Caenorhabditis elegans initiates the mPTP and shortens lifespan specifically during adulthood, in part via initiation of the mitochondrial unfolded protein response (UPRmt). Pharmacological or genetic inhibition of the mPTP inhibits the UPRmt and restores normal lifespan. Loss of the putative pore-forming component of F-ATP synthase extends adult lifespan, suggesting that the mPTP normally promotes aging. Our findings reveal how an mPTP/UPRmt nexus may contribute to aging and age-related diseases and how inhibition of the UPRmt may be protective under certain conditions.Mitochondrial activity determines aging rate and the onset of chronic diseases. The mitochondrial permeability transition pore (mPTP) is a pathological pore in the inner mitochondrial membrane thought to be composed of the F-ATP synthase (complex V). OSCP, a subunit of F-ATP synthase, helps protect against mPTP formation. How the destabilization of OSCP may contribute to aging, however, is unclear. We have found that loss OSCP in the nematode  Sustained opening of the mPTP leads to outer mitochondrial membrane rupture, release of Ca2+ into the cytosol, and cell death  increases . The mPTll death . Cyclospll death . Geneticll death , and CsA elegans . Thus, t2+ currents , mitochondrial phosphate carrier (PiC), and translocator protein (TPSO) due to genetic ablation studies showing that the mPTP can still occur in their absence . Due to currents . Some mocurrents . Despitecurrents , leadingcurrents . Other gcurrents , and thecurrents .in vitro , a subunit of the F-ATP synthase that regulates ATPase rotational activity to provide efficient ATP production , has emein vitro . In micein vitro . In mousin vitro . Conversin vitro . Thus, OER) and the cytoplasmic heat shock response (HSR), the mitochondrial unfolded protein response (UPRmt) is capable of initiating a broad-range transcriptional response that, among other functions, aids in the refolding of mitochondrial matrix proteins  during adulthood caused a loss of MMP as measured by the mitochondrial dye, tetramethylrhodamine methyl ester (TMRM), while RNAi of other OXPHOS subunits from complex I, IV, and V had no effect on the MMP  expression values of various OXPHOS subunits collected at young adulthood (YA) showed that OSCP/atp-3 did not display higher expression compared to other subunits, and that its expression during larval stages (L1) and YA was also comparable  , which we termed the post-developmental UPRmt (pdvUPRmt)  of development (dvUPRmt) , mrps-5 , and tomm-22   , and comdvUPRmt) . The excg adults , the timg adults . In contdvUPRmt) . These r elegans . Treatingans  , did notane)  led to lifespan extension (atp-3 RNAi during post-development experienced a high incidence of matricide (data not shown). To circumvent this outcome, we administered OSCP/atp-3 RNAi to young adults for 48 hr of adulthood and observed approximately a 38% decrease in lifespan independent of matricide (atfs-1(tm4525) mutants were exposed to OSCP/atp-3 RNAi during adulthood for 48 hr, we observed only about a 19% decrease in lifespan, suggesting that the initiation of the pdvUPRmt via atfs-1 contributes to reduced lifespan  and every few hours thereafter into adulthood. GFP expression was examined 48 hr after RNAi initiation  of the inner mitochondrial membrane and loss of ANTs helps prevent the mPTP , peripheral stalk or supernumerary subunits , induced little to no UPRmt  induced a mild to moderate UPRmt, though none as robustly as loss of OSCP/atp-3. We observed that post-developmental loss of the α/atp-1 ATPase subunit exhibited the second most robust pdvUPRmt , a catalytic ATPase that converts ADP to ATP (F1), and peripheral stalk and supernumerary subunits that help bridge these two portions together . OSCP/atst UPRmt . In contno UPRmt . Loss ofpdvUPRmt  but did PRmt  as well as peripheral and supernumerary subunits (color coded gray) via RNAi in adults, we observed nearly complete inhibition of the OSCP/atp-3 RNAi-mediated pdvUPRmt  had no effect or slightly increased the dvUPRmt and the pdvUPRmt  or the proton-driving rotor c-ring/Y82E9BR.3 rescued the loss in MMP, suppressed the rise in cytosolic Ca2+, and rescued the shortened lifespan caused by RNAi of OSCP/atp-3 . Furthermore, we observed that activation of the pdvUPRmt helps drive aging. Suppression of the mPTP/UPRmt via genetic or pharmacological interventions is protective. Loss of other OXPHOS subunits or administration of FCCP during adulthood does not cause a loss of the MMP, a rise in cytosolic Ca2+, or activation of the pdvUPRmt, suggesting that the activation of mPTP/UPRmt is specifically due to loss of OSCP/atp-3. In contrast, it does not appear that the loss of OSCP/atp-3 during development activates an mPTP. Thus, loss of OSCP/atp-3 can have drastically different effects depending on the life stage or cellular milieu of the organism.While loss of the F-ATP synthase subunit OSCP/mt contributes to health and longevity  plates. NGM plates were seeded with E. coli HT115 (DE3) bacteria obtained from the Ahringer and Vidal RNAi Library were used  but not yet gravid, approximately 45 hr for wild-type . Nematodes were shifted to 20°C once they reached adulthood.To achieve synchronous nematode populations, day 1 adult nematodes were allowed to lay eggs for 2 hr on seeded NGM plates. For convenience, nematodes were developed at 25°C on E. coli OP50 at 25°C until the young adult stage and then transferred to RNAi plates at 20°C. Nematodes were collected after 48 hr for analysis or for lifespans, moved onto E. coli OP50 for the remainder of their life.For developmental treatments, synchronized eggs were moved onto plates seeded with RNAi bacteria and developed at 20°C for 72 hr. Nematodes were then either collected for analysis or for lifespans, remained on RNAi bacteria for the remainder of their life for survival analysis. For post-developmental treatments, synchronized eggs were developed on plates with Approximately 300 adult nematodes were collected; nematode pellets were resuspended in 300 μL RNA Lysis Buffer and frozen. Pellets was thawed, vortexed, and snap frozen three times. Zymo Research Quick-RNA MiniPrep kit was used to extract RNA.Day 1 adult nematodes were allowed to lay eggs for 2 hr on seeded NGM plates to obtain a synchronous aging population. 5-fluoro-2-deoxyuridine (FUdR) was omitted from plates due to its potentially confounding effects . Worms aPlates were prepared by spotting seeded NGM plates with a TMRM solution diluted in water to a final concentration of 0.1 μM in the plates. Water was used as a solvent control. Plates were allowed to dry for 24 hr before use. For developmental experiments, synchronized eggs were placed on plates for 72 hr and then nematodes were collected for analysis. For post-developmental experiments, young adult nematodes were placed on plates for 48 hr and then collected for analysis.Plates were prepared by spotting seeded NGM plates with a CsA (stock solution in DMSO) solution diluted in 100% ethanol. Comparable amounts of DMSO and ethanol were used as solvent controls. Plates were allowed to dry for 24 hr before use. For developmental experiments, synchronized eggs were placed on plates for 72 hr and then nematodes were collected for analysis. For post-developmental experiments, young adult nematodes were placed on plates for 48 hr and then collected for analysis; for lifespans, worms were moved to regular NGM plates for the remainder of their life after 48 hr on drug-treated RNAi bacteria.Worms were anesthetized with 2 mM levamisole and mounted on 2% agarose pads on glass slides. Fluorescence micrographs of GFP and TMRM were taken using a Zeiss Imager A2 at 5× magnification with 600 ms exposure using the ZEN software. GFP expression was enhanced using the brightness/contrast tool in Photoshop. The same parameters were used for all images.Confocal micrographs of mitochondrially targeted GFP and the cytosolic calcium sensor D3cpv  were takApproximately 30–50 adult worms were collected in S-basal buffer. Supernatant was removed and nematodes were flash-frozen. Worms pellets were resuspended in 2% SDS sample buffer with 2.5% β−mercaptoethanol and samples were boiled for 10 min. Samples were subjected to SDS-PAGE using 4–12% SDS gels (Invitrogen) and transferred to Immun-Blot PVDF Membrane (Bio-Rad) using Bio-Rad western blot criterion apparatus. Membranes were blocked with 5% non-fat dry milk blocking solution; concentrations for antibodies were 1:1000 for primary antibodies and 1:2000 for secondary antibodies.t-test. Asterisks denote corresponding statistical significance: *p<0.05; **p<0.01; ***p<0.0001. Error bars were generated using the standard error of the mean (SEM), typically from three pooled biological replicates. GraphPad Prism 7 was used to plot survival curves. Log rank (Mantel–Cox) test in Prism was used to determine significance between the control and experimental groups.Significance between control and experimental groups was determined by using two-tailed Student’s GFP intensity of worms was quantified using ImageJ 1.52A. The ‘integrated density’ of GFP expression and length of worms was measured using ImageJ tools. Integrated density value was normalized by number of worms and average length of worms. The final value is in arbitrary units. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Both reviewers found the manuscript to be of high impact to the field. Particularly intriguing is the finding that disruption of a complex V component activates the mitochondrial unfolded protein response and affects longevity via mechanisms that appear distinct from disruptions of the other complexes, as previously reported.Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Jessica Tyler as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "The mitochondrial permeability transition pore activates a maladaptive mitochondrial unfolded protein response" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The authors provided evidence to suggest that RNAi knockdown of the mitochondrial ATP synthase subunit atp-3 in adults triggers mitochondrial permeability transition pore (mPTP), resulting in activation of the mitochondrial unfolded protein response (UPRmt) and shortening of lifespan. This is particularly intriguing as this observation in adult animals contradicts the well-established findings in the field that RNAi of various mitochondrial electron transport chain subunits, including atp-3, during development leads to a transient activation of the UPRmt during larval but not adult stage, and extension of adult lifespan. Further investigation into UPRmt during development vs in adulthood will be important for understanding this important pathway in aging. This study will also contribute to helping clarify the controversial field of mPTP.Essential revisions:The reviewers appreciated the potential importance of this work, but more evidence are needed to support the major claims put forth.A) Additional experimental evidence are required to demonstrate that atp-3 RNAi indeed induces mPTP.2+, however, the measurements of the membrane potential and the cytosolic ca2+ levels were missing from many key experiments. For example, in figure S2, the authors showed that the activation of the pdvUPRmt is reversible, however, whether the loss of membrane potential and the increase in cytosolic ca2+ were also reversed was not shown. The same measurement should also apply to the situation when the pdvUPRmt induced by atp-3 RNAi was suppressed by the RNAi against the ATPases subunits (a and b), the two UPRmt regulators , atfs-1(the downstream TF), or by the pharmacological inhibitors mPTP, and in the long-lived mutants ).1. The authors stated at the beginning of the manuscript that the opening of the mPTP is characterized by a loss of mitochondrial membrane potential and an increase in cytosolic ca2+ measurement after FCCP treatments should be included to demonstrate the efficiency of the drug treatment.2. Along with the previous point, the authors showed that loss of FCCP, a mitochondrial uncoupler, did not induce a UPRmt in adults, therefore excluding the possibility that loss of mitochondrial membrane potential is not sufficient to induce pdvUPRmt. However, it has been shown that FCCP treatment induces the UPRmt in mammalian cells . Therefore, controls with measurement of the mitochondrial membrane potential and the ca3. It is also difficult to understand the phenotypical differences between RNAi of atp-1 and atp-3. Loss of atp-1 and atp-3 can both induce the pdvUPRmt, however, atp-1 RNAi had no effect on the membrane potential but could partially suppress the pdvUPRmt induced by atp-3 RNAi.The reviewers pointed out papers in the literature that argued against the proposal that the mPTP is associated with the ATP synthase. Given that this is a controversial topic, clear evidence must be provided to convince the readers otherwise.B) Epistasis analysis between dvUPRmt and pdvUPRmt will be key to understand the interesting opposing observations.1) Mechanistically, why the inductions of dvUPRmt and pdvUPRmt have such huge differences in the regulation of organismal aging? The authors have not excluded the possibility that the mPTP is not formed when dvUPRmt is induced.2) Inhibition of multiple OXPHOS components such as CCO-1 during development activates UPRmt and extends worm lifespan. However, inhibition of CCO-1 at or after the L4 stage does not activate UPRmt or extend lifespan. Here, the authors demonstrate that inhibition of atp-3/OSCP during development activates the UPRmt and extends lifespan. However, inhibition of atp-3/OSCP at the young adult stage also activates the UPRmt, but shortens lifespan. Which is dominant? Is cco-1(RNAi)-dependent lifespan extension suppressed by atp-3(RNAi) administered at the young adult stage?This line of investigation is necessary to provide a clearer picture about how activation of UPRmt influences aging.C) The data on genetic interactions with other longevity mutants appear premature. The reviewers suggested completely removing that part from this manuscript, so that the current manuscript can focus on A and B above.– The section titled "Longevity mutants display exceptional mitochondria partially via FOXO/daf-16" is intriguing but much too underdeveloped. I agree that it is interesting that several longevity mutants suppress the post-developmental UPRmt.-1 promoter is induced by DAF-16, confounding the results. Mitochondriall staining independent of mtGFP should be performed such as TMRE. The imaging should also be of higher magnification. mtDNA quantification should be provided as an imaging-independent indicator of mitochondrial mass. The authors should also determine if the "exceptional mitochondria" in these worms provide more mitochondrial activity by measuring oxygen consumption etc. The data suggesting that mitophagy contributes to the loss of mitochondrial mass are also underdeveloped. Images should be provided and mitochondrial mass should be quantified via a mtGFP independent mechanism. I would suggest simply removing this section as it largely detracts from the rest of the manuscript.– However, the subsequent claims about "exceptional mitochondria" and increased mitochondrial mass using mtGFP as a marker are not convincing. For example, the ges Essential revisions:The reviewers appreciated the potential importance of this work, but more evidence are needed to support the major claims put forth.A) Additional experimental evidence are required to demonstrate that atp-3 RNAi indeed induces mPTP./atp-3. The use of CsA is an established method to help confirm the identity of the mPTP in many models, such as isolated mitoblasts and cell lines. We found that CsA was able to reverse all mPTP-like characteristics due to adult loss of OSCP/atp-3, which included loss of MMP, rise in cytosolioc ca2+, lifespan shortening, and mitochondrial rounding/fragmentation . These finding significantly strengthen our claim that our observations are due to the mPTP.To address this critique, we decided to use the well characterized mPTP inhibitior, cylosporin A (CsA), to determine if it could reverse the mPTP characteristics caused by adult loss of OSCPmt can be suppressed due to loss of additional F-ATP synthase subunits. Epistatic experiments also reverse nearly all mPTP-like characteristics due to adult loss of OSCP/atp-3, which include loss of MMP, rise in cytosolioc ca2+, lifespan shortening, and mitochondrial rounding/fragmentation . These finding support a model in which F-ATP synthase, especially the proton-driving c-ring rotor, is likely to form the mPTP.To additionally address this critique, we further examined our epistasis experiments that showed that the pdvUPR2+, however, the measurements of the membrane potential and the cytosolic ca2+ levels were missing from many key experiments. For example, in figure S2, the authors showed that the activation of the pdvUPRmt is reversible, however，whether the loss of membrane potential and the increase in cytosolic ca2+ were also reversed was not shown. The same measurement should also apply to the situation when the pdvUPRmt induced by atp-3 RNAi was suppressed by the RNAi against the ATPases subunits (a and b), the two UPRmt regulators , atfs-1(the downstream TF), or by the pharmacological inhibitors mPTP, and in the long-lived mutants ).1. The authors stated at the beginning of the manuscript that the opening of the mPTP is characterized by a loss of mitochondrial membrane potential and an increase in cytosolic ca2+ measurement after FCCP treatments should be included to demonstrate the efficiency of the drug treatment.2. Along with the previous point, the authors showed that loss of FCCP, a mitochondrial uncoupler, did not induce a UPRmt in adults, therefore excluding the possibility that loss of mitochondrial membrane potential is not sufficient to induce pdvUPRmt. However, it has been shown that FCCP treatment induces the UPRmt in mammalian cells . Therefore, controls with measurement of the mitochondrial membrane potential and the ca2+ levels , demonstrating that loss of MMP per se is not sufficient to recapitulate the mPTP.We verified that FCCP did induce a loss of MMP during adulthood, demonstrating the drug efficacy, while also failing to increase cytosolic ca3. It is also difficult to understand the phenotypical differences between RNAi of atp-1 and atp-3. Loss of atp-1 and atp-3 can both induce the pdvUPRmt, however, atp-1 RNAi had no effect on the membrane potential but could partially suppress the pdvUPRmt induced by atp-3 RNAi.atp-1 subunit. In addition to its loss not inducing a loss of MMP, its loss also did not lead to a rise in cytosolic ca2+  and had minor effects on lifespan . Thus, its ability to induce a moderately robust pdvUPRmt does not necessitate that it display mPTP characteristics. We conclude from these findings that mPTP activation is very specific to the loss of OSCP/atp-3.We further tested the role of the a/The reviewers pointed out papers in the literature that argued against the proposal that the mPTP is associated with the ATP synthase. Given that this is a controversial topic, clear evidence must be provided to convince the readers otherwise.We added more context in the introduction that references that experts in the field of the mPTP largely consider F-ATP synthase to be the probable pore-forming component. This is not without dispute though, as we also further discuss in the introduction. Thus, while our findings do not conclusively ascertain that the F-ATP synthase is the mPTP, they are supportive of the prevailing F-ATP synthase models of the mPTP.B) Epistasis analysis between dvUPRmt and pdvUPRmt will be key to understand the interesting opposing observations.1) Mechanistically, why the inductions of dvUPRmt and pdvUPRmt have such huge differences in the regulation of organismal aging? The authors have not excluded the possibility that the mPTP is not formed when dvUPRmt is induced.2+ release and found that loss of OSCP/atp-3 during development does not lead to a release of cytosolic ca2+ , a canonical feature of the mPTP. This finding, in addition to our previous findings that the dvUPRmt is not responsive to CsA or genetic mPTP factors , lead us to conclude that the dvUPRmt is not likely coupled to the mPTP.We performed additional experiments analysing cytosolic ca2) Inhibition of multiple OXPHOS components such as CCO-1 during development activates UPRmt and extends worm lifespan. However, inhibition of CCO-1 at or after the L4 stage does not activate UPRmt or extend lifespan. Here, the authors demonstrate that inhibition of atp-3/OSCP during development activates the UPRmt and extends lifespan. However, inhibition of atp-3/OSCP at the young adult stage also activates the UPRmt, but shortens lifespan. Which is dominant? Is cco-1(RNAi)-dependent lifespan extension suppressed by atp-3(RNAi) administered at the young adult stage?This line of investigation is necessary to provide a clearer picture about how activation of UPRmt influences aging.cco-1 RNAi followed by post-developmental treatment with OSCP/atp-3 RNAi did not significantly alter the long-lived phenotype cause by developmental cco-1, suggesting that the cco-1 regulation of longevity is “dominant”. We agree further investigation into how the dvUPRmt and the pdvUPRmt are regulated is an area of high priority for future studies.This interesting experiment proposed by the reviewers led to an interesting result. We found that developmental treatment with COX5B/C) The data on genetic interactions with other longevity mutants appear premature. The reviewers suggested completely removing that part from this manuscript, so that the current manuscript can focus on A and B above.We agree and have accordingly removed this portion.


Results: Under basal conditions, action potential (AP) morphology were similar in PDE2 OE and WT. ISO stimulation significantly increased the incidence of afterdepolarizations and spontaneous APs in WT, which was markedly reduced in PDE2 OE. The ISO-induced increase in ICaL seen in WT was prevented in PDE2 OE. Moreover, the ISO-induced, Epac- and CaMKII-dependent increase in INaL and Ca2+-spark frequency was blunted in PDE2 OE, while the effect of direct Epac activation was similar in both groups. Finally, PDE2 inhibition facilitated arrhythmic events in ex vivo perfused WT hearts after reperfusion injury. Conclusion: Higher PDE2 abundance protects against ISO-induced cardiac arrhythmia by preventing the Epac- and CaMKII-mediated increases of cellular triggers. Thus, activating myocardial PDE2 may represent a novel intracellular anti-arrhythmic therapeutic strategy in HF.Background: Phosphodiesterases (PDE) critically regulate myocardial cAMP and cGMP levels. PDE2 is stimulated by cGMP to hydrolyze cAMP, mediating a negative crosstalk between both pathways. PDE2 upregulation in heart failure contributes to desensitization to β-adrenergic overstimulation. After isoprenaline (ISO) injections, PDE2 overexpressing mice (PDE2 OE) were protected against ventricular arrhythmia. Here, we investigate the mechanisms underlying the effects of PDE2 OE on susceptibility to arrhythmias. Methods: Cellular arrhythmia, ion currents, and Ca Heart failure (HF) remains one of the leading causes of mortality worldwide. Patients with HF are at risk for lethal ventricular arrhythmias due to structural and electrical remodeling . Beyond 2+ cycling coupling the excitation-contraction processes [Na) through voltage-gated Nav1.5 channels triggers the opening of voltage-gated L-type Ca2+ channels, Cav1.2 (LTCC). The resulting L-type calcium current (ICaL) subsequently initiates the activation of ryanodine receptors (RyR2), allowing Ca2+ release from the sarcoplasmic reticulum (SR) into the cytosol which finally evokes myofilament contraction. Cardiomyocyte relaxation is mainly mediated by SR Ca2+-ATPase type-2a (SERCA2a), which pumps cytosolic Ca2+ back to the SR. Potassium extrusion from the myocyte via different K+ channels, generating the transient outward current (Ito), the delayed rectifier , and inward rectifier (IK1) currents, contributes to repolarization thereby restoring the resting membrane potential.Regular heart rhythm and contraction require a coordinated action potential (AP) generation followed by coordinated intracellular Carocesses . Cellula2+ cycling proteins, is modulated by the cAMP-dependent protein kinase A (PKA) as well as Ca2+-calmodulin-dependent protein kinase II (CaMKII) upon β-adrenergic stimulation [2+ cycling proteins such as RyR2 and the SERCA2a-inhibiting phospholamban (PLB) facilitating SR Ca2+ release and reuptake. CaMKII and PKA have been known to influence ion channel proteins like Nav1.5, Cav1.2, and Kv4 [NaL, ICaL, and Ca2+ leaks from the SR via RyR2 have been shown to prolong AP duration and trigger early (EAD) and delayed afterdepolarization (DAD), as well as spontaneous APs (sAP) promoting the development of ventricular fibrillation associated with SCD [The activity of ion channels, as well as Camulation ,14,15,16 and Kv4 ,14,15,16with SCD ,17.Phosphodiesterases (PDE) play a key role in regulating myocardial cAMP and cGMP levels. Among the family of cardiac PDEs, phosphodiesterase 2 (PDE2) has the unique property to be stimulated by cGMP via its GAF domains to primarily hydrolyze cAMP . Thus, PIn previous work, we studied the effects of chronically increased PDE2 activity in the heart using the transgenic mouse line with cardiac-specific overexpression of the wild-type murine PDE2A3 isoform . We observed that PDE2 OE mice showed lower susceptibility to arrhythmia after acute provocation by double isoprenaline (ISO) injections, displaying less premature ventricular contractions and ventricular tachycardia than control mice (WT) . Under pNaL, ICaL, and SR Ca2+ leak, were identified as underlying mechanisms. Thus, cGMP-mediated activation of myocardial PDE2 may represent a novel intracellular anti-arrhythmic therapeutic strategy in HF.In this study, we focused on investigating the role of PDE2 concerning the susceptibility to cardiac arrhythmias and on deciphering the underlying cellular mechanisms. We reveal that increased PDE2 activity protects against ISO-induced cellular arrhythmic events. Blunting of the ISO-induced, ‘exchange protein directly activated by cAMP’ (Epac) and CaMKII-mediated increases in IMice with cardiac-specific PDE2 overexpression (PDE2 OE) displayed significantly fewer arrhythmic events upon β-adrenergic stress triggered by isoprenaline (ISO) injections, as well as after myocardial infarction via left anterior descending coronary artery (LAD) ligation . In orde20, APD50) in PDE2 OE compared to WT. Conversely, upon ISO stimulation, there was a trend towards increased APD90  at 1 Hz from the same series of experiments are depicted in ed APD90 E–G.2+-handling in cardiomyocytes of PDE2 OE and WT littermates. First, L-type calcium current (ICaL) was measured under basal conditions and upon ISO stimulation (CaL voltage-membrane (IV) relationship as WT cardiomyocytes under basal conditions  as cardiomyocytes from WT littermates  can promote the generation of arrhythmia by AP prolongation and initiation of early afterdepolarizations [NaL amplitude upon β-adrenergic stimulation. Under basal conditions, INaL was similar in cardiomyocytes of PDE2 OE and WT mice. ISO incubation  significantly enhanced INaL in WT cells whereas this effect was not present in PDE2 OE  did not affect the ISO-mediated INaL increase  to produce diacylglycerol (DAG) and inositol triphosphate (IP3) leading to PKC and CaMKII activation [2+ sensitivity. Therefore, we evaluated the consequences of PDE2 overexpression on expression levels of selected cardiac kinases in lysates of isolated ventricular cardiomyocytes , which in turn hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIPtivation . These kmyocytes . Westernmyocytes . Interesmyocytes .2+ cycling proteins showing reduced phosphorylation levels of RyR2 and PLB at CaMKII-specific phosphorylation sites in PDE2 OE but similar protein expression compared to WT [+/Ca2+ exchanger type 1 (NCX1), Na+/K+ ATPase (NKA), and phospholemman (PLM), which are important for the maintenance of intracellular Na+ and Ca2+ homeostasis. Protein levels of NCX1, NKA, and PLM did not differ between PDE2 OE and WTThe phosphorylation level of PLM at Ser68 was similar in both genotypes  or the sustained K+ current component (Isus). Elevated K+ current densities of the slow component  were observed in PDE2 OE at membrane potentials above +40 mV compared to WT (+ current (IK1) was significantly reduced in PDE2 OE at membrane potentials below −100 mV, compared to WT  has previously been shown to provide marked protection against catecholamine induced ventricular arrhythmia under basal conditions, as well as after myocardial infarction (MI), leading to improved survival after MI . In thisto, ICaL, INaL, and IK1 amplitudes at physiological membrane potentials. In murine cardiomyocytes, under basal conditions PDE2 accounts only for ~3% of the total cAMP hydrolytic activity [Triggered cardiac arrhythmias are associated with frequent occurrence of early and delayed afterdepolarizations , as well as spontaneous action potentials (sAP), triggering inhomogeneous excitation, reentry and ventricular tachycardia . Indeed,activity ,28. ThusIn contrast to other PDEs, PDE2 activity is increased upon chronic β-adrenergic stimulation ,35. Unde+ and L-type Ca2+ channels play a pivotal role in the initiation and propagation of the action potential in cardiomyocytes. In heart failure, impaired inactivation of Na+ channels induces a sustained Na+ current (late INa) [+ concentration caused by increased INaL could lead to increased sarcoplasmic reticulum (SR) Ca2+ content via altered Na+/Ca2+ exchanger activity, thus promoting spontaneous SR Ca2+ release, which can trigger arrhythmia. Furthermore, increased INaL and ICaL promote AP prolongation in heart failure (HF), increasing the susceptibility to arrhythmia generation [Ca,L, INaL, and spontaneous SR Ca2+ sparks. The reduction in these cellular pro-arrhythmic triggers contributes to reduced DADs and sAPs at cellular level (this study) and the development of arrhythmia in vivo [ex vivo heart perfusion with BAY 60-7550 provoked increased incidence of arrhythmia. Further studies are needed to study the effects of PDE2 on the function of the Na+/Ca2+ exchanger and Na+/K+ ATPase (NKA), because they are important for maintaining intracellular Na+ and, consequently, regulation of the Na+/Ca2+ exchanger and Ca2+ homeostasis [Voltage-gated Naate INa) . Higher neration . The exineration . In the  in vivo . Accordieostasis .CaL under basal conditions, whereas no effect on ICaL was observed in murine ventricular myocytes [CaL current density in ventricular cardiomyocytes. In rat atria, PDE2 inhibition with BAY 60-7550 increased basal beating but did not affect β-AR-induced sinoatrial tachycardia [in vivo administration of BAY 60-7550 (i.p.) significantly increased heart rate in dogs and rats [It has been demonstrated that PDE2 affects L-type calcium currents in mice, but that its modulation is different in atrial versus ventricular cardiomyocytes. In human and murine atrial myocytes, PDE2 inhibition with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) resulted in stimulation of Imyocytes ,43. Accohycardia . Furtherand rats . Correspand rats . FurtherNaL and SR Ca2+ leak in PDE2 OE. The cAMP-independent CaMKII stimulation via direct activation of Epac enhanced the INaL and Ca2+ spark frequency in PDE2 overexpressing cells confirming the physiological action of Epac/CaMKII in PDE2 OE. In HF, pathological CaMKII signaling promotes cardiac arrhythmia by influencing ion channel activities including INa, ICaL and various potassium currents [NaL, as well as SR Ca2+ leak from RyR2 via phosphorylation in HF, thereby provoking arrhythmia [2+ leak via phosphorylation of RyR2-Ser2814 [NaL [NaL observed in PDE2 OE was specifically mediated via the Epac/CaMKII pathway, but not by PKA. In previous work, we could show that the steady-state phosphorylation levels of RyR2 at its CaMKII phosphorylation site Ser2814 were significantly lower in PDE2 OE, whereas the status at its PKA-phosphorylation site Ser2808 was not altered [2+-handling proteins RyR2, PLB, and SERCA2a were similar between PDE2 OE and WT. Although basal Ca2+ transient amplitude and SR Ca2+ load did not differ between PDE2 OE and WT, the ISO-related increase in Ca2+ transient amplitude was lower in cardiomyocytes from PDE2 OE [The observed antiarrhythmic PDE2 effects were mainly mediated by reduced activity of the cAMP-dependent Epac/CaMKII axis. While basal expression of PKA was similar in PDE2 OE and WT mice, CaMKII expression was significantly increased in PDE2 OE, potentially due to adaptive upregulation with respect to increased cAMP hydrolysis upon β-adrenergic stimulation. In addition, CaMKII phosphorylation was clearly reduced at basal conditions, suggesting a PDE2 association with CaMKII in defined subcellular pools. Thus, pharmacological inhibition of CaMKII (and PKA) did not affect the blunted ISO-induced increase in Icurrents . Especiarhythmia ,46. It h-Ser2814 . An incr814 [NaL . Hence,  altered . In addi PDE2 OE .Other studies have revealed PDE2 regulation of a local cAMP pool that acts anti-hypertrophically by activating PKAII, leading to phosphorylation of nuclear factors of activated T cells (NFAT) and reducing nuclear localization, which reduces the expression of hypertrophic genes . Furtherin silico models incorporating detailed cAMP and CaMKII signaling that can also simulate spontaneous SR calcium-release events and afterdepolarizations, precluding direct validation of the antiarrhythmic effects of PDE2 overexpression in our computational model. This limitation should be addressed in future studies developing novel computational models integrating spatial calcium handling and β-adrenoceptor and CaMKII signaling. Future work should also assess the potential antiarrhythmic effects of PDE2 overexpression in the presence of disease-related remodeling of cAMP and CaMKII signaling . Further studies are needed to reveal the influence of other PDE2 isoforms on the observed antiarrhythmic effects. Mitochondrial dysfunction has been shown to contribute to arrhythmia development [The limitation of potential PDE2 spillover should be carefully considered. Artificial PDE2A3 expression within subcellular compartments cannot be excluded. Our in silico analysis suggests that PDE2 overexpression may produce similar changes in cardiomyocyte calcium handling in large mammals as we observed experimentally in mice. However, at present there are no elopment . PDE2 iselopment . Furtherelopment .NaL and SR Ca2+ leak, as well as reduced increase in ICaL were identified as underlying mechanisms. Consequently, the increase in PDE2 found in heart disease may constitute an important cardiac defense mechanism from β-adrenergic stress. Thus, the activation of myocardial PDE2 may represent a novel intracellular anti-arrhythmic therapeutic strategy in HF.In conclusion, higher PDE2 abundance protects against isoprenaline-induced cardiac arrhythmia by preventing ISO-induced afterdepolarizations and spontaneous action potentials. Mechanistically, blunting of ISO-induced, Epac and CaMKII-mediated increases in IFor a detailed description of methods see The experiments comply with the ARRIVE guidelines and conform to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and was approved by the Dresden University Committee on the Use and Care of Animals. The PDE2 transgenic mouse line (PDE2 OE) with C57Bl/6 background exhibiting a cardiac-specific ~10 fold PDE2 overexpression under the control of the murine α-myosin heavy chain promoter was employed in this study ,52. The ex vivo perfused hearts after reperfusion injury [w/v). The hearts were rapidly excised and perfused with a Krebs-Henseleit buffer solution  with physiological catecholamine concentration  at 37 °C by gravity flow in a Langendorff system. After stabilization for 15 min, left ascending coronary artery was ligated for 30 min. ECG were measured within 30 min after reperfusion . Arrhythmic events were quantified using LabChart v7. Infarct size was determined after perfusion with Evans Blue (0.5% w/v in PBS)  and staining with 2,3,5-triphenyltetrazolium chloride (1% w/v in PBS)  from 2 mm heart slices fixed in 4% formaldehyde.A modified protocol was used to detect arrhythmia of n injury . Briefly2+ current , K+ currents and the late Na+ current (INaL), as described previously [Whole-cell voltage-clamp was used to measure L-type Caeviously . To asse2+ spark measurements were performed on a laser scanning confocal microscope . Fluorescence images of Fluo-4 AM  loaded ventricular myocytes were recorded in the line-scan mode after field stimulation (1 Hz).CaLysates of isolated cardiomyocytes were run on 10% SDS-PAGE and blotted onto nitrocellulose membranes. For protein quantification the following specific primary antibodies were used: Epac1 , Epac2 , CaMKIIδ total , CaMKII pThr286 PKAIIα , PKA-C , PKA IIα reg (C20) , PKCα (H-7) , NCX1 , NKA , PLM , EEF2 , GAPDH .The Heijman et al.  model oft-tests with equal variances and normal distribution, Welch’s t-test with unequal variances and normal distribution or One-way ANOVA followed by Sidak multiple comparison test or a non-parametric Kruskal Wallis test followed by a Dunn’s multiple comparison test were used to determine statistical differences according to the experimental setting. p values of less than 0.05 were considered statistically significant.Results are presented as mean ± SEM. Statistical analysis was performed with GraphPad Prism software . Either unpaired Student’s


Development of more sensitive imaging techniques has caused an increase in the number of diagnosed small renal tumors. Approximately 2–3% of these lesions are proved to be angiomyolipomas (AML), a rare benign tumor of the kidney sometimes causing pain and hematuria. The most required approach is observation, but in the case of recurrent symptoms or larger tumors, which may cause bleeding, a more active treatment is required. We present two cases of symptomatic AML tumors of different sizes in the kidney: one treated with transarterial embolization (TAE), and the other with percutaneous cryoablation (CRA). The lesions were diagnosed on the basis of contrast-enhanced computed tomography (CT) scan and magnetic resonance imaging (MRI). Both treatments proved to be effective and safe for treating renal AMLs. A follow-up carried out, based on contrast-enhanced CT scan, confirmed complete treatment of AML and decreased lesion size. There are myriad minimally invasive approaches for the treatment of renal AMLs, and the preservation of renal function remains a priority. The most popular treatment option is the selective renal artery embolization. Owing to its limited invasiveness, CRA could be an attractive option for the preventive treatment of AML. Renal angiomyolipoma (AML) is a benign mesenchymal tumor in its classic variant and comprises fat, smooth muscle, and blood vessels. AML accounts for 0.3–3% of all renal masses , 2. AML Historically, 64% of AML were presented symptomatically, most commonly with progressive flank pain, palpable mass, or rupture . ContempThe diagnosis of AML approaches 100% sensitivity with computed tomography (CT) scan or ultrasound. The presence of fat within the lesion indicated on CT scan by a negative attenuation of –10 Hounsfield units or lower, and the sonographic appearance of a hyperechoic signal and acoustic shadowing, is pathognomonic of AML neoplasm . AbsenceThe systematic review undertaken by the European Association of Urology (EAU) Renal Cell Carcinoma Guidelines Panel found that AMLs are mostly managed by active surveillance as most of them do not grow (in 89% of cases) . Even ifTransarterial embolization (TAE) is a first line of treatment for active hemorrhage and symptomatic lesions. TAE is associated with a mean 38.3% (–3.4 cm) reduction in AML size . The aimAblation (percutaneous or laparoscopic) has been performed using a variety of techniques, such as radiofrequency ablation (RFA), microwave ablation, and cryoablation (CRA), for tumors less than 6 cm in size .Castle et al.  report aMicrowave ablation is a relatively novel type of thermal ablative procedure that has emerged in recent years; however, currently insufficient data exist on this modality for recommending its routine use for renal AML .For the treatment of renal AMLs, cryoablation has been reported in two studies only with limited number of patients and short-term follow-up , 20. TheThis report presents two cases of symptomatic renal AML treated with two different techniques.®; GEM Srl, Viareggio, Italy) with Lipiodol®  to make the glue radiopaque and modulate the delay of polymerization . Other tumor markers  were within normal range. Contrast-enhanced CT scan revealed a 4.5×3.5×7.0-cm oval mass with heterogeneous density in based scan , located in the left kidney . It contrization .No significant change in creatinine levels appeared before and after the procedure. In the follow-up at 6 months of procedure, no bleeding area was observed and the AML was reduced. The patient remained without complaints 2 years after the procedure. The size of the left renal AML was indicated by MRI to be reduced , with onIn March 2020, A 71-year-old female was referred for treatment of an incidental right renal lesion in the presence of macroscopic hematuria. Her history included a lung adenocarcinoma with bone metastases in chemotherapy. Her laboratory data such as complete blood cell count and creatinine demonstrated no significant CHANGES. For appearance of macroscopic hematuria, a contrast-enhanced CT scan was performed revealing a 3.0×2.7-cm oval mass with heterogeneous density in based scan located in the upper pole of the right kidney . It contFor the management of AML, active surveillance remains the first line of option in properly selected asymptomatic patients with small AMLs. However, some of them may cause symptoms like pain or hematuria and may require treatment. The frequency of symptoms increases with the size of the mass. Even if the mass grows, its size increases very slowly and bleeding is a rare event (2.2%) . The mosFor the treatment of symptomatic AML, cryoablation appears to be a valid alternative to TAE or partial nephrectomy. The choice of treatment must consider the preservation of renal function and the clinical context.


Crystallization of the cytoplasmic surface ATPase molecules of isolated scallop SR vesicles was studied at various calcium concentrations by negative stain electron microscopy. In the absence of ATP, round SR vesicles displaying an assembly of small crystalline patches of ATPase molecules were observed at 18 µM [Ca2+]. These partly transformed into tightly elongated vesicles containing ATPase crystalline arrays at low [Ca2+] (≤1.3 µM). The arrays were classified as ‘’tetramer’’, “two-rail” (like a railroad) and ‘’monomer’’. Their crystallinity was low, and they were unstable. In the presence of ATP (5 mM) at a low [Ca2+] of ~0.002 µM, “two-rail” arrays of high crystallinity appeared more frequently in the tightly elongated vesicles and the distinct tetramer arrays disappeared. During prolonged (~2.5 h) incubation, ATP was consumed and tetramer arrays reappeared. A specific ATPase inhibitor, thapsigargin, prevented both crystal formation and vesicle elongation in the presence of ATP. Together with the second part of this study, these data suggest that the ATPase forms tetramer units and longer tetramer crystalline arrays to elongate SR vesicles, and that the arrays transform into more stable “two-rail” forms in the presence of ATP at low [Ca2+].The Ca After incubation for 37 min, tightly elongated vesicles still had comparable two-rail or tetragonal crystalline arrays  in the presence of ATP  with ATP were also observed in the tightly elongated vesicles after 16 min incubation with the low [Ca2+] (~0.002 µM) and ATP (data not shown). After 2.5 h incubation with ATP, “two-rail” arrays degraded, instead tetragon arrays dominated  Ca2+-AT (see in ). By conominated d,e. The ~0.01 µM . In the ~0.01 µM . Based o2+-ATPase of the rabbit SR  (>180 µM) without ATP, presumably began to change into assemblies of ~100 Å square tetragons when the vesicles were incubated at a low [Ca2+] (≤1.3 µM) without ATP, and developed into tetragonal arrays hout ATP f,g, and hout ATP a–c like [Ca2+] >10 µM withhout ATP d,d’ are crystals , i.e., Avanadate  (an inhi+-ATPase ) was fur+-ATPase ,27.2+-ATPase molecule is proposed to have a secondary structure comprised of ten transmembrane and five ‘stalk’ domains with two large cytoplasmic regions  had been performed in the calcium buffer solution. The total calcium was composed of the added (10–550 µM) and contaminated (16 ± 7 µM (n = 4)) calcium; the contaminated calcium was measured by the method of atomic absorption. The association constants for EGTA-calcium with and without ATP were taken as 1.9539 × 106 and 2083 × 106 M−1, respectively [3 and 1.239 × 104 M−1 [For electron microscopic study of the negatively stained SR, the SR (0.3 mg of protein/mL) was incubated with 100 mM imidazole buffer (pH 7.0) containing 0.12 KCl, 5 mM MgCl earlier ,30,31; iectively . In the  104 M−1 . One dro2+ concentration (<1.3 µM) transforms tightly assembled crystal patches of Ca2+-ATPase molecules into “two-rail” or tetragonal spiral arrays in isolated round scallop SR vesicles prepared at a high Ca2+ concentration, and that elongation of the spiral arrays transforms some of the vesicles into tightly elongated vesicles. (ii) At a low Ca2+ concentration of ~0.002 µM, ATP increases the stability and regularity of the “two-rail” arrays increasing their appearance frequency, and this is accompanied by the disappearance of distinct tetramer-arrays. (iii) Thapsigargin (a specific inhibitor of the Ca2+-ATPase) prevents both the crystallization of ATPase molecules and the elongation of vesicles in the presence of ATP. (iv) In both round and tightly elongated vesicles, Ca2+-ATPase can form crystals: tightly assembled crystal patches and/or crystallized cylinders. Taken together, these results suggest that crystal structures formed by membrane proteins, membrane endoskeletons, might have physiological roles in the formation of the microscale structures of cells.The data obtained in the present paper suggest that (i) in the absence of ATP, low Ca


Due to their presumed lack of contacts with the overlaying tectorial membrane (TM), the putative stimulation mechanism for these stereocilia is by means of the viscous drag of the surrounding endolymph. However, despite numerous efforts to characterize the TM by electron microscopy and other techniques, the exact IHC stereocilia-TM relationship remains elusive. Here we show that Ca2+-rich filamentous structures, that we call Ca2+ ducts, connect the TM to the IHC stereocilia to enable mechanical stimulation by the TM while also ensuring the stereocilia access to TM Ca2+. Our results call for a reassessment of the stimulation mechanism for the IHC stereocilia and the TM role in hearing.Mammalian hearing depends on sound-evoked displacements of the stereocilia of inner hair cells (IHCs), which cause the endogenous mechanoelectrical transducer channels to conduct inward currents of cations including Ca Hearing requires inner hair cell (IHC) stereocilia deflection, believed to result from hydrodynamic coupling due to the lack of contact with the tectorial membrane (TM). Here the authors show that IHC stereocilia are TM-embedded, and calcium rich structures in TM may facilitate sound transduction. These currents activate the OHC-based cochlear amplifier, essential for hearing faints sounds7 and cause the IHCs to produce electrical discharges in the auditory nerve9.Sound transduction into nerve signals happens when sound-evoked vibrations of the stereocilia of the inner hair cells (IHCs) and outer hair cells (OHCs) cause an indiscriminate entry of K2+], which a recent discovery of high [Ca2+] in the tectorial membrane (TM)10 helped estimate at 40–150 µM11, contributes to the regulation of hearing sensitivity.Patch-clamp recordings from isolated hair cells have established that extracellular [Ca12, their motions are proportional to the basilar membrane (BM) displacements14.Because the stereocilia of OHCs are attached to the TMHowever, the IHC stereocilia stimulation mechanism is poorly understood.16.Comparison of cochlear microphonic (CM) recordings in OHC-less and control guinea pigs in the early 70s suggested that the IHC stereocilia must be freestanding from the TM because their responses depended on the BM velocity17. However, the obvious constraints imposed by the insertion of the recording electrodes18 might complicate the interpretation of these data.This generally agrees with the intracellular recordings from the IHCs that showed phase difference relative to CM recordings19. Today, the preservation of the TM-stereocilia relationship in fixed tissues remains a challenge20.While electron micrographs of the organ of Corti (OC) from the guinea pig freeze-dried samples have showed a good tissue preservation, the TM was detached from both the OHC and IHC stereocilia2+ ducts connect the TM to the stereocilia of IHCs and OHCs.Here we use reflected light and fluorescence confocal microscopy to investigate the TM-stereocilia relationship in the guinea pig. We show that IHC and OHC stereocilia are similarly TM-embedded and that Ca21 and show that the TM-embedment enables the stereocilia of OHCs and IHCs to directly access the TM mechanical stimulation and Ca2+.Our results challenge the classic anatomical representation of the OC22, the temporal bone preparation from the guinea pig makes it possible to investigate sound transduction in functional and largely intact OC26. Its CM recordings exhibit amplitudes and nonlinearity  was brighter than the TM, making it easier to distinguish an interface line between the two structures, with no obvious gap between them Fig. . This is32 , which causes the RL to move towards the BMImmediately after the acoustic overstimulation stopped, the TM relaxed faster than the RL, causing a TM-RL gap to briefly appear  application, the TM rested tightly on the RL. However, upon 100 µM EGTA application through a thin glass electrode, the TM swelled rapidly, causing its normally “biconvex” shape to look more circular . In row 1 OHCs, the Ca2+ ratio was 0.91 ± 0.03 . In row 2 OHCs, the Ca2+ ratio was 0.88 ± 0.03 . In row 3 OHCs, the Ca2+ ratio was 0.94 ± 0.01 . The Ca2+ ratio differences between the stereocilia types were not statistically significant .Since the Caios Fig. . In IHCs2+ indicator produced a signal in the cell bodies  a 1 micron-wide spot within a Ca2+ duct-rich region and in same-location IHC stereocilia near the center of the bundle , it recovered rapidly in both regions of interest . The difference was not statistically significant . The recovery halftime in Ca2+ duct region was 2.89 ± 0.11 s . For the stereocilia bundles, the recovery halftime was significantly shorter, i.e., 2.58 ± 0.10 s .After photobleaching the Caest Fig. . On aver2+ in the stereocilia and Ca2+ ducts was freely mobile while the reminder could have a role in maintaining the integrity of the TM and sensory structures as discussed above.The high mobile fraction of as much as 75–76% suggests that much of the Ca2+equilibrium exists between the Ca2+ ducts and the stereocilia thanks to their attachments, the faster flow of stereocilia Ca2+ into the cell would drive the ducts’ Ca2+ into the stereocilia due to the Le Chatelier principle.Assuming that some form of Ca2+ducts attach the TM to the stereocilia of IHCs and OHCs and support that the Ca2+ducts ensure these stereocilia access to TM Ca2+.Together, the data described above indicate that the CaThe TM-embedment of the IHC stereocilia raises the question of how these stereocilia are stimulated.41), with the cationic dye di-3-aneppdhq10 and then investigated the sound-evoked motions of same-location TM and stereocilia by high-speed confocal imaging42.To address this issue, we stained the TM  randomly distributed across the TM Fig. . Remarka42, which allows for acquisition of 12 images at 12 equidistant phases of the acoustic stimulus. Row 1 OHC stereocilia bundles  yield elliptical trajectories, for the TM  while in row 3 OHC region .In the IHC region, the TM moved nearly transversally towards the BM, with a major peak-to-peak amplitude of 116 ± 7 nm  and the difference relative to the same-location TM counterpart was significant . In row 2 OHCs , which was significantly larger relative to same-location TM counterpart . In row 3 OHC stereocilia , which in this case was also significantly larger compared to same-location TM motion amplitude .In agreement with past studies OC Fig. . In the Plotting the average trajectories on their approximative location on the composite image of the OC from Fig. As shown by Fig. n = 21 same-location TM and IHCs from nine preparations; p = 0.0005, Wilcoxon signed-rank test). In the row 2 OHC region, the stereocilia motion amplitude was 45 ± 7 nm compared to 28 ± 4 nm for same-region TM . In the row 3 OHC region, the stereocilia motion amplitude was 54 ± 13 nm compared to 38 ± 9 nm for same-region TM . The larger IHC radial response is consistent with the fact that the cochlear amplifier boosts the IHC motion radially43.Radially, the stereocilia motion amplitude remained larger relative to same-location TM Fig. . In the Remarkably, the stereocilia radial motion BF compared poorly to that of the TM Fig. . In IHCs44, which would cause the IHCs and OHCs as well as the associated TM regions to move antiphasically46.A key argument frequently advanced by supporters of the IHC stimulation by viscous drag is that the IHC vs. OHC motions exhibit a phase difference as large as 180°To address this issue, we examined the motion phases for the stereocilia bundle tip and same-location TM for frequencies between 150 and 500 Hz Fig. . The phan = 21 same-location TM and IHCs from nine preparations; Fig. n = 21 same-location TM and IHCs from nine preparations; Fig. p = 0.1, LMM, n = 21 same-location TM and IHCs from nine preparations). The phase-frequency function had a slope of −0.024 rad/Hz and the frequency dependency of the phase was statistically significant .For the IHC stereocilia bundle tip, the phase decreased from −1.61 ± 0.09 to −10.89 ± 0.08 rad . The associated slope was −0.024 rad/Hz and the frequency dependency of the phase was statistically significant .In the row 2 OHC region, the stereocilia bundle and TM phases decreased from −1.71 ± 0.08 to −10.91 ± 0.11 rad  and the associated slope was −0.024 rad/Hz. The frequency dependency of the phase was statistically significant .In the row 3 OHC region, the phase for the stereocilia bundle and TM decreased from −1.76 ± 0.09 to −11.06 ± 0.15 rad . In addition, the phase dependency in frequency and structure type were statistically significant with p = 1 × 10−28 and 0.001, respectively (LMM).In the IHC region, the stereocilia phase decreased from 1.67 ± 0.08 rad to −6.67 ± 0.47 rad with a slope of −0.0231 rad/Hz . In addition, the effect of the frequency on the phase was statistically significant .In the row 2 OHC region, the phase associated to the stereocilia decreased with a slope of −0.0228 rad/Hz from 1.36 ± 0.27 to −6.78 ± 0.37 rad . In addition, the effect of the frequency and structure type on the phase were statistically significant .In the row 3 OHC region, the phase associated to the stereocilia bundle decreased with a slope of −0.0212 rad/Hz from −0.30 ± 0.42 to −7.68 ± 0.63 , zFluor is much more photostable i.e., brighter than Cy5 for identical spectral characteristics.To our surprise, zFluor produced not only a strong labeling in the TM but also in the hair cell membrane as well as their stereocilia and cuticular plates Fig. .Fig. 6IH55. That zFluor produced a strong labeling both in the hair cell membranes, their cuticular plates and stereocilia suggests that zFluor could be a reliable potentiometric dye.Historically, only potentiometric dyes have been known to stain hair cells2+ ducts and which contacted the IHC and OHC stereocilia in a similar fashion as Ca2+ ducts 58. Ca2+ ducts could resolve this issue by directly funnelling high TM Ca2+ concentration to the stereocilia.The presence of Ca2+ removal weakened and disrupted the attachments of the TM to the RL and stereocilia. It is already known that proteins such as stereocilin attach the TM to the OHC stereocilia12. However, the expression of otoancorin near the IHC cuticular plate59 suggests that this could be one of the proteins that attach the IHC stereocilia to the TM. Our study suggests that the function of these proteins could be Ca2+-dependent and will most likely spur interest into the Ca2+ dependency of their function.Interestingly, Ca2+ ducts will certainly be addressed by future studies, we also expect that their discovery will inspire research into the physiological role of the TM, whose function has been until recently thought to be mostly mechanical.While the exact molecular nature of the CaThis study complied with all relevant ethical regulations for animal testing and research. All the animal procedures described in this study were approved by the Regional Ethics Committee in Linköping, Sweden (Permit number DNR 5111-2019).Young adult guinea pigs of either sex  were anesthetized with a sodium pentobarbital intraperitoneal injection  and then decapitated.2 and 5% CO2) cell culture medium . Because of the special design of the custom experimental chamber, the cochlea and the middle ear were immersed in the medium while the outer ear was shielded to allow for natural sound stimulation with a computer-controlled speaker inserted in the ear canal. With a fine scalpel, two small openings were made at the apex and base of the cochlea10. A plastic tube was then fitted into the basal opening to allow perfusion of scala tympani with the medium above gently flowing (∼0.6 ml/h) from an external (4 ml) tank. The apical opening allowed the perfusion medium to flow out and was used for confocal imaging and electrophysiological recording of sound-evoked responses of OC. Because of the immersion of the middle ear, the effective sound stimulus intensity decreased by at least 20 dB SPL23. The values given in the present paper were therefore corrected accordingly.Immediately, the temporal bone was carefully and rapidly excised and mounted in a custom-built chamber, after which the bulla was gently opened with a fine scalpel and scissors in order to gain access to the cochlea. Right after this step, the entire preparation was immersed in an oxygenated  as well as a fluorescent dye or EGTA (100 µM) where indicated and bevelled at an angle of 20° to 3–7 MΩ. The electrode was then made to carefully penetrate the Reissner’s membrane with a stepping manipulator. Dye and pharmacological agents dissolved in the electrolyte as above were administrated either electrophoretically or by a gentle pressure injection, where indicated, with a Picospritzer. The CM data acquisition was achieved using custom LabView software.For the recording of CM responses (Ix1 amplifier (Dagan Instruments) of the OC, a thin capillary glass (World Precision Instruments) was pulled with a standard puller and filled with an endolymph-mimicking solution containing 1.3 mM NaCl, 31 mM KHCO2+ fluorescence confocal imaging with the ratiometric indicator Cal-520L®/Cy5-Dextran Conjugate also known as RatioWorksTM  as well the dextran-conjugated zFluor, where indicated . For sound-evoked motion measurements by high-speed confocal imaging42, the 0.8-NA water immersed lens was used after staining the stereocilia along with the TM with the membrane dye di-3-aneppdhq10 (Thermo Fischer).The OC was imaged in the preparation above with a laser scanning confocal microscope (LSM 780 (ZEN 2012 black edition software), Zeiss, Germany). The microscope and the preparation are housed in a light protected Faraday enclosure stabilized by a vibration-damping table. A 40x, 0.8 NA water immersion lens (Nikon NIR APO) was used for reflected light confocal imaging of the OC and CaWhen imaged under the cross-section view, the hair cells appear as a single IHC and three OHCs. In terms of coordinate, this z-position is not far from the injection site because this is generally where the electrode can smoothly penetrate the Reissner’s membrane i.e., when it is perpendicular to the Reissner’s membrane and parallel and close to the stria vascularis. Because the preparation rests on a rotative mount, this orientation is easy to find when the preparation is tilted such that the apical cochlear opening is optimally oriented relative to the lens.2+ ratiometric imaging, Cal-520L/Cy5 dextran conjugate  was loaded into a thin glass microelectrode. The indicator solution was then released from the electrode by application of a small pressure on the back end of the electrode  upon which the indicator rapidly partitioned in the TM and other water-accessible spaces from the injection site.For Ca2+ratio quantification for the stereocilia bundles, we used the MATLAB-built in “drawpolygon” function to delimit the stereocilia in the reference images with successive clicks of the mouse until the contour of such a region of interest (ROI) is fully outlined. Then from the corresponding ratiometric images, we used the coordinates of these ROIs to extract and quantify Ca2+ ratio values for the stereocilia of the different hair cell types.For Ca2+ duct-rich region nearby, 50 pre-bleaching images were acquired in fast scanning mode with no interframe delay before the ROIs were photobleached with maximum laser power and then immediately 300 post-bleaching images were acquired in fast scanning mode with no interframe delay. The FRAP data were then processed in MATLAB and individual FRAP traces were fitted with a single-phase exponential model60 for statistical analysis of FRAP data in different preparations.For Cal-520L FRAP experiments, the data acquisition was performed with the lens above. After marking 1 micron-wide ROIs at the center of the stereocilia bundle and in the Ca42 for different points of interest are given throughout the text. In order to reliably quantify the sound-evoked motion amplitudes, phases and other parameters of interest for the TM and stereocilia in different preparations, sound-evoked motion trajectories from each individual measurement were fitted with the ellipse equation61 in MATLAB (The MathWorks) and were also subjected to sine wave fitting with software packages already used in other publications60.The procedure for obtaining the sound-evoked trajectoriesAs the confocal microscope setup used in this study was optimized to work with the left ears, only one temporal bone preparation could be used per animal. Consequently, the number of animals and the number of preparations are the same and are understood to be interchangeable throughout the manuscript. The number of individual measurements as well as the number of preparations  is given throughout the text. For the frequency-tuning of sound-evoked motion amplitudes and associated phases, the motion measurements were performed for the 150–500 Hz. To optimize the frequency resolution especially near the BF where the motion is largest, the frequency steps were reduced to 20 Hz closer to the BF and maintained at 50 Hz outside this region. For figure plotting, the data were then subjected to the interp1 MATLAB function for interpolation every 10 Hz. Data processing, analysis, and quantification were performed using custom scripts in MATLAB (The MathWorks), unless indicated otherwise.Different types of statistic tests were used throughout this study depending on the data sets considered.62. The random effect in the model was the experiment ID whereas the fixed effects were the frequency and structure type. The dependent variable was the phase. The same model was used for the different hair cell regions. Calculations were performed using the lme4 and nlme packages in RStudio (version 1.2.1335)63.The statistical analysis of the phase across frequency for the motion of the stereocilia and same-location TM structure types was performed on the data before the interpolation step above. Accordingly, it was necessary to take into account the fact that such measurements were performed repeatedly for these structures for several frequencies. Inevitably, such repetitions introduce correlations that have to be dealt with by LMM25, where indicated. For statistical analysis of Ca2+ ratios in the four different stereocilia types, Kruskal–Wallis test was used. These calculations were performed in MATLAB (The MathWorks).For statistical analysis of motion amplitudes of the stereocilia and same-location TM at a single frequency  and FRAP parameters for the IHC stereocilia and same-location TM, the kind of correlation issue described above doesn’t arise here. Consequently, for such situations, Wilcoxon signed-rank test was deemed appropriate for paired samplesFurther information on research design is available in the Reporting SummaryDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3


RYR2, encoding the cardiac ryanodine receptor (RyR2). We report a novel RYR2 variant, p.Asp3291Val, outside the four CPVT mutation hotspots, in three CPVT families with numerous sudden deaths. This missense variant was first identified in a four-generation family, where eight sudden cardiac deaths occurred before the age of 30 in the context of adrenergic stress. All affected subjects harbored at least one copy of the RYR2 variant. Three affected sisters were homozygous for the variant. The same variant was found in two additional CPVT families. It is located in the helical domain 2 and changes a negatively charged amino acid widely conserved through evolution. Functional analysis of D3291V channels revealed a normal response to cytosolic Ca2+, a markedly reduced luminal Ca2+ sensitivity and, more importantly, an absence of normal response to 8-bromo-cAMP and forskolin stimulation in both transfected HEK293 and HL-1 cells. Our data support that the D3291V-RyR2 is a loss-of-function RyR2 variant responsible for an atypical form of CPVT inducing a mild dysfunction in basal conditions but leading potentially to fatal events through its unresponsiveness to adrenergic stimulation.Catecholaminergic polymorphic ventricular tachycardia (CPVT) is triggered by exercise or acute emotion in patients with normal resting electrocardiogram. The major disease-causing gene is  Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare inherited life-threatening disorder characterized by adrenergic induced bi-directional or polymorphic ventricular tachycardia (PVT) in absence of any detectable structural heart abnormalities . It mostRYR2, the gene encoding the cardiac ryanodine receptor type 2 (RyR2), are the major cause of the CPVT dominant form with detection of a RYR2 mutation in 60 to 65% of patients, including a high rate of de novo mutations [CALM1, CALM2, CALM3) leading to severe de novo or familial CPVT have been discovered [CASQ2), triadin (TRDN) and trans-2,3-enoyl-CoA reductase-like (TECRL) [Missense mutations in utations ,11,12,13scovered ,16,17,18 (TECRL) ,20,21. 2+ release from the sarcoplasmic reticulum (SR) to the cytosol, an essential process in the cardiac excitation-contraction coupling (ECC). Another mechanism occuring in cardiomyocytes termed ‘store-overload induced Ca2+ release’ (SOICR) has been described as the spontaneous release of Ca2+ through RyR2 that occurs when the SR Ca2+ content reaches a critical level [RyR2 is a large homotetrameric channel located in the sarcoplasmic reticulum (SR) membrane. It handles the Caal level ,23. The al level . 2+ release resulting in Ca2+ waves that trigger membrane depolarizations, premature ventricular beats (PVB), and polymorphic ventricular tachycardia (PVT) during physical activity or emotional stress [2+ release deficiency syndrome’ (CRDS) [The extent of RyR2 variants responsible for cardiac arrhythmias and cardiomyopathies as well as the various underlying pathogenic mechanisms remains unknown ,25,26,27l stress ,25. Thesl stress ,29. In a’ (CRDS) ,32,33,34’ (CRDS) ). RYR2 variant, located outside the mutation hotspots, found in three families with atypical CPVT. The largest family was a four-generation one with homozygous and heterozygous carriers of the variant, where eight family members died suddenly before the age of 30, in the context of acute emotion or exercise. Here, we assessed the Ca2+ release activity of the RyR2 variant, as well as its response to adrenergic stimulation at the cellular and single channel level. In this study, we report a novel CPVT was suspected in three families due to a history of sudden death in young family members during physical exercise or emotional stress. Clinical evaluation of the patients included 12-lead ECG, 24 h Holter ECG, EST, isoprenaline infusion and echocardiography. Relatives were considered affected when an EST or isoprenaline test showed bi-directional premature ventricular beats (PVBs), bigeminy, bi-directional or PVT . Blood samples were collected for genetic analyses after obtaining signed consent for a study approved by the local ethics committee of the Saint-Louis Hospital . The study was conducted according to the principles of the Helsinki Declaration.RYR2 (NM_001035), CASQ2 (NM_001232), and TRDN (NM_006073) were screened by direct Sanger sequencing and linkage analysis was performed in family 1 with intragenic or closely linked microsatellites to the RYR2 locus . Several genes  implicated in arrhythmias were screened for the proband of family 2 by Multiplex Amplification of coding regions and then analyzed by high-throughput sequencing. The RYR2 variant was genotyped by Sanger sequencing in all family members.Genomic DNA was isolated from whole blood using a standard protocol. All coding exons of ®-Blunt, and the aspartic acid was replaced by a valine by site-directed mutagenesis. The mutated KpnI/FseI fragment of the pCR®-Blunt plasmid was then amplified by overlapping PCR to be re-inserted into the full-length RYR2 plasmid using a recombination kit . A GFP-free hRyR2 plasmid was prepared for single channel experiments using a recombination kit . The GFP sequence was removed from the full-length RyR2 plasmid by AfeI/FspAI digestion. This plasmid was then sealed with a GFP-free RyR2 PCR fragment with overlapping ends to the linearized AfeI/FspAI digested RyR2 plasmid.The plasmid containing the human RyR2 sequence (X98330) tagged with enhanced green fluorescent protein (eGFP) was kindly provided by Dr. Spyros Zissimopoulos (UK) ,36 and cHuman embryonic kidney 293 (HEK293) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)  supplemented with 10% FBS and 1% penicillin/streptomycin. Lipofectamine 3000  was used to transfect 60–70% confluent HEK293 cells with either eGFP-WT-hRyR2 or eGFP-D3291V-hRyR2 plasmids. HL-1 cardiac cells  were grown in culture flasks coated with 0.02% gelatin and 10 μg/mL fibronectin, and were maintained in Claycomb media  supplemented with 10% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, and 0.1 mM norepinephrine. For transfection, HL-1 cells were cultured into 35 mm µ-dish with glass bottom , coated with gelatin and fibronectin. Cells were grown up to ~30% confluency and then transfected using X-tremGENE HP DNA Transfection Reagent  according to manufacturer’s instructions. Briefly, 1 μg of eGFP-WT-hRyR2 or eGFP-D3291V-hRyR2 plasmids was mixed with 100 μL of FBS and antibiotic free OPTI-MEM medium, followed by addition of 2 μL X-tremGENE and incubated at room temperature for 20 min. Culture medium in 35 mm µ-dishes was replaced with fresh Claycomb media, and the transfection solution was then applied directly to the cells and incubated at 37 °C for 48 h. Using this method, the transfection efficiency of HL-1 cells was ~20–30%. 2 dishes and then transfected with 2 µg of eGFP-WT-hRyR2 or eGFP-D3291V-hRyR2 plasmids. Forty-eight hours after transfection, cells were treated with either 0 μM or 250 μM of 8-bromo-cAMP (cAMP)  for 20 min and were then lysed with a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and 5 mM EDTA. Cell lysates were incubated for one hour at 4 °C under gentle stirring and then centrifuged at 16,000 rpm for 10 min at 4 °C. Protein concentrations were determined using a PierceTM BCA Protein Assay kit . Total proteins (20 µg) were separated by 3–8% Tris Acetate polyacrylamide gel electrophoresis and then transferred into a nitrocellulose membrane for 90 min at 35 V. Membranes were saturated with 5% milk-0.1% Tween-Phosphate Buffered Saline (PBS) for 45 min and then incubated overnight with antibodies against RyR2 (1/1000)  and α-tubulin (1/2000)  Membranes were washed four times in 0.1% Tween-PBS and then incubated with IR-fluorescent secondary antibodies  . Membranes were washed again twice in 0.1% Tween-PBS and twice in PBS before IR-fluorescence imaging. RyR2 and α-tubulin expression was quantified with the ImageJ software .HEK293 cells were seeded in 25 cmTM PLUS Western Blot Stripping Buffer  and then washed with PBS. Membranes were saturated again with 5% milk-0.1% Tween-Phosphate Buffered Saline (PBS) for 45 min and then incubated overnight with antibodies against pSer2808-RyR2 (1/1000)  and α-tubulin (1/2000). Membranes were washed four times in 0.1% Tween-PBS and then incubated with HRP secondary antibodies (1/5000) . Membranes were washed again four times in 0.1% Tween-PBS and then revealed using a chemoluminescent kit, SuperSignalTM West Femto Maximum Sensitivity Substrate .For S2808 phosphorylation assessment, membranes were then stripped for 10 min using Restore2+ dye Fura2-AM (5 µM) for 45 min at 37 °C, 5% CO2 in Krebs–Ringer–Hepes (KRH) buffer composed of (in mM): 125 NaCl, 25 HEPES, 5 KCl, 1.2 MgCl2, 6 glucose, pH 7.4. Cells were then perfused with KRH buffer containing different Ca2+ concentrations  at room temperature. At the end of each measurement, 10 mM caffeine was added in order to select only the responsive cells. Single-cell intracellular Ca2+ measurements were performed in Fura-2-loaded cells using a Nikon Eclipse Ti-U inverted fluorescent microscope  equipped with a dual excitation filter wheel (340 nm/380 nm), a 515 nm emission filter and a Hamamatsu ORCA-D2 CCD camera . Ca2+ traces were obtained by sequential acquisition of one image every 2 s for 3 min using a 20X S-Fluor 0.75 NA (WD = 1mm) objective. Image acquisition and analysis were performed with the HCimage software . Fluorescence intensities in individual cells were determined using region of interest (ROI) analysis with ImageJ software . Ca2+ signals were measured in the caffeine-responsive cells using F340/380 ratios after subtraction of Fura-2 background fluorescence. For experiments with cAMP treatment, cells were pretreated with 250 µM of 8-bromo-cyclic AMP  for 20 min. at room temperature before Ca2+-KRH perfusion. HEK293 and HL-1 cells were seeded in 35 mm µ-dish with glass bottom . After 48 h, transfected HEK293 and HL-1 cells (280 ng plasmid/dish) were loaded with the fluorescent Ca2+-dependency of single channel open probability (Po), the cytosolic compartment (cis chamber) was virtually grounded and filled with a 250 mM HEPES and 120 mM Tris solution at pH 7.4, and then, microsomes were added to the cis chamber. The luminal compartment (trans chamber) was filled with 250 mM HEPES and 50 mM Ca2+ at pH 7.4, and the voltage was applied in this compartment. To examine the luminal Ca2+-dependency, the solution in cis and trans contained 250 mM CsCH3SO3, 0.755 mM CaCl2, 1 mM BAPTA, 20 mM HEPES, pH 7.4. Immediately upon observing single-channel activity, we replaced the solutions to establish the specific free Ca2+ concentrations. Ca2+-buffer solution  compositions were calculated using the MaxChelator software. Membrane voltage was controlled using an Axopatch 200B . The current signal was digitized at 20 kHz through a Digidata 1322A interface  and subsequently filtered at 2 kHz, unless specified otherwise. Data acquisition and analysis were carried out using pClamp software . Single-channel studies were carried out with crude microsomes obtained from HEK293 cells transfected with WT-hRyR2 or D3291V-hRyR2 plasmids. Lipid planar bilayers were made of a mixture of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine in a 5:4:1 ratio (50 mg/mL in decane) . Microsomes were incorporated into the lipid bilayers formed across a 100 μm hole in a thin teflon partition separating two aqueous compartments. To examine the cytosolic Cat-test, ANOVA) were performed with OriginPro Software . A p-value of 0.05 was considered significant.Cellular data and single channel results are expressed as mean ± SEM unless otherwise stated. Results for fitting histograms and the Hill equation are given as the mean ± SD. Statistical tests . Hence, all RYR2 exons were screened, and a new variant was identified on exon 68, c.9872A>T, leading to a change of an aspartate to a valine at position 3291. This variant, p.Asp3291Val (D3291V), was absent in all databases (Genome Aggregation Database: gnomAD 2.1.1).When the proband was initially diagnosed with CPVT following her cardiac arrest, exons of RyR2 encompassing hot spot mutations and all coding exons of In accordance with the microsatellite haplotypes, the variant was found at the homozygous state in three sisters of the second generation  and at the heterozygous state in all of the other positive-phenotype members. Surprisingly, one of the three homozygous sisters (II.5) never had syncope, but her EST test was stopped because of numerous bidirectional PVBs.Her sister II.1 had exercise-related syncope on several occasions and an isoprenaline test showed bidirectional PVBs and a short run of VT. The last homozygous sister II.2 also had exercise-related syncope at 18 and her EST revealed PVT. Two siblings died suddenly during physical activity at age 24 (II.3) and 17 (II.6) and one sibling died during acute emotional stress at 16 (II.8). A brother (II.4) who was a heterozygous carrier and had coronary disease died while sleeping at the age of 54. The eight children of the three homozygous sisters were all obligate heterozygous carriers, and one of them died while swimming at the age of 22 (III.11). The grandmother (I.2) also had three children from a second marriage. One of them (II.11) died at age 54, and one affected daughter (II.10) lost her two sons  when they were 20 and 15 years old while swimming and biking, respectively.Most of the negative-phenotype members of the second and third generations did not carry the variant, except for six members, III.6, III.13, III.15, III.18, III.20, and III.21, who were heterozygous carriers with a negative EST at the age of 32, 21, 25, 49, 46, and 39, respectively. Two girls  had a negative exercise stress-test at first examinations at the age of 11 and 16 but developed bigeminy during EST later at the age of 21 and 27. In the fourth generation, only 2 of the 14 young carriers had a positive phenotype: IV.3 who had bigeminy during an EST even under the administration of nadolol (80 mg) and IV.13 who presented with an atrioventricular block (AVB) and bradycardia at birth and salvos of non-sustained ventricular tachycardia during a Holter recording at the age of 2 c.RYR2 at the heterozygous state. The maternal grandmother (I.2) was positive for this variant and remained asymptomatic for her whole life. Nevertheless, a 24 h Holter recording performed at the age of 80 revealed many PVBs and bigeminy. Her echocardiogram was normal. The mother (II.1) who died at age 45 was thus an obligate carrier of the variant.The proband (III.2) had syncope at age 20 and 25. While on a β-blocker, her EST performed revealed many PVBs and ventricular bigeminy originating from the right ventricular outflow track (RVOT), which disappeared during recovery, but no typical bidirectional PVT was observed. MRI and echocardiography were normal, and the implanted heart monitor did not record any abnormality. Her mother (II.1) died suddenly at age 45 in the context of emotion, attending the funeral of a friend, and her brother (III.1) died at 8 years old while swimming. The proband’s DNA was screened using a genetic testing panel for cardiac diseases and the only variant found as potentially pathogenic was D3291V in The Family 1 and Family 2 originate from the same region in Northern France, and they probably share a common ancestor as they harbor the same mutated haplotype (not shown).The same variant was recently identified in a girl from another family from the same geographical area. She died suddenly while swimming at the age of 10 without prior symptoms. No family inquiry has occurred yet.Caenorhabditis elegans  of RyR2, where no pathogenic mutation has been reported a. This r elegans b.In contrast to RyR1, the C-terminus of the RyR2 HD2 could not be assessed by structural crystallography since number of armadillo repeats in this domain were not visible . This do2+ concentrations (0.1 to 1 mM) in order to assess the SOICR activity. The spontaneous Ca2+ release was recorded using fluorescence microscopy  or 5 µM forskolin to mimic this condition. As expected, at 0.5 mM Ca2+ concentration. Of note, cAMP treatment revealed significant differences between WT-RyR2 and D3291V-RyR2 cells both for the fractions of oscillating cells at 0.5 mM Ca2+  and the number of oscillations per minute at 0.5, 1, and 2 Ca2+   and decay time at 50%—were similar for WT and D3291V-RyR2 cells  a. In bas5, 1 mM) b. NevertR2 cells e–g.2+ concentrations . Nevertheless, D3291V-RyR2 HL-1 cells did not show a significative reduction of the CTD50 at all concentrations . As expected, after cAMP treatment, the decay time 50% measurements of WT-RyR2 cells revealed a 52.8% and 59.4% reduction at 0.5 and 1 mM Ca2+, respectively (p = 0.003 and p = 0.000001 at 0.5 and 1 mM Ca2+). D3291V-RyR2 HL-1 cells showed a significant reduction of the decay time compared to untreated D3291V-RyR2 HL-1 cells only after cAMP treatment at 0.5 mM Ca2+ (36%)  f. It is 2+ (36%) h while n2+ as the charge carrier  of the channel. WT and D3291V-RyR2 channels were both maximally active (Po = 0.9) at 20–30 µM and had similar cytosolic Ca2+ EC50′s (WT: 438 nM ± 75 vs. D3291V: 320 nM ± 41). This cytosolic Ca2+ sensitivity data was collected at a high luminal Ca2+ level. However, there were differences between the two channels and their luminal Ca2+ sensitivity. 2+ concentrations on channel activity. Contrary to their counterparts, D3291V-RyR2 channels showed decreased channel activity at 5 µM, 100 µM and 1 mM luminal Ca2+. This activity was characterized by low Po and short open time durations at sub-millimolar levels of luminal Ca2+. Inspection of the WT-RyR2 single-channel recordings showed that the duration of the open events increased to several hundreds of milliseconds as luminal Ca2+ was increased. In agreement with previous reports, WT and D3291V-RyR2 channels showed a reduction in single-channel conductance by millimolar levels of luminal Ca2+ concentration, as reflected by the amplitude of the traces , whose lineage diverged from teleosts before the teleost genome duplication .The D3291V variant involves a highly conserved negatively charged residue among the hRyR homologs and RyR proteins from other animal species, thus reinforcing the hypothesis that this variant is associated with functional change in the hRyR2 gene product. This negative residue is conserved even in spotted gar , a neuromuscular disorder . This pa2+ imaging to assess the D3291V-RyR2 pathogenicity. We recorded the effect of this variant on SOICR by monitoring spontaneous RyR2 activity in HEK293 and HL-1 cells. Unlike most of the CPVT mutations expressed in HEK293 cells, we did not identify any difference between the WT-RyR2 and D3291V-RyR2 transfected cells under basal conditions [We performed Canditions ,51. As tnditions . In ordenditions . As in HThe cAMP is a well-known second messenger that activates the protein kinase A (PKA) in response to adrenergic stimulation and RyR2 channels are known to be a PKA target. Three phosphorylation sites  have been implicated in the regulation of the RyR2 adrenergic response, even if the RyR2 phosphorylation remains a subject of controversy . Our WesRecently, it was shown that the phosphorylation domain of RyR2 embraces the catalytic subunit of PKA and results in the necessary conformational changes for PKA binding and subsequent catalytic activity. It was also shown that the CPVT-associated mutations near the phosphorylation domain can induce structural changes that may affect PKA activity . MoreoveIn addition, the strong in vitro effect of the mutant suggests that residues in HD2 may interact directly with the phosphorylation domain. This domain could not be crystallized in RyR2, unlike RyR1, due to intrinsic flexibility ,56,57. N2+ conductance nor its activation by cytosolic Ca2+, in accordance with the unchanged Ca2+ release in HEK293 transfected cells. In contrast, we found a reduced D3291V-RyR2 response to luminal Ca2+ activation. RyR2 luminal Ca2+ regulation mechanisms are still a matter of debate. One proposed mechanism involves Ca2+ binding directly to the luminal activation site [2+ passing through opened RyR2 and acting at cytosolic Ca2+ binding sites of the same channel [2+ binding to luminal proteins such as calsequestrin 2, which are associated with RyR2, but this complex is absent in HEK293 cells and consequently absent in our single channel experiments [At the single channel level, the D3291V variant did not alter the channel Caion site  whereas  channel ,60,61. Aeriments .2+ sensitivity would be more likely explained by an alteration of the tertiary structure modifying the exposure of a putative luminal Ca2+ binding site.Since the residue 3291 is located in the cytoplasmic region of the channel and the cytosolic sensitivity is normal, the observed reduced intra-SR Ca2+ luminal sensitivity and a remarkable blunted response to adrenergic stimulation. It could be classified as a LOF variant but its mechanism probably differs from the LOF RyR2 mutations recently linked to ‘RyR2 Ca2+ release deficiency syndrome’ [In summary, we identified a new CPVT RyR2 variant with a reduced Cayndrome’ . Indeed,2+ activation as occurs in our variant [2+ activation, A4860G+/− and D4646A +/−, have been studied [Interestingly, reported LOF RyR2 mutations resulted in a loss or a reduction in luminal Ca variant ,33. Furt studied ,63.2+ release event through calcium-induced calcium release (CICR), a small quantity of Ca2+ in the SR. As the CICR occurred, this residual quantity of Ca2+ would increase. Once the threshold activation of these hypoactive RyR2 channels was reached, these channels would release Ca2+ and induce EADs, as was also reported for D4646A+/− murine model [2+ current ICa,L, and the phospholamban phosphorylation would lead to an increased SR Ca2+ store. We can hypothesize that the hypoactive D3291V-RyR2 channels will not be able to regulate the Ca2+ store level due to their loss of luminal sensitivity.Zhao et coworkers, after studying A4860G+/− mice, hypothesized that the hypoactive channels would leave, at each Cane model ,63. A si2+ released in D3291V-RyR2 HL-1 cells decreased compared to WT cells. Furthermore, 6% of D3291V-RyR2 HL-1 cells displayed EADs only with cAMP treatment. These data are congruent with the hypothesis that Ca2+ may accumulate in the SR under adrenergic stress and reach the threshold of D3291V-RyR2 activation  that would eventually lead to membrane depolarizations triggering potential arrhythmias.During cAMP treatment, the amplitude of Ca2+ waves and triggered activity. When β-adrenergic receptors are activated, the increase in cAMP activates PKA, which phosphorylates several targets that then produce enhanced Ca2+ entry through the LTCC and accelerate Ca2+ pumping to the SR through SERCA. The Ca2+ stored in the SR is then released as Ca2+ sparks or invisible Ca2+ leak, which are large enough to induce changes in the membrane voltage, but can limit SR overload, acting as a “safety valve”. The mutation we report lacks this “safety” mechanism. Thus, the result may be SR overload in conditions of stress. As the RyR mutant has abnormal sensitivity to luminal Ca2+, this store overload could be the origin of arrhythmogenic Ca2+ waves.One may question how a mutation that impairs the adrenergic response may induce arrhythmogenic CaNevertheless, the mechanisms responsible for the phenotypes associated with RyR2 variants should not be overinterpreted. The complexity is due to the central role of RyR2 channel in cardiomyocytes, its regulation by a high number of partners. In addition, recent unexpected observations, such as the electrophysiological remodeling of the surface membrane currents in the D4646+/− mice, the observed septum hypertrophy , as well2+ sensitivity and the normal Ca2+ conductance of the mutated channel could explain its limited severity in basal conditions and in absence of stress, thus explaining the presence of adult homozygous carriers and of numerous young heterozygous carriers with a negative stress-test. While the mutation does cause dysfunction in SR Ca2+ regulation, it may not be sufficient to induce a clinical phenotype in the absence of adrenergic stress. This baseline dysfunction could then be exacerbated in the context of adrenergic stress which would eventually induce typical CPVT positive stress-test with aging, or in association with modifier factors. Third, the blunted response of the D3291V-RyR2 channels to adrenergic stimuli is a new mechanism leading to sudden death. Lastly, this study points out the role of RyR2 helical domain 2 in adrenergic response and sudden death.In conclusion, due to the cosegregation of a RyR2 variant with the disease in a large family and the long-term follow up of all family members, we were able to identify the D3291V-RyR2 variant as pathogenic. First, this shows that variants outside the CPVT hotspots, considered by the diagnostic laboratories as of unknown significance, can be pathogenic and associated with a high risk of sudden death. Secondly, the apparently normal cytosolic Ca2+ sensitivity and an impaired adrenergic response in HEK293 and HL-1 cells. The absence of many cardiac-specific proteins in HEK293 cells lead us to study the effect of the variant in HL-1 cells which express RyR2 and regulatory proteins. Nevertheless, the two different cellular models are lacking the genomic background of the patient, and even though HL-1 cells express cardiac proteins, they do not completely reflect the structure and function of human cardiomyocytes. It would be interesting to generate hiPSC-derived cardiomyocytes to study the genetic background of the patient, the balance between heterozygous and homozygous carrier of this mutation, and the function of the D3291V-RyR2 in presence of its physiological partners. A murine model would be helpful to understand how such a hypoactive channel could lead to the occurrence of the typical clinical features of CPVT and the mechanisms involved in the development of ventricular fibrillation.We are reporting in this study a loss-of-function RyR2 mutation which is characterized by a reduced luminal Ca