The maintenance of an elongated myocyte morphology following the post-hatch transition from ectothermy to endothermy in birds is discussed in relation to cardiac load, myocyte ploidy, and cardiac regeneration potential in adult cardiomyocytes. Overall, the paper shows how little we know about cellular Ca2+ dynamics in the bird heart and suggests how increased research efforts in this area would provide vital information in our quest to understand the role of myocyte architecture in the evolution of the vertebrate heart.Bird cardiomyocytes are long, thin and lack transverse (t)-tubules, which is akin to the cardiomyocyte morphology of ectothermic non-avian reptiles, who are typified by low maximum heart rates and low pressure development. However, birds can achieve greater contractile rates and developed pressures than mammals, whose wide cardiomyocytes contain a dense t-tubular network allowing for uniform excitation–contraction coupling and strong contractile force. To address this apparent paradox, this paper functionally links recent electrophysiological studies on bird cardiomyocytes with decades of ultrastructure measurements. It shows that it is the strong transsarcolemmal CaThis article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’. Please see glossary at the end of the paper for definitions of specialized terms. When comparing animals of similar body size, birds have a nearly twofold larger heart mass than mammals –21. This2) and a small cell volume (approx. 10 pl), leading to a large surface-area-to-volume ratio ryanodine binding studies which showed the density and Ca2+ sensitivity of bird (pigeon and finch) RyRs are similar to those of mammals (rat) [CaL was considerably smaller [CaL is temperature-dependent, the authors point out that CICR would also occur in atrial myocytes at the body temperature of the quail [in vivo but is absent exvivo that enhances atrial ICaL conductance in quail heart.The L-type Cam the SR . In thismyocytes . Thus, ta et al.  showed tls (rat) . Interes smaller . The stuhe quail . Additio2+ signal occurring in the periphery of the myocyte. Presently we lack dynamic imaging studies which show the propagation of the peripheral signal through ejSR/cSR to the centre of the bird cardiomyocyte. However, such studies have been performed on the elongated/spindle-shaped myocytes from mammalian atrial cells [2+ signal is not effective at triggering CICR from centrally located non-junctional RyRs under normal conditions. Enhancing the amplitude of ICaL, RyR sensitivity to the cytosolic Ca2+ trigger, or increasing the Ca2+ content of the SR, have each been shown to be sufficient to trigger centripetal Ca2+ propagation and a global Ca2+ transient in mammalian atrial myocytes [ICaL and the large SR Ca2+ content (discussed below), combined with the organization of CRUs in the bird myocardium are all indicative of a rapid and near-uniform global Ca2+ transient during routine excitation–contraction coupling. However, this must be substantiated experimentally.The presence of functional crosstalk and CICR in quail cardiomyocytes align with ultrastructural and radio-ligand binding studies to provide a clear mechanism for a strong and rapid Caal cells ,86. Intemyocytes ,87. Thismyocytes . The lar (a)2+ is returned to the SR by the pumping activity of SERCA which facilitates the strong and rapid contractions of the bird heart. SR vesicles from adult turkey ventricular homogenates demonstrated robust SERCA activity [2+ transient in turkey heart preparations [Cytosolic Caactivity  that corarations . Refilliarations ,90 and warations  when com2+ without spontaneously releasing it. SR Ca2+ content grows during development in the chicken heart with late-stage embryonic chicken myocytes having a steady-state content of approximately 400 µmol l−1 Ca2+ [−1 Ca2+ steady-state content assessed via caffeine application in adult quail ventricular myocytes [2+ content measured in ectotherms (200–500 µmol l−1 Ca2+) [−1 Ca2+) [2+ content (greater than 1000 µmol l−1 Ca2+) of the fish SR [−1 Ca2+) when both are assessed by the application of 10 mM caffeine [−1 Ca2+ during 100 steady-state loading pulses when ICaL was not of sufficient magnitude to trigger CICR in the quail atrium [2+ storage capacity has been dramatically reduced during the evolution of the mammalian myocyte.Similar to ectotherms , the birl−1 Ca2+ . This comyocytes . These v−1 Ca2+) ,93 and g−1 Ca2+) ,94,95. T fish SR ,93 greatcaffeine . Maximall atrium . Thus, i2+ than mammalian SR  SR hold a larger quantity of Caview see ). On thed mammal , other  heart without spontaneous release is not known. Luminal Ca2+ sensing is strongly dependent on the SR Ca2+-buffering capacity  and on interactions with luminal proteins (e.g. triadin and junctin) [2+ storage capacity must be coupled to a low release sensitivity in birds and ectotherms. Release of the entire SR Ca2+ content  and dysfunction (i.e. arrhythmias) often associated with cytosolic Ca2+ overload in ischemia–reperfusion scenarios [2+ overload and errant SR Ca2+ release underlie many human cardiomyopathies, understanding how bird cardiomyocytes regulate SR Ca2+ storage and release could provide novel avenues for therapeutics.Perhaps the most interesting aspect of the large Cativators –105. In l−1 Ca2+ . The reajunctin) . Howeveral loads ,93) woulcenarios ,107. As . 42+ activation time in an avian heart cell model [The narrow, elongated cardiomyocyte morphology dominates the animal kingdom including cephalopods  table 1. In fishll model . Thus, ein ovo and attain endothermy post-hatch [The elongated myocyte morphology is also associated with cardiac regeneration. Neonatal mammalian, embryonic bird, fish, urodele amphibian and reptile hearts ,117,118 st-hatch . Differest-hatch . In the st-hatch ,121. In st-hatch . Such chst-hatch .The mechanisms linking endothermy and non-proliferating cardiomyocytes is an active area of research that has recently been linked to another key feature of cardiomyocyte architecture—polyploidy or genome duplication. By and large, all bird and mammal embryonic and neonatal cardiomyocytes, and most ectotherm cardiomyocytes (independent of age) are mononuclear and diploid ,123,124.. 5The elongated cardiomyocyte of ectothermic vertebrates is internally remodelled with a superhighway of CRUs in post-hatch birds to increase cardiac force and cardiac frequency beyond that of their ectothermic ancestors. Clearly this strategy is equally as successful as the hypertrophied and t-tubulated myocytes of adult mammals for powering the robust cardiac function necessitated by endothermy. Indeed, the force generated per cross-sectional area of the ventricular wall is similar in mammals and birds , emphasi2+ flux during excitation–contraction coupling are scarce, and knowledge of the spatial and temporal properties of the intracellular Ca2+ transient is lacking. These are necessary to improve our structural and functional understanding of the hearts of this group of endothermic vertebrates. Birds also comprise the only taxon that transitions from ectotherm to endotherm during development. Studies of their elongated myocytes throughout this transition are uniquely placed to shed insight into a key area of human cardiac research—the drivers of polyploidization at the expense of proliferation in heart regeneration.However, detailed functional studies of bird cellular CaCurrently, one-in-seven (14%) of the world's bird species are threatened with extinction (greater than 4000 species)  and so c


Fam83h) is essential for dental enamel formation. Fam83h mutations cause human amelogenesis imperfecta (AI), an inherited disorder characterized by severe hardness defects in dental enamel. Nevertheless, previous studies showed no enamel defects in Fam83h‐knockout/lacZ‐knockin mice. In this study, a large deletion of the Fam83h gene (900 bp) was generated via a dual sgRNA‐directed CRISPR/Cas9 system in rabbits. Abnormal tooth mineralization and loose dentine were found in homozygous Fam83h knockout (Fam83h−/−) rabbits compared with WT rabbits. In addition, reduced hair follicle counts in dorsal skin, hair cycling dysfunction and hair shaft differentiation deficiency were observed in Fam83h−/− rabbits. Moreover, X‐rays and staining of bone sections showed abnormal bending of the ulna and radius and an ulnar articular surface with insufficient trabecular bone in Fam83h−/− rabbits. Taken together, these data are the first report of defective hair cycling, hair shaft differentiation and abnormal bending of the ulna and radius in Fam83h−/− rabbits. This novel Fam83h−/− rabbit model may facilitate understanding the function of Fam83h and the pathogenic mechanism of the Fam83h mutation.Family with sequence similarity 83 members H ( Hence, the combination of CK‐1 and keratin has an effect on desmosome formation,Fam83h as one of the major genes with a role in causing amelogenesis imperfect (AI).Fam83h contributes to AI in clinical, its actual functions, pathogenesis and association with enamel defects are still unknown.A genome‐wide search identified Fam83h‐knockout/lacZ‐knockin miceFam83h mutated and WT mice.Fam83h has debatable effects on the development of teeth and the formation of enamel in mice model. Therefore, to further investigate the function of Fam83h and precisely recapitulate human AI, the new animal models are needed in the further study.It is reported that most of AI patient exhibited yellow‐brown teeth, localized enamel defects and sustained defective mineralization.Fam83h knockout (KO) rabbits using the CRISPR/Cas9 system, the abnormal dental mineralization, hair cycling disorder and hair shaft differentiation deficiency were determined in those Fam83h−/− rabbits. In conclusion, this is the first report of defects in hair cycling and hair shaft differentiation in the dorsal skin of Fam83h−/− rabbits. Thus, the present study provides further insight into the relationship between Fam83h mutations and AI and also a novel animal model for delayed hair cycling and defective hair shaft differentiation for the pre‐clinical study in the future.Of note, rabbits are considered as a better animal models than mice in recapitulating some human diseases because rabbits have greater similarity in terms of physiology, anatomy and genetics to humans than mice.22.1New Zealand rabbits used in this study were obtained from the Laboratory Animal Centre of Jilin University . All animal studies were conducted according to experimental practices and standards approved by the Animal Welfare and Research Ethics Committee at Jilin University .2.2The 3xFLAG‐NLS‐SpCas9‐NLS vector  was linearized with NotI, and it was in vitro transcribed using the mMessage mMachine SP6 kit (ThermoFisher Scientific) and the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. The design of targeted single‐guide RNAs (sgRNAs) has been described previously.The Puc57‐T7‐sgRNA vector was amplified by PCR using the T7 primers (T7‐F: 5'‐GAAATTAATACGACTCACTATA‐3′ and T7‐R:5‐AAAAAAAGCACCGACTCGGTGCCAC‐3′). Then, PCR products were in vitro transcribed using T7 RNA polymerase  and purified using a miRNeasy Mini kit (Qiagen) according to the manufacturer's instructions.2.32 for short‐term culture. Finally, 30–50 injected zygotes were transferred into the oviduct of recipient rabbits.The embryo microinjection and embryo transfer procedures were as previously described.2.4Fam83h‐KO and WT rabbits was isolated from a small piece of ear tissue using the TIANamp Genomic DNA kit  according to the manufacturer's instructions. Mutation detection was performed by PCR using the following primers (Table The DNA of rs Table . PCR pro2.5http://crispr.mit.edu/). PCR products were subjected to T7 endonuclease I assay  and Sanger sequence analysis (Sangon Biotech). The primers are listed in Table Potential off‐target sequences were selected by using the CRISPR Design tool (2.6The T7EI assay was performed as described previously.2.7Fam83h‐KO rabbits using TRNzol‐A+ reagent (Tiangen) and then treated with DNase I (Fermentas). Primers used for qRT‐PCR are shown in Table −ΔΔCT formula was used to analyse gene expression. Gapdh was used as a reference gene. All experiments were repeated three times for each gene. The data are expressed as the mean ± SEM.Total RNA was isolated from rabbit dorsal skin of WT and 2.8Fam83h‐KO and WT rabbits and fixed in 4% paraformaldehyde at 4°C. Then, the tissue was dehydrated in increasing concentrations of ethanol , cleared in xylene and embedded in paraffin for histological analysis under a light microscope.Haematoxylin and eosin (H&E) and Masson's trichrome staining were performed as previously described.For von Kossa staining, renal tissue sections were treated with 5% silver nitrate solution and exposed to incandescent light for 1 h. Then these were treated with 5% sodium thiosulfate for 1 min, and calcium salts were often stained black or brown‐black.2.9X‐ray radiography scans of the femur were taken using a YEMA Radiography System with a digital camera (Varian) attached to an X‐ray radiographer (Rotanode). Images were taken at 50 KV with 3 mAs exposure.2.10Fam83h‐KO rabbit blood was collected at 15 months old. Serum samples were obtained by centrifugation. Serum phosphorus levels were measured using a Phosphorus Assay kit (Changchun Huili) with the peacock chlorine colorimetric method. Serum ALP levels were assayed using an ALP Assay kit (Changchun Huili) with the disodium phenyl phosphate method. Serum creatinine was measured using a Creatinine Assay kit (Changchun Huili) with the picric acid method. Serum BUN was measured using a BUN Assay kit (Changchun Huili) with the urease indoxyl method.WT and 2.11Fam83h‐KO and WT rabbits were attached to specimen stubs using double stick conductive tabs and sputter‐coated with gold using a polaron scanning electron microscope E‐1010 (Hitachi). Samples were imaged using an S‐3400 N scanning electron microscope (Hitachi). The length of the hair was measured with a Vernier calliper.Scanning electron microscopy was performed as previously described.2.12p < 0.05 was considered statistically significant.Data are expressed as the mean ± SEM, with at least three individual measurements in all experiments. The data were analysed by one‐way ANOVA using GraphPad Prism 6.0 software. A probability of 33.1Fam83h gene in rabbits, a pair of sgRNAs targeting the fifth exon of rabbit Fam83h was designed  carried biallelic Fam83h mutations.To disrupt the function of the d Figure . Mixed CFam83h mutation, F1 and F2 individuals were obtained by brother–sister breeding. The genotypes of F2 Fam83h KO rabbits were confirmed by PCR and Sanger sequence analysis  was elevated in Fam83h−/− rabbits  cases,Fam83h is known to play an important role in the formation of enamel, the underlying mechanism has not yet been elucidated in detail.The Fam83h mutated mice showed defective dental development and discoloured incisors, even, sparse coat and premature deaths; In this study, the Fam83h−/− rabbits appeared multisystemic lesions, which included abnormal tooth mineralization, defective hair follicle and shaft development. Thus, the comparation of Fam83h mutated mice, rabbits and AI humans regarding the phenotypes were listed in Table Previous studies showed that most of AI patients exhibited yellow‐brown teeth, localized enamel defects and sustained defective mineralization;Fam83h−/− rabbits. Consistent with the findings from previously reported, the incisors of Fam83h null mice showed a smaller cross‐sectional area of pulp chamber than the other genotype at comparable levels.Previous studies showed that human dental pulp is a highly vascular tissue that has an highly regenerative capacity due to its unique blood supply.Fam83h knockout and WT rabbits, which is different in Fam83h null mice dying at approximately 2 weeks and generally weak in Fam83h‐knockout/lacZ‐knockin mice.Fam83h−/− rabbits exhibited abnormal bone bending and disordered phosphate homeostasis, especially the upregulation of ALP and the downregulation of inorganic phosphate, and the phosphate calcium deposition in kidney medulla of Fam83h−/− rabbits. This is consisted with previous study showing that a Fam83h mutation inhibited mineralization via the Wnt/β ‐ catenin signalling pathway.Furthermore, the similar lifespan and reproductive ability between Fam83h KO mice had a slightly scruffy coat,Fam83h plays a pivotal role in skin homeostasis.Fam83h in epithelial cells (included tongue epithelium), suggesting potential functions of Fam83h in epithelial cells.Fam83h−/− rabbits in this study  to keratin,Fam83h−/− rabbits, showing the defects in hair cycling and hair shaft differentiation.A recent study reported that y Figure . FurtherFam83h−/− rabbits.Fam83h regulates the development of hair, skin and teeth by regulating the expression of the acidic hair keratin genes.Fam83h in the hair growth cycle and in hair shaft differentiation is still unclear. Thus, further investigation about the function of Fam83h in ameloblast differentiation and cytoskeleton network homeostasis are need in the following study.As previously reported, the BMP2/BMP4/MSX2/FOXN1 acidic hair keratin pathway was involved in hair cycling.Fam83h−/− model that shows dental defects similar to what is observed in AI: defective hair cycling, hair shaft differentiation and abnormal bending of the ulna and radius. This novel Fam83h knockout rabbit model may facilitate understanding the function of Fam83h and the pathogenic mechanism of AI and skin homeostasis.In summary, this is the first report of a rabbit Yuning Song: Formal analysis ; funding acquisition ; writing – original draft ; writing – review and editing . Yuxin Zhang: Conceptualization ; data curation ; methodology ; writing – original draft ; writing – review and editing . Jie Yang: Formal analysis ; investigation ; validation . Haobin Yao: Data curation ; investigation ; project administration . Zhongtian Zhang: Investigation ; writing – original draft ; writing – review and editing .No conflict of interest to disclose.Figure S1‐S3Click here for additional data file.Table S1‐S4Click here for additional data file.


Twenty-four hours after AKI induction, cardiomyocyte contraction was decreased in mice with the partial deletion of klotho expression (heterozygous hypomorphic klotho named +/kl). This was accompanied by alterations in Ca2+ transients during systole and an impairment of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) function in +/kl mice after AKI induction. Moreover, Ca2+ spark frequency and the incidence of Ca2+ pro-arrhythmic events were greater in cardiomyocytes from heterozygous hypomorphic klotho compared to wild-type mice after AKI. A decrease in klotho expression plays a role in cardiorenal damage aggravating cardiac Ca2+ mishandling after an AKI, providing the basis for future targeted approaches directed to control klotho expression as novel therapeutic strategies to reduce the cardiac burden that affects AKI patients.Acute kidney injury (AKI) is associated with an elevated risk of cardiovascular major events and mortality. The pathophysiological mechanisms underlying the complex cardiorenal network interaction remain unresolved. It is known that the presence of AKI and its evolution are significantly associated with an alteration in the anti-aging factor klotho expression. However, it is unknown whether a klotho deficiency might aggravate cardiac damage after AKI. We examined intracellular calcium (Ca Klotho is an aging suppressor gene that encodes a single-pass transmembrane protein expressed in multiple tissues, with the highest expression in the renal tubules, parathyroid glands and choroid plexus of the brain ,2. Kloth2+) mishandling [2+ handling in ventricular cardiomyocytes, where endogenous klotho overexpression or increasing its bioavailability exogenously through recombinant murine klotho administration improves cardiomyocyte function and impedes deleterious cardiac remodelling associated with renal damage [kl/kl mice, significantly worsens cardiomyocyte function, inducing a deleterious ionic remodelling that predisposes them to suffer heart failure and fatal ventricular arrhythmias even in the absence of any additional renal insult [+/kl mice worsens cardiomyocyte function after AKI.Cardiorenal syndrome (CRS) is defined as a complex pathological condition in which kidney and heart function is impaired, whereby acute or chronic dysfunction in one organ can induce acute or chronic dysfunction in the other . This syhandling ,19,20,21l damage ,23,24. Ol insult ,24. Howep < 0.05 for +/kl-FA vs. +/kl mice). Increased KW indicates renal hypertrophy, which is probably a compensatory mechanism for the renal damage caused by FA crystallisation in renal tubules. We did not observe significant differences in heart weight (HW), HW/BW or HW/TL in any of the experimental groups.Macroscopic and biochemical parameters were analysed to assess macroscopic renal and cardiac damage associated with folic acid (FA)-induced AKI, and whether they could be aggravated by klotho deficiency . AKI micp < 0.001) relative to each control group independent of genotype. FA-treated wild-type (+/+) mice had higher phosphate concentrations than its control group (p < 0.001). Similarly, heterozygous hypomorphic klotho (+/kl) mice treated with FA showed higher phosphate levels than its control group, with the +/kl mice (p < 0.001) being significantly lower than wild-type +/+ FA-mice (p < 0.001). In FA-treated mice, there was a significant increase in plasma FGF23 levels independently of the genotype (p < 0.001 vs. +/+ and +/kl), although +/kl-FA mice showed significantly lower FGF23 levels than +/+-FA mice.The results related to renal damage are shown in p < 0.001 for KIM-1, and p < 0.01 for NGAL), with KIM-1 expression still higher in +/kl-FA compared to +/+-FA mice (p < 0.001). Klotho expression at the renal level was significantly decreased in AKI mice, regardless of genotype . All these data together  clearly indicate that an FA-induced AKI model causes damage to the kidneys independently of the genotype of the mice.Furthermore, to confirm kidney damage in our experimental model, we measured the expression levels of kidney injury molecule-1 (KIM-1) and NGAL , both considered kidney damage markers, as well as renal Klotho mRNA expression . We founkl groups  compared to their corresponding controls  in cardiomyocytes from the +/kl vehicle group was significantly higher compared with cardiomyocytes from +/+ vehicle animals  and the SR Ca2+ leak during diastole. 2+ sparks in unstimulated cardiomyocytes from each experimental group. Under AKI conditions, cardiomyocytes from heterozygous +/kl-FA mice had a strong increase in frequency of Ca2+ sparks compared to those +/+ mice after AKI or +/kl mice in the absence of AKI , full duration at half maximum (FDHM) and full width at half maximum (FWHM) in cardiomyocytes from each experimental group , such as the presence of Ca2+ waves or automatic Ca2+ transients, in the absence of electrical stimulation. Representative images of both forms of SCR in ventricular cardiomyocytes from AKI-mice are shown in 2+ transients; bottom panel: representative of a Ca2+ wave). The occurrence of the SCR was significantly increased in cardiomyocytes obtained from mice with AKI , automatic Ca2+ transients in the absence of electrical stimulation (black arrow) or the absence of Ca2+ transients during electrical stimulation (orange arrow). Interestingly, the unique circumstance of klotho expression deficiency induced a significant increase in the frequency of Ca2+ release-dependent pro-arrhythmogenic activity at baseline conditions  is able to induce pathological hypertrophy development, which can be attenuated with exogenous klotho administration [2+ handling can be altered as a primary effect on the cardiomyocytes. We observed that relevant alterations in cardiac Ca2+ handling can be detected in the conditions of partial deficiency of klotho expression where an AKI is induced. In fact, these alterations are quite similar to those observed in experimental models of heart failure with alterations in cellular contraction, a decrease in systolic Ca2+ release and an increase in the diastolic Ca2+ leak [2+ release in cardiomyocytes isolated from mice with kidney damage, independently of the genotype of the animals; although mice with reduced klotho expression had higher Tau, which probably means a slower SERCA2a action and Ca2+ reuptake velocity into SR, compared to wild-type mice under basal condition. Moreover, when AKI is induced in heterozygous hypomorphic +/kl mice, the Ca2+ reuptake velocity, which is an indirect measure of SERCA2a activity, compared with heterozygous +/kl mice without AKI was significantly decreased probably as a consequence of the reduction in systolic Ca2+ release observed in these mice. In uremic conditions, some disturbances in cellular Ca2+ metabolism are frequently observed due to the effects of uremic toxins, especially at the renal level, although more studies are needed to elucidate its consequences in the CRS setting as in cardiomyocytes. However, it is logical to think that uremic toxins as phenylacetic acid, with known capacity to block plasma membrane Ca2+-ATPase [Here, we performed an experimental nephrotoxic model of FA-induced AKI, in which an abrupt loss of renal function has been shown to have detrimental repercussions on cardiac structure and function . Alteratstration ,27,28. Ha2+ leak ,30. Here+-ATPase , could b2+ handling observed in the present study are translated into alterations in contraction with a significantly worsening cellular shortening in those animals with partial klotho depletion with AKI. Thus, the cell shortening of cardiomyocytes from heterozygous hypomorphic AKI-mice was lower than observed in control heterozygous hypomorphic mice. Taken together, partial klotho depletion aggravates cardiomyocyte contraction after AKI, most likely because of the relevant alterations in the systolic Ca2+ handling. Moreover, cardiomyocytes isolated from AKI mice had a lower SR-Ca2+ load than vehicle-treated mice, demonstrating that AKI induces a decrease in SR Ca2+ load. Similar results of decreased SR Ca2+ load were obtained in studies carried out in ventricular cardiomyocytes from other experimental models of renal damage such as 5/6 nephrectomy as a model of CKD development [2+ leak observed during diastole after AKI induction may explain the decrease in the Ca2+ load contained in the SR, in turn compromising Ca2+ release during electrical stimulation and, therefore, cellular contraction.All the disturbances in Caelopment . In this2+ sparks was significantly increased in the heterozygous hypomorphic +/kl group and was aggravated after AKI induction. This diastolic Ca2+ leak is a consequence of an increase in RyR2 activity estimated indirectly through the quantification of the frequency of Ca2+ sparks [2+ sparks during diastole has been associated with cardiac dysfunction, especially with the predisposition to arrhythmias [2+ released can induce the activation of adjacent RyR2 producing spontaneous Ca2+ waves capable of provoking automatic ventricular contractions [2+ events. Partial klotho depletion predisposed cardiomyocytes to enhance automaticity due to the increased sensitivity of RyR2 to Ca2+ leak during diastole. Furthermore, when heterozygous klotho-deficient mice suffered AKI, both Ca2+ spark frequency and the automaticity activity of cardiomyocytes were even more significantly increased compared to their baseline condition. This aspect has important translational clinical relevance as it may explain why patients are at high risk of cardiac events, specifically fatal arrhythmias following AKI. As shown here, the higher predisposition to fatal arrhythmias could be a consequence of altered intracellular Ca2+ handling in their ventricular cardiomyocytes, which is observed only a few hours after AKI.It was observed that the frequency of Ca+ sparks . An incrhythmias ,34, sincractions ,35. Our Previous studies in experimental AKI models have shown that renal and plasma klotho levels decrease dramatically 72 h after FA injection . In addikl-FA animals would be an interesting option to be tested in future studies. Supporting this idea, exogenous klotho supplementation has been recently shown to have a relevant cardioprotective action in CKD [2+ mishandling with a reduction in RyR2 hyperactivity and reduction of its phosphorylation at Ca2+/calmodulin-dependent protein kinase type II (CaMKII) [Finally, current treatments for AKI are unsatisfactory and not directed to avoid cardiac complications. Thus, more studies are needed to elucidate new mechanisms that can be traduced into novel therapeutic targets and subsequently strategies that might significantly reduce cardiac complications in AKI patients. In this sense, the exogenous supplementation of recombinant soluble klotho in +/n in CKD ,22,24 orn in CKD ,42. This(CaMKII) ,43, toge(CaMKII) ,45,46. T2+ handling induced by AKI. This opens the possibility to therapeutic strategies aimed at increasing the bioavailability of klotho as a preventive tool against the occurrence of fatal cardiac events that frequently occur in patients with AKI.In conclusion, we have demonstrated that partial klotho deficiency aggravates the altered cardiac Cakl/kl) mice were kindly provided by Dr Kuro-o and bred in our animal facilities. Klotho-deficient mice were generated with transgene integration into the klotho locus, leading to disruption of the 5′-flanking region and the loss of expression as previously described [kl mice to obtain transgenic mice carrying the heterozygous kl mutation in the F1 generation; these mice were then backcrossed with +/kl mice and, in the F2 generation, transgenic mice carrying the kl/kl homozygous mutation together with +/+ and +/kl were obtained and their phenotypes were analysed [kl mice and obtained all the phenotypes, but for the present study we used heterozygous hypomorphic mice (+/kl), and their +/+ littermates of 20–25 g in weight. Animals were group housed in cages at the Experimental Animal Centre, Hospital Universitario 12 de Octubre Madrid, Spain, and were maintained at a controlled and constant temperature (23–25 °C) on a 12 h light/dark cycle with ad libitum access to water and a standard diet. Mice were genotyped by tail DNA and PCR as follows: an initial denaturation at 94 °C for 2 min, followed by 30 cycles of denaturation at 94 °C for 30 s; annealing at 56 °C for 30 s; and extension at 72 °C for 1 min. A common primer (TGGAGATTGGAAGTGGACG) and a wild-type-specific primer (TTAAGGACTCCTGCATCTGC) amplified a 458 bp fragment from the wild-type klotho allele. The common primer and a mutation-specific primer (CAAGGACCAGTTCATCATCG) amplified a 920 bp fragment from the Klotho mutant allele.Klotho-hypomorphic  with FA at a dose of 250 mg/kg diluted in 0.3 M sodium bicarbonate (vehicle). Animals were sacrificed 24 h after AKI induction. Different tissues were collected from each animal, such as heart, kidneys and tibia, and blood was collected in heparinised tubes. The blood was centrifuged for 10 min at 2000 rpm to obtain plasma. Hearts and kidneys were collected and kept at −80 °C in a freezer until processed for further analysis.To evaluate the possible development of renal and/or cardiac hypertrophy as a consequence of FA-induced AKI, data on BW, KW and HW, as well as TL were obtained after the sacrifice of the animals. Just prior to sacrifice, blood samples from the mice were collected for plasma collection. To study kidney damage, we analysed urea and BUN plasmatic levels . In addition, the Phosphate Assay Kit  was used to analyse plasmatic levels of phosphate. All assays were used according to the manufacturers’ recommendations. Plasma levels of cFGF23  were assessed by ELISA kits according to the manufacturer’s instructions.® 480 II instrument  at optimized thermocycling settings. Relative gene expression was normalized to ribosomal 36b4 (RPLP0) as a housekeeping gene, evaluated using the 2−ΔΔCt method and reported as fold change. The sequences (5′–3′) of the primers used were as follows: Klotho-Forward primer TGTGACTTTGCTTGGGGAGTT and reverse primer TCTTCTTGGCTACAACCCCG; KIM-1-Forward primer AGGCGCTGTGGATTCTTATG and reverse primer AAGCAGAAGATGGGCATTGC; NGAL-Forward primer GGCCAGTTCACTCTGGGAAA and reverse primer TGGCGAACTGGTTGTAGTCC; RPLP0-Forward primer AGATGCAGCAGATCCGCAT and reverse primer GTTCTTGCCCATCAGCACC.Total RNA was isolated from kidney tissue using the RNeasy Mini Kit  according to the manufacturer’s instructions. RNA was reverse transcribed using the cDNA High-Capacity cDNA Reverse Transcription Kit . Quantitative RT-PCR was performed using FastStart Essential DNA Green Master  in a 10 µL total reaction volume on a LightCycler2+-free Tyrode’s solution supplemented with EGTA (0.2 mmol/L) was used to retrograde perfuse the hearts over 2–3 min at room temperature. After that, the hearts were digested using a type II collagenase solution  and CaCl2 (0.1 mmol/L) for 3 to 5 min. The hearts were then removed from the Langendorff apparatus and ventricles were cut into small pieces to disperse the isolated ventricular cardiomyocytes. Digested hearts were filtered through a 250-µm nylon mesh and centrifuged at room temperature at 300 rpm for 3 min. Finally, the resulting pellet was resuspended in Tyrode’s solution with CaCl2 (1 mmol/L) and BSA (2mg/mL). Only electrically excitable, rod-shaped cardiomyocytes with clear cross-striations, which were also Ca2+-tolerant in Tyrode solution with increasing Ca2+ concentration , were used for imaging studies of intracellular Ca2+. Tyrode’s solution contained (in mM): 130 NaCl, 5.4 KCl, 0.4 NaH2PO4, 0.5 MgCl2, 25 HEPES and 22 Glucose. The pH was adjusted to 7.4 with LiOH.Twenty-four hours after AKI induction by FA injection, the mice were anesthetised and sacrificed with sodium pentobarbital-heparin . The hearts were rapidly removed and cannulated via the ascending aorta onto a Langendorff perfusion system. A Ca2+ was imaged by confocal microscopy  at a speed of 1.5 ms/line (1000 lines per image) to record Ca2+ transients and Ca2+ spark images and at 3 ms/line  for caffeine and arrhythmia protocols. All Ca2+ images were corrected for background fluorescence. For Ca2+ transients, the fluorescence value (F) was normalised by baseline fluorescence (F0) to obtain F/F0. The decay time constant of the Ca2+ transients, Tau, was obtained by adjusting the decay trace of the fluorescence to a single exponential and was used to indirectly estimate SERCA2a function. K SERCA2a refers to the SR-dependent fraction of the rate constant of decay of the systolic Ca2+ transient and was measured by subtracting the rate constant of decay of the caffeine-evoked transients from that of the systolic Ca2+ transients [2+ load of the SR was estimated in intact ventricular myocytes by a quick administration of 10 mmol/L caffeine to deplete Ca2+ stores. To quantify spontaneous Ca2+ sparks, Fluo-3AM-loaded quiescent myocytes were maintained without electrical stimulation and Ca2+ spark regions were determined as the specific sites where the fluorescence signal briefly increased by at least four times the standard deviation of the fluorescence. Total spark-mediated Ca2+ leak was calculated by multiplying spark frequency × peak (F/F0) × duration (FDHM) × width (FWHM). SCR was quantified by the percentage occurrence of Ca2+ waves or automatic Ca2+ transients without electrical stimulation. Two seven-pulse cycles of a 2 Hz electrical stimulation field with a rest period were then applied to record pro-arrhythmogenic activity. Arrhythmic Ca2+ events were considered to be any spontaneous abnormal release of Ca2+ as waves, missing transients or automatic contractions during the protocol. All Ca2+ images were recorded using the following recording solution containing (in mM): 140 NaCl, 4 KCl, 1.8 CaCl2, 1.1 MgCl2, 10 HEPES and 11 Glucose with pH adjusted to 7.4 using LiOH. All Ca2+ images were processed and analysed using home-made routines with IDL  and Image J  software programs.Isolated ventricular cardiomyocytes were loaded with the cell-permeant fluorescent calcium dye Fluo-3 AM  for 30 min at room temperature. Myocytes were field stimulated by two parallel platinum electrodes at 2 Hz. Cell shortening was measured using the difference in cardiomyocyte length between electrical stimulation and the resting period and was expressed as the percentage of the cell’s shortening length. Intracellular Caansients . The Ca2χ2 test was used when appropriate. Group measurements were expressed as mean ± SEM. Values of p < 0.05 were considered statistically significant. All analyses were performed using an OriginPro 9.0  or GraphPad Prism v6.0 .All collected data were first tested for normality using the Shapiro–Wilk test. Statistical significance was accomplished by applying an analysis of variance (ANOVA) with a Newman–Keuls multiple comparison test for normally distributed data or the Kruskal–Wallis test with Dunn’s correction for non-normally distributed data. The


Anticancer drugs (chemotherapeutics) used in cancer treatment (chemotherapy) lead to drug resistance. This study was conducted to investigate the possible effect of iron on calcium homeostasis in epithelial ovarian cancer cells (MDAH-2774) and cisplatin-resistant cells of the same cell line (MDAH-2774/DDP).To develop MDAH-2774/DDP cells, MDAH-2774 (MDAH) cells were treated with cisplatin in dose increases of 5 μM between 0 μM and 70 μM. The effect of iron on the viability of MDAH and MDAH/DDP cells was determined by 3--2,5-diphenyltetrazolium bromide test at the end of 24 h incubation.3R)1/2/31/2, sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)1/2/3, Na+/Ca2+ exchange (NCX)1/2/3, and plasma membrane Ca2+ ATPase (PMCA)1/2/3/4] associated with Ca2+ differences in expression were determined by quantitative reverse transcription-polymerase chain reaction. Changes in 3R2, RYR1, SERCA2, NCX3, PMCA1,IP and PMCA3 gene expressions were observed in iron treatment of MDAH/DDP cells, while changes were detected in iron treatment of MDAH cells in 3R1/2/3, RYR1/2, SERCA1/2/3, NCX2/3,IP and PMCA1 expressions.At increasing iron concentrations in MDAH and MDAH/DDP cells, the mRNA gene of fifteen genes [inositol 1,4,5-triphosphate receptor This changes in the expression of calcium channels, pumps, and exchange proteins in the epithelial ovarian cancer cell line and in cisplatin-resistant epithelial ovarian cancer cells suggest that iron may have an important role in regulating calcium homeostasis. Due to differences in the expression of genes that play of an important role in the regulation of calcium homeostasis in the effect of iron, drug resistance can be prevented by introducing a new perspective on the use of inhibitors and activators of these genes and thus cytostatic treatment strategies. Cancer is an important health problem affecting many people around the world. For this reason, great interest and economic effort is spent to discover new strategies and methods for the prevention and treatment of cancers worldwide . Althoug2+]i). The intracellular Ca2+ concentration is kept at very low levels (~10−7 mol/L), but the release of a very small amount of Ca2+ from the intracellular organelles (~10−5 mol/L) or the flow of Ca2+ into the cell from outside (~10−5 mol/L) the cell can generate important signals by activating the signal transduction cascades in the cell [2+ increases [Ca2+]i, while active Ca2+ carriers are responsible for returning [Ca2+]i to rest. Intracellular Ca2+ homeostasis is regulated through diverse calcium channels, pumps and exchange proteins. In non-excitable cells, ER is the main organelles that store Ca2+ in the cell, and the channels that give Ca2+ from these organelles into the cell; inositol 1,4,5-triphosphate receptors (3RsIP) and ryanodine receptors (RYRs) i, and significantly augmented the cisplatin-induced cell death of cisplatin resistant ovary carcinoma cells without effecting cisplatin-induced cell death of cisplatin sensitive ovary carcinoma cells. In another study by Pelzl et al. i) in all eukaryotic cells. Cytotoxic Ca2+ overload has such a central role in cell death and the PMCA takes a role for the delicate balance between cell survival, and cell death. In general, the decreased PMCA expression leads to cytotoxic Ca2+ overload and Ca2+ dependent cell death both in apoptosis and necrosis. Therefore, PMCA is suggested to have a paradoxical role in cell survival in drug resistance. Our results also showed that the upregulation of PMCA1 at 10 µM, and 20 µM iron and PMCA3 at 5 µM, 40 µM and 80 µM in MDAH/DDP cells. Upregulation of PMCA1 and PMCA3 mRNA levels in cisplatin resistant EOCs under the influence of iron may suggest that PMCAs may be a separate pathway for resistant cells to escape from cell death and/or, perhaps a new route through which they efflux drugs out. In addition, some isoforms of PMCA in colorectal and breast cancers have been shown to have significant changes in gene expression [PMCA4 compared to normal colon cancers.pression –42 and cBarabas and Faulk  have sho3R1IP, 3R2IP, 3R3IP, RYR1, RYR2, SERCA1, and SERCA3 genes in iron treatment of MDAH cells, while changes were determined in 3R2IP, RYR1, SERCA3, PMCA1, and PMCA2 gene expression in iron treatment of MDAH/DDP cells. These results suggest that changes in the expression of calcium channels, pumps and exchange proteins in the EOC cell line and cisplatin-resistant EOC cells may have an important role in iron regulation of calcium homeostasis. Due to the differences in gene expressions involved in regulation of calcium levels under the effect of iron, drug resistance can be prevented by introducing a new perspective on the use of inhibitors and activators of these genes and thus cytostatic treatment strategies.In conclusion, changes were observed in expression of


Recent studies have also identified rapid alkalization factor (RALF)-family peptides and their receptors as critical components for pollen tube tip growth and integrity. The functional relationship of RALF and calcium signaling modules remains largely unclear. Here we report that disruption of RALF signaling pathway abolished the cytosolic Ca2+ gradient in the pollen tube, indicating that Ca2+ signaling is downstream of the RALF signaling pathway. We identified MILDEW RESISTANCE LOCUS O (MLO) family proteins MLO1, 5, 9, 15, as Ca2+ channels required for Ca2+ influx and pollen tube integrity. We further reconstituted the biochemical pathway in which signaling via RALF and RALF receptors activated MLO1/5/9/15 calcium channels. Together, we conclude that RALF peptides derived from pollen tube bind to their receptors to establish pollen tube Ca2+ gradient through activation of the MLO channels. Our finding has thus provided a mechanistic link between the RALF signaling pathway and Ca2+ signaling in controlling pollen tube integrity and growth.Pollen tube tip growth requires intricate Ca As soon as a pollen grain germinates on the stigma, a tip-focused cytosolic Ca6 In particular, RALF4/19 bind to the pollen tube receptor-like kinases, ANX1/2 (ANXUR1 and 2) and BUPS1/2 (BUDDHA’S PAPER SEAL 1 and 2),9 and their co-receptor LLG2/3 (LORELEI-like-GPI anchored protein 2 and 3)10 to activate downstream processes required for pollen tube integrity. One of such downstream components is the receptor-like cytoplasmic kinase (RLCK), MARIS, which acts as a positive regulator of pollen tube integrity/growth.11 The RALF family peptides also bind to the LEUCINE-RICH REPEAT EXTENSIN (LRX) family proteins that play a role in cell wall integrity and remodeling to sustain normal growth of pollen tube.13In addition to calcium signals, RALF peptides derived from pollen tube are critical in maintaining pollen tube integrity and growth.2+ is elevated in response to several RALFs in the root.16 Furthermore, in the anx1/2 double mutant, cytosolic Ca2+ spiking in the pollen tube is reduced.17 These results suggest that tip-focused calcium signal in the pollen tube may be downstream of RALF peptide signaling pathway.Regarding functional relationship of RALF-RALF receptor pathway and calcium signaling, studies showed that cytosolic Ca19 root thigmomorphogenesis,20 and reproduction.22 Our latest study shows that MLOs function as Ca2+ channels unique to plants,23 indicating that MLOs in pollen tubes may also play important roles in Ca2+ signaling. Indeed, the tip-focused Ca2+ gradient of mlo5mlo9 double mutant was much lower than that of WT plants,22 suggesting that MLOs are positive regulators of pollen tube Ca2+ signal.MLO proteins are involved in plant powdery mildew susceptibility,2+ signals, but it remains unknown how they functionally interact to control pollen tube integrity/growth. Here we report that the disruption of any component of the RALF signaling pathway, including RALF4/19, ANX1/2, BUPS1/2, LLG2/3, or MARIS, abolished cytosolic Ca2+ gradient in the pollen tube, illustrating a functional link between the RALF-RLK-RLCK pathway and Ca2+ signaling. We further revealed that MLO1/5/9/15 are Ca2+ channels activated by ANX1/2-BUPS1/2-LLG2/3-MARIS pathway in response to RALF4/19.Multiple factors and pathways described above appear to impinge on the regulation of pollen tube Ca2+ increase in root cells.16 We thus tested whether RALF4/19, which are essential for pollen tube integrity, can alter pollen tube Ca2+ signal. Using a transgenic plant expressing Ca2+ indicator (GCaMP6s) driven by the Ubiquitin 10 promoter,24 we observed a sharp Ca2+ increase in pollen tubes when applying 500 nM RALF4 or 19 to the medium  impaired but did not completely abolish RALF4 activity.10 We thus generated a series of transgenic plant lines harboring C-terminal truncated RALF4 driven by RALF4 promoter in the ralf4ralf19 double mutant background  inhibited the AtMLO1, 5, 9 or 15-mediated inward currents  of firefly luciferase was fused to MARIS/MARISR240C and the C-terminal portion (cLUC) was fused to MLO1. As a positive control, CBL2 was fused to nLUC and CIPK9 was fused with cLUC.32 Strong fluorescence signals were detected in the leaf cells expressing MARIS/MARISR240C-nLUC and MLO1‐cLUC, as well as in the cells expressing positive control, whereas no discernible signals were observed in the cells expressing negative controls  assay in ols Fig. , suggestR240C, wild type MARIS did not activate AtMLO1/5/9/15-mediated Ca2+ influx when co-expressed in COS7 cells  and female gametophytes (ovule) during pollen tube guidance and reception.37 Although it is well-established that RALF signaling plays a critical role in plant growth, immunity, and reproduction,41 little is known about how RALFs generate Ca2+ signal in any of these processes. Our study on pollen tube-synergid interaction23 suggest that MLO family Ca2+ channels are downstream of RALF peptide signaling, which shed light on the mechanistic link between RALF signaling and the encoding mechanisms of calcium signatures. Considering the functional diversity of MLO family proteins in plants,42 it is possible that MLOs also function downstream of other RALF-RLK signaling events beyond plant reproduction.Like RALF peptides, Ca40 In leaf pavement cells, another CrRLK1L, FERONIA, binds demethylesterified pectin to activate the intracellular ROP GTPase signaling pathway, connecting mechanical stress to cell morphogenesis in plants.44 It appears that RLKs-MARIS-MLOs may be a more directly connected pathway and ROP signaling may be associated through a crosstalk mechanism.Although we found that MLOs are calcium channels downstream of the RALF-RLK-MARIS pathway, it remains unknown if other channels, such as CNGC18, may also play a role in the same process. Another question is how calcium signaling is connected with cell wall integrity: while the mutants lacking any of the components in the RLKs-MARIS-MLOs pathway are defective in pollen tube integrity and calcium signaling, it remains unclear if calcium signaling is directly linked to cell wall integrity. In this regard, signaling processes involving Rho-like GTPase from Plant (ROP) may be particularly relevant. In pollen tubes, ROPs promotes exocytosis that facilitates secretion of RALF4/19 for mechanical signal amplification and cell wall rigidification.R240C  subfamily, including FERONIA, ANX1/2 and BUPS1/2, have not been studied despite the works showing their ligand binding properties of the extracellular domains.46 In our studies, we found it very challenging to successfully express and purify these kinase domains in heterologous systems. In the general framework of RLK signaling, the finding of MARIS phosphorylation by BUPS1 suggests that ANX1/2 may play a primary role in ligand perception while BUPS1/2 may function predominantly in transducing the ligand signal by phosphorylating and activating MARIS, an RLCK. In an attempt to connect MARIS to its potential substrates MLOs, however, we were not able to detect the phosphorylation of MLO1/5/9/15 by MARIS or MARISR240C . The seeds for mlo1 (CS881485), mlo5 (SALK_118934C) and mlo9 (SALK_073198) were purchased from Arabidopsis Biological Resource Center. The ralf4ralf19, llg2llg3, bups1bups2 mutants were generated by CRISPR as reported.10 The plant harboring Ca2+ indicator GCaMP6s is from the Wolf B. Frommer’s lab,24 and anx1anx2, maris mutants are kindly provided by Ueli Grossniklaus and Aurélien Boisson-Derniera.17Seeds were sterilized with 10% (v/v) bleach and sown on agar plates containing 1/2 MS medium  Phyto agar, and 1% (w/v) sucrose, pH adjusted to 5.8 with KOH). Plates were incubated at 4 °C for 3 days for stratification and then seeds were transferred to the soil pots in 22 °C growth room with a 16-h light/8-h dark cycle  plants via Agrobacterium tumefaciens GV3101 using the floral dip method.47 Transgenic plants were selected on 1/2 MS plates containing 35 mg/L hygromycin.The coding DNA sequence (CDS) of MARIS was PCR-amplified from Columbia-0 (Col-0) cDNA and fused to the 48 and cloned into a dual-promoter vector, pBudCE4.1 (Invitrogen), with each CDS for MLOs or ANX1/BUPS1/LLG2/MARIS for co-expression in HEK293T or COS7 cell.The CDS of GCaMP6s was amplified from HBT-GCaMP6-HA2 incubator at 37 °C with controlled moisture. HEK293T or COS7 cells were transfected using Lipofectamine™ 3000 Transfection Reagent Kit (Invitrogen). Plasmids for transfection were extracted from E. coli (DH5α) using QIAGEN Plasmid Mini Kit (Qiagen), and 2 μg plasmid DNA was added into each well of 6-well plates (Nunc) containing the cells (70%–80% confluent). To confirm that the cells were successfully transfected, green and/or red fluorescent signals were examined using an inverted fluorescence microscope (Zeiss AxioObserver Z1 Inverted Microscope) before patch clamp and Ca2+ imaging experiments were performed 48 h after transfection.Mammalian cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum in a 5% CO29 Clampex10.7 software  was used for data acquisition and Clampfit 10.7 was used for data analysis.The whole-cell patch-clamp experiments were performed using an Axopatch-200B patch-clamp setup  with a Digitata1550 digitizer  as previously described.2+ currents across the plasma membrane of HEK293T cells, the standard bath solution contained 140 mM N-Methyl-d-Glucamine (NMDG)-Cl, 10 mM CaCl2, 10 mM glucose, and 10 mM HEPES, adjusted to pH 7.2 with Ca(OH)2. The standard pipette solution contained 140 mM Cs-glutamate, 6.7 mM EGTA, 3.35 mM CaCl2, and 10 mM HEPES, adjusted to pH 7.2 with CsOH. Free Ca2+ in the pipette solution was 175 nM, as calculated using the Webmaxc Standard (web.stanford.edu/∼cpatton/webmaxc/webmaxcS.htm). A ramp voltage protocol of 2-s-duration from ‒180 mV to +30 mV  was applied 1 min after accessing to a whole-cell configuration, and currents were recorded every 20 s for 5 repeats in total for each cell. The 5 current traces were used for statistical analysis for average current-voltage curves.To record Ca5 The interval of data acquisition was 2 s. The standard solution for Ca2+ imaging contained 120 mM NaCl, 3 mM KCl, 1 mM MgCl2, 1.2 mM NaHCO3, 10 mM Glucose, 10 mM HEPES, pH 7.5. About 60 s after initiation of imaging procedure, the bath was perfused using a peristaltic pump with the standard solution supplemented with 10 mM Ca2+ and/or RALFs to elicit Ca2+ entry through active channels.HEK293T or COS7 cells expressing GCaMP6s and various combinations of candidate channel proteins were monitored by a Zeiss AxioObserver Z1 Inverted Microscope (Ivision 4.5 software) using a 20× objective as previously reported.2+]cyt elevation experiment, pollen grains were germinated in the pollen germination medium (PGM) containing 18% sucrose, 0.01% boric acid, 1 mM MgSO4, 1 mM CaCl2, 1 mM Ca(NO3)2 and 0.5% agarose, pH 7.0.49 After 6-h incubation at 22 °C and 100% relative humidity, pollen tubes expressing GCaMP6s were monitored by a Zeiss AxioObserver Z1 Inverted Microscope (Ivision 4.5 software) using a 20× objective, and various RALFs were added to the PGM as indicated.For RALF-induced pollen tube [CaFor pollen germination imaging, pollen grains were spread on the surface of PGM, and then monitored immediately using a Zeiss AxioObserver Z1 Inverted Microscope (Ivision 4.5 software) using a 20× objective.13The pFastBac constructs expressing RALF4, RALF19 and LRX8 were kind gifts from Julia Santiago of University of Lausanne, and RALF4/19 peptides were purified as reported.2+ column , and incubated with TEV protease (New England Biolabs) to remove the tags. Peptides were further purified by size-exclusion chromatography on a Superdex 200 increase 10/300 GL column , equilibrated in 20 mM sodium citrate, pH 5.0, 150 mM NaCl. The peptides were diluted with sterile pure water before use.High Five cells were infected with virus with a multiplicity of infection (MOI) of 3 and incubated for 1 day at 28 °C and 2 day at 22 °C at 110 rpm on an orbital shaker. The secreted peptides were purified from the supernatant by NiAgrobacterium tumefaciens GV3101 carrying proper constructs were grown in Luria-Bertani medium overnight until optical density (OD) at 600 nm was about 1.0. A. tumefaciens cells were collected and suspended in infiltration buffer  at a final OD600 = 0.5. For coexpression of two proteins, same amounts of A. tumefaciens cells carrying proper constructs  and A. tumefaciens carrying p19 helper plasmid  were mixed. The cells were incubated at room temperature for 2 h before infiltration. Leaves were observed 36‒48 h after infiltration under a Zeiss 710 confocal microscope. The leaves expressing split-luciferase complementation constructs were sprayed with 1 mM d-luciferin (Neta Scientific) and the luciferase activities that indicate protein‒protein interactions were detected by a BioRad CCD imaging system.R240C were expressed as His-tag proteins using pET28a. Proteins were expressed in Rosetta (DE3) and purified using standard procedures. Eluted proteins were desalted with a buffer containing 20 mM Tris-HCl, pH7.5 and 1 mM DTT before use. For the kinase assay, 2 or 3 μg of each protein was incubated in the kinase reaction buffer containing 20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT, 10 µM adenosine triphosphate (ATP) and 5 µCi γ-32P-labeled ATP at 28 °C for 1 h, and the reaction was terminated in the SDS-PAGE loading buffer at 65 °C for 5 min, followed by 12% SDS-PAGE, autoradiography and Coomassie Brilliant Blue (CBB) staining.ANX1, BUPS1 kinase domains and MLO cytosolic regions including the middle loop domain or the C-tails were expressed using pMAL-c2x vector with C-terminal MBP fusion. MARIS and MARIS0)/F0 was used, where F0 denotes the average baseline fluorescence determined by the average of F over the first 10 frames of the recording before the treatment.Using the imageJ (1.51j8 version) software, GCaMP6s signals were analyzed overtime at several regions of interest. To calculate the fractional fluorescence changes (ΔF/F), the equation ΔF/F = (F − FMicrosoft Excel in office 365 and GraphPad Prism 7.0 were used for calculation and statistical analysis of the data; Adobe Illustrator CC 2019 was used for image assembly; Clampfit 10.7 was used to analyze and process data from electrophysiological experiments.VS1VS2VS3VS4VS5Fig. S1figS2figS3figS4Table S1video description


Review of peripheral blood showed blasts of variable sizes with a moderate amount of basophilic cytoplasm . An integrated diagnosis of AML with maturation was established (French–American–British Classification Subtype M2).An 81‐year‐old male with a history of diabetes, hypertension, hypercholesterolemia, previous DVT not on anticoagulation, and arthritis presented to the emergency department with a chief complaint of generalized weakness. Complete blood counts revealed white blood cells WBC) 165.3 × 10C 165.3 ×PML‐RARA and AML with RUNX1‐RUNX1T1, occasionally seen in other subtypes of AML and myelodysplastic syndrome (MDS) in the literature as well [RUNX1‐RUNX1T1: blasts with basophilic cytoplasm, salmon‐pink  cytoplasmic granules and perinuclear Hof besides Auer rods as well as granulocytic dysplasia in the background. However, no recurrent cytogenetic and molecular genetic abnormalities as defined by World Health Organization (WHO) classification are detected, while a CSF3R mutation seen. Although mutations in the colony stimulating factor 3 receptor (CSF3R) occurs rarely in AML, comprising only about 1% of adults with AML, it is suggested that CSF3R can be a potential therapeutic target in AML [Auer rods are a morphologic feature in leukemic blasts defining myeloid differentiation. The presence of Auer rods in neutrophils and monocytes are rarely reported, most commonly in association with acute promyelocytic leukemia (APL) with t in AML .CSF3R mutation. Whether the association between the morphologic features and the CSF3R mutation can be established warrants additional studies. In addition, a potential diagnostic pitfall to be taken into consideration in this case is that no reverse transcription‐polymerase chain reaction (RT‐PCR) was done to further exclude RUNX1‐RUNX1T1 rearrangement, although the likelihood of having undetected rearrangement by karyotyping and FISH studies is extremely low.In summary, we presented for the first time a case of AML with a few rare morphologic features including the presence of Auer rods in non‐blast cells coinciding with a potential therapeutic target of What is the new aspect of your work?RUNX1‐RUNX1T1, including Auer rods in non‐blast cells, coinciding with CSF3R mutation, which is a rare mutation in AML while representing a potential novel therapeutic target.Here, we for the first time presented a case of AML with morphologic features resembling AML with What is the central finding of your work?CSF3R‐mutated AML with no recurrent cytogenetic abnormality, showing unusual morphologic features resembling AML with RUNX1‐RUNX1T1, including Auer rods in non‐blast cells.A rare case of What is (or could be) the specific clinical relevance of your work?CSF3R mutation for targeted therapy.The rare morphological features present in this AML could potentially serve as a trigger to identify cases of AML with The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.The authors received no specific funding for this work.Not applicable.Not applicable.Not applicable.


We tried to identify novel molecular subtypes of acute myeloid leukemia (AML) associated with histone methylation and established a relevant scoring system to predict treatment response and prognosis of AML.Gene expression data and clinical characteristics of patients with AML were obtained from The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database. Molecular subtyping was carried out by consensus clustering analysis, based on the expression of 24 histone methylation modification regulators (HMMRs). The clinical and biological features of each clustered pattern were taken into account. The scoring system was constructed by using differential expression analysis, Cox regression method and lasso regression analysis. Subsequently, the scoring system in the roles of prognostic and chemotherapeutic prediction of AML were explored. Finally, an independent GSE dataset was used for validating the established clustering system.NPM1 mutation, tumor mutation burden, somatic mutations, pathway activation, immune cell infiltration and patient survival. The scoring system, M-RiskScore, was established. Integrated analysis demonstrated that patients with the low M-RiskScore displayed a prominent survival advantage and a good response to decitabine treatment, while patients with high M-RiskScore have resistance to decitabine, but they could benefit from IA regimen therapy.Two distinct subtypes of AML were identified based on the expression of the 24 HMMRs, which exhibited remarkable differences in several clinical and biological characteristics, including HMMRs expression, AML-M0 distribution, NPM1 mutant patients. M-RiskScore was a useful prognostic biomarker and a guide for the choice of appropriate chemotherapy strategy.Detection of HMMRs expression would be a potential strategy for AML subtyping. Meanwhile, targeting histone methylation would be a preferred strategy for either AML-M0 or  Acute myeloid leukemia; Histone; Methylation; Classification; Chemotherapy. MeanwhiTwo main systems that have been used to classify AML into subtypes are the French-American-British (FAB) classification and the newer World Health Organization (WHO) classification . FAB claEpigenetics refers to the covalent modification of DNA, RNA and histones without changing the DNA sequence to affect the expression of genes . EpigeneNPM1, survival, TMB, somatic mutations, and pathways activation and immune cell infiltration. Besides, based on the clustering, we established a novel scoring system, M-RiskScore, which not only acts as an independent prognostic predictor but also guides an appropriate and effective chemotherapy strategy.In this study, we established a comprehensive classification approach for AML based on the expression level of 24 HMMRs in AML patients. At the same time, we identified two distinct histone methylation modification patterns that exhibit remarkable differences in several clinical and biological characteristics, including AML-M0 distribution, mutations of 22.1https://portal.gdc.cancer.gov/) and Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). LAML cohort from the TCGA database was used as the training dataset. GSE110087, GSE84334, GSE103424 and GSE71014 cohorts were acquired from the GEO database. Among them, the first three cohorts were used for analyses of chemotherapy in AML, and the last one was for validation.Gene expression, mutation and clinical annotation data of AML cases were obtained in The Cancer Genome Atlas (TCGA) database , 7 erasers  and 4 readers , were chosen and identified from the related studies for further analyses .2.2https://www.cbioportal.org/).Correlation analysis of the gene expression among the 24 histone methylation modification regulators in AML was explored by the “corrplot” package. The Wilcoxon rank-sum test was used to investigate the expression difference of histone methylome regulators in AML patients with different RiskStatus. Information on somatic mutation and copy number variations (CNV) of included genes were generated from cBioPortal website , a technique for reducing the dimensionality of such datasets, increasing interpretability but at the same time minimizing information loss , was conTumor mutational burden (TMB), a new promising biomarker that emerged recently, is classically defined as the number of non-synonymous exonic mutations per megabase (Mb) . The tothttps://www.gsea-msigdb.org/gsea/index.jsp) for GSVA analysis. The result was considered to be statistically significant while its p-value was less than 0.05. Moreover, we explored the somatic gene mutations in the different clusters by the “maftools” package.To discriminate the biological activity difference between the clustered AML subtypes, we performed gene set variation analysis (GSVA) enrichment analysis by using “GSVA” R packages, which is an unsupervised and non-parametric method for estimating the variation in biological process and pathway activity in the samples of an expression dataset . The gen2.5We explored immune cell infiltration patterns between the distinct AML clusters with MCP-counter and CIBERSORT methods. MCP-counter is a methodology based on transcriptomic markers that assess the proportion of immune and stromal cell populations in the tumor microenvironment (TME) from transcriptomic data. There are 10 cell populations estimated by MCP-counter, including T cells, CD8+ T cells, cytotoxic lymphocytes, B lineage, NK cells, Myeloid dendritic cells, Neutrophils, Endothelial cells and Fibroblasts . CIBERSOCD244, PDCD1, PD-L1, CTLA4, CD80, CD86, CD28, TIGIT, PVR, CD96, SIRPA, CD47, LGALS9, HAVCR2, ICOS, ICOSLG, TNFSF18, TNFRSF18, KLRD1, KLRC1) were retrieved from a previous study  among the histone methylation modification patterns of AML cases. Then, we performed a univariate Cox regression analysis to identify the prognostic genes with a p-value less than 0.01 for further analysis.The lasso regression analysis was subsequently applied to construct the histone methylation modification-related score (RiskScore) by “glmnet” and “survival” packages. In this analysis, a lasso penalty was used to account for shrinkage and variable selection. The optimal value of the lambda penalty parameter was defined by performing 10 cross-validations. The calculation formula for the acetylation-related score was as follows:According to the median of the RiskScore, AML patients were divided into two groups (high-risk or low-risk group). We then performed the survival analysis based on this grouping strategy. A receiver operating characteristic (ROC) curve, which is a plot of the sensitivity versus 1 − specificity of a diagnostic test , was con2.7To improve the clinical application of RiskScore, we constructed the nomogram based on the Cox regression model. There were 5 components of the nomogram, including gender, FAB subtype, RiskStatus, age, and RiskScore. Decision curve analysis was performed to compare the net benefits of different prognostic models . The concordance index, calibration plot, and ROC curve were used to verify the nomogram.Nomograms are a pictorial representation of a complex mathematical formula . MedicalModel performance was evaluated through calibration and discrimination . Bias coDecision curve analysis (DCA) was further performed to measure and compare the clinical utilities of the different prognostic models. DCA is a method for evaluating the benefit of a diagnosis test across a range of patient preferences for accepting the risk of undertreatment and overtreatment to facilitate decisions about test selection and use .2.8GSVA analysis, TMB analysis, Somatic gene mutations analysis and immune cell infiltration analysis were performed to exhibit the biological characteristics of the high-RiskScore group and low-RiskScore group, respectively.33.1The overview of this work is shown in the form of a flowchart . The cliA total of 24 HMMRs were finally identified in this study, including 13 writers, 7 erasers and 4 readers. The dynamic reversible process of histone methylation mediated by HMMRs was summarized in Given the interplaying among HMMRs, we performed a co-expression analysis and demonstrated that there were two positive co-expression zones (red boxes), in which the bigger one displayed that SETD2, a histone methyltransferase, has a positive correlation with ATRX, KDM5A, NSD1 and KMT2A in gene expression, respectively C. Meanwh3.2K value of the consensus matrix was set as 2  than Cluster B, with the altering frequency 62.5% against 48.15% . Furthermore, the tumor mutation burden quantification analysis also confirmed that Cluster A was markedly correlated with a higher TMB level A, althouThe tumor microenvironment (TME), composed of tumor cells, stromal cells, immune cells and multiple secreted factors, plays a crucial role in tumor progression . We thenEnlightened by the results of immune cell infiltration, we compared the expression of some immune cell markers, chemokines and cytokines between Cluster A and Cluster B to figure out more about the immune characteristics of histone methylation modification patterns in AML. As shown in 3.4To further evaluate the changes in transcriptome of Cluster A and Cluster B, we conducted the empirical Bayesian approach of the limma package to separate the differentially expressed genes (DEGs) between these two clusters. The volcano plots exhibited that there were lots of DEGs between Cluster A and Cluster B, in which 61 DEGs even with the log2 (fold change) value up to ±3 A. The clTo directly predict the individual therapeutic effect and prognosis of the patients with AML basing on the histone methylation modification patterns, we established a scoring system to quantify the histone methylation modification alteration of AML patients based on the indicated DEGs in 3.5To explore the potential practical value of our M-RiskScore system in predicting the outcome of AML patients, we divided all AML patients into the high and low M-RiskScore groups when the medium value was set as the cutoff value. Prognosis analysis showed that the low M-RiskScore group exhibited a better survival rate than the high M-RiskScore group A. ImpresTo validate the prediction accuracy of M-RiskScore, two receiver-operating characteristics (ROC) curves were created. As shown in To further improve the prognostic application of M-RiskScore in AML, we performed the nomogram associating M-RiskScore with other prognostic markers of AML, including FAB subtype, gender, RiskStatus and age F. Result3.6DNMT3A, KIT and WT1. In contrast, in the high M-RiskScore group, NPM1, DNMT3A and RUNX1 were observed the most frequently mutated genes  is an apoptosis-inducing tumor suppressor that is inactivated by methylation in a variety of tumor types, which may play a role in chemotherapy-induced cell death in AML , but theFAB classification is the first and the most comprehensive classification method of AML in morphology . HoweverNPM1 is well-known to be associated with leukemogenesis and is an important marker for the WHO classification of AML. We find here that patients with the high M-RiskScore have frequently NPM1 mutation (as the top three mutated genes), hinting that NPM1 mutation would be associated with the inactive gene expression of HMMRs in AML. Coincidently, it is reported that SP2509, a KDM1A antagonist, induces more apoptosis in mutant NPM1-expressing AML cells than those with mixed-lineage leukemia fusion oncoproteins (NPM1-mutated AML cells (NPM1 and the inactive expression of HMMRs. We believe that these HMMRs are promising targets for NPM1-mutated AML therapy.The mutation status of proteins . SimilarML cells . But morRUNX1 (RUN family transcription factor 1), which also is a distinct factor of the WHO classification of AML  and/or WT1 (WT1 transcription factor) would be the oncogenic cause of AML patients with low M-RiskScore, thus they should be included as the potent entities for AML classification, as studies have shown that mutated KIT and WT1 are critically associated with leukemogenesis  and the first-line chemotherapeutic drug for most patients with chronic myelogenous leukemia (CML) , exhibitOur results further demonstrate that patients with low M-RiskScore have a better response to the chemotherapy in an AML-cohort, implying that the lower the M-RiskScore is, the better chemotherapeutic response would outcome, especially to the treatment of decitabine, rather than IA therapy regimen. As the cohorts used in our study here do not contain abundant patient samples and differences between different races, there is no statistical difference in the current analysis. Nevertheless, our results still indicate that M-RiskScore is a novel and accurate prognostic or diagnostic marker for AML patients.5In conclusion, our study demonstrates that histone methylation modification profile plays a curial role in the diagnosis and prognosis of AML, and our M-RiskScore system could be a valuable tool not only for the classification of AML but also for the therapy strategy of the patients with AML.Dade Rong and Xiaomin Chen: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.Jing Xiao: Conceived and designed the experiments; Analyzed and interpreted the data; contributed reagents, materials, analysis tools or data; Wrote the paper.Daiyuan Liu and Xiangna Ni: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.Xiuzhen Tong and Haihe Wang: Conceived and designed the experiments; contributed reagents, materials, analysis tools or data; Wrote the paper.Guangdong Basic and Applied Basic Research Foundation [No. 2019A1515010294].Xiuzhen Tong was supported by Guangdong Provincial Key Laboratory of Tumor Interventional Diagnosis and Treatment [2021B1212040004].Jing Xiao was supported by National Natural Science Foundation of China [No. 81472730].Haihe Wang was supported by Data will be made available on request.The authors declare no conflict of interest.No additional information is available for this paper.We are grateful to Wen-Chien Chou , Luciano Castiello , Chieh-Lin Jerry Teng  and Stephan Rainer Bohl  for shared the genetic and clinical information of the patients with AML.Not applicable.


It plays a key role in regulating excitation–contraction coupling and modulating the systolic and diastolic phases. Defective handling of intracellular Ca2+ can cause different types of cardiac dysfunction. Thus, the remodeling of Ca2+ handling has been proposed to be a part of the pathological mechanism leading to electrical and structural heart diseases. Indeed, to ensure appropriate electrical cardiac conduction and contraction, Ca2+ levels are regulated by several Ca2+-related proteins. This review focuses on the genetic etiology of cardiac diseases related to calcium mishandling. We will approach the subject by focalizing on two clinical entities: catecholaminergic polymorphic ventricular tachycardia (CPVT) as a cardiac channelopathy and hypertrophic cardiomyopathy (HCM) as a primary cardiomyopathy. Further, this review will illustrate the fact that despite the genetic and allelic heterogeneity of cardiac defects, calcium-handling perturbations are the common pathophysiological mechanism. The newly identified calcium-related genes and the genetic overlap between the associated heart diseases are also discussed in this review.Calcium (Ca Indeed, Ca2+ plays a key role in excitation–contraction coupling (ECC) and electrophysiological signaling in the heart [2+ storage in the cardiomyocytes. The regulation of the release of Ca2+ ions from SR Ca2+ stores is mediated by the ECC, which then propagates and binds to the myofilaments to initiate the systole phase (contraction). The diastole phase (relaxation) is then ensured by the reuptake of Ca2+ into the SR Ca2+ store via sarcoendoplasmic reticulum Ca2+ ATPase (SERCA2a) or extruded from the cell via the Na+/Ca2+ exchanger [2+-induced Ca2+ release (CICR) and is mediated by the ryanodine receptors (Ca2+ activation of Ca2+ release channels) [2+ homeostasis in cardiomyocytes is regulated by the phosphorylation and dephosphorylation of several key Ca2+-handling proteins.In the heart, calcium  . Intrace2+ is the cornerstone of cardiac electrophysiology and contraction, calcium mishandling has been associated with contractile dysfunction, arrhythmia, and cellular hypertrophy [Given that Caertrophy . 2+-regulating genes. This review will define both diseases and describe their genetic etiology by reporting the different Ca2+-related genes.In this review, we focus on the genetic etiology of cardiac diseases related to calcium mishandling. We will approach the subject by focalizing on two clinical entities: catecholaminergic polymorphic ventricular tachycardia (CPVT) as a cardiac channelopathy and hypertrophic cardiomyopathy (HCM) as a primary cardiomyopathy. HCM is considered the most common primary cardiomyopathy, usually due to heterozygous mutations in sarcomeric genes. HCM may lead to severe complications such as heart failure and sudden cardiac death (SCD). On the other hand, catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare genetic arrhythmia that is mainly defined by bidirectional or polymorphic ventricular tachycardia occurring during physical activity or emotional stress. CPVT patients are predisposed to life-threatening arrhythmias. CPVT is caused by mutations in CaWe conducted a literature search on PubMed for cardiac disorders related to calcium handling with a special focus on catecholaminergic polymorphic ventricular tachycardia (CPVT) and hypertrophic cardiomyopathy (HCM). Different keyword combinations were applied: “catecholaminergic polymorphic ventricular tachycardia” AND “calcium” OR “calcium handling” OR “calcium mishandling” OR “calcium homeostasis”, “catecholaminergic polymorphic ventricular tachycardia” AND “RYR2, CASQ2, TECRL, TRDN, CALM1, CALM2, CALM3, PKP2 OR ANK2”,“hypertrophic cardiomyopathy” AND “calcium” OR “calcium handling” OR “calcium mishandling” OR “calcium homeostasis”, “hypertrophic cardiomyopathy” AND “calcium” OR “calcium handling” OR “calcium mishandling” OR “calcium homeostasis”, “hypertrophic cardiomyopathy” AND “RYR2, ALPK3, TNNC1, JPH2 OR PLN”. The relevance of the articles was determined by screening titles and abstracts. About sixty articles were selected for each heart disease. https://www.ncbi.nlm.nih.gov; accessed 15 September 2022), Uniprot , GeneCards , GeneMANIA , and OMIM .Gene data, namely the gene symbol, location, exon count, protein function, and associated OMIM diseases, were collected from several databases: NCBI-gene .All variants were described following the recommendations of the Human Genome Variation Society  detected in digitalis toxicity [2+ from the SR are associated with the CPVT phenotype, namely the RYR2 and CASQ2 proteins [2+ release channel complex located in the SR. Although RYR2 serves as a Ca2+ release channel, the SR Ca2+-binding protein, CASQ2, plays a dual role by regulating RYR2 function and serving as a buffer for SR Ca2+ [The life-threatening arrhythmias in CPVT are mainly caused by unregulated Cam the SR . Indeed,toxicity ,15. Thustoxicity ,15. Consproteins ,16. Of n SR Ca2+ .2+-handling genes have been linked to CPVT including mainly the RYR2 gene and, less frequently, the CASQ2, TRDN, CALM1, CALM2, CALM3, TECRL, PKP2, and ANK2 genes [RYR2 mutations in CPVT patients ranges from 35 to 79%, whereas mutations in the CASQ2 gene account for 3–5% of CPVT cases [To date, a handful of CaK2 genes . Indeed,VT cases . The CPVRYR2) gene encodes the cardiac ryanodine receptor in the SR. The encoded RYR2 protein is the major Ca2+ channel protein in the membrane of the SR, which acts as intracellular Ca2+ storage in cardiomyocytes. Ca2+ can be released from the SR to the cytosol by the RYR2 channels [2+ release in cardiomyocytes is triggered by increased Ca2+ levels due to the activation of CACNA1C channels. A dysfunctional RYR2 channel leads to diastolic Ca2+ leak from the SR and contributes to the development of DADs [The ryanodine receptor 2  from the RYR2 channel, leading to an increase in intracellular Caactility . Functioing CPVT . RYR2 are found in approximately 79% to 95 % of CPVT1 cases with an autosomal dominant pattern of inheritance [RYR2 mutations are less frequent and linked to other ventricular arrhythmia syndromes [Gain-of-function mutations in eritance ,20. LossRYR2 mutations increase the likelihood of spontaneous RYR2 openings and Ca2+ leak from the SR during diastole, triggering malignant arrhythmias [RYR2 mutations have been associated with CPVT [RYR2 mutations are located in four well-conserved domains including the pore, pseudo-voltage sensor, and central domains. Indeed, these domains are implicated in channel activation and gating. A potential link between mutation localization and phenotype severity has been emphasized [CPVT-linked hythmias . To dateith CPVT . The majphasized ,21. Therphasized .CASQ2) gene encodes the calsequestrin protein localized in the SR of cardiac and slow skeletal muscle cells. CASQ2 is Ca2+-binding protein that stores Ca2+ for muscle function. Ca2+ ions are bound by clusters of acidic residues at the protein surface [2+ through the RYR2 channel. Thus, CASQ2 significantly contributes to the cardiac ECC and regulates the heartbeat rate [The calsequestrin 2  was the first reported mutation associated with an autosomal-dominant inheritance of CPVT [Mutations in utations . CASQ2 c of CPVT .CASQ2 result in a lack of control of the RYR2 channel and, consequently, a constant release of Ca2+ into the cytoplasm, leading to arrhythmias [2+ release, calsequestrin is anchored to RYR2 by triadin and junction proteins. It has been proposed that the interaction between CASQ2 and RYR2 may contribute to the refractory period of Ca2+ release occurring after each physiological CICR but the mechanism is not fully understood [CASQ2 mutations is impaired Ca2+ buffering. However, missense mutations, such as the CASQ2 c.98G>A; p.(Arg33Gln), have been linked to Ca2+ buffering decrease and an alteration of CASQ2/RYR2 interaction [Mutations in hythmias . Indeed,derstood ,20. The Casq2-null mice exhibited normal Ca2+ release from the SR and contractile function under basal conditions. However, mutant mice had an increase in SR volume and an absence of Casq2-binding proteins such as triadin-1 and junction [2+ release and triggered arrhythmias [junction . Exposurhythmias .TECRL) gene encodes the trans-2,3-enoyl-CoA reductase protein, which is an endoplasmic reticulum protein mainly expressed in the heart and skeletal muscle [2+ homeostasis [2+ regulation of major cardiac proteins such as RYR2, CASQ2, and CALM [The trans-2,3-enoyl-CoA reductase-like  from a 5-year-old homozygous patient (TECRLHom-hiPSCs), his heterozygous father (TECRLHet-hiPSCs), and a non-carrier family member (CTRL-hiPSCs) were generated [TECRL leads to the skipping of exon 3. Moreover, the TECRLHomhiPSC-CMs closely replicated the disease phenotype and the mutant cells showed an increase in triggered electrical activity upon catecholaminergic stimulation [To assess the functional consequence of the enerated . hiPSCs mulation . 2+ properties, including a high diastolic Ca2+, smaller amplitude and slower decay of cytosolic Ca2+ transients, and a prolonged action potential duration [TECRL mutations [TECRL c.587G>A; p.(Arg196Gln) mutations can cause a decrease in Ca2+ stores in the SR and an increase in diastolic cytoplasmic Ca2+ concentration in cardiomyocytes, leading to a CVPT phenotype [Analysis of intracellular calcium dynamics of the TECRLHom-hiPSCs revealed altered Caduration ,27. ThisTECRL c.331+1G>A mutation was subsequently reported by Jaouadi et al. (2020) in a consanguineous family with three deceased children, each at 8 years old [TECRL c.331+1G>A mutation in the last deceased child. Both parents were found to be heterozygous for the variant. The father was asymptomatic with a structurally normal heart and no history of cardiac arrhythmias, whereas the mother had a history of syncopes and a clinical suspicion of Brugada syndrome [The ears old . The thrsyndrome . Intrigusyndrome .TECRL mutations displayed strict CPVT features or CPVT-specific features combined with a long QT interval but not an isolated LQTS [TECRL mutations displayed divergent cardiac phenotypes within a single genetic locus [Although the first reported patients with ted LQTS ,30, the TECRL gene in CPVT patients with severe and early-onset clinical presentation at the homozygous and compound heterozygous state , c.893T>C; p., c.926C>A; p.(Ser309*), and c.869C>A; p.(Pro290His)). The authors concluded that variants in TECRL may be causative of up to 5% of CPVT patients [Moscu-Gregor et al. (2020) have identified four additional mutations in the patients .TECRL mutations presented a highly lethal form of arrhythmias, with a median age of symptom onset at 8 years of age [Overall, patients with s of age ,32. 2+ influx in the calcium release complex [TRDN gene lead to a significant decrease in protein expression causing Ca2+ overload in the SR, which may explain the development of CVPT [Triadin (TRDN) is one of the major transmembrane proteins located in the junctional SR playing a role in ECC regulation and Ca complex . Mutatio of CVPT ,35. RYR2 and CASQ2 genes, Roux-Buisson et al. (2012) identified three mutations in the TRDN gene, which cosegregated with the disease according to an autosomal recessive pattern in two families: a c.del53_56ACAG; p.(Asp18Alafs*13) homozygous deletion in the first family and compound heterozygous mutations c.176C>G; p.(Thr59Arg) and c.613C>T; p.(Gln205*) in the second family [TRDN gene was associated with less than 1% of CPVT5 cases [From a cohort of 97 CPVT patients with no mutations in the d family . Thereafd family . OverallT5 cases .2+ sensing and signal transducing. Three distinct calmodulin genes  are distributed within the human genome that encode the identical protein but differ at the nucleotide level. The three calmodulin genes share about an 80% identity within their coding regions. Calcium-induced activation of calmodulin modulates the function of cardiac ion channels including CACNA1C, SCN5A, and RYR2 [Calmodulin proteins (CALM) are members of the EF-hand calcium-binding protein family and play an essential role in Caand RYR2 ,38.CALM1, CALM2, and CALM3 genes have been associated with CPVT.Mutations in CALM1 c.161A>T; p.(Asn53Ile) mutation that segregated with the disease in 10 affected family members [CALM1, c.293A>G; p.(Asn97Ser), was subsequently identified in a 23-year-old woman with a history of resuscitated cardiac arrest at 4 years of age due to ventricular fibrillation while running. Both substitutions showed compromised calcium binding [Nyegaard et al. (2012) identified a heterozygous  members . A de no binding . CALM2 mutations in two patients with overlapping features of LQTS and CPVT [CALM2 variant, c.407A>C; p.(Gln136Pro), was identified in an 8-year-old girl with a presumptive diagnosis of LQTS and CPVT who died suddenly during exercise despite treatment with β-blockers [2+-binding affinity, likely causing Ca2+ signaling dysfunction [Makita et al. (2014) identified two heterozygous missense and CPVT . The c.3blockers . The twofunction . CALM3 mutation, c.308C>T; p., in a 31-year-old woman among a cohort of 12 CPVT patients with no mutations in the other known CPVT genes. The CALM3 mutation was shown to activate RYR2 Ca2+ release channels, generating Ca2+ waves and depleting the SR Ca2+ store [Gomez-Hurtado et al. (2016) identified a heterozygous 2+ store ,41. More2+ store ,41.CALM mutations may present overlapping features of LQTS and CPVT.Thus, both de novo and inherited mutations have been reported and patients harboring PKP2) gene encodes the desmosomal plakophilin-2 protein. Mutations in the PKP2 gene were associated primarily with arrhythmogenic right ventricular cardiomyopathy (ARVC) [Pkp2 in cardiomyocytes to demonstrate that the lack of Pkp2 reduces expression levels of Ryr2, Ank2, Cacna1c, Trdn, and Casq2 proteins, leading to disruption of intracellular Ca2+ homeostasis and isoproterenol-induced arrhythmias, even in the absence of overt structural heart disease [PKP2 gene in genotype-negative patients with CPVT. PKP2 mutations were found in 27.7% of CPVT cases and 5.3% of exercise-related sudden unexplained death in the young cases. Cardiac imaging or autopsy demonstrated a structurally normal heart in all patients [The plakophilin-2 (y (ARVC) . Subsequ disease . Tester patients . ANK2) gene encodes an ankyrin-B protein that is located mainly in the transverse-tubule SR sites of the cardiomyocytes [+/Ca2+ exchanger, Na+/K+ ATPase, and inositol trisphosphate (InsP3) receptor. Thus, ankyrins have key roles in membrane trafficking and regulation of different ion channels in the heart [ANK2 loss-of-function mutations in patients with variable expressivity of cardiac dysfunction including bradycardia, sinus arrhythmia, idiopathic ventricular fibrillation, and CPVT [ANK2 c.4864C>A; p.(Leu1622Ile), c.5437G>A; p.(Glu1813Lys), and c.T4547A; p. mutations [ANK2 mutations were shown to abolish the ability of ankyrin-B to restore defective Ca2+ dynamics. The authors also noted abnormal localization and expression of the Na+/Ca2+ exchanger, Na+/K+ ATPase, and InsP3 [The ankyrin 2  were expressed in a Ca2+-dependent manner [2+ loading seems to contribute to both cardiac hypertrophy and arrhythmogenesis [Nevertheless, there is a lack of strong evidence to show whether these alterations are a causal factor of HCM or consequential clinical manifestations. With the advent of patient-specific iPSC models, Lan et al. (2013) have provided evidence that the elevation of intracellular Caelopment . Indeed,t manner . Furtherogenesis .2+ in ECC, several studies have investigated the implication of mutations in calcium-related genes in HCM pathogenesis. The TNNC1 gene was among the first genes encoding a Ca2+-sensitive/handling protein to be neatly linked to HCM [ALPK3, RYR2, PLN, and JPH2, have been implicated in the pathogenesis of HCM [Given the aforementioned observations and key role of Cad to HCM . Subsequs of HCM . It shous of HCM . The HCM2+-sensitive troponin C subunit (TNNC1). TNNC1 is a sarcomeric Ca2+ sensor that binds to the cytosolic divalent cation at the specific Ca2+ binding site to enhance its interaction with TNNI3. This complex reduces the inhibitory function of TNNI3, releasing it from actin, and causes the troponin–tropomyosin complex to move into the actin groove, exposing myosin binding sites [TNNC1 plays a critical molecular role in the initiation of myofilament contraction [Troponin is a central regulatory protein of striated muscle contraction located on the actin filament. The cardiac troponin complex is a heterotrimeric myofilament composed of three subunits: an elongated troponin T subunit (TNNT2), an inhibitory troponin I subunit (TNNI3), and a Cang sites . Accorditraction . TNNC1 gene are rare, occurring in ~0.4% of HCM patients [TNNC1, c.86T>A; p.(Leu29Gln), was identified by Hoffmann et al. in 2001 [2+-dependent structural change in cardiac TnC in trabeculae under basal conditions and abolish the effect of force-generating myosin cross-bridges. [TNNC1 gene , c.91G>T; p.(Ala31Ser), c.251G>A; p.(Cys84Tyr), c.402G>T; p.(Glu134Asp), c.435C>A; p.(Asp145Glu), and c.363dupG; p.(Gln122Alafs*30)) have been associated with HCM by the screening of the TNNC1 gene in a cohort of 1025 HCM patients [Mutations in the patients . The fir in 2001 . Using ibridges. . Six addpatients ,75,76,772+ sensitivity of force development for c.23C>T, c.251G>A, and c.435C>A and force recovery for c.23C>T and c.435C>A mutations. The TNNC1 c.402G>T mutation showed no changes in these parameters [TNNC1 c.363dupG is located in the EF-hand 3 domain and was found to destroy the H-helices of troponin C that are required for the interaction with troponin I [TNNC1 c.91G>T mutation directly affects Ca2+ sensitivity and may alter Ca2+ handling, leading to arrhythmogenesis [Functional studies showed an increased Carameters . The fraoponin I . Moreoveogenesis .RYR2) gene are typically associated with CPVT, ventricular arrhythmias due to calcium release deficiency syndrome, and arrhythmogenic right ventricular cardiomyopathy/ dysplasia  [RYR2 mutation c.3372G>A; p.(Pro1124Leu), in an HCM patient who did not have a sarcomeric mutation [Mutations in the ryanodine receptor type 2 (ysplasia . In 20061107Met) . This mu1107Met)  and in C1107Met) , questiomutation . Functiomutation . ALPK3) gene encodes the alpha-protein kinase 3 that may act as a transcriptional regulator through the phosphorylation of cardiac transcription factors [ALPK3 gene is early expressed in the cardiac crescent and later remains highly expressed in cardiomyocytes throughout life [ALPK3 was identified as an important cardiac pseudokinase that inserts into the nuclear envelope and M-band of the sarcomere [The alpha kinase 3  and variable facio-thoraco-skeletal features [ALPK3 carriers diagnosed in utero or at birth presented a mixed DCM/HCM phenotype with progression to an HCM phenotype with age [ALPK3 mutations to include (i) missense mutations, (ii) compound-heterozygous and autosomal-dominant patterns of inheritance, and (iii) adult-onset HCM with a less severe clinical presentation [Homozygous myopathy . Pediatrmyopathy ,86,87,88features . It is nwith age . Althougentation ,90.ALPK3 gene appears to be essential for the normal formation of the intercalated disc and the organization of cardiomyofibril in humans and mice [ALPK3- truncating mutation displayed defective Ca2+ handling in addition to sarcomeric disorganization and impaired intercalated disc integrity [The and mice ,88,91. Integrity ,88.2+ flux during contraction demonstrated that ALPK3-mutant cardiomyocytes displayed several changes in intracellular Ca2+ including significantly increased irregular Ca2+-transients, which may explain the cellular hypertrophy [ALPK3–hiPSCs-derived cardiomyocytes demonstrated an extended extracellular field potential duration, indicating that the loss of ALPK3 disrupts membrane repolarization [A functional study of the Caertrophy . Indeed,rization . 2+ is elevated in ALPK3-deficient cardiomyocytes, agreeing with what has been observed in other hiPSC models of HCM [These results suggest that intracellular Cas of HCM ,88. JPH2) gene encodes junctophilin 2, a major component of the junctional membrane complex involved in Ca2+ homeostasis and ECC [JPH2 plays a crucial role in maintaining the proper structure of the cardiac dyad, which is necessary for effective CICR [JPH2 gene in HCM was first reported by Landstrom et al. (2007) with the identification of three mutations , c.421T>C; p.(Tyr141His), and c.494C>T; p.(Ser165Phe), in three unrelated patients with HCM negative for sarcomeric or Z-disc mutations [JPH2 are considered a rare cause of HCM and are found in less than 1% of index cases [The junctophilin 2 ( and ECC . Junctop and ECC . JPH2 plive CICR . The imputations . The chautations . Of noteex cases . PLN) gene encodes phospholamban, a major substrate for the cAMP-dependent protein kinase in cardiac muscle. In its unphosphorylated state, PLN is an inhibitor of cardiac muscle SERCA2a. This inhibition is abolished upon phosphorylation of the PLN protein. The consecutive activation of the Ca2+ pump results in enhanced muscle relaxation rates. Thus, PLN is a key regulator of cardiac diastolic function [The phospholamban  mutation is recognized as a causative HCM mutation [PLN–HCM mutations of 0.65% by comparing different studies reporting PLN mutations in HCM cohorts [Mutations in the ause DCM . Howeverause DCM ,101. Nevmutation . This mumutation . The aut cohorts .2+-related genes linked to CPVT and HCM with their underlying physiopathological mechanisms. Reporting HCM patients with mutations in Ca2+-related genes stems from the fact that abnormal Ca2+ handling is a common feature of structural heart diseases. Studying calcium handling in healthy and diseased hearts may improve our understanding of Ca2+-mediated cardiac diseases and aid the advancement of therapeutic strategies.This review aimed to summarize the Ca