1 (6C1/ATTO-488), guinea pig anti-human Kv1.3, rabbit anti-human Orai1  followed by anti-guinea pig (Dy350 goat anti-guinea pig IgG/Thermo Fisher) and anti-rabbit (Alexa Fluor 594 goat anti-rabbit IgG/Thermo Fisher) secondary antibodies. To stain for intracellular ion channel epitopes, cells were permeabilized with BD Cytofix/Cytoperm kit (BD Biosciences) as per manufacturer’s instructions. Cells were then stained for rabbit anti-human STIM1 (Proteintech) primary antibodies followed by secondary antibodies (Alexa Fluor 594 goat anti-rabbit IgG/Thermo Fisher). Specificity of these antibodies was previously reported by our laboratory . All flot test (paired or unpaired), Mann-Whitney rank sum test , and ANOVA or ANOVA on Ranks as indicated. Post hoc testing on ANOVA was done by multiple pairwise comparison procedures using the Holm-Sidak, Dunn’s or Tukey’s methods depending on sample normality and variance. Statistical analysis was performed using SigmaPlot 13.0 (Systat Software Inc.). p value of less than or equal to 0.05 was considered as statistically significant.Statistical analyses were performed using Student’s 2+ signaling and effector functions (+ PBTs (in vitro experiments to study in detail the effect of pembrolizumab on ion channels in HNSCC T cells. We tested the effect of a short exposure (6 h) to pembrolizumab (αPD-1 antibody) (10 μg/ml) on activated CD8+ PBTs. Whole-cell currents were measured before and after αPD-1 treatment to assess KCa3.1 and Kv1.3 activity , αPD-1 treatment increased the Ca2+ response of T cells (2+ response to αPD-1 treatment in vitro were defined as “no-response” (NR) patients (n = 6/9), (2+ in the PR group (2+ at baseline (before αPD-1 treatment) of CD8+ PBTs from PR patients was 67% lower than NR patients; even lower than that of HDs CD8+ PBTs  induced by αPD-1 versus the baseline ΔCa2+ of all HNSCC patients , . αPD-1 tPR group . InteresD8+ PBTs . Indeed,patients . We perf+ channel function. While we have no cancer cells in our in vitro setting, CD8+ PBTs of HDs and HNSCC patients can be a source of PD-L1 as both express it  increased KCa3.1 function, while Kv1.3 function was increased only at the higher concentration  on KCa3.1 and Kv1.3 in CD8+ PBTs of HDs. Cells were incubated with PD-L1 for 72 h followed by treatment with αPD-1 antibody (10 μg/ml) for 6 h. Our data showed that PD-L1 reduced KCa3.1 activity by 65.44%, but, at this concentration, had no effect on Kv1.3  decrease in ΔCa2+ as compared to control cells . We recently showed that CD8+ PBTs of HNSCC patients have reduced levels of CaM which diminished their KCa3.1 activity  followed by measurement of KCa3.1 in presence and absence of PI3P (100 nM) delivered intracellularly via patch pipette  in HNSCC patients (2+ fluxing ability of CD8+ PBTs from HNSCC patients predicts the response to pembrolizumab. It is possible that HNSCC patients whose T cells display lower Ca2+ fluxing abilities in vitro had a higher exposure to circulating PD-L1 (either soluble or via tumor-derived exosomes) in vivo, and they will ultimately benefit the most from αPD-L1 and αPD-1 blockade. An in vitro study showed that PD-L1 decreases TCR dependent Ca2+ fluxes in a dose dependent manner in human T cells overexpressing the PD-1 receptor  explains the response to αPD-1 and αPD-L1 antibodies without pre-treatment with PD-L1-Fc  we used (10 μg/ml) suppressed KCa3.1, but not Kv1.3, currents in CD8reatment . The effreatment . We repo in TILs . However in TILs  where th in TILs . While wPD-L1-Fc , 3. It ius PD-L1 .+ T cells are inhibited by the non-selective PI3K blocker LY294002, while PI3P (whose formation from PI is facilitated by PI3K) reverses this inhibition  and Akt/Protein Kinase B)], while ligation of PD-1 results in a decrease in TCR proximal signaling through inhibition of PI3K activity . PI3K hahibition . NDPK-B hibition . Similar channel . We havepatients . In this by Ca2+ .in vitro studies showed that αPD-1 induces an increase in Kv1.3, KCa3.1 and Ca2+ fluxing that ultimately will lead to improved anti-tumor response. Ca2+ signaling in fact regulates multiple effector functions of T cells, including the production of cytokines like IL-2 and IFN-γ, and cytotoxicity (in vivo administration of αPD-1 blockade followed by surgical resection increases Ca2+ fluxing abilities of CD8+ T cells by increasing the KCa3.1 and Kv1.3 channel function in head and neck cancer patients (2+ levels of T cells resulting in increased cytotoxicity (Overall, our toxicity . These apatients . Furthertoxicity .+ channels as early modulators of PD-1 signaling and point to them as therapeutic targets in restoring anti-tumor immunity in HNSCC patients. This study paves the way for further investigations about new therapeutic strategies in cancer that incorporate K+ channel activators along with immune checkpoint inhibitors.In conclusion, the data presented in this manuscript highlight the crucial role of K


Intracellular Ca2+ is intricately compartmentalized, and the Ca2+ flow is tightly controlled by a network of Ca2+ transport and buffering proteins. Impaired Ca2+ signaling is strongly associated with endoplasmic reticulum stress, mitochondrial dysfunction and autophagic defects, all of which are etiological factors of NAFLD. In this review, we describe the recent advances that underscore the critical role of dysregulated Ca2+ homeostasis in lipid metabolic abnormalities and discuss the feasibility of targeting Ca2+ signaling as a potential therapeutic approach.The liver plays a central role in energy metabolism. Dysregulated hepatic lipid metabolism is a major cause of non-alcoholic fatty liver disease (NAFLD), a chronic liver disorder closely linked to obesity and insulin resistance. NAFLD is rapidly emerging as a global health problem with currently no approved therapy. While early stages of NAFLD are often considered benign, the disease can progress to an advanced stage that involves chronic inflammation, with increased risk for developing end-stage disease including fibrosis and liver cancer. Hence, there is an urgent need to identify potential pharmacological targets. Ca The liver is a major site for lipid processing and storage. As such, liver dysfunction can lead to progressive lipid accumulation, a condition often predisposed to more severe diseases. Among the various hepatic lipid metabolic disorders, non-alcoholic fatty liver disease (NAFLD) is now the most common chronic liver disease worldwide, affecting approximately 25% of the population . First dMechanistically, NAFLD represents an imbalance of metabolic activities that regulate the lipid uptake, synthesis, export and degradation. Free fatty acids are central to the pathogenesis of NAFLD. The major supply of lipids delivered to the liver comes in the form of free fatty acids following lipolysis of triglycerides in the adipose tissue. This action is highly regulated by insulin . The liv2+) is the most abundant mineral in the human body, primarily found in bones and teeth (in the form of hydroxyapatite). It plays a key role in maintaining the bone mass to support the skeletal system. The body is also constantly using Ca2+ in a wide range of biological activities, including muscle contraction and nerve impulse transmission , via Ca2+-permeable channels, e.g., TPCs and transient receptor potential mucolipins (TRPMLs).The lysosome is a significant intracellular Cachangers . The relTRPMLs are evolutionarily conserved, nonselective cation channels containing six membrane-spanning domains and function in tetrameric forms. In mammals, the TRPML channel family has three members, with each member having partially overlapping biological properties but different tissue distribution and subcellular localization. TRPML1 is expressed ubiquitously in all tissues, whereas TRPML2 and TRPML3 show more functional specificity in various cell types .2+ mobilizing messenger NAADP [+ release channels that can be activated by [PIP2] and by voltage [2+ release [TPCs are members of the voltage-gated ion channel superfamily. Structurally, TPCs function as homodimers, with each subunit containing two six-transmembrane domain repeats connected by a linker region . The TPCer NAADP . However voltage ,79. Desp voltage . In addi release ,82.One of the major functions of the endolysosomal system is membrane trafficking through the interconnected vesicular network, acting as a sorting station to process internalized cargo. In the liver, cholesterol-enriched low-density lipoprotein (LDL) is taken up by the LDL receptor on the plasma membrane and internalized through endocytosis. The LDL-bound LDL receptor is then sorted in the endosome: the receptor proteins are transported back to the plasma membrane, while LDL cholesterol that exits from the late endosomes, and lysosomes is either processed to bile acids or to cholesterol esters for storage . In a reThe lysosome not only acts as the cell’s recycling center but also mediates a tightly regulated adaptive response to energy-demanding conditions widely known as autophagy. Autophagy is a well-conserved degradative process through which cellular organelles and macromolecules are disassembled during times of nutrient deprivation and other threats to cellular homeostasis. Three types of autophagy have been categorized based on the mechanisms of cargo sequestration: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). The detailed mechanistic distinctions of these autophagic pathways have been discussed in several recent elegant reviews and will not be covered here ,86.2+ is a critical modulator of autophagy. It has been proposed that, during nutrient deprivation, Ca2+ is released from the lysosome through TRPML1, which creates high Ca2+ microdomains, leading to activation of the Ca2+-sensitive serine/threonine phosphatase calcineurin and dephosphorylation of transcription factor EB (TFEB) [2+/CaM-dependent protein kinase kinase β (CaMKKβ) and AMPK, an observation independent of TFEB [Growing evidence indicates that lysosomal CaB (TFEB) . TFEB isB (TFEB) . In nutrB (TFEB) . Interes of TFEB , further2+ signaling in canonical autophagy is more established, studies specifically investigating the role of Ca2+ regulation in lipophagy, especially in the context of liver disease, are relatively few. As discussed in The process by which intracellular lipid droplets are catabolized by autophagy is also referred to as lipophagy . LipophaDuring the course of NAFLD progression, in addition to hepatocytes, which are the primary target of lipotoxicity and oxidative stress, hepatic nonparenchymal cells are also crucial contributors. The nonparenchymal cells represent about 20% of the total liver mass and are composed of Kupffer cells, hepatic stellate cells (HpSCs), liver sinusoidal endothelial cells and other cell types. These specialized cells interact with hepatocytes and play a key role in liver regeneration and inflammatory responses.2+ levels, thereby promoting (V)-type H+-ATPase-dependent lysosome acidification and lysosomal lipolysis. This results in an increase in the alternative M2 macrophages, a protective response to prevent NAFLD progression [Liver-resident Kupffer cells are the major hepatic macrophages in healthy livers and essential components of the innate immunity. Kupffer cells are central in modulating local inflammation, tissue remodeling and metabolic processes . Upon ligression .2+ mobilization. In activated HpSCs, ER Ca2+ transport proteins InsP3R1, SERCA2b and Ca2+ buffers calnexin and calreticulin are upregulated [2+ transients are induced downstream of the cell surface GPCRs  receptors), which mediate HpSC contractility, proliferation and gene expression [2+ signaling abrogates nuclear calmodulin kinase II-mediated Cdc25 phosphorylation and, thus, results in cell cycle arrest [2+ homeostasis causes ER stress and, eventually, HpSC apoptosis. The accumulation of intracellular Ca2+ increases the phosphorylation of the c-Jun N-terminal kinase (JNK) and p38, followed by the activation of Ca2+-dependent cysteine proteases calpains and caspase-12/caspase-3 processing [2+ in both the initiation and resolution of HpSC activation.The cytokines released by Kupffer cells and monocyte-derived macrophages activate HpSCs, the main effector cells that initiate fibrogenic events. Upon activation, HpSCs undergo morphological and functional changes, transdifferentiating into a myofibroblast-like phenotype. Activated HpSCs produce contractile cytoskeletal proteins and components of the extracellular matrix, which may further lead to cirrhotic changes associated with hepatic dysfunction in advanced disease . The actegulated . Ca2+ trpression ,102,103.e arrest . In contocessing . These r2+ being a critical intracellular messenger for lipid metabolism can be acknowledged. Hepatic lipid accumulation leads to SERCA dysfunction, resulting in the elevation of cytoplasmic Ca2+ and ER stress. Therefore, treatment options that target Ca2+ signaling to maintain optimal Ca2+ homeostasis is a rational approach and can potentially be beneficial. Indeed, the overexpression of SERCA2b in obese mice alleviates ER stress and hepatic steatosis [2+ channels/pumps are responsible for normal physiological functions, a challenge for drug development would be to ensure these compounds are specific to the relevant pathways and do not interfere with normal cellular function of the target tissue or even other off-target tissues. The overexpression of SERCA2b or CDN1163 treatment in normal lean mice does not impair energy homeostasis and alter the body fat composition [2+ is within the normal physiological levels.Based on what we have discussed in the previous sections, a general idea of Cateatosis . The treteatosis . As numeposition ,106, ind2+ increase is also responsible for attenuation of the autophagy flux. Park et al. therefore proposed that the inhibition of cytoplasmic Ca2+ influx by using Ca2+ channel blockers may be beneficial for ameliorating the autophagy defects and the pathological consequences in obesity. They show that, in mice on a high-fat diet, the administration of verapamil, a non-dihydropyridine Ca2+ channel blocker commonly used in the treatment of high blood pressure and heart arrhythmias, can restore autophagy flux in the liver and reduce obesity-induced pathologies, including hepatic steatosis and insulin resistance [2+ channel blockers can achieve similar results. A number of those have proven to have no or even negative effects on glucose or lipid metabolism [In addition to ER stress, lipotoxicity-induced cytoplasmic Casistance . Despitetabolism . Further2+ channels or transporters, have shown the ability to modulate Ca2+ signaling and potentially improve metabolic abnormalities. The glucagon-like peptide-1 (GLP-1) receptor agonists exendin-4 and exenatide can alleviate hepatic steatosis by correcting impaired SOCE in hepatocytes [Artemisia vestita, has also been reported to reduce hepatic lipid accumulation and ameliorate insulin resistance in obese mice through the upregulation of SERCA2b [2+-signaling modulators—in particular, those that regulate SERCA activity in the treatment of NAFLD-associated metabolic pathologies.A number of pharmacological agents, although not specifically designed to target Caatocytes ,110. Liratocytes . Maresinatocytes ,113. Jac SERCA2b . These s2+, as an intracellular second messenger, modulates a myriad of cellular processes. These actions involve an intricate set of regulatory proteins that allow interplay between the Ca2+ stores  and transmit coordinated Ca2+ signals. During the past decade, significant progress has been made towards the understanding of Ca2+ signaling in regulation of the hepatic lipid metabolism. In this review, we have summarized some of the crucial mechanisms among different cellular organelles and highlighted the association between disrupted Ca2+ homeostasis and NAFLD development  [2+ for mitochondrial respiration [2+-binding chaperone, calreticulin, determines the sensitivity of the ER cholesterol-sensing mechanism through the sterol regulatory-element binding protein (SREBP) pathway, which is crucial in maintaining membrane fluidity and ER stress levels [In this review, we mainly discussed the involvement of Ca(PACS-2) . Calnexipiration . Anothers levels . Their i


Tg(myl7:GA), and optimized a reconstitution protocol to boost aequorin luminescence. The analogue diacetylh-coelenterazine enhanced light output and signal-to-noise ratio. With this cardioluminescence model, we imaged the time-averaged calcium levels and beat-to-beat calcium oscillations continuously for hours. As a proof-of-concept of the transgenic line, changes in ventricular calcium levels were observed by Bay K8644, an L-type calcium channel activator and with the blocker nifedipine. The β-adrenergic blocker propranolol decreased calcium levels, heart rate, stroke volume, and cardiac output, suggesting that larvae have a basal adrenergic tone. Zebrafish larvae treated with terfenadine for 24 h have been proposed as a model of heart failure. Tg(myl7:GA) larvae treated with terfenadine showed bradycardia, 2:1 atrioventricular block, decreased time-averaged ventricular calcium levels but increased calcium transient amplitude, and reduced cardiac output. As alterations of calcium signalling are involved in the pathogenesis of heart failure and arrhythmia, the GFP-aequorin transgenic line provides a powerful platform for understanding calcium dynamics.Zebrafish embryos and larvae have emerged as an excellent model in cardiovascular research and are amenable to live imaging with genetically encoded biosensors to study cardiac cell behaviours, including calcium dynamics. To monitor calcium ion levels in three to five days post-fertilization larvae, we have used bioluminescence. We generated a transgenic line expressing GFP-aequorin in the heart,  The stocks of native-, hcp-, fcp-, h-, and f-CTZ analogues  were prepared at 5 mM in methanol. All CTZ were stored at −80 °C in 5 µL aliquots and were used at 50 µM final concentration. For aequorin reconstitution, Tg(myl7:GA) zebrafish larvae at 3, 4, and 5 dpf were rinsed in nominally zero Ca2+ E3 (E30Ca) medium . Subsequently, larvae were incubated in 1 mL of E30Ca medium containing 25 µM nifedipine for 30 min at room temperature in the dark (nifedipine period). Larvae whose heart was completely stopped after this treatment were washed out five times to remove nifedipine and incubated in E30Ca medium containing 50 µM CTZ for 2 h at room temperature in the dark (CTZ incubation period). Finally, the larvae were incubated in complete E3 medium  for 30 min at 28.5 °C (heart recovery period). A stock of Tg(myl7:GA) larvae were embedded in 100 µL of 0.3% low melting point agarose prepared in E3 medium and transferred to an 8-well glass bottom plate . When the agarose solidified, 100 µL of E3 medium was added and a small portion of agarose surrounding the heart was cut out to improve diffusion of drugs or Triton X-100. Bioluminescence images were acquired as previously described [2+, emits all remaining luminescence counts.After aequorin reconstitution, escribed  with a cescribed ) equippes878Tg(myl7:GCaMP) adult zebrafish were outcrossed to wild-type strain and fertilized eggs at 1-cell stage were injected with 2 ng of the morpholino oligomer tnnt2a (5′-CATGTTTGCTCTGATCTGACACGCA-3′). Larvae at 24 h post-fertilization were placed in 0.003% N-phenylthiourea to prevent pigmentation. For nifedipine titration, 3 dpf larvae were incubated in E3 or E30Ca medium containing 10, 25, or 100 µM nifedipine for 30 min. Then, larvae were embedded in 1% low melting point agarose and transferred to an 8-well glass bottom plate . To study the recovery of Ca2+ dynamics with GCaMP, the aequorin reconstitution protocol described above was applied to 3 dpf larvae, without addition of CTZ. Fluorescence images were acquired at a rate of 200 Hz with a CSU X1 spinning disc confocal microscope  equipped with a Hamamatsu ORCA Flash4.0 sCMOS camera  in 16 bits with 2×2 binning. Chemical compounds were dissolved in DMSO to prepare stocks of 10 mM nifedipine , 20 mM Bay K8644 (Tocris 1544), 10 mM propranolol , 50 mM terfenadine (Tocris 3948), and 7.5% N-phenylthiourea .Embryos at 24 h post-fertilization were placed in 0.003% N-phenylthiourea in E3 medium to prevent pigmentation. At 3 dpf, larvae were transferred to a 6-well plate, 10 larvae per well, with 5 mL of N-phenylthiourea solution containing 10 µM terfenadine or 0.02% DMSO, for 24 h. For bioluminescence experiments, aequorin reconstitution was done during the last 3 h of terfenadine or DMSO treatment.0:t is the fluorescence at a given time and F0 is the minimum diastolic fluorescence value of the whole recording. For characterization of the Ca2+ transients, ΔF/F0 data were analysed with Clampfit 10.7  to determine peak amplitude ((Fsystole − F0)/F0), rise time 10% to 90%, and decay time 90% to 10%  . For GCa% to 10% .Signal). Then, ROIs of identical size were placed in six different locations far from the larva to obtain average background intensity values (Background) and their SD. The SNR for each frame was calculated as:For bioluminescence image analysis and calculation of SNR, a time-projection of the image stack was performed to draw the ROIs over the ventricle and atrium  was transformed into Luminescence rate . The Total counts  were obtained as the sum of luminescence along the experiment. Lconsumed, the sum of all L values from time zero to any given time point, was calculated. It represents the aequorin that has already been spent in each time point. Finally, Lmax was calculated as Ltotal − Lconsumed at each time point. Lmax represents the aequorin available at each time point: the sum of counts from that time point to the end of the experiment.Luminescence diastole) and end-systolic (Dsystole) diameters, as: The hemodynamic parameters were analysed in Fiji-ImageJ by measuring the major and minor diameters (D), and the area (A) on the ventricular cavity at end-systole and end-diastole in three transmitted light or fluorescence images in each larva, as indicated. The major and minor diameters were measured in the longitudinal and transverse directions of the ventricle, respectively. Images were acquired at 50 Hz in a wide-field fluorescence microscope . Ventricular fractional shortening (FS) was calculated from major or minor end-diastolic  and end-diastolic volume (EDV) were calculated as the volume of an ellipsoid of revolution, as reported : Volume The stroke volume and cardiac output were: t-test, as indicated. One-way ANOVA and two-way ANOVA with Holm–Sidak post hoc correction for multiple comparisons and multiple t-tests were used where indicated. Correlation between two datasets was analysed using linear regression. Datasets of total counts were transformed into log10 values. Data are presented as mean ± SD and p < 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. N indicates the number of embryos or larvae used per dataset. Data were analysed in Graphpad Prism 6 .The Shapiro–Wilk statistic was used to test for normality. Differences between two groups were analysed using the unpaired or paired two-tailed myl7. Upon Ca2+ binding to aequorin, there is energy transfer between the excited state of CTZ and the GFP so that light emission is shifted to the green  zebrafish larvae were incubated in 50 μM diacetyl h-CTZ in E3 medium for 2 h, spontaneous Ca2+-dependent bioluminescence from the heart was not detected  medium s878 zebrafish line (2+ transients were indeed abrogated by the treatment with nifedipine and restored after the heart recovery period.A protocol was devised to blunt Ca) medium B. Figure for 2 h B. At theish line C. The CaTg(myl7:GA) larvae subjected to the aequorin reconstitution protocol. The HR was similar before and after the aequorin reconstitution protocol in 3 dpf , 4 dpf , and 5 dpf larvae  (p = 0.832), 4 dpf , and 5 dpf  (2+ transients in 3 dpf s878Tg(myl7:GCaMP) larvae indicated that Ca2+ transients and their kinetics were similar in the controls and in larvae subjected to the aequorin reconstitution protocol  . In addi= 0.878) . Analysiprotocol . These rTg(myl7:GA) larvae were incubated in 50 µM diacetyl h-CTZ for 2 h with or without the previous incubation with 25 µM nifedipine. Cardioluminescence, the spontaneous bioluminescent flashes due to beat-to-beat Ca2+ oscillations during the cardiac cycle, was detected only in the ventricle of larvae preincubated with nifedipine    . In cont= 0.824) .2+ sensitivity and membrane permeability. We, therefore, tested various analogues  larvae from 3 to 5 dpf reconstituted with native-, h-, fcp-, hcp-, f- and diacetyl h-CTZ were evaluated  line, we used two drugs known to increase and decrease Ca2+ levels: the LTCC activator Bay K8644 and the LTCC antagonist nifedipine. 2+ levels. The pharmacological approach in zebrafish usually requires higher concentration of drugs than single-cell studies, since drugs must diffuse through the agarose layer, the skin, and tissues to reach the heart. Thus, we used 100 µM Bay K8644 to trigger an increase in luminescence. Then, addition of 5% Triton X-100 caused the release of the remaining aequorin light. It is worth noting that luminescence values have no meaning in terms of Ca2+ levels until they are transformed into L/Lmax. This ratio, which is proportional to Ca2+ concentration [2+ transients, images were acquired at 9 frames/s (9 Hz)   B. The Cactively) C. 2+ levels in 3 dpf larvae imaged at 1 Hz  (p = 0.0002) (p = 0.002) (Tg(myl7:GA) line was sensitive enough to detect both increases and decreases of Ca2+ levels, as was shown by modulating Ca2+ influx with Bay K8644 and nifedipine. In contrast with Bay K8644, nifedipine (25 μM) decreased the time-averaged Ca= 0.015) D. As ima 0.0002) D,E. As e= 0.002) F. Theref2+ transients, although it was affected by motion artifacts. A shoulder in the decay phase was observed compared to stopped hearts  larvae and tested their Ca2+ levels by cardioluminescence. 2+ levels (p = 0.01)  G. These 2+ levels were not assessed. Terfenadine is a potent hERG blocker and can induce prolongation of the QT interval as well as 2:1 atrioventricular block [2+ levels in this model. The treatment with terfenadine caused bradycardia and a decrease in the atrio-ventricular HR ratio (0.51 ± 0.11) in 94% of the larvae, indicating a 2:1 atrio-ventricular block  (2+ levels (measured at 1 Hz image acquisition) also decreased (as with propranolol), the amplitude of the Ca2+ transients markedly rose (2+ amplitude and stroke volume.Both acute incubation with propranolol  and 24 hecrease) . While tecrease) . Since tecrease) . As the dly rose . Thus, p2+ levels in non-contracting hearts [2+ imaging in the heart with intensiometric fluorescence biosensors requires stopping contraction with drugs or morpholinos against myosin to avoid motion artifacts [para-amino blebbistatin also increased autofluorescence. To overcome these issues, here we have used bioluminescence imaging of aequorin fused to GFP (GA) to monitor continuously Ca2+ levels in experiments of up to 2 h in 3–5 dpf larvae. In these experiments, the heart was performing its mechanical function and the mechano-electrical feedback was preserved.Imaging the heart of zebrafish larvae with a HR of about 3–4 Hz 180–240 bpm) requires acquiring images at high speed. In a previous report using widefield fluorescence microscopy with LED illumination at 50 images/s, we obtained excellent SNR and spatial resolution, but recording was limited to short duration periods (~10 s) to avoid photobleaching [0 bpm reqg hearts . Indeed,rtifacts ,21. Anot2+ is oscillating continuously. It has been previously reported that nifedipine-treated larvae restored the frequency and amplitude of heart Ca2+ transients after drug washout [2+ medium to temporarily abolish Ca2+ transients during reconstitution. No deleterious effects on heart function were observed after this aequorin reconstitution procedure  increased the SNR and provided information about the time-averaged Ca2+ levels, as was shown with both nifedipine and propranolol. This highlights the versatility of GA bioluminescence to monitor oscillatory Ca2+ transients or averaged levels.As the SNR decreased at high acquisition frequencies . Incubation of 3 dpf zebrafish larvae with the antihistamine drug terfenadine for 24 h has been reported to induce heart failure ,45, invoillation ; increasizations ; and bloizations . In zebrizations . Our res+ levels . In addi+ levels . As a re2+ ions [2+-response curve confers it an excellent SNR. However, in Ca2+ microdomains near sarcolemmal Ca2+ channels or ryanodine receptors on the sarcoplasmic reticulum surface (Ca2+ sparks) [2+ concentrations. Thus, L/Lmax may be dominated by these microdomains and may not represent the average cytoplasmic Ca2+ levels. In fact, the experiments with Bay K8644, which induced a 20-fold change in L/Lmax, suggest the existence of such microdomains. Since the bioluminescence reaction of aequorin is triggered by three Ca2+ ions , the ste sparks) , a small2+ levels because atrial bioluminescence was under the limit of detection of our imaging system. Nevertheless, atrial bioluminescence was observed when Ca2+ levels were increased with Bay K8644. Imaging, in contrast to photometry, allowed us to quantify ventricular Ca2+ by setting appropriate ROIs, eliminating any contribution from the atrium. Luminescence may also be close to noise levels when Ca2+ levels decrease by drugs  or in a pathological model, precluding accurate measurements. In the future, the sensitivity issue may be overcome by increasing aequorin’s Ca2+ affinity with mutagenesis or with appropriate CTZ analogues [The main limitation of aequorin luminescence methods compared to fluorescence is their low photon yield. The atrium, being thinner than the ventricle, contains fewer cells and thus, less GA. Therefore, we focused on the ventricular Canalogues  and by t2+ is desired  or a bioluminescent (GA) biosensor would depend on the research question. Bioluminescence would be preferred when a continuous record of Ca2+ physiology and pathophysiology in the embryonic zebrafish heart by bioluminescence, as a complementary method to the popular fluorescent Ca2+ biosensors. The devised reconstitution protocol increased the amount of functional aequorin without causing deleterious heart effects. Ventricular Ca2+ dynamics in beating hearts were imaged continuously for hours in 3–5 dpf larvae. Both the time-averaged Ca2+ levels and individual Ca2+ transients were monitored and, as a proof-of-concept, we studied changes induced by drugs acting on LTCC and sympathetic input. Tg(myl7:GA) larvae also revealed a decrease in time-averaged Ca2+ levels, but an increase in Ca2+ transient amplitude and stroke volume in a model of heart failure induced by terfenadine. Fluorescence and luminescence imaging have each particular benefits and constraints but are orthogonal techniques able to interrogate different aspects of pathophysiological processes. Cardioluminescence in particular avoids autofluorescence and photobleaching and allows monitoring heart Ca2+ for longer periods of time.In conclusion, we describe a transgenic model to study Ca