An increase in mitochondrial matrix free Ca2+ concentration ([Ca2+]m) predisposes mitochondria to opening of the permeability transition pore (mPTP). Opening of the pore can be delayed by cyclosporin A (CsA), possibly by inhibiting cyclophilin D (Cyp D), a key regulator of mPTP. Here, we report on a novel mechanism by which CsA delays mPTP opening by enhanced sequestration of matrix free Ca2+. Cardiac-isolated mitochondria were challenged with repetitive CaCl2 boluses under Na+-free buffer conditions with and without CsA. CsA significantly delayed mPTP opening primarily by promoting matrix Ca2+ sequestration, leading to sustained basal [Ca2+]m levels for an extended period. The preservation of basal [Ca2+]m during the CaCl2 pulse challenge was associated with normalized NADH, matrix pH (pHm), and mitochondrial membrane potential (ΔΨm). Notably, we found that in PO43− (Pi)-free buffer condition, the CsA-mediated buffering of [Ca2+]m was abrogated, and mitochondrial bioenergetics variables were concurrently compromised. In the presence of CsA, addition of Pi just before pore opening in the Pi-depleted condition reinstated the Ca2+ buffering system and rescued mitochondria from mPTP opening. This study shows that CsA promotes Pi-dependent mitochondrial Ca2+ sequestration to delay mPTP opening and, concomitantly, maintains mitochondrial function.Regulation of mitochondrial free Ca Under physiological conditions, a moderate increase in [Ca2+]m is believed to stimulate key enzymes of the Krebs cycle and oxidative phosphorylation and to drive mitochondrial ATP production to match cellular energy demand [2+]m causes opening of the mitochondrial permeability transition pore (mPTP), a key factor in initiation of cell death [2+]m is a primary mediator in cardiac ischemia and reperfusion (IR) injury, as Ca2+ overloading can lead to apoptosis [Regulation of intra-mitochondrial free calcium  [m: −180 to −200 mV) across the inner mitochondrial membrane (IMM). The ΔΨm in turn is generated by the flow of electrons and proton pumping along the respiratory chain complexes [2+]m increases, this depolarizes ΔΨm, which is compensated by enhanced H+ pumping/extrusion to alkalinize the matrix. Therefore, powerful, dynamic buffering of matrix pH (pHm) and Ca2+ are required to enable sufficient recovery of ΔΨm and to avoid overloading the matrix with a high [Ca2+]. Inorganic phosphate (Pi) has been recognized as a major player in maintaining the trans-matrix pH gradient when accompanied by the effective cotransport of H+ m are the Na+/Ca2+exchanger (NCLX) [+-independent Ca2+ exchanger/Ca2+-hydrogen exchanger (CHE) [2+ would disrupt the delicate balance of the [Ca2+]m and lead to impaired bioenergetics and to opening of the mPTP m, potentially by increased buffering of [Ca2+]m m buffering system have not been investigated systematically. It is worth noting that in a previous study from Chalmers and Nicholls [2+ loading capacity of mitochondria without changing the relationship between free [Ca2+]m and total [Ca2+]m during continuous Ca2+ infusion in isolated rat liver and brain mitochondria. Altschuld et al. m accumulation. Our study sought to answer these questions by (i) examining the effect of CsA during repeated CaCl2 challenges over an extended time-period on mitochondrial Ca2+ buffering, and (ii) by examining the underlying changes in bioenergetics during excessive Ca2+ overload.Adenine nucleotides (AdN: ATP and ADP) have been implicated in the inhibition of Ca opening ,28. A pr [Ca2+]m . Oligomy opening . Cyclosp opening ,32. CsA Nicholls , it was d et al.  proposedi et al.  demonstr2+ buffering and compared its effect with a known matrix buffering component, the AdN pool (OMN+ADP), by monitoring [Ca2+]e, [Ca2+]m, and key mitochondrial bioenergetics variables, ΔΨm, pHm, and NADH (redox state), under conditions of repeated Ca2+ loading. Furthermore, we determined the effect of CsA on the rescue of buffering capability and bioenergetics of failing mitochondria just before mPTP opening. We found that CsA enhanced the sequestration of mitochondrial Ca2+, maintained [Ca2+]m at a steady-state level, and markedly delayed mPTP opening. In addition, CsA preserved ΔΨm, NADH, and pHm during CaCl2 bolus challenges. However, in the absence of Pi, this CsA-induced matrix Ca2+ sequestration was abrogated, and in turn led to the early mPTP opening. The results described herein reveal a novel way by which CsA modulates matrix Ca2+ sequestration to maintain [Ca2+]m, despite increased Ca2+ loading. CsA-mediated Ca2+ sequestration is likely achieved via a Pi-dependent [Ca2+]m buffering system that delays Ca2+-induced mPTP opening.To address our objective, we investigated systematically the effect of CsA on mitochondrial CaAM, tetramethylrhodamine methyl ester perchlorate (TMRM) and 2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECFAM) were purchased from Life Technologies .All chemical reagents were purchased from Sigma-Aldrich , unless stated otherwise. Fluorescent probes Fura-4F, Fura-4FAlbino Hartley guinea pigs of both sexes weighing between 250 to 350 g were procured from Kuiper Rabbit Farm . All procedures were carried out in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals  and were approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.2PO4, 5 mM MOPS, 1 mM EGTA, and 0.1% bovine serum albumin (BSA) at pH 7.15 (adjusted with KOH). The suspension was homogenized at low speed for 20 s in ice-cold isolation buffer containing 5 U/mL protease (from Bacillus licheniformis) and the homogenate was centrifuged at 8000× g for 10 min. The supernatant was discarded, and the pellet was suspended in 25 mL isolation buffer, and centrifuged at 850× g for 10 min. The supernatant was centrifuged further at 8000× g to yield the final mitochondrial pellet, which was suspended in isolation buffer and kept on ice until experimentation. All isolation procedures were performed at 4 °C and all experiments were conducted at room temperature. Protein concentration was determined by the Bradford method and the final mitochondrial suspension was adjusted to 12.5 mg protein/mL with isolation buffer.Mitochondria were isolated from guinea pig hearts as described previously ,35,36. BThe functional integrity of mitochondria was determined by the respiratory control index (RCI) as described before ,37. Mito2+ handling and bioenergetics in normal and Ca2+-overloaded mitochondria, as shown in 2+ handling and delay mPTP opening. To further substantiate CsA-mediated buffering of matrix Ca2+, Protocol B was designed to test the effectiveness of CsA and AdN on rescuing a failing mitochondrial Ca2+ buffering system from imminent mPTP opening. There were five experimental groups: vehicle (DMSO), CsA, ADP, OMN, and OMN+ADP. Experiments were also conducted in the presence of deionized H2O as another vehicle (not shown). Each group was subjected to two different experimental protocols (Protocol A and Protocol B) that differed in the order of treatment and addition of CaCl2 boluses to the mitochondrial suspension in experimental buffer.Two protocols (Protocol A and Protocol B) were used to assess the effect of CsA and AdN on mitochondrial Ca2HPO4, 20 mM MOPS, 1 mM EGTA, 0.1% BSA, and EGTA ~0.036–0.040 µM at pH 7.15 (adjusted with KOH). At t = 30 s, mitochondria were treated with DMSO (1 µM), ADP (250 µM), OMN (10 µM), OMN+ADP or CsA (0.5 µM); at t = 60 s, mitochondria were energized with PA (0.5 mM). At t = 180 s, CaCl2 bolus  was added and subsequent CaCl2 boluses added at 5 min intervals until pore opening (Delayed opening of mPTP (Protocol A): At t = 0 s, the experiment was initiated by suspending 0.5 mg of isolated mitochondria into the experimental buffer containing 130 mM KCl, 5 mM K opening A. Note t2 (20 μM) as described in Protocol A; rescue of mitochondria from mPTP opening with the different treatments was carried out at 1 min of the last CaCl2 bolus in which mitochondria Ca2+ uptake was observed before pore opening (2+ via the Na+/Ca2+ exchanger (NCLX) was prevented by conducting all the experiments in Na+-free conditions. That is, the respiration buffer, mitochondrial substrates, and all reagents/drugs were Na+-free to prevent activation of the NCLX. Some experiments were conducted in the presence of 10 μM CGP 37157 (Tocris Bioscience), an NCLX inhibitor, which ascertained there was no potential Na+ contamination in the respiration buffer from other sources  , ΔΨm, redox state (NADH), and pHm. Fura-4F penta-potassium salt  was used to measure [Ca2+]e. For [Ca2+]m measurements, mitochondria were incubated with Fura-4F AM  for 30 min at room temperature (25 °C) followed by a final spin and resuspension to remove any residual dye. ΔΨm was assessed using the cationic lipophilic dye TMRM  in a ratiometric excitation approach e and [Ca2+]m were made using the fluorescent Ca2+ indicator probe Fura-4F with dual-excitation wavelengths (λex) at 340/380 nm and a single emission wavelength (λem) at 510 nm. Ca2+ fluorescent intensities with Fura-4F are not influenced by background noise , so a background subtraction was unnecessary [340/F380) were used to calculate [Ca2+] using the equation described by Grynkiewicz:  in the buffer was calculated using an online version of MaxChelator program (http://www.stanford.edu/~cpatton/maxc.html) and accordingly, a standard curve was generated for the Fura-4F signal to the free [Ca2+] in the experimental solution by fitting to the Grynkiewicz equation, as described above in Equation 1 e or [Ca2+]m at any given time, t. Global trend-fits were performed in MATLAB  and parameters p1 , p2 , p3 (time lag), p4 (decay time constant), and p5 (steady-state slope) were estimated and optimized using the lsqnonlin and fmincon functions.The ability of mitochondria to sequester Cal et al. . Briefly2+ buffering capacity for the second Ca2+ pulse (a cumulative of 40 µM added Ca2+) was then calculated e/dt and d[Ca2+]m/dt are the rates of change of extra-and intra-mitochondrial free [Ca2+], respectively. d[Ca2+]e/dt and d[Ca2+]m/dt were estimated by evaluating the analytical derivative of Equation (2) using parameter estimates obtained from the trend fits e during repetitive additions of 20 µM CaCl2 boluses at 5 min (300 s) intervals to allow characterization of the detailed kinetics of steady-state Ca2+ dynamics (influx and buffering). 2+]e during CaCl2 pulse challenges, with different treatments. Panels A, B, C and panels D, E, F depict the Ca2+ dynamics profile using Protocols A and B, respectively. Each panel consists of five traces representing different treatment groups  in the presence of approximately 40 μM EGTA (carried over from the isolation buffer). In response to each CaCl2 pulse, an increase in Fura-4F fluorescence intensity was observed, which then returned to a baseline; the steady-state (ss) level is marked by the flat response as mitochondria take up and sequester the added Ca2+. The opening of mPTP is evident by cessation of mitochondrial Ca2+ uptake and a sharp rise in the extra-matrix dye fluorescent intensity. Ca2+ concentrations were determined from the fluorescence ratios using Equation (1).To determine the effect of CsA on matrix Ca2+]e (ss[Ca2+]e) as a function of cumulative added CaCl2 at each pulse   did not significantly increase the ss[Ca2+]e until the sixth to seventh pulse , 140 µM (0.57 ± 0.06 µM vs. 0.77 ± 0.06 µM), and 180 µM (0.71 ± 0.02 µM vs. 0.95 ± 0.1 µM) CaCl2, respectively  . In the  OMN+ADP . Mitochor groups . Importath pulse B, suggesectively B,C. The 2+]e dynamics in the situation in which mitochondrial matrix Ca2+ nearly reached threshold, as determined by the predicted opening of mPTP; in this case we added CsA just before the anticipated mPTP opening. Using Protocol B  were sufficient to induce the release of matrix Ca2+. Addition of ADP or OMN reversed the initial pore opening and delayed matrix Ca2+ release by one to two pulses compared to DMSO. Addition of OMN+ADP also showed a significant reversal of Ca2+ release with reduction in the ss[Ca2+]e (0.74 ± 0.18 µM). Thus, there was a considerable increase in the CRC by OMN+ADP compared to DMSO   D,E, it fectively .2+ uptake, thereby inhibiting a consequent increase in free [Ca2+]e during CaCl2 pulse challenges, leading to an increase in the CRC of the mitochondria. This sustained low ss[Ca2+]e and concomitant increase in Ca2+ uptake are likely explained by enhanced [Ca2+]m buffering to maintain basal [Ca2+]m, which results in a preserved ΔΨm for Ca2+ uptake and greater CRC. To investigate further the potential for CsA on mediating Ca2+ buffering, it was necessary to examine the effects of CsA on matrix [Ca2+]m dynamics in the next set of experiments.Altogether, these results demonstrate that CsA enhances mitochondrial Ca2+ was assessed with Fura-4 AM as described in Materials and Methods. We explored the effect of CsA on [Ca2+]m, under identical conditions and protocols as shown in 2+ buffering was measured as a function of a decrease in Ca2+ fluorescence, reaching a steady-state at approximately 270 s after each bolus of CaCl2 added. The magnitude of mitochondrial Ca2+ uptake for the first CaCl2 pulse (20 µM) was similar in all groups; however, on subsequent additions of CaCl2, the ADP- and/or OMN-treated groups showed faster declines in [Ca2+]m with lower mitochondrial steady-state [Ca2+]m (ss[Ca2+]m) and delayed mPTP opening compared to DMSO : (ΣCa2+uptake-[Ca2+]m)/[Ca2+]m, as described previously m, indicated a greater Ca2+ buffering capacity of mitochondria in the presence of CsA. Consistent with this notion, the calculated matrix Ca2+ buffering capacity (mβCa) in CsA-treated mitochondria was about ten-fold higher compared to DMSO and two-fold higher than with OMN+ADP  becomes overwhelmed by the added boluses of CaCl2 ; but increased Ca2+ uptake without efflux or sequestration can decrease ΔΨm by flooding the matrix with positive charges. To strengthen the thesis that CsA increases the capacity of mitochondria to sequester Ca2+, we next investigated the effect of CsA on mitochondrial bioenergetics. ΔΨm, NADH, and pHm were assessed using the same protocols as described in 2+]m to changes in bioenergetics over time. mPTP opening was marked by a sudden rise in the TMRM signal, indicating maximal depolarization of Ψm. Correspondingly, the oxidation of NADH was marked by a decrease in matrix NADH signal intensity when mPTP opens. m, NADH, and pHm for each experimental condition. The rate of ΔΨm depolarization and NADH oxidation correlated well with the induction of mPTP, as seen in the CRC data. The loss of CRC coincided with total ΔΨm dissipation and NADH oxidation. DMSO-treated mitochondria (control) exhibited rapid Ca2+-induced ΔΨm depolarization and NADH oxidation (black trace) after only a few CaCl2 pulses. Addition of OMN+ADP significantly delayed the Ca2+ induced ΔΨm depolarization and NADH oxidation when compared to DMSO, with 533.3 ± 26.7 nmol Ca2+/mg protein vs. 200 ± 23 nmol Ca2+/mg protein and 546.7 ± 18.9 nmol Ca2+/mg protein vs.173.3 ± 13.3 nmol Ca2+/mg protein Ca2+ capacity, respectively  (m) is known to modulate mitochondrial Pi concentration and thus influence the matrix Ca2+ buffering e, [Ca2+]m, and ΔΨm after adding CsA at different time points. Adding CsA at all three tested time points, markedly delayed the large increase in [Ca2+]e due to mitochondrial Ca2+ release. However, the effect of CsA to prolong Ca2+ uptake, which eventually maintains ss[Ca2+]e at baseline, diminished as the interval before CsA addition and [Ca2+]e accumulation was lengthened  of the succeeding Ca2+ pulses, compared to adding CsA at 2 min (0.67 ± 0.03 µM) and 3 min (0.84 ± 0.05 µM)  and the Ca2+ threshold for pore opening was significantly higher when CsA was added at the early time points  compared to the late time point of 3 min  D; inset.e points B. The raof 3 min E, inset.m profile at the same rescue time points . Adding CsA reversed the Ca2+-induced ΔΨm depolarization even after a large depolarization  is a required component for mitochondrial matrix Ca2+ buffering e and interestingly, after cumulative additions of CaCl2 to 80 ± 15 µM, there was a significant decrease in mitochondrial Ca2+ uptake during additional CaCl2 pulses , pore opening was not prevented in either group (data not shown). The addition of exogenous Pi to the buffer triggered a rapid reversal of Ca2+ release (decrease in ([Ca2+]e) in parallel with complete restoration of ΔΨm  plays two important roles: (i) Activation of Ca2+-dependent dehydrogenases for oxidative phosphorylation at low concentrations [2+ by sequestration of excess Ca2+ at high concentrations [2+]m is a leading factor in inducing mPTP opening. It is well established that repetitive mitochondrial Ca2+ loading triggers a gradual increase in [Ca2+]m, leading to a loss of IMM integrity that results in dissipation of ΔΨm and release of Ca2+. CsA is known to delay pore opening, in part, by inhibiting the PPIase activity of Cyp-D [2+]m by Pi-induced matrix Ca2+ buffering has not been addressed before. In this study, we investigated the effects of CsA on [Ca2+]m regulation during repeated Ca2+ loading and its functional significance in mPTP opening. Additionally, we determined if changes in [Ca2+]m induced by CsA correlated with changes in mitochondrial bioenergetics under identical experimental conditions and if matrix Pi was required for the observed CsA effects.Matrix free m owing to increased mitochondrial Ca2+ buffering capacity; (ii) the effectiveness of CsA to maintain basal ss[Ca2+]m correlates well with preserved mitochondrial bioenergetics; (iii) the buffering effect of CsA in a Pi-replete buffer was more pronounced than the known buffering effect of OMN+ADP; (iv) CsA-induced buffering was abolished in Pi-depleted mitochondria and Pi-free experimental medium. We conclude that the CsA-mediated delay in mPTP opening could, in large part, be attributed to CsA-induced activation of a Pi-dependent mitochondrial Ca2+ buffering system (MCBS), which maintains a low free [Ca2+]m and preserves mitochondrial bioenergetics.Since the key postulate was that CsA contributes to mitochondrial Ca2+]e and [Ca2+]m in response to boluses of CaCl2 in the presence of vehicle (DMSO), CsA, ADP, OMN, or OMN+ADP over time. Our experimental approaches allowed us to define the contribution of CsA in the regulation of [Ca2+]m when CsA was given before the CaCl2 boluses (Protocol A) and at the threshold for pore opening under condition of increased free [Ca2+]m accumulation (Protocol B). Our results clearly indicate that the effect of CsA on delaying mPTP opening is due largely to its efficacy in maintaining free ss[Ca2+]m by activating the MCBS in a Pi-dependent manner, and thereby preclude early mitochondrial Ca2+ overload and delay induction of mPTP opening. Sustained low ss[Ca2+]e in the CsA-treated group indicated increasing mitochondrial Ca2+ uptake driven by the enhanced sequestration of free [Ca2+]m to maintain a transmembrane Ca2+ gradient and a charged ΔΨm that facilitated additional Ca2+ uptake  The quantity of Ca2+ that can be retained, and ii) the Ca2+ threshold level for release when Ca2+ exchangers are blocked or maximally operated [i as a physiological buffer in regulation of [Ca2+]m has been extensively studied [i-mediated Ca2+ sequestration in mitochondria is believed to be achieved by formation of amorphous Ca2+–Pi complexes in the matrix [2+]m at a low level. Hence, sustained [Ca2+]m cyclically promotes more Ca2+ uptake via the MCU due to better preservation of both the Ca2+ gradient and ΔΨm.It is well established that mitochondria are able to sequester large amounts of Caa2+ load ; howeveroperated . The rol studied ,44,45,49e matrix ,50,51, wi plays an essential role in matrix Ca2+ buffering, Pi has also been suggested to induce mPTP opening [2+–Pi precipitation with complex I inhibition and reduced ATP synthase rate during Ca2+ overload [i] decreased the mitochondrial Ca2+ loading capacity m, which in turn would contribute to more Ca2+ uptake and retention by increasing the electrochemical driving force for Ca2+ influx.The importance of mitochondrial matrix Cai et al. ,45. They2+ by mitochondria e and ss[Ca2+]m for an extended period of cumulative CaCl2 additions, and showed a higher Ca2+ threshold for mPTP opening without Pi compared to with Pi  . Thu+ levels . Consequ and pH)  to have P opened . Maintaim levels transiently increased but immediately returned to baseline with each added CaCl2 bolus. This transient depolarization and NADH oxidation with each addition of CaCl2 was not observed in Protocol A. The reason for this is unclear. Nonetheless, the observed transient oxidation of NADH helped to restore ΔΨm after Ca2+ induced transient depolarization before the next CaCl2 bolus  significantly delayed the mPTP opening when compared to ADP or OMN+ADP (Protocol A); (ii) overturned the high amplitude increase in [Ca2+]m (Protocol B); (iii) maintained pHm, redox state (NADH) and basal ΔΨm, which maintains the driving force for more Ca2+ uptake and sequestration; and (iv) activates Pi-dependent mitochondrial Ca2+ sequestration to delay mPTP opening.The salient observation of this study is that CsA mitigated mPTP opening by promoting the maintenance of a low [Ca2+]m below the threshold for mPTP activation. This concept is shown in the scheme presented in i-dependent matrix Ca2+ buffering, which maintains matrix free Ca2+ and enables massive Ca2+ loading capacity, without diminishing the driving force for Ca2+ influx by maintaining ΔΨm. CsA may delay mPTP opening by enhancing Pi-dependent matrix Ca2+ buffering and by inhibiting Cyp D m homeostasis represent a promising strategy to reduce cardiac injury due to Ca2+ overload by delaying mPTP opening and preventing induction of apoptosis.Our study provides a novel insight into how CsA-mediates a delay in mPTP opening by activating the MCBS, which lowers ss[Cang Cyp D ,32. The


While an overall, monophasic increase in [Ca2+]cyt during RV infection has been shown, the nature of the RV-induced aberrant calcium signals and how they manifest over time at the single-cell level have not been characterized. Thus, we generated cell lines and human intestinal enteroids (HIEs) stably expressing cytosolic and/or ER-targeted genetically-encoded calcium indicators to characterize calcium signaling throughout RV infection by time-lapse imaging. We found that RV induces highly dynamic [Ca2+]cyt signaling that manifest as hundreds of discrete [Ca2+]cyt spikes, which increase during peak infection. Knockdown of nonstructural protein 4 (NSP4) attenuates the [Ca2+]cyt spikes, consistent with its role in dysregulating calcium homeostasis. RV-induced [Ca2+]cyt spikes were primarily from ER calcium release and were attenuated by inhibiting the store-operated calcium entry (SOCE) channel Orai1. RV-infected HIEs also exhibited prominent [Ca2+]cyt spikes that were attenuated by inhibiting SOCE, underlining the relevance of these [Ca2+]cyt spikes to gastrointestinal physiology and role of SOCE in RV pathophysiology. Thus, our discovery that RV increases [Ca2+]cyt by dynamic calcium signaling, establishes a new, paradigm-shifting understanding of the spatial and temporal complexity of virus-induced calcium signaling.Like many viruses, rotavirus (RV) dysregulates calcium homeostasis by elevating cytosolic calcium ([Ca Since Ca2+ signaling modulates a wide array of cellular processes, it is not surprising that many different viruses exploit Ca2+ signaling to facilitate their replication, and the resulting dysregulation of Ca2+ signaling causes pathogenesis. Rotavirus (RV), a member of the Reoviridae family, is one of the first viruses shown to elevate cellular Ca2+ levels and has become a widely-used model system to characterize mechanisms by which viruses dysregulate host Ca2+ homeostasis1. RV is a clinically important enteric virus that causes severe diarrhea and vomiting in children, resulting in over approximately 258 million diarrhea episodes and 198,000 deaths in 20162. Hyperactivation of cyclic nucleotide  and Ca2+ signaling pathways is a common strategy among enteric pathogens3. Thus, understanding how RV exploits Ca2+ signaling is key to understanding and combating RV-induced diarrhea.Eukaryotic signal transduction pathways employ a variety of signaling molecules to regulate cellular processes. Calcium , which stimulated subsequent research into how RV alters cellular Ca2+ levels4. RV causes a 2-fold steady-state increase in cytosolic Ca2+, which is due to increased Ca2+ release from the endoplasmic reticulum (ER) and increased Ca2+ influx through host Ca2+ channels in the plasma membrane (PM)5. Elevated cytosolic Ca2+ activates autophagy, which is critical for RV replication, and has wide-ranging consequences to host cell functions, including disruption of the cytoskeleton and activation of chloride and serotonin secretion to cause diarrhea and vomiting5.RV was first reported to elevate cytosolic  and passed through a Qiashredder (Qiagen). Samples were boiled for 10 min at 100 °C in SDS-PAGE sample buffer and separated on Tris-glycine 4–20% SDS-PAGE gels (BioRad). Detected protein bands for each blot were quantified using ImageJ software for gel densitometry measurements of NSP4:GAPDH.RV proteins were detected by immunoblot analysis as previously described, with the following modificationsMA104 cells and HIEs were fixed using the Cytofix/Cytoperm kit (BD Biosciences) according to manufacturer instructions. Primary antibodies were diluted in 1X Perm/Wash overnight at 4 °C. The next day, the cells were washed three times with 1X Perm/Wash solution and then incubated with corresponding secondary antibodies for 1 hr at room temperature. Nuclei were stained with NucBlue Fixed Cell Stain (Life Technologies) for 5 min at room temperature and washed with 1X PBS for imaging.37. Briefly, MA104 cells or the MA104-shRNA expressing cells were seeded and grown to confluency in 6 wells plates. Wells were infected at 10-fold dilutions in duplicate for 1 hr and media replaced with an overlay of 1.2% Avicel in serum-free DMEM supplemented with DEAE dextran, and 1 μg/mL Worthington’s Trypsin, and, for indicated experiments, DMSO vehicle or SOCE drugs38. The cells were incubated at 37 °C/5% CO2 for 48–72 hrs before overlay was removed and cells stained with crystal violet to count plaques.Plaque assays were performed as described previously with the following modificationsTotal RNA was extracted from HIEs wells (in hW-CMGF+ or differentiation media for 4 days) or MA104 cells grown to confluency in a 6-well plate using TRIzol reagent (Ambion). Total RNA was treated with Turbo DNase I (Ambion) and cDNA was generated from 250 ng RNA using the SensiFAST cDNA synthesis kit (Bioline). Quantitative PCR was performed using Fast SYBR Green (Life Technologies) with primers designed using NCBI Primer-Blast  with results presented as mean ± standard deviation. Comparisons used an unpaired Student’s t-test, the nonparametric Mann-Whitney test, or a One-way Analysis of Variance (ANOVA) with Tukey’s post hoc multiple comparisons test where appropriate. Differences were considered statistically significant for p < 0.05. All authors had access to the study data, reviewed, and approved the final manuscript.2+ over several hours during the peak of RV replication39; however, the kinetics of this increase and whether it is a monophasic increases or manifests as discrete Ca2+ transients are not known. To address these questions, we developed a series of cell lines stably expressing GECIs and used these cell lines to perform live-cell Ca2+ imaging over the course of a RV infection23. For the long-term imaging experiments, MA104 cells stably expressing cytosolic GCaMP5G (MA104-GCaMP5G) were seeded into chamber slides and either mock- or RV-infected with strain SA114F (MOI 10), and GCaMP5G fluorescence imaged for ~18 hr (2–20 hpi)  from 3–8 hpi by western blot  for the whole field-of-view (FOV) (~455 µm2) for three replicate infections and time lapse images were acquired once per minute  shows that the increase in cytosolic Ca2+ manifests as discrete and dynamic Ca2+ fluxes from individual or small groups of cells  show that while individual cells display unique characteristics of Ca2+ signaling, they all exhibit similar patterns of Ca2+ signaling . The mean Ca2+ transient amplitude of mock-infected cells was 0.3% (± 0.5% standard deviation) 28. Cells were infected at MOI of 10, 1, or 0.1, and we performed time-lapse Ca2+ imaging and single-cell analysis of the resulting Ca2+ signaling. Infection of cells with native SA114F at different MOIs showed the expected infectious dose-dependent increase in the number of RV-positive cells  and again observed active and dynamic Ca2+ signaling. While virtually every cell exhibited multiple Ca2+ transients over the course of 10 mins, the Ca2+ spike frequency and amplitude were variable from cell-to-cell . OSUv is pathogenic in gnotobiotic piglets but the tissue-culture attenuated OSUa is non-pathogenic40. Mutations in the OSUa NSP4 protein are associated with reduced elevation in cytosolic Ca2+ levels in recombinant NSP4-expressing Sf9 cells40, but the Ca2+ signaling phenotype of these two viruses had not been studied in the context of an infection. Immunofluorescence of OSUa- and OSUv-infected MA104 cells at MOI 1 show a similar number of infected cells , and a third GCaMP6s cell line stably expressing a non-targeted scrambled shRNA. Cells were infected with SA114F (MOI 0.01), and we found that cells expressing NSP4-targeted shRNAs exhibited knockdown of NSP4 protein levels  and used live-cell imaging to measure Ca2+ signaling from ~2–18 hpi. The scrambled shRNA-expressing cells exhibited a similar degree of dynamic Ca2+ signaling as observed in parental MA104 cells , and at 1 HPI replaced the media with either normal media containing 2 mM Ca2+, media without Ca2+ (supplemented with 1.8 mM EDTA), or media supplemented with Ca2+ for a 10 mM final concentration. The imaging data shows that decreasing extracellular Ca2+ strongly reduced the number and duration of Ca2+ signaling , in which R-GECO1.2 is a red fluorescent cytoplasmic GECI and GCEPIAer is a green fluorescent ER-targeted GECI41. As above, infection with SA114F (MOI 1) induced highly dynamic cytoplasmic Ca2+ signaling by ~8 hpi, as illustrated in two representative single-cell traces , and the sodium-calcium exchanger (NCX)12. To determine which pathway(s) were important for the dynamic Ca2+ spikes in RV infection, we used pharmacological blockers targeting each pathway . MA104-GCaMP5G cells were infected with SA114F (MOI 1), and then treated with different concentrations of the blockers at 1 hpi and imaged to measure GCaMP5G fluorescence. None of the blockers exhibited cytotoxic effects to uninfected cells (data not shown). Cells treated with DMSO as a vehicle control exhibited the dynamic Ca2+ spikes as above . We also noted that RV-infected cells treated with 10 μM KB-R7943 underwent cell death more frequently than any other treatment, which was marked by a rapid increase in cytosolic Ca2+ and then lysis  for the ability to block thapsigargin-induced SOCE and found that all of them showed a similar inhibition of Ca2+ entry after ER store depletion , and Synta66 treatment also caused a modest increase in NSP4-20  cultures from human intestinal stem cells and are non-transformed cells, which make them a biologically relevant system to study the GI epithelium45. Thus, we sought to determine if the dynamic cytosolic Ca2+ signaling observed in MA104 cells were also observed in HIEs with RV infection.Although MA104 cells provide a robust model for RV replication and form a single epithelial sheet ideal for microscopy studies, they are neither of human nor of intestinal cell origin. Human intestinal enteroids (HIEs) have been developed as a model 2+ in the enteroids, we treated 3D jHIE-GCaMP6s stabilized in a diluted Matrigel, with carbachol, a known Ca2+ agonist. Carbachol treatment of jHIE-GCaMP6s significantly increased GCaMP6s fluorescence 200–300% over the mock-treated jHIE-GCaMP6s  using lentivirus transduction. To test the response of GCaMP6s to cytoplasmic CaP6s Fig. . Thus, j2+ dynamics during RV infection as observed in MA104-GCaMP5G cells. jHIE-GCaMP6s enteroids were mock- or RV-infected with the human RV strain Ito, seeded into chamber slides in diluted Matrigel, and imaged every 2–3 minutes for phase contrast and GCaMP6s fluorescence throughout the RV infection (~16 hrs). At 24 hpi, the HIEs were fixed and immunostained for RV antigens to confirm successful infection, which is evident by both infected cells within the HIEs as well as strong positive staining of the dead cells sloughed from the HIEs . Yet, what is paradigm changing is that at the individual cell level, RV does not merely cause a steady increase in cytosolic Ca2+, but rather activates a cacophony of discrete Ca2+ signaling events. Further, by generating GECI-expressing HIEs, this study is the first characterization of RV-mediated Ca2+ signaling in normal, human small intestinal enterocytes. We found that the prominence of the Ca2+ spikes in RV-infected HIEs is similar to that in MA104 cells, underlining that this is a biologically relevant phenomenon.A hallmark of RV infection, and several other viruses, is an elevation in cytosolic Ca2+ as a series of discrete, transient Ca2+ signals is an important new insight into the cellular pathophysiology of RV infection. Transient increases in cytosolic Ca2+ serve as pro-survival signals by activating phosphoinositide 3-kinase (PI3K) and by calcineurin-dependent NFAT activation. Further, Ca2+ oscillations stimulate mitochondrial Ca2+ uptake that enhances ATP synthesis, and this increase in mitochondrial metabolism contributes to cell survival pathways. In contrast, strong sustained elevation of cytosolic Ca2+ drives pro-apoptotic signaling through mitochondrial Ca2+ overload20. Thus, even though the mean cytosolic Ca2+ level is progressively increasing in the RV-infected cell, early activation of the intrinsic apoptotic cascade may be prevented because it occurs as hundreds of transient Ca2+ signals over hours. This premise is consistent with studies showing that early activation of PI3K during RV infection delays apoptosis47. Concomitantly, the elevated Ca2+ signaling activates cellular pathways, such as autophagy, that promote RV replication and assembly of progeny virus33. Whether the initial Ca2+ dynamics enhance mitochondria bioenergetics or ATP synthesis early during RV infection has not been studied, but a loss of mitochondria membrane potential and decrease in ATP output occur at late stages of infection48. Ultimately the massive increase in Ca2+ signaling damages the cell and triggers cell death, and cell lysis was observed in our time-lapse imaging, but this data cannot differentiate whether this was through apoptosis, necrosis, and/or pyroptosis49. Thus, RV exploitation of discrete Ca2+ signals, rather than a sustained increase in cytosolic Ca2+, may function in concert with other RV anti-apoptotic proteins, such as NSP1, to forestall the onset of cell death and enable sustained viral replication.The characterization of the RV-induced increase in cytosolic Ca2+ imaging of individual cells throughout the RV infection. The increased Ca2+ spikes are the predominant feature of this imaging, and the SA11-mRuby reporter virus demonstrates that the onset of the increased Ca2+ signaling correlates with RV protein synthesis as well as input virus dose. Yet, despite individual cells exhibiting very distinct Ca2+ signaling traces, a general pattern emerges: [i] early in infection Ca2+ signaling remains at basal levels; [ii] onset of increased Ca2+ signaling is characterized by relatively low-amplitude Ca2+ spikes; and then [iii] very high-amplitude Ca2+ spikes become predominant, which results in elevated cytosolic Ca2+ levels. The progression of the RV-induced Ca2+ signals have important implications for characterizing how Ca2+-regulated cellular processes are influenced by RV infection. For example, RV induces autophagy through increased cytosolic Ca2+, thereby activating calcium/calmodulin-dependent kinase kinase-β (CaMKKβ)33. This raises several questions: When after onset of the aberrant Ca2+ signaling is CaMKKβ activated? How many Ca2+ signals are needed to induce autophagy? Similar questions can be asked about the role of these Ca2+ signals in RV-induced apoptosis, cytoskeletal rearrangement, and the serotonin and chloride secretion that causes RV diarrhea51. Tracking these dynamic relationships poses a challenge that may be addressed by further engineering GECI-expressing cell lines/HIEs to express other biosensors such that both processes can be measured throughout the infection.Due to GECI photostability, we were, for the first time, able to perform Ca2+ stores based on a blunted cytosolic Ca2+ release in response to agonists  or thapsigargin treatment to prevent SERCA-mediated refilling52. However, other results show increased in radioactive 45Ca2+ loading into the ER in RV-infected cells, which is hypothesized to be due to an increase in Ca2+ binding proteins 11. Thus, controversy remains about whether RV causes a decrease in the ER Ca2+ store. To address this question, we developed MA104-RGECO1.2/GCEPIAer cells to directly measure cytosolic and ER Ca2+ together during the RV infection. RV induces a dynamic decrease in ER Ca2+ levels that occurs in conjunction with the increase in cytosolic Ca2+ signaling. In most instances, the cytosolic Ca2+ spike correlated with a decrease in ER Ca2+, indicating release of ER Ca2+ substantially contributes to the increased cytosolic Ca2+ signaling. Further, despite the 30% reduction in steady-state ER Ca2+, the dynamic nature of the ER Ca2+ signaling suggests that SERCA pumps continually work to refill the ER. The observed depletion of ER Ca2+ levels is consistent with the blunted cytosolic response to Ca2+ agonists like ATP, the NSP4 function as a Ca2+-conducting viroporin in the ER, and the activation of the ER Ca2+ sensor STIM110. In contrast, it is more difficult to reconcile the previously observed increase in 45Ca2+ loading into the ER with the 30% reduction in steady-state ER Ca2+ levels detected by GCEPIAer imaging in this study. It has been hypothesized that increased 45Ca2+ loading may be due to increased ER Ca2+ buffering capacity, caused by the high levels of RV VP7 and/or chaperones BiP and endoplasmin11. However, our data suggest this is unlikely to be the case because this would sequester Ca2+ and render GCEPIAer unresponsive to changes in ER Ca2+ 24, yet this is not the case because GCEPIAer remains dynamic throughout the infection. Alternatively, the increase 45Ca2+ may reflect loading into the ER-derived autophagy-like microdomains that surround viroplasms, which we observed form after the initial depletion in ER Ca2+ and during a partial recovery of ER stores53. These ER microdomains are the site VP7 assembly onto nascent RV particles, which requires high Ca2+, so Ca2+ sequestration in these microdomains may occur independently of the rest of the ER. Future studies using GCEPIAer and viroplasm-targeted GECIs are needed to determine whether the ER and viroplasm-associated membranes are functionally distinct compartments.Many studies show that RV infection (or NSP4 expression) reduces the ER Ca2+ homeostasis through the ion channel function of iNSP4 and Ca2+ agonist function of the secreted eNSP4 enterotoxin10. Our data show that NSP4 governs the dynamic Ca2+ signaling induced by RV infection since NSP4 knockdown significantly abrogated the number and amplitude of the Ca2+ spikes. Unfortunately, it is not possible to determine the relative roles of iNSP4 versus eNSP4 in the induction of the Ca2+ spikes from these data because the shRNA decreased total NSP4 synthesis, and therefore both pathways would be attenuated. The importance of NSP4 for the Ca2+ signaling is also demonstrated by the extremely different Ca2+ signaling profiles of OSUa- and OSUv-infected cells. These differences correlate with the attenuated elevation of cytosolic Ca2+ caused by recombinant OSUa NSP4 both when expressed in Sf9 cells  and exogenous treatment of cells 40. The attenuated NSP4 phenotype is the result of mutations in the NSP4 enterotoxin domain, indicating that this domain is critical for induction of the Ca2+ spikes by OSU40. However, it is important to note that these two viruses are not isogenic so the genetic backgrounds of the OSUa and OSUv NSP4 are different, requiring further Ca2+ imaging studies using recombinant RV bearing these attenuating NSP4 mutations to fully dissect the relative importance iNSP4- and eNSP4-mediated Ca2+ signaling and whether there is a difference in Ca2+ signaling between pathogenic and non-pathogenic RV strains.The pleiotropic functions of NSP4 are responsible for the RV-mediated dysregulation of host Ca2+ signaling, yet these signals are maintained through host Ca2+ channels and signaling pathways both in the ER and PM. Removal of extracellular Ca2+ significantly attenuated the Ca2+ spikes, demonstrating that Ca2+ influx is crucial for these signals. Three classes of Ca2+ channels  have been implicated RV-induced Ca2+ influx23. Our results using different pharmacological blockers indicate SOCE is the primary Ca2+ influx pathway that supports the RV-induced dynamic Ca2+ spikes, both in MA104 cells and in HIEs. Blocking SOCE significantly reduced the number and amplitude of the RV-induced Ca2+ spikes. Since elevated Ca2+ levels are critical for RV replication, the attenuated Ca2+ signaling caused by the SOCE blockers significantly reduced RV yield33. Interestingly, blocking SOCE in HIEs significantly reduced the Ca2+ spikes, but the effect was less pronounced than in MA104 cells, suggesting other pathway(s) may exist that support RV-induced Ca2+ spikes in HIEs. Further, RV can also infect other cell types, including monocytes and macrophages, hepatocytes, cholangiocytes, and enteroendocrine cells, all of which express a different repertoire of Ca2+ channels56. Thus, characterizing the nature of RV-induced Ca2+ signaling in these cell types will help elucidate whether these signals are involved in diseases caused by RV.NSP4 is the trigger of the dynamic Ca2+ release and Ca2+ entry through the PM to the Ca2+ spikes observed in the long-term Ca2+ imaging studies. Our results from dual imaging of both cytosolic Ca2+ and ER Ca2+ showed that many of the cytosolic Ca2+ spikes coincide with ER Ca2+ troughs, indicating that ER Ca2+ release contributes to a large number of these events. However, some cytosolic Ca2+ spikes were observed that did not correspond with a decrease in ER Ca2+, which would suggest these events involve Ca2+ entry. ER Ca2+ release could occur either through iNSP4 channel activity or activation of the IP3-Receptor Ca2+ channel, and our studies with Orai1 blockers indicate that SOCE is responsible for Ca2+ entry-mediated cytosolic spikes. Nevertheless, future studies with genetic knockout of IP3-Receptor or Orai channels, as well as engineered mutation of NSP4 using the new RV reverse genetics system, will be needed to dissect the contribution of iNSP4, IP3-Receptors, and SOCE to the cytosolic Ca2+ signaling. Furthermore, these studies will help determine whether Ca2+ signals generated from these different channels selectively regulate downstream pathways important for RV replication .One unresolved question is the relative contribution of ER Ca2+ homeostasis by a massive and progressive increase in discrete Ca2+ signaling events, mainly from ER Ca2+ release. Many viruses elevate cytosolic Ca2+ and alter ER Ca2+, leading us to question whether dynamic Ca2+ spikes, as seen in RV infection, is a common manifestation for virus-induced Ca2+ signaling. If so, the host channels that support these Ca2+ signals, such as Orai1, may represent novel targets for broadly acting host-directed antiviral therapeutics.In summary, RV dysregulates host CaSupplementary Figures and Video LegendsSupplementary Video 1Supplementary Video 2Supplementary Video 3Supplementary Video 4Supplementary Video 5Supplementary Video 6Supplementary Video 7Supplementary Video 8Supplementary Video 9Supplementary Video 10


Acute Myeloid Leukemia (AML) is the most common form of leukemia in adults with an incidence of 4.3 per 100,000 cases per year. Historically, the identification of genetic alterations in AML focused on protein-coding genes to provide biomarkers and to understand the molecular complexity of AML. Despite these findings and because of the heterogeneity of this disease, questions as to the molecular mechanisms underlying AML development and progression remained unsolved. Recently, transcriptome-wide profiling approaches have uncovered a large family of long noncoding RNAs (lncRNAs). Larger than 200 nucleotides and with no apparent protein coding potential, lncRNAs could unveil a new set of players in AML development. Originally considered as dark matter, lncRNAs have critical roles to play in the different steps of gene expression and thus affect cellular homeostasis including proliferation, survival, differentiation, migration or genomic stability. Consequently, lncRNAs are found to be differentially expressed in tumors, notably in AML, and linked to the transformation of healthy cells into leukemic cells. In this review, we aim to summarize the knowledge concerning lncRNAs functions and implications in AML, with a particular emphasis on their prognostic and therapeutic potential.  Acute myeloid leukemia (AML) is an aggressive malignancy of the hematopoietic system caused by malignant transformation of hematopoietic stem- or progenitor cells. AML is the most common form of acute leukemia in adults with an incidence of 4/100,000 cases per year . HistoriArbitrary defined as transcripts of over 200 nucleotides without coding potential, lncRNAs were originally considered as dark matter due to their low expression and poor conservation across species compared to their messenger RNAs counterparts ,14,15,16Because of their heterogeneity, much debate remains to find the best classification system for lncRNAs that are currently defined according to their genomic location. Indeed, lncRNAs can be divided into sense  or antisAs illustrated in Nuclear lncRNAs are implicated in epigenetic and transcriptional regulations by recruiting activator or repressor chromatin-modifying complexes and transcription factors onto target genes ,31. MostRegulation of gene expression is crucial during hematopoietic differentiation . LncRNAsConcerning the potential role of lncRNAs as diagnostic biomarkers, the most inspiring example is PCA3 (Prostate Cancer Antigen 3) for prostate cancer diagnosis. It is a prostate specific lncRNA overexpressed in prostate cancer patients. Found in the urine of most cancer patients compared to healthy patients, it is undetectable in other tumor types ,48. AlthIn AML, several lncRNAs have been characterized and described as oncogenes  or tumorThe best described function of lncRNAs is their involvement in epigenetic and transcriptional regulation. They are known to act as guides, leading to the recruitment of proteins or complexes to chromatin sites to regulate downstream gene expression ,31. INK4bp15 locus . P. P145]. Amongst the top upregulated LSC-associated lncRNAs, Bill et al. identified the promising lncRNA DANCR whose knock-down in LSCs resulted in decreased stem-cell renewal and quiescence, suggesting its oncogenic role. The delivery of nanoparticles containing siRNAs against DANCR prolonged the survival of AML mouse model even after serial transplantation . Another supportive case of targeting lncRNAs by siRNAs is UCA1, where molecular evidence has established that there is a close relationship between UCA1 and Adriamycin resistance in pediatric AML cases . By usinAnother synthetic oligonucleotide-based molecular product used to knock-down lncRNA expression has been developed: the Antisense oligonucleotide (ASO). ASO (as GapmeRs) are single-stranded DNA oligonucleotides duplexing by base complementarity their target lncRNAs to promote their degradation by RNase H ,153. For example, ASO has been used in De Clara et al. study to knock-down the newly identified XLOC_109948 lncRNA. In this study, they showed that low XLOC_109948 expression is correlated with a good prognosis especially for NPM1-mutated AML patients and that its downregulation using ASO in NPM1-mutated AML cells treated with Ara-C or ATRA enhances apoptosis suggesting a role for XLOC_109948 in drug sensitivity . Targeting lncRNAs implicated in chemoresistance with interference strategies could improve drug response and outcome. Curna Inc., MiNA Therapeutics, RaNA Therapeutics Inc. and others are taking steps towards the development of oligonucleotide-based strategies . The greCatalytic degradation is another therapeutic strategy. Ribozymes are single-stranded RNA able to target and cleave RNA in a site-specific manner ,157. AntAptamers could also provide greater specificity. These single stranded DNA folded into secondary and tertiary structures are able to bind a wide range of molecules, hiding specific-binding structures to limit activity. The chemical structure of aptamers could be modified to enhance their stability and half-life. Aptamer-based -therapeutics are undergoing clinical trials for different indications  . In 2004Through their ability to bind proteins to regulate chromatin organization, transcription, and translation, Fatemi et al. proposed innovative therapeutic strategies based on Small molecule Inhibitors able to disrupt lncRNA-protein interactions ,161. A sRe-expression of the specific tumor suppressor lncRNA may be induced by common gene therapy strategies, packaging the whole transcript into viral or non-viral delivery tools. Recombinant viral systems used in gene therapy have been extensively reviewed previously . Howeverα oncoprotein in APL cells and restores the process of myeloid differentiation in those cells [Even though its implication as AML clinic target was not proposed yet, HOTAIRM1 lncRNA seems to be a promising candidate in APL treatment. Indeed, as aforementioned, enhanced HOTAIRM1 expression induces degradation of PML-RARse cells . RestoriNew Genome editing strategies such as CRISPR/Cas9 are developing very fast and seem to be a powerful tool to knock-in or knock-out lncRNA candidates . ModifieA great challenge in cancer therapy is to selectively kill tumor cells without harming healthy cells. As H19 lncRNA is highly upregulated in several cancers, a plasmid containing diphtheria toxin under the control of H19 regulatory sequences has been designed to selectively target tumor cells which overexpressed H19 compared to normal cells. Diphtheria Toxin is massively produced and kills H19-overexpressing tumor cells selectively. This construct leads to an overall reduction in tumor size and is under clinical trials for ovarian (NCT00826150), pancreatic (NCT00711997) and bladder cancers (NCT00595088) . This coFinally, given their tissue specific expression, using the same strategy with the promoter of a myeloid specific lncRNA could lead to the development of a new tool to target selectively myeloid lineage to treat AML patients.There is no doubt as to the role of lncRNAs in leukemia development, progression, and drug resistance. However, due to the limited number of studies in AML, their application as therapeutic agents in clinical routine is still at its beginnings. Few results have been replicated across cohorts, probably due to no-standardized sample collection and quantification techniques, but also as a consequence of AML biological complexity, characterized by spatio-temporal relationships between the coding and non-coding genome. Adequate sample sizes, well-designed cohort studies, and validation of the results in independent cohorts are needed to confirm their clinical usefulness. The functional and molecular characterization of lncRNAs is also needed to select the most promising lncRNA targets and to design the best IncRNA therapeutic tool with appropriate efficacy and safety profile. Translating this knowledge into clinical practice still represents a tremendous challenge.


The major aim of this review is to provide a definitive overview of the links and cross talk between β-adrenergic stimulation, mechanical load, and arrhythmogenesis in the setting of HF. We will initially review the role of Ca2+ in the induction of both early and delayed afterdepolarizations, the role that β-adrenergic stimulation plays in the initiation of these and how the propensity for these may be altered in HF. We will then go onto reviewing the current data with regards to the link between mechanical load and afterdepolarizations, the associated mechano-sensitivity of the ryanodine receptor and other stretch activated channels that may be associated with HF-associated arrhythmias. Furthermore, we will discuss how alterations in local Ca2+ microdomains during the remodeling process associated the HF may contribute to the increased disposition for β-adrenergic or stretch induced arrhythmogenic triggers. Finally, the potential mechanisms linking β-adrenergic stimulation and mechanical stretch will be clarified, with the aim of finding common modalities of arrhythmogenesis that could be targeted by novel therapeutic agents in the setting of HF.Heart failure (HF) is associated with elevated sympathetic tone and mechanical load. Both systems activate signaling transduction pathways that increase cardiac output, but eventually become part of the disease process itself leading to further worsening of cardiac function. These alterations can adversely contribute to electrical instability, at least in part due to the modulation of Ca HF can be defined as HF with preserved ejection fraction (HFpEF), when diastolic dysfunction plays a major role, or HF with reduced ejection fraction (HFrEF). HF with reduced ejection fraction has been associated with elevated sympathetic tone and mechanical load . Both syThe first documentation of alterations in the sympathetic signaling in chronic HF was when a decrease in concentrations of the sympathetic nervous neurotransmitter, norepinephrine, was shown in the failing human heart . Since tThe hyperadrenergic state is in large part caused by an imbalance of autonomic reflex responses to early alterations in cardiac and peripheral hemodynamics . In HF, Arrhythmias are a major cause of mortality in HF patients, and sudden cardiac death has previously been linked with a higher NYHA class . Further2+ dynamics, β-adrenergic stimulation and stretch at the level of the single cardiac myocyte. It is hoped that information gained in this field will ultimately lead to novel strategies that could improve our therapeutic arsenal against HF.In this review, we will focus on the roles that altered sympathetic stimulation as well as mechanics may have on arrhythmogenic phenotype in patients with HF with reduced ejection fraction, concentrating on alterations of Ca2+.Before discussing arrhythmogenic mechanisms in HF we need to understand the basic mechanisms of arrhythmogenesis and the link to CaFigure 1). These oscillations in the membrane potential can lead to either triggered activity and/or functional block which may encourage re-entry circuits , while the Ca2+-activated Cl- current (IClCa) appears to contribute in some species  which may also contribute to the arrhythmogenic nature of these afterdepolarizations  will likely be negated by the neighboring cells not experiencing afterdepolarizations (the sink). Elegant work from the laboratory of James Weiss has investigated this phenomenon and has shown that chaotic EADs are able to synchronize globally when the tissue is smaller than a critical size. However, when the tissue is large enough, complete synchronization of EADs can no longer occur and this results in regions of partial synchronization that shift in time and space, that can act as foci for arrhythmia . Furtheractivity .2+ control of repolarization, and therefore also of arrhythmogenesis is more of a local rather than a global phenomenon. Both functional and structural microdomains dictate local Ca2+ concentrations, gradients and effector proteins. As these domains appear to be affected in HF, especially when we consider β-adrenergic stimulation and stretch we must also consider how this local control occurs.Over recent years it has become apparent that Ca2+ is centrally involved in many processes including excitability, contraction, and regulation of gene expression. Such diversity of functional roles postulates the existence of dedicated microdomains in which Ca2+ signals are generated independently of cytosolic Ca2+ concentrations and sensed by macromolecular signaling components localized to these microdomains. Besides a functional component, Ca2+ microdomains are often physically delimited by specialized membrane structures and subcellular compartments. Specialized structures include dyadic junctions between transverse membrane invaginations (T-tubules) and SR, sarcolemmal domains outside dyads such as lipid rafts and caveolae, and intracellular structures such as tethered junctions between SR and mitochondria. Interestingly, compartmentalization of proteins that generate or regulate microdomain Ca2+ signaling is dynamic, often as a cause or consequence of disease. For example, the LTCC, or nNOS coupled to RyR, can translocate from the dyad to the sarcolemma which alters their function, presumably by coupling to different signaling complexes  causes dyssynchrony of subcellular Ca2+ release during systole  . Individase unit . When anvate RyR . Not all systole . During 2+ release on Ca2+-regulated membrane currents. Negative feedback through Ca2+ release-dependent inactivation of LTCC serves as a mechanism to limit Ca2+ influx during the initial phase of the AP   .2+ is NOX2, a membrane-bound NADPH oxidase that resides in T-tubules. NOX2 is induced by fast pacing and stretch, and activates RyR via S-glutathionylation  coupling underscoring the complexity of cardiac NO signaling . NO exerinotropy . The mec of LTCC . nNOS isLTCC . Molecularly, the PKA signaling cascade is clearly defined. Upon activation, β-adrenergic agonists stimulate adenylate cyclase via Gs-coupled proteins raising cAMP levels that activates PKA (2+ influx) and PLB (accelerating SR Ca2+ uptake), results in enhancement of SR Ca2+ load. In cardiac myocytes, the compartmentation of cAMP signaling has been attributed to different β-AR subtypes that have distinct subcellular locations. β2 receptors are preferentially located at T-tubules where they co-localize with LTCC in caveolae, while β1 receptors are distributed more globally across T-tubules and surface sarcolemma. Using FRET sensors for cAMP, it was demonstrated that selective β1 stimulation generates cAMP signals that propagate throughout the cell, whereas the β2 AR signal is locally confined in T-tubules and specifically regulates LTCC during CICR . Patients with CPVT carry mutations in RyR that increase the open probability of the receptor, or in calsequestrin (Casq2) where SR Ca2+ buffering is hindered and/or the interaction of Casq2 and RyR is altered , and the enhanced SR Ca2+ by catecholamines , and modeling and voltage-clamp analysis explained the ICaL facilitation by a reduction of Ca2+ dependent inactivation of ICaL. The feedback of DAD on ICaL caused significant BVR. Furthermore, because of the prolonged AP after a DAD, new Ca2+ waves could be generated before the end of repolarization and form EADs.In conditions of excessive Caaneously . A study2+ sparks and waves that occur due to spontaneous (not triggered) openings of RyR clusters during diastole is well known , particularly in settings of HF with prolonged AP and increased CaMKII activity.The phenomenal observation of Call known . Triggerpression . CaMKII pression , which c2+ window currents through Ca2+-dependent feedback. The mechanism is independent of spontaneous Ca2+ events (unlike the mechanisms discussed above) but reflects local feedback of SR Ca2+ release on LTCC during CICR. In the classical view, EAD are caused by voltage-dependent recovery of inactivated LTCC. Inactivation and recovery of LTCC also have a Ca2+-dependent component . NOX2 activation has been proposed as the principle mechanosensor underlying the initial fast response of Ca2+ sparks to stretch  have been functionally demonstrated in ventricular myocytes at the whole-cell and single-level  channel family. The activation of TRP channels is polymodal, and some members are directly activated by membrane deformation , althougignaling . The micignaling . It has ignaling .In vivo, intrinsic force adaptation is modulated by sympathetic activation by imposing a higher load on the heart through modulation of vascular tone. The myocyte can respond to higher mechanical and adrenergic stress through activation of mechanotransduction and adrenergic signaling networks, as discussed above and depicted in Figure 2, but interactions have not been systematically investigated. The mechanotransduction pathway shows both the rapid preload-induced NOX2 and slower afterload-induced NO branch, that most probably operate independently .At the gross structural level, the geometry of the heart changes as a result of HF, becoming less elliptical and more spherical . HF is aK1, which contributes to maintaining the resting membrane potential as well as contributing to terminal repolarization  show altered Na+ and Ca2+ haemostasis. The modified Ca2+ handling is characterized by decreased Ca2+ transients, enhanced diastolic SR Ca2+ release and diminished SR Ca2+ reuptake, which all contribute to altered Ca2+ concentrations ‘seen’ by the NCX , which is detrimentally altered in HF. ROS also as having their own independent effects on RyR and other components of the Ca2+ handling machinery (dependent on the source of the ROS). In HF, just as in normal physiology, ROS has a number of different sources including NOX2, mitochondria and uncoupled NOS  and protein phosphatases, however, the distribution of these is out of the scope of the present manuscript .2+ entry via the LTCC is the initial trigger for Ca2+ release from the SR, therefore it is important to discuss potential changes in this current in HF. At a global level, the majority of studies have shown that there is no alteration in whole cell Ca2+ current recorded from myocytes from HF patients or in animal models, although single channel studies have shown that the availability and open probability of the LTCC is increased in human HF myocytes  are localized to the T-tubules, in HF β2-receptors were redistributed from the transverse tubules to the cell crest, leading to a change in β2-receptors associated compartmentation of cAMP  interventions over the next few paragraphs that could be utilized to reduce the arrhythmogenic burden in HF patients.Despite advances in our knowledge of the pathophysiology underlying HF over the last 10 years, as outlined above, only a limited number of compounds have become available for the treatment of this debilitating disease . Given tβ-Blockers have been a mainstay of pharmacological treatment for HF for a number of years and are included in guidelines for treatment of HF, in part due to their antiarrhythmic properties . In addi+ channels  is an alternative approach to alter mechanical loading and stretch and will assist to resynchronize ventricular wall motion. A recent meta-analysis indicated that this intervention may significantly reduce the risk of ventricular arrhythmias when compared with patients receiving ICDs. Interestingly the same study also showed that patients who were not-responsive to CRT may have had a significantly higher risk of ventricular arrhythmias . FurtherTaking all of these data together it appears that alterations in mechanical load should be considered as a potential therapeutic strategy.2+ microdomains in HF which could be the target for pharmaceutical intervention. For example, specific cardiac targeting of BIN1 could be an attractive option, although there are only limited data currently available and small molecules altering the activity of this protein are currently lacking. The potential for altering the T-tubule structure as a therapeutic intervention has recently been reviewed by Another therapeutic strategy would be to potentially restrict remodeling in the first place. As previously mentioned, it appears there are a number of scaffolding proteins that are involved in the structural  remodeling of the CaIt could also be possible to directly target proteins involved in arrhythmia formation under conditions of enhanced stretch or intense ß-adrenergic stimulation. For example, the potential to target stretch activated channels was recently reviewed by TI which actually contributes to afterdepolarization could also be an interesting target. Inhibition of NCX as a target has been discussed previously  with the results being highly anticipated.An alternative approach would be to alter the activity of SERCA in HF. A number of pre-clinical studies showed that there was anti-arrhythmic potential for increasing SERCA activity in HF using adeno-associated virus technology . DespiteAn alternative avenue would be to target downstream signaling pathways that are involved. Perhaps one of the most investigated of these is the CaMKII pathway, perhaps due to the multimodal action of this kinase, and the fact that targeting this molecule may lead to selective modulation of proarrhythmic RyRs . Interes2+ transient amplitude in isolated failing ventricular myocytes and reduced premature atrial contractions and afterdepolarizations in atrial cardiomyocytes  did not affect the catecholamine induced increases in late Na+ current, indicating that Epac alone plays a crucial role in these arrhythmias (Finally, targeting Epac has also been proposed as a potential target for arrhythmias driven by β-adrenergic stimulation, although there is discussion about its role. A recent study showed that treatment with an Epac activator, 8-CPT, enhanced the late Nahythmias .We still have a long way to go in treating ventricular arrhythmias that occur as a result of HF, however, concentrating on various microdomains within the cardiac myocytes that are under control of multiple pathways could be of great interest. Furthermore, linking the potential electro-mechanical and mechano-electro feedback loops could aid us in treating arrhythmias that occur as a result of this debilitating disease.Is stretch sympathetic to HF? Only time and research will fully answer this question, but in the mean time we will have to be.Both authors contributed to manuscript preparation and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.