At mitochondria-ER contacts (MERCs) these proteins are able to interact with major Ca2+ transporters, thus controlling mitochondrial Ca2+ homeostasis and downstream Ca2+ signalling pathways. Beyond the regulation of cell survival, this Bcl-2-dependent control over the mitochondrial Ca2+ dynamics has far-reaching consequences on the physiology of the cell. Here, we review how the Bcl-2 family of proteins mechanistically regulate mitochondrial Ca2+ homeostasis and how this regulation orchestrates cell death/survival decisions as well as the non-apoptotic process of cell migration.Bcl-2 family proteins are considered as one of the major regulators of apoptosis. Indeed, this family is known to control the mitochondrial outer membrane permeabilization (MOMP): a central step in the mitochondrial pathway of apoptosis. However, in recent years Bcl-2 family members began to emerge as a new class of intracellular calcium (Ca Apoptosis is a form of regulated cell death by which complex multicellular organisms orchestrate the regulated removal of unwanted or damaged cells. It is well established that apoptosis plays critical roles in development, tissue homeostasis and the response to cellular stress . AberratMitochondria play a central role in apoptosis execution. Actually, these genuine intracellular powerhouses contain in their intermembrane space (IMS) several cytotoxic proteins including Omi, SMAC/Diablo, and cytochrome c ,5,6,7,8.This mitochondrial outer membrane permeabilization (MOMP) is under the tight control of the Bcl-2 family of proteins . InitialBcl2-related genes are found only in multicellular animals and thus they are referred to as markers of multicellularity, evolutionarily selected to regulate apoptotic cell removal in development and sustain tissue homeostasis in metazoans [C. elegans in which the Bcl-2 homolog CED-9 was mutated. Loss-of-function of the ced9 gene resulted in widespread death of embryonic cells [bcl2 homologs solidified their critical role in apoptosis regulation [2+ ions [etazoans ,18. Thisic cells ,20. Subsgulation ,22,23. Hgulation ,26,27,28gulation ,31,32,33[2+ ions ,35,36,372+ ions are important secondary messengers participating in many cellular functions [2+ is able to enhance mitochondrial bioenergetics by promoting the activities of pyruvate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase [2+ is also required for the efficient execution of apoptosis, while Ca2+ overload induces the opening of the elusive mitochondrial permeability transition pore (mPTP), leading to necrotic cell death [2+ in the balance between life and death underlies the need for tight regulation of mitochondrial Ca2+ pools. Bcl-2 family of proteins participates in this process through direct interactions with various intracellular Ca2+ transporters or channels, which have profound consequences for mitochondrial Ca2+ homeostasis and downstream Ca2+ signalling pathways. Here, we review the role of Bcl-2 proteins in mitochondrial Ca2+ homeostasis and how this regulation orchestrates not only survival/death decisions but also non-apoptotic processes like cell migration.Caunctions . Ca2+ isrogenase . In contll death ,41. The 2+ ions [2+ is required to cross both layers in order to reach the matrix. The inner mitochondrial membrane (IMM) mitochondrial Ca2+ uniporter (MCU) has a low affinity for Ca2+, with a Kd of 10 µM [2+ directly from the cytosol, but rather require the direct transfer of Ca2+ from other stores through membrane contact sites (MCS), in order to be able to maintain their Ca2+ pools [2+ to mitochondria, the most understood pathway is the transfer of Ca2+ from the ER to mitochondria at mitochondria-ER contacts (MERCs) [Mitochondria are dynamic intracellular organelles that can store and exchange with the surrounding environment substantial amounts of Ca2+ ions . As mitoof 10 µM . This th2+ pools . While l2+ pools  can dire (MERCs) . Mitocho (MERCs) ,48. MERC (MERCs)  or PDZ d (MERCs)  in mamma (MERCs) ,52,53.2+ transfer. The ER-localized inositol 1,4,5-trisphosphate receptor (IP3R) and OMM-localized voltage-dependent anion channel (VDAC) are bridged by the glucose regulated protein-75 (GRP75), forming a tethering complex between both organelles [3 binds to IP3R leading to opening of the channel and subsequent Ca2+ release into cytosol and mitochondria, through VDAC [2+ transfer at the MERCs generates a high local concentration of Ca2+, called Ca2+ microdomains, enabling the MCU complex to uptake Ca2+ into the mitochondrial matrix [2+ signalling platform at MERCs organizes all of the appropriate machinery required to efficiently transfer Ca2+ from the ER to mitochondria. The high local concentration of Ca2+ generated at this interface enables the IMM- and MERCs-localized MCU [2+ into the matrix. The spatial organization and coordination of the ER-, OMM- and IMM-localized Ca2+ channels/receptors are therefore crucial in order for mitochondria to efficiently uptake Ca2+.At the MERCs, a specialized subdomain exists to enable the efficient ER to mitochondria Caganelles ,55. Uponugh VDAC   and in the rhythmicity of cardiomyocytes [The OMM is highly permeable to ions and low molecular weight molecules, due to the presence of VDACs, whereas the IMM-localized MCU complex enables Cae matrix ,64,65. Te matrix . They cae matrix . All thrmulation , whereasmyocytes .2+ uptake mainly through the control of VDACs permeability  uptake less Ca2+ into the mitochondria compared to control cells [2+ uptake was restored when KO MEFs were complemented with exogenous Bcl-xL targeted to the mitochondria but not to the ER. It has then been proposed that Bcl-xL promotes mitochondrial Ca2+ entry via direct interaction with the VDAC1 and VDAC3 channels [2+ regulation [2+ uptake and protects cells from apoptosis [More recently, the team of Chi Li demonstrated that ol cells . Notablychannels , and thagulation . In thispoptosis ,75.2+ uptake [2+ [2+ uptake and bioenergetics in a non-small cell carcinoma cell line [Actually, within the Bcl-2 family, VDACs participate in functional interactions with Bcl-2 and Mcl-1 as well. Several studies have shown that Bcl-2 interacts with VDAC1 N-terminal α-helix, thereby leading to a reduction in mitochondrial Ca+ uptake . This pr+ uptake . In conttake [2+ ,79. Finaell line .2+ trafficking highlight the complex interactions between anti-apoptotic Bcl-2 proteins and VDACs. Due to their role in cell survival and death, it can be hypothesized that under physiological conditions, anti-apoptotic Bcl-2 members enhance mitochondrial Ca2+ uptake to regulate mitochondrial metabolism and bioenergetics, whereas upon apoptotic stimulations they protect mitochondria from deleterious massive Ca2+ overload by interacting with VDACs. This hypothesis is notably supported by findings in the heart of transgenic mice describing that Bcl-2 decreases mitochondrial Ca2+ efflux via the Na+/Ca2+ exchanger, NCLX, to maintain mitochondrial ATP production [The discrepancies regarding mitochondrial Caoduction .2+. ER-dependent Ca2+ release controls basal cytosolic Ca2+ levels and mitochondrial Ca2+ uptake through the direct transfer of Ca2+ ions at MERCs [2+ through ER-Ca2+ channels. IP3Rs and ryanodine receptors (RyRs) are the two major families of ER Ca2+ channels [3R isoforms (IP3R1-3), which are often co-expressed in most mammalian cell types. The three isoforms differ in their affinity for the IP3 ligand; IP3R2 exhibiting the highest sensitivity while IP3R3 has the weakest [3R2 isoform is the most effective in delivering Ca2+ to the mitochondria [3, the natural ligand for IP3R, is produced upon G-protein coupled receptor (GPCR) activation by ligands such as histamine or ATP at the plasma membrane. GPCR activation leads to hydrolysis of phosphatidyl inositol-4,5-bisphosphate (PIP2) by phospholipase C resulting in the production of IP3. IP3 then diffuses through the cell and binds to the IP3-binding domain of IP3R oligomers resulting in the opening of the Ca2+ channel, which subsequently allows Ca2+ flux into the cytosol and mitochondria [The ER is the major storage organelle for cellular Caat MERCs ,48,82. Achannels . In vert weakest . Intereschondria . IP3, thchondria ,86.3R [3R-dependent Ca2+ release, the binding sites and the functional consequences of this regulation are different. For instance, Bcl-2 is able to bind to the central modulatory and transducing domain II (MTD II) of IP3R, which requires the N-terminal BH4 motif of Bcl-2 [2+ release from the ER and inhibits the transfer of toxic Ca2+ insults to the mitochondria. Conversely, Bcl-xL interacts with IP3R through its BH3-binding groove [3R C-terminus that are able to bind to Bcl-xL with high affinity. This interaction leads to IP3R opening and subsequent ER-Ca2+ release. Interestingly, the mode of action of Bcl-xL appears to be concentration-dependent because increasing Bcl-xL levels lead to a secondary IP3R inhibition, which occurs through the binding of Bcl-xL at the Bcl-2 interaction site in the MTDII domain [3R by Bcl-xL seems to be biphasic. At low levels, ER-based Bcl-xL favors the release of Ca2+ ions from IP3R and transfer to the mitochondria thus enhancing mitochondrial bioenergetics by activating the Ca2+-dependent dehydrogenases of the Krebs cycle. In contrast, at high protein concentration levels, Bcl-xL inhibits IP3R-dependent Ca2+ release and subsequent apoptosis initiation [3R but preferentially binds to VDAC1, controlling its permeability [3R. The corresponding residue in Bcl-xL is an aspartate at position 11 (Asp11). Mutating Lys17 into Asp in Bcl-2 leads to complete loss of IP3R binding capacity, whereas changing of Asp11 into Lys in BH4 of Bcl-xL converts Bcl-xL into an IP3R binder and inhibitor [Actually, many anti-apoptotic Bcl-2 proteins including Bcl-2, Bcl-xL, Mcl-1 and Nrh, possess dual mitochondrial and ER localizations and are able to interact with IP3R ,35,87 . In breast cancer (BC) cells, Nrh is exclusively found at the ER where it is able to interact with the N-terminal IP3 binding domain of the IP3R1 via its BH4 motif [3R1 opening, which in turn dampens the unfolded protein response (UPR). Actually, the UPR is an adaptive reaction that prevents the accumulation of misfolded proteins in the ER lumen to maintain cell viability. If stressful conditions persist, the UPR can prime cells for cell death through the activation of the BH3-only protein Bim [3 binding domain protein, exert an additive inhibitory effect over IP3R at resting states [2+ accumulation in the mitochondria and subsequent apoptosis [An outsider of this Bcl-2-IPH4 motif . This intein Bim ,94. The tein Bim . Interesg states . Howeverpoptosis .2+ channels [2+ release into the mitochondria [2+ channels, Bcl-2 proteins tightly control ER to mitochondria Ca2+ transfer required for cell fate decisions.Finally, Bcl-2 proteins have also been proposed to interact with other ER-Cachannels . Both Bcchannels ,98. Inde2+ plays a pivotal role in the balance between cell survival and cell death events [2+ is required to maintain mitochondrial bioenergetics and metabolism, larger and toxic mitochondrial Ca2+ levels have been proposed to facilitate apoptosis [2+ homeostasis as well as the cell death and survival balance. During apoptosis, the number of mitochondria-ER contact sites increases [2+ uptake [2+ overload may also lead to IMM cardiolipin oxidation, increasing ROS production and mPTP opening [2+ fluxes described above, anti-apoptotic Bcl-2 proteins have been widely shown to inhibit apoptosis by decreasing ER-induced Ca2+ release or decreasing VDAC1-dependent Ca2+ uptake [3R and several Bcl-2 proteins and impact on the apoptotic Ca2+ signals transfer to the mitochondria. For instance, a BH4-domain-targeting peptide of Bcl-2, called Bcl-2/IP3 receptor disrupter-2 (BIRD-2), has been shown to have cell death-inducing effects in different cancer cell lines [2+ overload and caspase activation [Mitochondrial Cah events . While apoptosis  and to tpoptosis . As alrencreases ,101, fos+ uptake ,95, whic+ uptake . Mitocho opening . Due to + uptake   was identified . Bcl-wav+ levels . At the henotype .2+ transport was further emphasized in the motility of cancer cells [mcu-silencing in highly invasive triple-negative breast cancer (TNBC) cell lines resulted in altered F-actin cytoskeleton dynamics, cell polarization loss and impairment of the focal adhesion proteins dynamics [2+-dependent Calpain and Rho-GTPases activities [mcu also resulted in decreased cell motility and invasiveness as well as reduction of tumor growth [2+ uptake could probably control multiple downstream signalling pathways. High mtROS production is detrimental for cell survival, however, in cancer cells sub-lethal mtROS levels promote cell proliferation, migration and invasion [2+ homeostasis [mcl1-silencing or treatment with peptides that suppress VDAC-based Ca2+ uptake led to reduced mtROS generation. Bcl-xL and Bcl-2 were also shown to act as accelerators of cell motility, invasiveness and metastasis spreading. As it is the case for Mcl-1, mitochondrion-localized Bcl-xL, but not ER-based Bcl-xL, contributes to cell migration through the generation of reactive mtROS [2+ entry and mtROS production. Interestingly, one study linked this regulation with the effect of metalloprotease-processed CD95L (cl-CD95L) on TNBC accelerated metastatic dissemination and poor patient prognosis [2+ release from the ER to mitochondria at MERCs. In this particular case, mitochondria-targeted Bcl-xL and ER-targeted Bcl-2 were proposed to increase Ca2+ transfer between the ER and the mitochondria, thus accelerating ATP production and mtROS generation [The importance of the MCU-dependent Caer cells ,130. Inddynamics . These ptivities . In addiinvasion ,132. In invasion ,133,134.eostasis . Indeed,2+ in mediating cell death decisions has also been emphasized, but recent evidence support additional functions for mitochondrial Ca2+ on top of mitochondrial bioenergetics and cell death. With their capacity to be localized at the mitochondria-ER interface and to interact with keys channels or receptors on both ER and mitochondrial membranes, Bcl-2 proteins have emerged as key regulators of intracellular and mitochondrial Ca2+ homeostasis, and subsequently to several other processes such as cell migration. Due to this connection, numerous studies are currently directly targeting Bcl-2-IP3R or Bcl-2-VDAC interactions to modulate Ca2+ signalling and to control cell fate in different types of cancer cell models. Together, future studies identifying precisely how mitochondrial Ca2+ is regulated by Bcl-2 proteins may identify new strategies for therapeutic intervention.The role of Bcl-2 family of proteins in the initiation of apoptosis has been well studied, which has led to our current understanding of how cells integrate stress signals at the level of the mitochondria, leading to initiation of the death program. The role of Ca


Simultaneous imaging of Ca2+ transients and recording of action potentials (APs) demonstrated failure to generate Ca2+ transients when APs peaked at potentials more negative than –30mV. An AP property that closely correlated with failure of the Ca2+ transient was the integral of AP voltage with respect to time. Simultaneous recording of Ca2+ transients and APs with electrodes separated by 1.6mm revealed AP conduction fails when APs peak below –21mV. We hypothesize propagation of APs and generation of Ca2+ transients are governed by distinct AP properties: AP conduction is governed by AP peak, whereas Ca2+ release from the sarcoplasmic reticulum is governed by AP integral. The reason distinct AP properties may govern distinct steps of ECC is the kinetics of the ion channels involved. Na channels, which govern propagation, have rapid kinetics and are insensitive to AP width  whereas Ca2+ release is governed by gating charge movement of Cav1.1 channels, which have slower kinetics such that Ca2+ release is sensitive to AP integral. The quantitative relationships established between resting potential, AP properties, AP conduction and Ca2+ transients provide the foundation for future studies of failure of ECC induced by depolarization of the resting potential.Excitation-contraction coupling (ECC) is the process by which electrical excitation of muscle is converted into force generation. Depolarization of skeletal muscle resting potential contributes to failure of ECC in diseases such as periodic paralysis, intensive care unit acquired weakness and possibly fatigue of muscle during vigorous exercise. When extracellular K Successful ECC involves propagation of action potentials (APs) from the neuromuscular junction (NMJ) along the length of the fiber as well as into a network of membrane invaginations in muscle known as the transverse tubules (t-tubules) . Depolaroduction .2+ transient with depolarization of the resting potential beyond –60 mV -acquired weakness  as well d –60 mV .The mechanism underlying failure of ECC in the setting of depolarization of the resting potential remains unknown. The simplest possibility is that failure of ECC with depolarization is due to failure to generate or conduct APs. Consistent with this possibility, the decline in force is paralleled by reduction in extracellular recordings of compound muscle APs . One pos2+ indicator in muscle fibers in which the resting potential was depolarized by elevation of extracellular K+. The AP parameter that most closely correlated with elevation of intracellular Ca2+ was the area of the AP above –30 mV. We further determined that failure of AP propagation occurs when APs peak below –21 mV. Identification of the AP properties governing conduction and Ca2+ release along the length of the fiber provides a basis for future studies of depolarization-induced failure of ECC.To determine the mechanism underlying the failure of ECC, we measured generation and conduction of APs as well as the ΔF/F of a genetically encoded Ca+ concentration dependence of force generation previously reported (+ and measured force. Measurement of twitch force in the mouse extensor digitorum longus (EDL) following elevation of extracellular K+ revealed an initial increase in force (+ concentration), which was followed by a decline that became faster with higher levels of extracellular K+  and in 14 mM K+, where force had largely failed it averaged –57.1 ± 1.5 mV . These data suggest that a steep reduction of force is caused by depolarization over a narrow range of resting potentials  at which force production was near 0. These data suggest that failure of force production is not due to failure to initiate APs.To determine the mechanism underlying depolarization-induced failure of force generation, APs were recorded. AP amplitude decreased as the resting potential became more depolarized with elevation of extracellular K+ . What wa2+ transients, we simultaneously recorded APs while imaging Ca2+ in mice expressing GCAMP6f in skeletal muscle. These experiments were performed on the same muscles used to measure resting potential and AP properties following infusion of solutions containing elevated concentrations of K+. GCAMP6f is a high-affinity Ca2+ indicator with a Kd near 600 nM in cardiomyocytes such that it can detect changes in Ca2+ ranging from 100 nM to 5 µM .With infusion of 16 mM Ko –45 mV . Between+, there was an initial increase in the ΔF/F from a mean normalized value of 0.81 ± 0.10 to the normalized maximum of 1 .With depolarization during infusion of 16 mM Kequation . For theequation , which w2+ transient was within the range of values obtained from voltage-clamp studies of mouse muscle fibers . This difference led us to look for another AP parameter that more closely correlated with the reduction in ΔF/F.To identify properties of APs that would allow for accurate prediction of ΔF/F, the correlation between normalized AP amplitude and ΔF/F was examined. A loss of 11.6 ± 1.7 mV of resting potential was required to reduce AP amplitude from 90% to 10% of maximum. In contrast, ΔF/F was reduced from 90% to 10% of maximum with a loss of only 4.1 ± 2.4 mV of resting potential . The R2 value was larger for AP area vs. ΔF/F because AP area more closely mimicked the rapid decrease in ΔF/F  were used to track Cae muscle . Despitee muscle . The ΔF/e muscle .The last AP to trigger successful ECC was associated with a ΔF/F at the ROI 1.6 mm from the stimulating electrode of 87% ± 7% of the ΔF/F value for the ROI at a resting potential of –70 mV, and this fell to 16% ± 4% by the first AP that failed to trigger successful ECC . The pea2+ release from the SR.At the time of failure of AP conduction, ΔF/F near the stimulating electrode still averaged 96% ± 15% of the ΔF/F at that ROI at a resting potential of –70 mV . This re+  along the length of mouse EDL muscle fibers 26Sortm95.1(CAG-GCaMP6f)Hze/MwarJ, cat #028865) with mice expressing parvalbumin promoter-driven Cre Arbr/J, cat# 030218). Mice were sacrificed using CO2 or isoflurane inhalation followed by cervical dislocation.Mice expressing GCAMP6f  in skele2, 0.7 MgSO4, 26.2 NaHCO3, 1.7 NaH2PO4, and 5.5 glucose and maintained at pH 7.3–7.4 by aeration with 95% O2 and 5% CO2. Solutions containing elevated concentrations of KCl  had corresponding reduction in NaCl .All experiments were performed at 21–23°C within 4 hr of sacrifice. The same solutions were used for both force experiments and intracellular recording performed on EDL muscle fibers. The control solution contained (in mM) 118 NaCl, 3.5 KCl, 1.5 CaCl+ solution for 20 min, followed by high K+ solution (10–16 mM) for 45 min, and then washed again with normal K+ solution for 25 min to follow recovery. The EDL was stimulated with a twitch pulse every 5 min, and force was recorded.The EDL muscle was dissected, and the proximal tendon was tied with a 6-0 caliber silk suture to a bar attached to a custom recording chamber. The distal tendon was tied to a hook and attached to the force transducer (Aurora Scientific). The EDL was stimulated with two platinum electrodes placed parallel to the muscle in the bath. The force transducer was controlled by a 305C two-channel controller (Aurora Scientific) and digitized by a Digidata 1550B digitizer (Molecular Devices). A S-900 pulse generator (Dagan) was used to generate 1 ms 100 V pulse. The pulse generator was triggered using pCLAMP 11 data acquisition and analysis software with a sampling frequency of 10 kHz. No filtering was applied to the signal. Optimal length was determined by adjusting the tension of the muscle until maximal twitch force was achieved. During force recordings, the muscle was exposed to normal K+, after threshold was determined current injection was increased to 150% of threshold and perfusion of 16 mM K+ was initiated. In studies of Ca2+ release following failure of ECC, depolarization was achieved by increasing the duration of current injection to 5 ms. Fibers with resting potentials more depolarized than –74 mV in solution containing 3.5 mM KCl were discarded. Sampling frequency was 10 kHz with a 5 kHz-low pass filter.To prevent contraction, muscles were loaded with 50 μM BTS  dissolved in DMSO for 45 min prior to recording. Muscle was stained with 10 µM 4-(4-diethylaminostyrl)-N-methylpyridinium iodide . Muscle fibers were impaled with two sharp microelectrodes filled with 2 M potassium acetate solution containing 1 mM sulforhodamine 101  to allow for visualization. Resistances were between 15 and 30 MΩ, and capacitance compensation was optimized prior to recording. APs were evoked by a 0.2 ms injection of current ranging from 100 to 1000 nA. For recordings of APs during infusion of 16 mM K2+ I3 cube, band pass 450–490, long pass 515). Imaging was synchronized with triggering of APs using a Master-8 pulse generator . Frames were acquired at 30 frames per second with a sCMOS camera (CS2100M-USB) using ThorCam software . During infusion of solution containing high K+, APs were triggered every 5 s. Each AP was synchronized with capture of 48 frames at 30 frames/s. Images were analyzed using ImageJ (NIH).Muscle expressing GCAMP6f was imaged without staining  with a sampling rate of 5 kHz. The mean time to peak of the ΔF/F signal of APs in 3.5 mM Ca2+ was 44.3 ± 3.2 ms (n = 9 muscles). This was slower than the time to peak of muscle twitch tension , suggesting a slow rate of binding of Ca2+ to GCAMP6f. The fluorescence signal 33 ms after triggering the AP averaged 96.2% ± 0.8% of the peak signal. These data suggest that the sampling frequency of 30 Hz introduced a slight underestimation of the peak ΔF/F.To confirm that the sampling rate of 30 Hz was not missing an early peak in Ca2+ transient on AP properties, fibers were imaged using a ×40 objective and a single ROI 50–100 μm from the current injecting electrode was analyzed. For studies of conduction of APs along the length of the fiber, fibers were imaged using a ×5 objective. Placement of both electrodes in the same fiber was accomplished using the fluorescence signal triggered along the length of the fibers expressing GCAMP6f after triggering of an AP. ROIs were selected at regular lengths along the length of the muscle fiber being stimulated. The signal between stimuli in each ROI was used to record background, which was subtracted from peak signal detected after stimulation.For studies of the dependence of the Ca2+ image intensity vs. AP, and Ca2+ image intensity vs. resting potential were all fit to a Boltzmann equation:Data for AP peak vs. resting potential, CaOut represents the dependent variable (either AP peak or Ca2+ image intensity), V is the independent voltage variable , LV is the limiting value when V is very low (toward more negative), HV is the limiting value when V is very high (toward more positive), V50 is the value of V at which Out is halfway between HV and LV, and k is the slope factor. All voltages and the variable k are expressed in mV, and Ca2+ image intensity is in arbitrary units between 1 for maximum intensity for each experiment and 0.where + and control fibers followed over time in 3.5 mM K+, the paired Student’s t-test was used with n as the number of fibers. The 12 fibers recorded from over time during infusion of 16 mM K+ came from five different mice, and the 5 control fibers maintained in 3.5 mM K+ came from three different mice. The numbers of animals and fibers for comparisons are described in the corresponding figure legends and text. All data are presented as mean ± SD. p<0.05 was considered to be significant.Two types of statistical comparisons were made. For recordings at a single time point, repeated ANOVA with n as the number of mice was used. For comparisons within individual fibers followed over time during infusion of 16 mM K 2+ transients. It makes an investigation of excitation contraction coupling failure associated with depolarization of the resting potential.This is a rigorous electrophysiological paper that investigates relationships between resting potential, action potential properties and conduction and Ca public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Mone Zaidi as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "The Role of Action Potential Waveform in Failure of Excitation Contraction Coupling" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:All three reviewers responded positively to the study but had serious issues to raise concerning interpretation of the findings, the need for some further experiments, with a consequent large number of issues being raised as enumerated in the comments to follow. The reviewers have also indicated their reservations in broad terms as follows:1) Reviewer 1 regards the observations as empirically helpful, but is very concerned about the further interpretation that need to address in particular (a) whether there is a possible tubular action potential conduction failure despite persistent surface membrane excitation and (b) the relative inactivation properties of Na channel function and of excitation contraction coupling.2) To Reviewer 2 a large negative aspect of the manuscript in its current form was over-interpretation and the discussion about how these findings challenge fundamental aspect of our understanding of action potentials.3) Although Reviewer 3 was enthusiastic about the study and the new finding  but made extensive and important comments. In particular they also felt that the conclusions are not fully justified. In particular there was a lack of information regarding the calcium indicator, commenting that due to the limitation of the calcium indicator that has been used, at least one other calcium indicator must be tested to confirm the conclusions.4) Note that all three reviewers had independently raised such queries concerning the possibility of action potential failure, and interpretation of the all or none concept.Despite the rather extensive changes that these points entail, the three reviewers felt they wished to give the authors the opportunity for further work and revisions, whilst clearly on the understanding that a revised version of the work would be subject to careful scrutiny in relationship to the recommendations that were made.Reviewer #1 (Recommendations for the authors):2+ transients. The findings provide a helpful and constructive empiric demonstration showing the point at which the processes of action potential activation and triggering of the ca2+ transient fail.A) These are well conducted and elegant experiments, both in their electrical recordings and in their determinations of caB) My problem is their taking this interpretation further, as their present experimental design does not consider:1) The possibility of a persistent surface action potential generation despite a tubular action potential conduction failure. This will give the appearance of persistent regeneration Na channel activity reflecting the surface action potential wave, combined with an excitation contraction coupling failure. I wonder if changes of this kind are evidenced in the late component waveform changes as illustrated in Figure 1C.2) If it is possible to resolve (1), the authors also need to draw attention to the presence of inactivation properties in both the Na channel mediating the action potential, and the excitation contraction coupling process, as evidence in inactivation properties of its underlying charge movement. Both these relationships are steep, and fall over similar voltage ranges.2+ transient activation?3) Is it possible to obtain potentiometric optical records of tubular action potential propagation or its possible failure to demonstrate a persistence of a tubular action potential in the face of a failure in ca2+ transientand its underlying triggering intramembrane charge movement [2-3].4) Having addressed (3), one then needs to distinguish the inactivation properties and its voltage dependence respectively of Na channel inactivation [1] and the inactivation of excitation contraction coupling. The latter should be considered in terms of the corresponding inactivation properties of tension generation, the caC) If it is possible to resolve the point (B) above, the outcome of this paper indeed distinguishes between action potential failure and excitation contraction coupling failure. If it is not possible to resolve this point, then it remains possible to report an empiric finding relating resting potential modification and skeletal muscle function.References:[1] Adrian RH, Marshall MW. Sodium currents in mammalian muscle (nih.gov)[2] The effects of calcium deprivation upon mechanical and electrophysiological parameters in skeletal muscle fibres of the frog. – Lüttgau – 1979 – The Journal of Physiology – Wiley Online Library[3] Analysis of 'off' tails of intramembrane charge movements in skeletal muscle of Rana temporaria. – Huang – 1984 – The Journal of Physiology – Wiley Online LibraryReviewer #2 (Recommendations for the authors):+] affect twitch force. On the one hand, Figure 1, which is about twitch force and action potential, brings nothing new because it has been shown many times. On the other hand, the data on the relationships between resting potential, action potential and ca2+ transient are new and very interesting. However, it must be expanded; and, at the present time there are serious major concerns that must be dealt with.The objective of this study is to further understand the mechanism by which increases in extracellular , then the action potential shape will change according to how the kinetics of the Na+ channels are affected by the changes in ion concentration and/or membrane potential. However, in the new physiological condition, the ‘all or none’ concept remains.In regard to action potential, the ‘all or none’ concept means that regardless of the stimulation strength, once threshold has been reached the action potential shape is always the same. However, this is true for a given physiological condition. When there is a change in the physiological condition, e.g., change in [Na+] or  relationship shifted to higher [K+] as temperature is raised. Furthermore, the K+-induced twitch potentiation is much greater at 37{degree sign}C (Yensen study) than at 25{degree sign}C (Cairns study). In other words, no one can use the data in this study to understand the role of the K+ effects during fatigue. It is now possible to carry out experiments at higher temperature.3. Another issue with Figure 1 and the entire studies is the experimental temperature of 25{degree sign}C when muscle temperatures during exercise exceed 37degC. Data at 25{degree sign}C are completely and physiologically irrelevant because the K2+ indicator. There are major differences between various ca2+ indicators providing µM to mM changes in intracellular [ca2+] during contraction, and the biggest problem is to estimate how ca2+ transient occurs during twitches. In this study GCAMP6f is used as a ca2+ indicator with its own limitations, but for which little is given and discussed, albeit references are provided. This is the most important and interesting part of the study and correlation between action potential and ca2+ transients have never been demonstrated. Here, the section need major strengthening as follows:4. Ca2+] in sarcomere, ca2+ values being in the µM range. However, indicators with very low affinity for ca2+ provide [ca2+] values close to mM. Why the differences? Most likely because [ca2+] near the releasing site must be very high to provide fast ca2+ diffusion into sarcomere and such concentration can only be measured with low ca2+ affinity indicator because those with nM affinity are saturated and tend to provide [ca2+] within the sarcomere. So to be relevant, one needs information about its location in the sarcoplasm.– The indicator is a protein being expressed in muscles. Where is the indicator mainly located? Indicators with nM affinity only measured ? Why not take a picture or two before fibers are stimulated to get the resting [ca2+]?5. Why is a neighboring fiber is used for resting  is increased to 16 mM and demonstrate that the effects are much greater.6. Fiber damage, lines 225-231. Why are the changes in resting EM and AP overshoot consider to be due to fiber damage? This question is raised because muscle movement during stimulation was blocked by BTS, so microelectrode damage is not expected. Another possibility is a leak around the microelectrodes. If this was the case, one would not get resting EM and overshoot as measured. The last thing is a continuous depolarization following microelectrode penetration. This is a major issue with KCl filled microelectrodes because K+ over a few minutes  vs. after 45 min at elevated [K+] . I agree with the authors that the difference involved the slow inactivation process that takes several min to complete. Unfortunately, they raised the issue only in the discussion and are forcing us to discern the differences from different figures. I suggest that a figure is added to clearly show the overshoot-resting EM relationship in the same figure. This is clearly an important issue because we often look at a potential role of K+ in muscle fatigue during a 3-5 min fatigue period with the K+ effect over a 45 min period. Showing the differences in how depolarization and time affect overshoot is critical.7. Slow inactivation. Another important aspect of the data presented in this study is the difference in AP overshoot-resting EM. In the discussion the authors raised the issue that for a given resting EM, AP overshoot is greater when measured at 16 mM K2+] when they measured AP after inducing membrane depolarizations at high [K+]. There is evidence for slow inactivation of the ca2+ release mechanism involving both CaV1.1 and RyR1. So, it would be interesting to see if prolonged K+-induced membrane depolarization also affects over time the ca2+ transient; i.e., different [ca2+]-resting EM or AP relationships. This would really increase the interest for this study.8. It is unfortunate that the authors did not think about measuring  with repetitive stimulation by an increase in [ca2+] between contractions because it means that more ca2+ remains bound to ca2+ binding proteins so when ca2+ is released more remains in the fee state. You do have an increase in resting [ca2+] in Figure 3C and this possibility must be discussed in the Discussion because it can be a reason as to why there is a portion of the ca2+ transient – overshoot that cannot be explained by the overshoot decrease.14. Pedersen and others often explain the increase in twitch .4) Having addressed (3), one then needs to distinguish the inactivation properties and its voltage dependence respectively of Na channel inactivation [1] and the inactivation of excitation contraction coupling. The latter should be considered in terms of the corresponding inactivation properties of tension generation, the caNew experiments have been performed and a new figure has been generated. Inactivation of Na channels appears to be responsible for failure of ECC.C) If it is possible to resolve the point (B) above, the outcome of this paper indeed distinguishes between action potential failure and excitation contraction coupling failure. If it is not possible to resolve this point, then it remains possible to report an empiric finding relating resting potential modification and skeletal muscle function.We believe the new experiments performed have greatly strengthened our study and support our conclusion that depolarization-induced failure of ECC is due to failure of AP generation/conduction rather than inactivation of SR Ca release.Reviewer #2 (Recommendations for the authors):+] affect twitch force. On the one hand, Figure 1, which is about twitch force and action potential, brings nothing new because it has been shown many times. On the other hand, the data on the relationships between resting potential, action potential and ca2+ transient are new and very interesting. However, it must be expanded; and, at the present time there are serious major concerns that must be dealt with.The objective of this study is to further understand the mechanism by which increases in extracellular , then the action potential shape will change according to how the kinetics of the Na+ channels are affected by the changes in ion concentration and/or membrane potential. However, in the new physiological condition, the ‘all or none’ concept remains.In regard to action potential, the ‘all or none’ concept means that regardless of the stimulation strength, once threshold has been reached the action potential shape is always the same. However, this is true for a given physiological condition. When there is a change in the physiological condition, e.g., change in [Na+] or  relationship shifted to higher [K+] as temperature is raised. Furthermore, the K+-induced twitch potentiation is much greater at 37{degree sign}C (Yensen study) than at 25{degree sign}C (Cairns study). In other words, no one can use the data in this study to understand the role of the K+ effects during fatigue. It is now possible to carry out experiments at higher temperature.3. Another issue with Figure 1 and the entire studies is the experimental temperature of 25{degree sign}C when muscle temperatures during exercise exceed 37degC. Data at 25{degree sign}C are completely and physiologically irrelevant because the KWe agree and plan to perform these experiments in the future. We are working to modify our set up, and hope within the next year to begin experiments closer to physiologic temperature.2+ indicator. There are major differences between various ca2+ indicators providing µM to mM changes in intracellular [ca2+] during contraction, and the biggest problem is to estimate how ca2+ transient occurs during twitches. In this study GCAMP6f is used as a ca2+ indicator with its own limitations, but for which little is given and discussed, albeit references are provided. This is the most important and interesting part of the study and correlation between action potential and ca2+ transients have never been demonstrated. Here, the section need major strengthening as follows:4. Ca2+] in sarcomere, ca2+ values being in the µM range. However, indicators with very low affinity for ca2+ provide [ca2+] values close to mM. Why the differences? Most likely because [ca2+] near the releasing site must be very high to provide fast ca2+ diffusion into sarcomere and such concentration can only be measured with low ca2+ affinity indicator because those with nM affinity are saturated and tend to provide [ca2+] within the sarcomere. So to be relevant, one needs information about its location in the sarcoplasm.– The indicator is a protein being expressed in muscles. Where is the indicator mainly located? Indicators with nM affinity only measured ? Why not take a picture or two before fibers are stimulated to get the resting [ca2+]?5. Why is a neighboring fiber is used for resting  is increased to 16 mM and demonstrate that the effects are much greater.6. Fiber damage, lines 225-231. Why are the changes in resting EM and AP overshoot consider to be due to fiber damage? This question is raised because muscle movement during stimulation was blocked by BTS, so microelectrode damage is not expected. Another possibility is a leak around the microelectrodes. If this was the case, one would not get resting EM and overshoot as measured. The last thing is a continuous depolarization following microelectrode penetration. This is a major issue with KCl filled microelectrodes because K+. We have shortened discussion of damage-induced depolarization and have removed it from the figure.We agree with the reviewer that it is not critical for our study whether depolarization is induced by damage or high K+ over a few minutes  vs. after 45 min at elevated [K+] . I agree with the authors that the difference involved the slow inactivation process that takes several min to complete. Unfortunately, they raised the issue only in the discussion and are forcing us to discern the differences from different figures. I suggest that a figure is added to clearly show the overshoot-resting EM relationship in the same figure. This is clearly an important issue because we often look at a potential role of K+ in muscle fatigue during a 3-5 min fatigue period with the K+ effect over a 45 min period. Showing the differences in how depolarization and time affect overshoot is critical.7. Slow inactivation. Another important aspect of the data presented in this study is the difference in AP overshoot-resting EM. In the discussion the authors raised the issue that for a given resting EM, AP overshoot is greater when measured at 16 mM KWe agree with the reviewer that this is a critical issue. However, we have opted not to put the data in the same figure as we could not figure out how to do so without disrupting the flow of the manuscript. We hope that pointing the difference in the discussion will suffice.2+] when they measured AP after inducing membrane depolarizations at high [K+]. There is evidence for slow inactivation of the ca2+ release mechanism involving both CaV1.1 and RyR1. So, it would be interesting to see if prolonged K+-induced membrane depolarization also affects over time the ca2+ transient; i.e., different [ca2+]-resting EM or AP relationships. This would really increase the interest for this study.8. It is unfortunate that the authors did not think about measuring  with repetitive stimulation by an increase in [ca2+] between contractions because it means that more ca2+ remains bound to ca2+ binding proteins so when ca2+ is released more remains in the fee state. You do have an increase in resting [ca2+] in Figure 3C and this possibility must be discussed in the Discussion because it can be a reason as to why there is a portion of the ca2+ transient – overshoot that cannot be explained by the overshoot decrease.14. Pedersen and others often explain the increase in twitch [ca2+ signal is due to widening of the AP. We also mention the possibility that an increase in resting ca2+ is the cause.We now include mention of a recent modeling study, which suggests the increase in ca15. Lines 394. A non-linear relationship does not imply a poor correlation. You have in fact obtained excellent relationships, they are simply not linear. For such relationships you must fit the data for nonlinear relations and you will still get excellent correlation coefficient.What you are looking for is a linear relationship. Correct the discussion.Done.Reviewer #3 (Recommendations for the authors):2+ release. It shows that the release is not as binary as it is often portrayed in textbooks and by colleagues in our field. Rather this work demonstrates that the SR ca2+ release is highly dependent on the waveform of the activating action potential. This finding may be relevant for fatigue and for muscle weakness in some paralytic disorders.This manuscript is a very interesting piece of work, and it leads to a very important conclusion regarding SR caThe experimental approach is interesting, and experiments are well performed. Moreover, the authors are aware of some of the limitations and these limitations are mentioned. They are, however, not really taken into consideration when the data is interpretated. In the mind of the reviewer this leads to some degree of over-interpretation of the data. Two particular elements of concern exist that should be modified/addressed:2+ signals are (presumably) recorded within the same region where the electrodes are impaled. This means that it is not possible to exclude that the compromised action potentials at high K+ did not propagate along the fiber. It is therefore hard to exclude that some of the loss of force at elevated K+ is caused by all-or-none behavior due to failing propagation. It is recommended that statements like in L195-199 are modified because how do you know that the signals propagated? If they did not propagate the all-or-none is more accurate description perhaps. Please also re-consider that the signals recorded at the excitation zone naturally will depend on the size of the passive response to the action potential trigger current and hence the recorded signals with the voltage electrode will depend on both the regenerative action potential signals and the triggering current. In propagating signals, only the true action potential will remain and the passive response to the action potential triggering current would disappear.First, the discussion of whether the signals are all-or-none is misguided by the fact that the two electrodes are close to each other. While not mentioned in the methods (please correct this) the electrodes appear to be inserted at very close proximity and the ca2+ release is strongly dependent of the waveform of the action potential. This is new and sufficiently novel to justify the work.The paper is interesting due to the data and in the mind of the reviewer the whole narrative about these findings leading to new definition of an action potential and the challenge of the all-or-none theory is not really needed. Frankly, it takes away the focus of the nice scientific data that demonstrates that SR caThese are excellent comments that we took to heart. We have performed new experiments examining conduction along the length of the fiber. This has led to the addition of 2 new figures. We have also removed discussion of all-or-none APs.2+ is with a high affinity reporting system and with low sampling frequency. These two experimental design characteristics means that the peak of the ca2+ transients could have been missed, and it is therefore difficult to know how the transient peaks were affected when the action potential peaks declined. The conclusion that the waveform integral above -30 mV is the best predictor for ca2+ release is likely correct, but again the conclusions are rather dogmatic and should be more balanced taking into consideration the limitations of the method.Second, the imaging of ca2+ imaging technique.We have performed new experiments using a PMT tube, which provides for a 5 kHz sampling rate. These experiments confirmed that errors introduced by our slow sampling rate with the camera are modest. We have added a section to the discussion where we mention some of the limitations of our ca


Ultrasonic transducers performance can be seriously deteriorated by loss of adhesion between some constitutive elements such as the active element, the backing, or the matching layer. In the present work, the influence of bonding delaminations on the performance of a single-element ultrasonic transducer, which is composed of a piezoelectric disk, a backing, and a matching layer, is studied numerically and experimentally. Based on the positions between layers, two cases, i.e., delaminations between ceramic and backing or between ceramic and matching layer, are considered. Each case involves three different types of delaminations, which are marked as delamination type (DT)-I, II, and III. DT-I, a circular shape delamination, starts from the center and expands towards the peripheric zone; DT-II, an annular shape delamination, starts from the peripheric zone and expands towards the center; DT-III is a sector shape delamination with a given angle. The numerical simulations are performed by the finite element method and the influence of delaminations on the electromechanical admittance (EMA) of the transducer is investigated. 3D printed backings and matching layers are mounted on a PZT sample to assemble delaminated single-element transducers. An impedance analyzer is used for experimental measurements. Comparison between numerical and experimental results shows a reasonable agreement making changes in EMA an interesting indicator to inform about the occurrence and severity of delaminations in a single-element ultrasonic transducer. Ultrasonic transducers have been widely used for applications, including medical diagnosis and therapy, non-destructive evaluation (NDE), and underwater sonar. The proper functioning of the transducer itself is a key factor in the reliability of the entire system. However, due to the misuse of operators or material degradation, defects may occur, such as breakages in cables, cracks, damaged or weakened crystals, and delamination between layers ,2,3,4. TTraditional methods of detecting the defects in a transducer include visual inspections, electroacoustic tests, and phantom-based measurements. Visual inspection is practical for the detection of visible physical faults. Electroacoustic tests are performed in water with commercially used devices, such as FirstCall and the ProbeHunter system. Transmit–receive signals are collected and analyzed, then parameters for each element, such as the sensitivity, pulse width, center frequency, or capacitance are displayed ,5. The PIn the field of structural health monitoring (SHM), changes in the electromechanical impedance ,14,15,16To this end, a typical single-element ultrasonic transducer consisting of a piezoelectric disk, a backing, and a matching layer is studied numerically and experimentally. Depending on the geometric shape, three types of delaminations between different elements are considered. Their influence on the EMA is investigated and several quantitative indicators are proposed to reveal the occurrence and extent of delaminations.A classical single-element ultrasonic transducer  is compot) and has a diameter (ϕ) of 16 mm. Its resonance frequency (rf) is around 1.71 MHz. Material properties of PZ27 are given in ρ is the density, Z is the acoustic impedance.Here, the PZT sample consists of a soft Ferroperm Piezoceramics PZ27 disk from Meggitt A/S Kvistgaard, Denmark with an electrode on each of its surfaces and polarized along the thickness direction. The PZT sample is 1.13 mm-thick  filamenL) waves in the matching layer. Components are bonded by an acoustic coupler phenyl salicylate  (discrepancy of less than 4%). The thickness of the quarter-wave matching layer is given as e  that isY refers to how easily a circuit will allow a current to flow inside. It is the reciprocal of electrical impedance Z. An electrical circuit composed of the resistance (R), inductive reactance (LX), and capacitive reactance (CX) is depicted in G) and an imaginary part (susceptance B). Relationships between these terms can be expressed asIn the electrical circuit theory, the electrical admittance G(ω) is used to track structural changes in the system.Considering the backing and matching layer as a double-side mechanical loading on the ceramic disk, the structural changes in these passive elements or in the adhesive layers are reflected in its electrical admittance or so-called electromechanical admittance (EMA), owing to the mechanical loading effect of the passive elements. The health state of the transducer is closely related to the damping effect of the resonance characteristics; thus, the conductance ω is the angular frequency, N indicates the number of points, G(ω) is the state at any time, and G0(ω) is the reference state.Traditionally, the statistical based metrics, such as root mean square deviation (RMSD) and damage index (DI), have been widely employed to characterize the changes in EMA ,27,28,29Damage index is defined asRA) and bandwidth (RBW) can also be used to quantify the changes. As we know, conductance reaches its maximum value Gmax, i.e., RA, at the resonance frequency rf. The frequencies corresponding to 1f and 2f, then the −3dB bandwidth ciE as [For passive elements, the loss factors are added to their elastic modulus E as (8)E*=E is modeled between the electrodes and then a frequency domain analysis is performed. The frequency varies from 3 kHz to 2.4 MHz with a 3 kHz incremental step. The admittance is obtained asI(ω) is the current flowing inside the ceramic.Depending on the type of delamination simulated, a 2D or a 3D model is considered. The 2D model is an axisymmetric one. It is meshed with rectangular quadratic Lagrange elements of 9 nodes and a standard of 8 elements per wavelength is adopted to ensure an adequate accuracy . As a reTwo main cases of delaminations are studied in this work. In the first case, delaminations arise in the bonding layer between ceramic and backing, while they arise in the bonding layer between ceramic and matching layer in the second case. For each situation, three delaminations types (DT) are studied that are named respectively as DT-I a, a circSince the three types have different symmetric features, the first two types, i.e., DT-I and DT-II, have been studied through 2D axisymmetric modeling. However, for the DT-III, a three-dimensional (3D) model is required, using a symmetry boundary condition as indicated by the dotted line in η varying from 0% to 100% with a step of 6.25% and η is defined asSimulations are performed with the delamination ratio In this part, the influence of delaminations between ceramic and backing on EMA is studied. FE simulations are carried out for various delamination ratios and the results are shown in η changes. To better illustrate the changes, the peak at Ts1 is taken as an example and has been magnified as shown in η increases, the resonance peaks become thinner and higher. This is due to the fact that as delamination ratio increases, the damping effect of backing is reduced, leading to the increase of resonance peaks. This phenomenon appears not only on the thickness modes but also on the radial harmonics, although they are not as obvious as the former ones.Compared to radial harmonics, changes in the resonance peaks of thickness modes are more obvious as RA and η. Resonance amplitude RA increases but the bandwidth η. On the other hand, it can be observed that the three delamination types show different variation patterns. The pattern of DT-III type is between those of the other two. In terms of the damaging influence of delamination on the performance of the ultrasonic transducer, such as the influence on the bandwidth, DT-I delamination makes the most, DT-III less, and DT-II even less. For example, a half delamination of the adhesive layer gives a decrease of rf = 1.7 MHz) gradually appears and rises up. It seems that the two split peaks merge gradually towards the original T1 mode in the middle. This is because the matching layer is losing its function as η increases.A similar analysis is made for the case when delamination happens between ceramic and a matching layer. RA and In this case, we can no longer use the delamination indicators In summary, EMA measurements can be reversely exploited to predict and reveal the occurrence of delaminations. The location of the delamination can be distinguished by whether the two split thickness peaks have merged. If the delamination happens between ceramic and backing, the severity can be quantitatively evaluated through the variation of resonance amplitude and bandwidth of the first split thickness mode Ts1. If the delamination happens between ceramic and matching layer, the severity can be evaluated through the variation of the values of RMSD or DI over the frequency range covering two split thickness modes. Discrimination of delamination types is made possible by two or more measurements at different times.η values from 25% to 75% are printed and mounted one by one (for example η = 25%). As for the delaminations between ceramic and matching layer, only DT-III type is realized by cutting the matching layer to its three quarter, half, and one quarter sequentially. The admittance curves are measured by the HIOKI IM3570 [As mentioned in I IM3570  impedancRA and η, extracted from the first split thickness mode peak Ts1. A good agreement is observed between numerical and experimental results. The evolution of the delamination indicators RA and RA  method, a systematic study of the influence of bonding delaminations on the electrical admittance—referred to as the EMA—of a single-element ultrasonic transducer is presented. Three different types of delaminations between ceramic and backing or between ceramic and matching layer are considered. Numerical results show that changes in the EMA versus frequency curves are observed as delaminated area increases. Conversely, EMA can be exploited to detect defects inside. For the delaminations between ceramic and backing, the resonance amplitude In summary, EMA can help to detect, locate, and characterize defects in a single-element ultrasonic transducer by qualitative and quantitative assessments during its lifetime. Future work will extend this method to multi-element ultrasonic transducers.


Dear Editor,Overall outcomes are dismal in patients with relapsed/refractory (R/R) acute myeloid leukemia (AML). Alvocidib is a multi-cyclin-dependent kinase (multi-CDK) inhibitor with potent activity against CDK9. CDK9 forms a complex with cyclin T1, positive transcription elongation factor b, which exists in a superenhancer complex to regulate the activity of RNA-polymerase II. By inhibiting CDK9, alvocidib leads to the suppression of RNA-polymerase II-mediated transcription of myeloid cell leukemia-1 (MCL-1), a pro-survival BCL-2 family member that inhibits the intrinsic pathway of apoptosis and promotes leukemia survival , 2.5 months  and 11.8 months , respectively  recovery for R/R was 48  days compared with 45  days in the NDHR AML cohort.Treatment-emergent grade ≥3 non-hematologic adverse events after alvocidib are illustrated in Supplemental Table n = 67; NDHR: 13). In order to assess whether MCL-1 dependence may enrich for overall response to ACM, bootstrap resampling analysis was performed in order to infer population variance of MCL-1 priming results  [P < 0.001), whereas overall response was less frequently observed in the MCL-1 <30% cohort compared with non-responders  . There wP = 0.08). However, the study design was not powered to compare CRc rates between MCL-1-dependent cohorts and small numbers of patients likely precluded statistical significance. Salutary clinical activity was also evident in a high-risk MCL-1-dependent newly diagnosed AML exploratory cohort (CRc = 62%), which appear to be similar to ACM in unselected newly diagnosed poor-risk AML patients [This is the first clinical trial prospectively assessing MCL-1 dependence as a biomarker for AML. We have shown that 47% of R/R AML patients are MCL-1 dependent ≥30%) and may be candidates for MCL-1-directed therapies [0% and mart CRc = 2%, whichDespite recent treatment advances in R/R AML, there remains no standard-of-care salvage regimen, and R/R AML continues to be the highest unmet need in AML. We divided R/R AML patients into 3 distinct subgroups: refractory (induction failure or CR1 duration <3 months), early relapse (CR1 duration 3–12 months), and late relapse (CR1 duration 12–24 months). The majority of R/R AML patients had either refractory or early relapse (84%) compared with late relapse (16%) corroborating the poor-risk group of patients enrolling on this study. In fact, median OS was 16.9 months in patients with refractory MCL-1-dependent AML, which compares favorably to outcomes seen with conventional chemotherapy agents in refractory AML.Previous studies have demonstrated clinical activity of ACM in R/R AML. A randomized phase 2 study performed by the Eastern Cooperative Oncology Group (ECOG) investigated three treatment regimens for R/R AML: ACM, sirolimus plus mitoxantrone, etoposide, cytarabine, and carboplatin plus topotecan. Of the three regimens studied, ACM was the only regimen to meet the primary endpoint of the study with a CR rate of 28% . A compoMCL-1 is an anti-apoptotic member of the BCL-2 family that inhibits BAX/BAK-mediated mitochondrial permeabilization and cell death. MCL-1 is upregulated in AML patients, particularly during relapse, and is a major contributor to AML progression , 9. LeukIn conclusion, ACM showed an acceptable safety profile and demonstrated clinical activity in MCL-1-dependent R/R AML. This is the first prospective trial incorporating a novel BH3 profiling biomarker-based strategy to identify whether MCL-1 dependence can predict for response to CDK9 inhibition. Our findings suggest that prospective analysis of MCL-1 dependence is feasible and can potentially stratify patients into unique biologic subgroups, which may be applicable to other tumor subsets with high dependence on MCL-1 for survival. Future biomarker-based study designs are warranted to determine predictive signatures of response to therapeutic agents targeting BCL-2 family members and other regulators of apoptosis.Supplemental Methods


Taken together, we found that LINC00998 is a novel tumor-inhibiting lncRNA in human AML. The dysregulation of LINC00998/ZFP36/mTORC2 axis is linked to leukemogenesis and progression.Acute myeloid leukemia (AML) is a severe hematologic malignancy that threatens human health. Long non-coding RNA (lncRNA) is emerged as a key player in human cancer. Herein, we explored the role of LINC00998 in human AML. LINC00998 was significantly decreased in human AML, which was linked to relapse and poor prognosis. Stable overexpression of LINC00998 inhibited AML cell viability, colony ability, DNA synthesis rate and increased apoptosis. LINC00998 was mainly located in the cytoplasm, in which interacted with ZFP36 ring finger protein (ZFP36), a mRNA destabilizing factor, resulting in increased decay of mammalian target of rapamycin complex 2 (mTORC2), a well-known proto-oncogene in AML. Overexpression of mTORC2 partly blocked the tumor suppressive effects of LINC00998. Importantly, LINC00998 shortened  Acute myeloid leukemia (AML) is the most common acute leukemia, characterized by accumulation of immature myeloid cells and hematopoietic suppression in the bone marrow . At presFor a long time, the research on non-coding RNA (ncRNA) mainly focused on RNA less than 200 nucleotides, such as microRNA, rRNA and so on. Long non-coding RNA (lncRNA) is a type of endogenous RNA with a length of more than 200 nucleotides . It has http://gepia.cancer-pku.cn/) containing tumor and normal samples from the TCGA and the GTEx databases. LINC00998 was found to be specifically and differentially expressed in AML among 32 types of human cancers. We verified its downregulation in AML in our cohort, and further explored its function and mechanism. Thus, our study aimed to explore the expression, function and clinical significance of LINC00998 in AML, and further reveal the underlying regulatory mechanism of LINC00998.In the current study, we analyzed the GEPIA database (de novo AML (32 cases with relapse) and 59 healthy controls were enrolled from January 2015 to April 2021. The diagnostic criteria for primary and recurrent AML were based on the French-American-British (FAB) and the 2016 World Health Organization classifications [80 cases of patients with ications . Lymphop−ΔΔCt method.The protocol was described in the previous study . Total RThe protocols of functional assays were described in the previous study . In shorThe digestive enzymes were used to prepare AML cell suspension, and then the Annexin V-FITC/PI Apoptosis Assay Kit  was used to determine cell apoptosis. The results were recorded by flow cytometer and analyzed using Flowjo software.The full-length of LINC00998 was cloned into pSIN-EF1α-IRES-puro lentiviral vector, followed by transfection into U937 and THP-1 cells in the presence of 6 μg/mL polybrene. 72 h later, puromycin was added into RPMI-1640 medium to screen stable clones expressing LINC00998.The protocol was described in the previous study . Total pThe protocol was described in the previous study , but wit10 nude mice were randomly divided into 2 groups (n = 5 per group). LINC00998-overexpressing U937 cells were subcutaneously injected into each mice and grown for 4 weeks. The volume of the subcutaneous tumor was weekly measured.Then, all mice were sacrificed and tumor tissues were collected for qRT-PCR and western blot assays. Cell apoptosis was conducted using TUNEL Apoptosis Detection Kit based on supplier’s instructions .P < 0.05 was considered to have statistical difference.Student’s t test was used to analyze the difference between two groups. All functional experiments were repeated independently at least 3 times. Figures were plotted by Graphpad 7.0 software and statistics were done using SPSS 18.0 software. Herein, we explored the role of LINC00998 in AML. We found that LINC00998 was significantly decreased in human AML. Next, we performed a series of functional and animal assays, found that LINC00998 inhibited AML progression through regulation of mTORC2 mRNA stability via binding to ZFP36. Our data uncover the importance of LINC00998 in AML, and provide new insights into the pathogenesis of AML.https://lncatlas.crg.eu/) database, we found that LINC00998 was a cytoplasmic lncRNA , the qRT-PCR results verified the cytoplasmic location of LINC00998 in AML cells (The GEPIA data showed that LINC00998 was notably decreased in AML (n = 173) as compared to normal control (n = 70) . We thenML cells .Figure 1We established stable LINC00998-overexpressed U937 cell lines , and conThrough analyzing TCGA database, we found that LINC00998 was strongly negatively correlated with mTORC2 in AML (r = −0.705) . ConsistWe then tested how LINC00998 affected mTORC2 mRNA level. As shown in Lastly, we conducted subcutaneous injection of U937 cells into nude mice. As shown in in vitro and in vivo. Further, we revealed the mechanism of LINC00998 inhibiting AML progression. In detail, LINC00998 served as a partner of ZFP36, facilitating the binding of ZFP36 on mTORC2 mRNA 3`-UTR, leading to the decay of mTORC2 mRNA. Thus, our data suggest that LINC00998 is a novel negative regulator of AML tumorigenesis, and also highlight the functional implication of lncRNA as a protein binding partner.The etiology of AML is still not fully understood and ongoing research is underway. In this study, we described a novel tumor-inhibiting factor in AML. Endogenous LINC00998 was drastically reduced in human AML, linking to aggressive clinical features and dismal prognosis. The functional assays and nude mice study showed that LINC00998 inhibited AML cell survival Emerging evidence demonstrates that lncRNA is expected to be novel biomarker for early diagnosis, prognostic value and therapeutic target, due to the relative tissue-specific expression ,16. For LncRNA plays a role in a variety of mechanisms, among which its role as a protein binding partner has attracted increasing attention . The mosIn addition, some lncRNAs have been proven to be a key regulator in AML progression, such as ANRIL , HOTTIP Our work unveil the critical role of LINC00998 inhibiting AML growth via the ZFP36/mTORC2 axis. Restoration of LINC0998 may be a potential therapeutic protocol for AML patients.Click here for additional data file.


Acute myeloid leukaemia (AML) is the most common and aggressive form of acute leukaemia in adults. The most common driver mutations in AML are activating mutations in the FMS-like tyrosine kinase 3 (FLT3) gene, occurring in approximately one third of cases . Internap = 0.047, Fig. FLT3-, NRAS-, and BRAF-mutant AML [The landscape of treatment options for AML is rapidly changing, however there remains limited durable treatment options for molecular subtypes such as mutant-FLT3 AML. To identify novel therapeutic targets, we undertook quantitative phosphoproteomic profiling of primary AML blasts (Supplementary Methods) . Furthertant AML . In addip = 0.048, Fig. p = 0.024; Fig. p = 0.024, Fig. p = 0.017) and further decreased following combined FLT3 and DNA-PK inhibition  and the potent, selective DNA-PK inhibitor M3814 (DNA-PKIC50 = 0.0006 μM). M3814 is currently in clinical trial for advanced solid tumours (NCT02516813), and in combination with chemotherapy for relapsed/refractory AML (NCT03983824). Isogenic Ba/F3 cell lines dependent on FLT3 signalling for growth ) were more sensitive to NU7441 compared to either EV cells or WT-FLT3 cells grown in IL-3  or NRAS  mutations all displayed dose-dependent sensitivity to NU7441 and M3814  but not the WT-FLT3 lines  Figs. . SimilarThe combination of NU7441 and sorafenib enhanced the accumulation of cells in G1 phase and significantly reduced the proportion of cells in S and G2M phases, in FLT3-ITD MV4-11 cells but not WT-FLT3 HL-60 cells Fig. S. Similarp = 0.041, Figs. To investigate the clinical relevance of DNA-PK and FLT3 inhibitor combinations, drug sensitivity was assessed by Annexin V flow cytometry in human AML blasts ex vivo. NU7441 combined with sorafenib was synergistic in both wildtype and mutant-FLT3 blasts Figs.  and S8A.in vivo preclinical AML model. FLT3-ITD MV4-11 cells, stably expressing luciferase, were engrafted in wildtype-DNA-PK Nod-Rag-Gamma (NRG) mice (Supplementary Methods). Once mean leukaemia burden reached 2% in the peripheral blood, mice were randomised to receive vehicle, M3814 (150 mg/kg), sorafenib (5 mg/kg), or M3814 combined with sorafenib (150 mg/kg M3814 + 5 mg/kg sorafenib). As a monotherapy, M3814 had no effect on leukaemia burden , 43 days for mice receiving M3814 alone (p = 0.0029), and 42 days for mice receiving vehicle (p = 0.0018, Fig. The potentiation of DNA-PK and FLT3 inhibitors in mutant-FLT3 cells was further shown in an den Fig. . Howeverden Fig. . Biolumiden Fig. . ConsequHerein, the increased phosphorylation of DNA-PK-dependent NHEJ proteins, and decreased phosphorylation of BER pathway proteins, identified by phosphoproteomics in mutant-FLT3 AML blasts Fig.  togetherCollectively, we have shown the utility of quantitative phosphoproteomic profiling of AML blasts for identifying activated pathways and guiding the rational selection of drug target combinations. As DNA-PK displayed activation in the majority of AML blast samples, the use of multikinase inhibitors in combination with DNA-PK inhibitors may provide therapeutic benefit in a range of AML subtypes, not limited to mutant-FLT3 AML. Although the mechanism linking FLT3 with DNA-PK activation remains to be determined, our studies demonstrate that DNA-PK is an attractive therapeutic target in AML. Our preclinical results support the clinical evaluation of DNA-PK inhibitors in combination with FLT3 inhibitors as a novel therapeutic strategy for mutant-FLT3 AML.Supplementary MethodsSupplementary FiguresSupplementary Data


Here, we analyzed mitochondria and CRUs in hearts of adult (4 months old) and aged (≥24 months old) mice. Analysis by confocal and electron microscopy  revealed an age-related loss of proper organization and disposition of both mitochondria and EC coupling units: (a) mitochondria are improperly disposed and often damaged ; (b) CRUs that are often misoriented  and/or misplaced from the correct position at the Z line. Immunolabeling with antibodies that mark either the SR or T-tubules indicates that in aged cardiomyocytes the sarcotubular system displays an extensive disarray. This disarray could be in part caused by the decreased expression of Cav-3 and JP-2 detected by western blot (WB), two proteins involved in formation of T-tubules and in docking SR to T-tubules in dyads. By WB analysis, we also detected increased levels of 3-NT in whole hearts homogenates of aged mice, a product of nitration of protein tyrosine residues, recognized as marker of oxidative stress. Finally, a detailed EM analysis of CRUs (formed by association of SR with T-tubules) points to ultrastructural modifications, i.e., a decrease in their frequency  and size . The changes in morphology and disposition of mitochondria and CRUs highlighted by our results may underlie an inefficient supply of Ca2+ ions and ATP to the contractile elements, and possibly contribute to cardiac dysfunction in ageing.Ageing is associated with an increase in the incidence of heart failure, even if the existence of a real age-related cardiomyopathy remains controversial. Effective contraction and relaxation of cardiomyocytes depend on efficient production of ATP (handled by mitochondria) and on proper Ca The cardiovascular system, as many of the others vital systems of the human body, is a target of age-related cellular insults.Statistics indicate that the risk of heart failure (HF) doubles with each decade of life in individuals aged over 50 , making 2+ ions to the contractile elements. ATP is provided by mitochondria during cellular respiration, and the myocardium is well known to be heavily dependent on the oxidative metabolism [2+ needed for contraction is released by the sarcoplasmic reticulum (SR) during excitation–contraction (EC) coupling, the mechanism that allows transduction of the action potential of transverse (T)-tubules into release of Ca2+ from intracellular stores (the SR). In cardiac cells, EC coupling is based on a mechanism known as Ca2+-induced Ca2+ release (CICR), where Ca2+ entry through Ca.V. 1.2 placed in T-tubules  activates ryanodine receptors (RYRs), the SR Ca2+ release channels [The function of striated muscles  relies on appropriate supply of ATP and Catabolism ,7. Ca2+ channels .2+ release units (CRUs) and are formed by the association of the SR with T-tubules. In skeletal muscle CRUs are also named triads, as they are formed by three elements , which are placed at the transition between the I and the A band of the sarcomere. In cardiac muscle cells, CRUs have a different morphology and disposition [The intracellular units deputed to EC coupling are named Caposition ,10: T-tu2+, is also controlled by their cross-communication: indeed, the importance of a close structural association between intracellular Ca2+ stores (ER and SR) and mitochondria has been described and discussed extensively in the last couple of decades in both non-excitable and excitable cells [2+ released by the CRUs enters mitochondria via the mitochondrial Ca2+ uniporter (MCU) to stimulate the respiratory chain, while the reactive oxygen species (ROS) produced by mitochondria can regulate SR Ca2+ release [The function of mitochondria and CRUs, besides being important for providing independently ATP and Cale cells ,15,16,17le cells : Ca2+ re release .2+ levels caused by dysfunctional EC coupling may cause excessive Ca2+ uptake by mitochondria, which in turn may lead to mitochondrial dysfunction and possibly to death of cardiomyocytes [2+ overload as a key event in HF, as it determines improper ATP production and increased ROS production [Dysfunction of mitochondria and CRUs has been considered a major determinant of age-related cardiac dysfunction ,20,21. Omyocytes ,23. Markoduction .2+ release sites due to improper remodeling of the T-tubular network was reported in failing human heart [2+ content, intracellular Ca2+ levels, and SR Ca2+ release were all reduced in aged cardiomyocytes [2+ ATPases (SERCA) and phospholamban (two proteins involved in SR Ca2+ re-uptake), leading to prolonged activation of contractile proteins in cardiac muscles [2+ release, was proposed to contribute to age related cardiac alterations [Abnormalities in EC coupling have been described in various forms of human HF ,26,27. Can heart . Non unian heart ,20,21. Omyocytes . Advance muscles . Finallyerations .2+ ions to the contractile elements, and possibly contribute to the cardiac dysfunction.In this work, we have studied the morphology and intracellular disposition of both mitochondria and CRUs in cardiac cells of adult (4 months old) and aged (≥24 months old) mice. Our structural results collected either by confocal and electron microscopy (respectively CM and EM) and our biochemical data, obtained by western blot (WB) analysis, highlight age-related modification of the two machineries. A loss of proper organization of both mitochondria and EC coupling membranes suggests that age-related changes could cause inefficient supply of ATP and Can = 3) and aged mice . All procedures and experiments were conducted according to the National Committee for the protection of animals used for scientific purposes (D. lgs n.26/2014) and were approved by the Italian Ministry of Health (992/2015-PR). Animals were sacrificed by cervical dislocation as approved by the Italian D. lgs n.26/2014.Wild type (WT) C57Bl/6 mice were housed in microisolator cages at 20 °C in a 12 h light/dark cycle and provided free access to standard chow and water. All experiments were performed on cardiomyocytes from WT male mice hearts: papillary muscles were used because of their cardiomyocytes better alignment if compared to those of other regions of the heart. Animals were randomly assigned to two experimental groups: adult . Ultrathin sections (~50 nm) were cut using a Leica Ultracut R microtome  with a Diatome diamond knife  and double-stained with uranyl acetate replacement and lead citrate. Sections were viewed in a FP 505 Morgagni Series 268D electron microscope , equipped with Megaview III digital camera  and Soft Imaging System at 60 kV.WT hearts were fixed by left ventricle injection at room temperature (RT) with 3.5% glutaraldehyde in 0.1 M sodium cacodylate (NaCaCO) buffer (pH 7.4) and then stored in the fixative solution at 4 °C. Papillary muscles were then dissected from whole fixed hearts, post-fixed in 2% OsOHearts were fixed by left ventricle injection with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 2 h at RT. Papillary muscles were then dissected, rinsed twice in PBS, incubated for 1 h in PBS containing 1% bovine serum albumin (BSA), 10% goat serum, and 0.5% TRITON X-100 and incubated overnight at 4 °C with appropriate dilution of primary antibody in PBS/BSA. Samples were then rinsed three times in PBS and incubated with a specific fluorochrome-conjugated secondary antibody diluted in PBS solution 1 h at RT and mounted on coverslips with anti-bleach media. Primary antibodies used: mouse anti-RYR2 C3-33 ; rabbit polyclonal anti-TOM20 , rabbit polyclonal anti-Junctophilin 2 (y-15) ; mouse monoclonal anti-caveolin-3 (A-3) . Wheat Germ Agglutinin Alexa Fluor 594 Conjugate  was used to label T tubules. Secondary antibodies used were: Cy3-labeled goat anti-mouse IgG diluted 1:300; Cy3-labeled goat anti-rabbit IgG diluted 1:300; Cy5-labeled goat anti-mouse IgG diluted 1:200, all from Jackson Immuno Research Laboratories . Specimens were viewed and imaged using a scanning laser confocal microscope  interfaced with an inverted Zeiss Axiovert microscope.(1)Apparently empty cytoplasmic space and mitochondrial volume were determined in electron micrographs from transversal sections using the well-established stereology point-counting technique ,33 and r(2)The number of severely altered mitochondria was counted in electron micrographs from transversal sections and reported as percentage of the total number. In each specimen, 10 fibers were analyzed, and in each fiber, 5 micrographs were taken at 18,000× magnification. Mitochondria with any or several of the following ultrastructural alterations were classified as severely altered: (1) mitochondria with clear disruption of the external membrane; (2) severe vacuolization and disruption of the mitochondria internal cristae; (3) mitochondria containing rode-like inclusions, clear matrix and/or lamellar inclusions.(3)2. In each specimen, 10 fibers were analyzed, and in each fiber 5 micrographs were taken at 14,000× magnification. We defined as CRU a T-tubule with associated SR vesicle(s) either single, multiple, or surrounding it.Density of CRUs  was determined in electron micrographs from longitudinal sections and reported as average number over 50 μm(4)2 were determined in electron micrographs from longitudinal sections. In each specimen, 10 fibers were analyzed, and in each fiber 5 micrographs were taken at 56,000× magnification.Size and average number of couplons  over 50 μmFor all quantitative EM analyses electron micrographs of non-overlapping regions were randomly collected from longitudinal or transversal sections of internal fiber areas taken from cardiomyocytes of adult and aged WT male mice:(i)Couplons length  was measured in random micrographs from WT adult and aged cardiomyocytes. Dyads that did not show clear membrane outlines were not photographed.(ii)Average area of individual couplons was estimated for each WT sample group (adult vs aged) assuming that EM section cuts across an approximately “circular” junction produces a random cord of such circles. The average measured chord (y) is related to the diameter of the average circle (D) by the equation: y = πD/4. We have used this equation to calculate the average diameter of each couplon and from that the average area.(iii)The number of RYRs in each couplon was estimated in micrographs from WT adult and aged cardiomyocytes assuming that (a) the couplon is filled with RYR2s, which form ordered arrays touching each other as in  and (b) (iv)The average width of SR cisternae was measured for each WT sample group (adult vs aged) in a high number of SR profile showing clear membrane outline. Three to six lines  were randomly drawn across the SR and measured.For each couplon, we evaluated the following parameters:Morphometric data of couplons size  were obtained using the Analy-SIS software  of the EM digital camera .g, at RT. The supernatant was recovered and processed to quantify total protein content. Protein concentration was determined spectrophotometrically using a modified Lowry method, as previously performed [Whole intact hearts dissected from euthanized adult and aged mice, were placed in a dish, cut longitudinally in two halves, rinsed in PBS to eliminate excessive blood, homogenized in a lysing buffer containing 3% sodium dodecyl sulphate (SDS)  and 1 mM EGTA  using a mechanical homogenizer, and finally centrifuged for 15 min at 900× erformed . Total pt-test to compare means of the two groups (adult vs aged), except for data in Figure 3, where significance was evaluated using a Chi-square test. Normal distribution of data in Tables 1 and 2 was checked with the Shapiro–Wilk normality test (GraphPad Prism) and equality of variance was tested with an F test (GraphPad Prism). Statistical analysis in Tables 1 and 2 was determined using a Mann–Whitney U test (GraphPad Prism).Statistical analysis was determined using a two-tailed unpaired Student’s p < 0.01, or p < 0.05, where indicated.Data are shown as mean ± SEM. Statistical significance was set at We analyzed WT hearts from adult (4 months of age) and aged mice (≥24 months of age) using CM, EM, and WB approaches.We immunostained cardiomyocytes with antibodies marking the position of mitochondria (TOM20) and the position of membrane elements involved in EC coupling, i.e., SR and T-tubules  .Double-immunolabeling with primary antibodies against RYR2 and TOM20 revealed that in adult cardiomyocytes (a) the EC coupling apparatus has an ordered transversal disposition that creates a cross-striated pattern  the average area of contact between SR and T-tubule in each couplon and (b) the approximate number of RYR2s, which would be contained in a couplon of such size, assuming that the whole area of a couplon is filled with RYR2s . Couplons in aged cardiomyocytes are significantly smaller in size compared to those of adult and would contain a significantly lower number of RYR2 D and fou2+, which is finely controlled by CRUs during EC coupling [2+ uptake via MCU [The primary function of the heart is to generate the force needed for the generation of blood pressure and its circulation into vessels. To accomplish this primary physiological function of contraction, cardiomyocytes require Cacoupling , and ATPcoupling ,51. Mito via MCU ,53.Increasing age is a main risk factor for cardiovascular diseases and HF, which in turn represent the leading causes of death worldwide . As cell2+ release  [In the past 10 years, we have investigated age-related changes in CRUs and mitochondria in skeletal muscle fibers . Sedentahe CRUs) ,41, (b) he CRUs) , and (c)he CRUs) . These che CRUs) ,41,55. She CRUs) ,56,57,58he CRUs) ,55.(i)loss of architecture and damage of the mitochondrial network: (i) often mitochondria are found abnormally grouped instead of being packed in longitudinal columns ; (ii) mo(ii)de-modeling of the sarcotubular system: (i) often T-tubules are longitudinal and interrupted causing disarray of the precise cross-striation that characterizes their disposition in adult cardiac cells ; (ii) ex(iii)increased levels of oxidative stress, measured as 3-NT levels, that in principle could contribute to the damage of organelles ; and(iv)CRUs, the sites of EC coupling, are decreased in number, miss-placed from the correct position at the Z line , and smaIn this manuscript, we have taken advantage of the previous experience collected studying skeletal ,42,54,55These age-related modifications are summarized by the cartoon of The role of mitochondrial dysfunction in the pathophysiology of cardiac ageing has been reviewed by several authors ,62,63,64Others point to overgeneration of ROS as the central event causing damage to mitochondrial proteins and DNA and organelle dysfunction . The key2+ entry into mitochondria may underlie excessive production of ROS and mitochondrial dysfunction in ageing and disease [2+ entry would be mediated by MCU [2+ uptake into the mitochondrial matrix has been reported in patients and mice with mutations and knockout of a gatekeeper protein regulating the function of MCU [There is general agreement about the fact that excessive Ca disease ,69. Thisd by MCU ,53: inden of MCU ,71.In our aged hearts, we found that oxidative stress is elevated, as 3-NT, a well-recognized and established marker of excessive production of ROS/RNS in the cell, is markedly increased in specimens from aged mice compared to those of adults . Elevate2+ entry from the extracellular space and release by the SR activates contraction of myofibrils. Ca2+ entry and release are coupled by a mechanism known as CICR [2+ enters through the L-type Ca2+ channel (Ca.V. 1.2), also known as DHPR, located mainly in the T-tubule membranes, then (ii) Ca2+ entry triggers RYR2 to open allowing release of a larger amount of Ca2+ from the SR in the form of discrete release events called Ca2+ sparks [2+-waves, a transient rise of cytosolic Ca2+ concentration which spreads along the cardiac cells. In physiological conditions, though, the synchronous activation of many sparks induced by depolarization, each of them arising from a different CRU, generates a homogeneous transient that leads to uniform activation of the contractile machinery [In cardiomyocytes Ca as CICR : (i) upo+ sparks . In expeachinery ,78,79. Hachinery ,53,54.2+ sparks decrease with age, some authors have reported that the expression of both RYR2 channel and DHPR channel do not seem to be affected [Indeed, recent publications have proposed disruption of the systems mediating EC coupling as possible explanation for the impaired cardiac function and loss of contractility ,81. In aaffected . Howeveraffected .2+ release , and in partial misplacement of some couplons at the A band from the proper correct position at the Z line (2+ release and could in principle result in an inefficient CICR propagation [CASQ2 linked to catecholaminergic polymorphic ventricular tachycardia [The results presented in this article show (i) a progressive disarrangement of the T-tubular system , e Z line . These cpagation ,84. In ohycardia ,48,49.2+ ions to the contractile elements, and possibly contribute to cardiac dysfunction in ageing. However, we must underline the limitation of this study, which is mostly based on structural and biochemical analysis, but not yet supported by functional analysis and mechanistic insights. Indeed, molecular changes underlying the loss of proper architecture of these intracellular organelles are unknown and would definitely require additional investigation to be identified. We only reported some changes in expression of proteins involved in SR/T-tubules docking and in T-tubulation (The dysfunction of cardiac cells caused by ageing is responsible for HF. Our results highlight a loss of proper organization and disposition of both mitochondria and EC coupling systems, and fragmentation of CRUs. These results suggest that these age-related changes may cause inefficient supply of ATP and Cabulation , which cbulation , which i2+ ions to the contractile machinery, and with CRU–mitochondria signaling. In addition, as the intracellular arrangement of organelles is strictly dependent on the integrity and lateral alignment of contractile elements, we could also speculate that the main cause of the disarray of the mitochondrial and sarcotubular systems could be secondary to the structural disarray of contractile elements, visible as misalignment of Z lines (However, based on our results, we could speculate that the changes we described could interfere with proper CICR, with delivery of Ca Z lines  and as a Z lines .


Store-operated channels (SOCs) are highly selective Ca2+ channels that mediate Ca2+ influx in non-excitable and excitable  cells. These channels are triggered by Ca2+ depletion of the endoplasmic reticulum and sarcoplasmic reticulum, independently of inositol 1,4,5-trisphosphate (InsP3), which is involved in cell growth, differentiation, and gene transcription. When the Ca2+ store is depleted, stromal interaction molecule1 (STIM1) as Ca2+ sensor redistributes into discrete puncta near the plasma membrane and activates the protein Ca2+ release activated Ca2+ channel protein 1 (Orai1). Accumulating evidence suggests that SOC is associated with several physiological roles in endothelial dysfunction and vascular smooth muscle proliferation that contribute to the progression of cardiovascular disease. This review mainly elaborates on the contribution of SOC in the vasculature . We will further retrospect the literature implicating a critical role for these proteins in cardiovascular disease.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-022-00829-z. CRAC is considered as prototypic SOC. Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ influx mechanism, expressed in non-excitable and excitable cells, triggered by the depletion of intracellular Ca2+ stores (ER or SR). SOCE is thus necessary to allow the entry of Ca2+ ions to replenish depleted ER/SR and initiate intracellular Ca2+ signals  plays a pivotal role in pulmonary vascular remodeling [2+ entry is responsible for developing pulmonary arterial hypertension [2+ influx in PASMCs, especially Orai2 is found to be a hypoxia-inducible factor-1α(HIF-1α)-dependent [2+ entry in proliferative PASMCs. The authors further found that proliferative pulmonary artery smooth muscle cells have enhanced store-operated Ca2+ entry. Moreover, STIM2 was implicated in increased activity of store-operated Ca2+ entry [2+ entry may attribute to its effect on the augmentation of PASMC proliferation. However, STIM1 was not significantly changed in idiopathic pulmonary hypertension-PASMC [2+ entry and decreased hypoxia-induced PASMC proliferation, suggesting that STIM1 significantly impacts hypoxic pulmonary arterial hypertension [2+ entry, which may be associated with inhibition of TRPC1 and TRPC6 [The imbalance of pulmonary systolic and vasodilation is the cause of pulmonary hypertension. Importantly, pulmonary arterial remodeling is a pathological alteration. Mechanistically, it is involved in the proliferation of pulmonary artery smooth muscle cells (PASMC). Cytosolic free Camodeling . Furthermodeling . Accumulrtension –99. Wangependent . Followi2+ entry . Intereson-PASMC . Previourtension , and CaNrtension . Recentlnd TRPC6 . Similarnd TRPC6 . The corThe role of store-operated channels in cardiovascular diseases has been summarized in Fig. Accumulating evidence indicates that approaches aimed at store-operated Ca2+ entry in the cardiovascular system may be therapeutic. The pharmacological tools have demonstrated the contribution of SOCE constituents in cardiovascular diseases; while useful for initial investigation and analysis, they lack specificity. Although the constituents of SOC were shown to play a central role in Ca2+ signals, their absolute requirement for store-operated Ca2+ entry regulation is helpful to carry out further investigation. The Cav1.2 and colocation with SOC have been reported for the first time in vessel contraction. The effect of functional crosstalk in hypertension and atherosclerosis might be worthy of further study. Besides, Ca2+ originating from SOCE could modulate cell adhesion and ECC by influencing intercalated disk-residing proteins' functions. The role of SOCE in maintaining the intercalated disk structure and function via regulation of protein synthesis, trafficking, and targeting may be a future research direction of arrhythmia. There is a hope that future studies will reveal further signaling molecules regulating cardiovascular Orai1/STIM1 and TRPC abundance and its function. Evaluation of SOC channels will shed new light on the treatment of cardiovascular disease.