We probed the kinetics of Ca2+/H+ exchange (CHEm) in guinea pig cardiac muscle mitochondria. We tested if net mCa2+ flux is altered during a matrix inward H+ leak that is dependent on matrix H+ pumping by ATPm hydrolysis at complex V (FOF1-ATPase). We measured [Ca2+]m, extra-mitochondrial (e) [Ca2+]e, ΔΨm, pHm, pHe, NADH, respiration, ADP/ATP ratios, and total [ATP]m in the presence or absence of protonophore dinitrophenol (DNP), mitochondrial uniporter (MCU) blocker Ru360, and complex V blocker oligomycin (OMN). We proposed that net slow influx/efflux of Ca2+ after adding DNP and CaCl2 is dependent on whether the ΔpHm gradient is/is not maintained by reciprocal outward H+ pumping by complex V. We found that adding CaCl2 enhanced DNP-induced increases in respiration and decreases in ΔΨm while [ATP]m decreased, ΔpHm gradient was maintained, and [Ca2+]m continued to increase slowly, indicating net mCa2+ influx via MCU. In contrast, with complex V blocked by OMN, adding DNP and CaCl2 caused larger declines in ΔΨm as well as a slow fall in pHm to near pHe while [Ca2+]m continued to decrease slowly, indicating net mCa2+ efflux in exchange for H+ influx (CHEm) until the ΔpHm gradient was abolished. The kinetics of slow mCa2+ efflux with slow H+ influx via CHEm was also observed at pHe 6.9 vs. 7.6 by the slow fall in pHm until ΔpHm was abolished; if Ca2+ reuptake via the MCU was also blocked, mCa2+ efflux via CHEm became more evident. Of the two components of the proton electrochemical gradient, our results indicate that CHEm activity is driven largely by the ΔpHm chemical gradient with H+ leak, while mCa2+ entry via MCU depends largely on the charge gradient ΔΨm. A fall in ΔΨm with excess mCa2+ loading can occur during cardiac cell stress. Cardiac cell injury due to mCa2+ overload may be reduced by temporarily inhibiting FOF1-ATPase from pumping H+ due to ΔΨm depolarization. This action would prevent additional slow mCa2+ loading via MCU and permit activation of CHEm to mediate efflux of mCa2+.Mitochondrial (m) CaHIGHLIGHTS-2+ efflux via Ca2+/H+ exchange (CHEm) is triggered by matrix acidity after a rapid increase in [Ca2+]m by adding CaCl2 in the presence of dinitrophenol (DNP) to permit H+ influx, and oligomycin (OMN) to block H+ pumping via FOF1-ATP synthase/ase (complex V).We examined how slow mitochondrial (m) Ca-m and pHm after DNP and added CaCl2 were larger when complex V was blocked.Declines in ΔΨ-2+]m slowly increased despite a fall in ΔΨm but maintained pHm when H+ pumping by complex V was permitted.[Ca-2+]m slowly decreased and external [Ca2+]e increased with declines in both ΔΨm and pHm when complex V was blocked.[Ca-m hydrolysis supports a falling pHm and redox state and promotes a slow increase in [Ca2+]m.ATP-2+ influx due to a bolus of CaCl2, slow mCa2+ efflux by CHEm occurs directly if pHe is low.After rapid Ca During  therapy , which coverload  sufficieoverload . [Ca2+]mne (IMM) . The majmponents . Ca2+ inlic Mg2+ . Additio the IMM  and at tto occur .2+ efflux pathway is the Na+/Ca2+ exchanger (NCEm) (2+ efflux through a Na+-independent Ca2+ exchanger (NICE), i.e., a non-electrogenic Ca2+/H+ exchanger (CHE) that might be activated when the ΔpHm gradient across the IMM is altered. The amount of free (ionized) [Ca2+]m available for exchange depends on the extent of dynamic mCa2+ buffering (2+ influx via the MCU and efflux via the NCEm are largely voltage (ΔΨm) dependent, whereas Ca2+ transport via the CHEm, while pH-dependent, may be electrogenic (1 H+ for 1 Ca2+) or non-electrogenic (2 H+ for 1 Ca2+). However, CHEm can be indirectly dependent on the full IMM electrochemical gradient if there is a decrease in the IMM ΔpHm gradient (A primary mCar (NCEm) . In unicr (NCEm)  for slowuffering . mCa2+ igradient .m and mCa2+ uptake based on the Nernst equation; a more polarized ΔΨm permits greater mCa2+ uptake  gradient, ΔΨm, and on the concentration gradient for [Ca2+] across the IMM. ATPm hydrolysis with H+ pumping can occur at complex V (FOF1-ATPsynthase/ase) during cardiac ischemia  and lower ΔΨm.There is a well-known direct correlation between ΔΨ+ uptake . mCa2+ uischemia  in an atgradient , equals 2 when the IMM is fully charged (high ΔΨm), defined here by the presence of substrate in state 2 conditions without an induced inward H+ leak, promotes rapid voltage-dependent mCa2+ uptake via MCU  across the IMM. mCa2+ influx via the MCU can partially depolarize ΔΨm  CaCl via MCU . In contrize ΔΨm  due to t2+ influx/efflux in cardiac cell mitochondria. The conditions under which CHEm may occur in cardiac mitochondria are unknown. We proposed that an induced, net influx of H+ is coupled to net mCa2+ efflux by activation of CHEm in the face of continued mCa2+ uptake via the MCU in partially depolarized ΔΨm mitochondria. In addition, if the extra-mitochondrial milieu is acidic, pHm would slowly decrease as mH+ entry by mCHEm is exchanged for mCa2+ efflux in Ca2+ overloaded mitochondria. We postulated that CHEm is activated under conditions of slow a H+ influx and a high m[Ca2+], and especially when H+ pumping by complex V, stimulated by the lowered ΔΨm, is prevented. To carry out our aim, we examined the time dependent changes in ΔΨm, [Ca2+]m and pHm, and extra-mitochondrial [Ca2+]e and pHe, after a bolus of CaCl2 either by inducing an inward H+ leak that causes an outward pumping of H+ by complex V, or by altering the extra-mitochondrial pHe.Our aim was to mechanistically examine the slow mode kinetics of mCa2 in the absence or presence of increasing concentrations of the protonophore 2,4-dinitrophenol (DNP) in the absence or presence of the complex V inhibitor oligomycin (OMN) to block ATP hydrolysis-induced H+ pumping, and or Ru360 to block the reuptake of Ca2+ via the MCU. To understand how DNP, OMN, and Ru360 dynamically alter [Ca2+]m or [Ca2+]e after a bolus of CaCl2, we considered it crucial to also dynamically measure ΔΨm, pHm, and NADH, as well as mitochondrial respiration (extent of uncoupling), total [ATP]m, and ATPm/ADPm ratio. In another set of isolated mitochondrial experiments, we directly induced mCa2+ efflux via CHEm after CaCl2 loading by altering the Na+-free medium from a control pHe of 7.15 to either pH 7.6 or 6.9. We show that secondary Ca2+ influx vs. efflux is Δ[H+]m dependent.In one set of experiments, we challenged isolated energized mitochondria with a bolus of CaClm, [Ca2+]m, NADH redox state, pHm, [ATP]m, ADPm/ATPm ratio, respiration, and the number of animals per group, are furnished  at room temperature (21°C). The experimental buffer had a final protein concentration of 0.5 mg/mL. Specific fluorescent probes and spectrophotometry  were used to measure [Ca2+]m (indo-1AM) and buffer [Ca2+]e , NADH, an indicator of mitochondrial redox state (autofluorescence), pHm (BCECF-AM), and mitochondrial membrane potential (ΔΨm) assessed by rodamine-123 or TMRM (TM – Thermo Fisher Scientific). Respiration (Clark electrode) and ATPm (bioluminescence) and ATPm/ADPm ratio  were also measured. The experimental buffer, mitochondrial substrates, and drugs were Na+-free to prevent activation of NCEm by extra-mitochondrial Na+. The inactivity of the NCE was verified by comparing data from these experiments to data from experiments with added CGP-37157, a known mitochondrial NCEm inhibitor (data not shown).All experiments conformed to the Guide for the Care and Use of Laboratory Animal and were approved by the Medical College of Wisconsin Biomedical Resource Center animal studies committee. Detailed methods for mitochondrial isolation and measurements of ΔΨ or TMRM   was added, followed by a bolus of 40 μM CaCl2 at t = 210 s to initiate rapid mCa2+ uptake via MCU. Note that in guinea pig cardiac mitochondria, the respiratory control index (RCI) is higher in the presence of pyruvate alone (t = 300 s shortly after adding CaCl2 to block Ca2+ reuptake into mitochondria via MCU after the Ca2+ was extruded from mitochondria. At the end (1700 s) of each experiment, the potent protonophore, carbonyl cyanide m-chlorophenyl hydrazone  was given to completely abolish the ΔpH gradient and depolarize ΔΨm. Data for each pH group were collected in mitochondrial suspensions from the same heart; approximately 8–10 hearts were used for each fluorescent probe. At pH 7.15, adding 40 μM CaCl2, which increased extra-mitochondrial [Ca2+]e into the 1 μM range and increased the initial [Ca2+]m to approximately 500 nM  was added to the mitochondria suspended in the experimental buffer, followed by 0, 10, 20, 30, or 100 μM DNP, a mild protonophore, at t = 90 s, followed by the addition of de-ionized H2O, 10, or 25 μM CaCl2 at t = 225 s. The 90 s period allowed for full ΔΨm polarization and stabilization of pHm and NADH. In some experiments . Each drug or DMSO alone was added to a final volume of 10 μL. To test for mPTP opening, CsA was given before adding 20 or 30 μM DNP and 25 μM CaCl2 in several experiments conducted at pHe 7.15.Experiments were initiated at protocol . At the m on [Ca2+]e. Data were summarized for protonophore-induced changes in pHm on [Ca2+]m at 80 s (after adding PA), 215 s (after adding DNP), 275 s (early after adding CaCl2), and 700 s (late after adding CaCl2)  was considered significant. See Figure legends for statistical notations.Data were summarized at 500, 1000, and 1500 s for , 2 for e2) e.g., . All datm activation was observed by acidifying the extra-mitochondrial buffer (low pHe), which subsequently decreased the matrix pHm slowly over time (m and Na+/H+ (NHEm) inactivated by using Na+-free solutions and substrates, fast mCa2+ influx via the MCU, induced after adding 40 μM CaCl2 at pH 6.9, was followed by a slow mCa2+ efflux over time ∼(300–1700 s) as shown by the increase in extra-mitochondrial [Ca2+]e from <200 nM to nearly 4500 nM in the absence of Ru360  of increase in [Ca2+]e (mCa2+ efflux) over time (300–1700 s) was 1.5 ± 0.1 nM/s, ΔpH 0.4 units). This was greater than in the pH 6.9 minus Ru360 group (1.0 ± 0.2 nM/s over 300–1000 s), suggesting that approximately 1/3 of the mCa2+ extruded was retaken up across the IMM via the MCU. In contrast, mCa2+ efflux was not observed in the pH 7.6 medium without Ru360, and minimally at 1500 s at pH 7.6 with Ru360. There was less mCa2+ efflux at pH 7.15 ± Ru360 compared to pH 6.9 ± Ru360. However, even at pH 7.15 ± Ru360, there were similar steady declines in pHe while net slow Ca2+ efflux was noted only in the plus Ru360 groups, indicating Ca2+ re-uptake via MCU. Therefore, in the acidic extra-mitochondrial medium, slow decreases in pHm (H+ influx) were accompanied by slow increases in mCa2+ efflux, indicating CHEm activity. Eventually, matrix acidification was more pronounced in the pH 6.9 medium (ΔpH 0.62 units) in the absence of Ru360 than in all other groups so that over time as H+ influx was exchanged for Ca2+ efflux the IMM ΔpH gradient was eventually obliterated, halting Ca2+ efflux . Although adding CaCl2 at an external pHe of 6.9 led eventually to near complete dissipation of ΔΨm, when CsA was first added to the buffer, CsA prevented the gradual, slow extrusion of mCa2+ and declines in pHm and ΔΨm induced by adding CaCl2 at pHe 6.9 indicating a complete lack of CHEm activity , much less so in the pH 7.15 medium, and not at all in the pH 7.6 medium. Ca2+ efflux was accentuated in the presence of Ru360 given just after the added CaCl2 bolus   and addition of CCCP denotes complete depolarization (100%). Adding 10 μL of 0.1% DMSO (DNP vehicle) or 10 μM DNP had no significant effect when given before CaCl2, whereas adding 20, 30, or 100 μM DNP before 10 μM CaCl2 reduced the R123 ΔΨm signals by 12.7, 18.7, and 92.4% vs. DMSO  by 16.2, 33.0, and 99.0%, respectively, vs. DMSO (0%). Overall, before adding either 10 or 25 μM CaCl2, 20 and 30 μM DNP moderately decreased ΔΨm in the absence of OMN but greatly decreased ΔΨm in the presence of OMN, suggesting blocked proton pumping from complex V    , in a covs. DMSO , respectvs. DMSO , adding omplex V . If no Cd by CsA . Supplem2 without DNP (ΔΨm fully polarized) caused [Ca2+]m to increase rapidly from 80 nM (no added CaCl2) initially to 235 nM at 300 s, whereas after adding 25 μM CaCl2, [Ca2+]m rose more rapidly to 450 nM (2+]m remained unchanged over time (300–750 s) after adding 10 μM CaCl2 but fell slightly and gradually (non-significantly) over time after adding 25 μM CaCl2 ; after adding 25 μM CaCl2 in the absence of DNP  and converted to mCa2+ efflux in the presence of OMN as shown by the increase in [Ca2+]e  were larger than those in the absence of OMN  after adding 10 or 25 μM CaCl absence  or prese absence  of OMN sμM CaCl2  decreaseCaCl2  gradual increase in [Ca2+]m over time in the absence of OMN with a threefold greater slope after 30 μM DNP vs. DMSO  in [Ca2+]m over time  even in the presence of DNP, except for 100 μM DNP, when pHm fell markedly (ΔΨm fully depolarized) . m replotted from m as a function of [Ca2+]m at 700 s after adding DNP and CaCl2; these correlations show how [Ca2+]m decreases while pHm decreases in the presence, but not in the absence of OMN.Baseline matrix pHding DNP –D. In thased pHm . This efgradient . In the larized) . In the trix pHm  after adm. Adding the substrate PA increased the redox state (more reduced) as determined by high NADH autofluorescence .A reduced redox state is associated with maintenance of pHrescence . In the cence . Adding 20 or 30 μM DNP alone had no significant effect on [ATP]m, but adding CaCl2 resulted in a decrease in [ATP]m  of ΔΨm, [Ca2+]m, fast  d[Ca2+]m/dt, and pHm as a linear function of 0–30 μM DNP ± OMN at the 700 s time point. 2+ flux rates (JCHE) for CHEm (see section “Supplementary Results Supplementary Results m in maintaining homeostasis of Ca2+ against H+ under certain conditions in cardiac cell mitochondria that may mimic some sequelae of cardiac IR injury. Our results: (1) furnish direct evidence for CHEm activity by the secondary, slow increases in matrix Ca2+ efflux coupled to slow increases in matrix H+ influx, when both NCE and NHE activities are blocked, and particularly, when MCU-dependent mCa2+ re-uptake is blocked with Ru360; (2) demonstrate that respiration increases while ΔΨm decreases mildly, whereas pHm and redox state are relatively maintained when inducing a matrix inward H+ leak with DNP before adding CaCl2; adding CaCl2 results in a secondary, slow increase in [Ca2+]m that slowly depolarizes ΔΨm; (3) show that with permissive H+ influx, but inhibited outward H+ pumping at complex V, adding CaCl2 causes larger decreases in ΔΨm, pHm, and NADH and results in a slow decrease in [Ca2+]m; (4) indicate that blocking complex V with OMN to prevent H+ pumping causes ΔΨm to further decrease after adding CaCl2 because the influx of mCa2+ via the MCU is not opposed by H+ pumping at complex V; (5) suggest that the lack of a slow fall or rise in [Ca2+]m in the presence of 100 μM DNP is due to the loss of ΔΨm-dependent mCa2+ uptake by MCU; (6) point out that only in partially depolarized mitochondria does added CaCl2 result in a pHm-independent gradual increase in [Ca2+]m that is reciprocated by H+ pumping to maintain pHm; preventing matrix acidification is associated with a maintained redox state; and (7) show that the decrease in [ATP] in the absence of OMN supports ATP hydrolysis with H+ pumping. These two scenarios, ±OMN, are depicted graphically in We provide firm support for a role of CHE2+ uptake on ΔΨm has been examined extensively , can maintain the ΔpHm and support the pmf (ΔΨm + RT/FΔpHm)  is not countered by reciprocal H+ pumping to restore ΔpHm, the slow influx of H+ is exchanged for slow Ca2+ efflux via CHEm until the ΔpH gradient is dissipated.The dependence of rapid MCU-mediated mCaensively . But ourT/FΔpHm) . This vising ΔΨm . In cont2+ and H+ gradients across the IMM are largely dependent on ΔΨm and ΔpH gradients resulting from H+ pumping by respiratory complexes. Ionic homeostasis requires one cation efflux pathway to oppose another cation influx pathway and vice versa. Cation exchangers fulfill this need. Unlike mCa2+ uptake via MCU, which is dependent on ΔΨm and on the chemical gradient, exchange of Ca2+ and H+ via CHEm may or may not be dependent on ΔΨm  when complex V cannot pump H+ in the presence of OMN.Cat on ΔΨm . But theequation , calculaequation . JCHE flm likely occurs both in the absence or presence of OMN, our results suggest that the observed secondary, slow influx of mCa2+ influx (minus OMN) is due primarily to re-uptake by a Ru360 sensitive mechanism, presumably MCU, that may overwhelm any CHEm activity. This is because Ru360 blocked the slow rise in [Ca2+]m and the slow fall in [Ca2+]e, thus supporting MCU as the mediator of the slow mCa2+ influx. The JCHE flux equation only monitors differences in [H+] and [Ca2+] on either side of the IMM and does not rely on effects of the ΔpHm gradient on H+ pumping or the ΔΨm gradient on mCa2+ uptake via MCU. Thus the secondary, slow mCa2+ uptake after the initial CaCl2 bolus (+ pumping by complex V (minus OMN) to maintain the ΔpHm charge gradient and support the pmf although ΔΨm continues to fall due to the continued mCa2+ influx. On the other hand, inhibiting ATPm hydrolysis , thus exposing net Ca2+ uptake via MCU. The slow increases in [Ca2+]m that we observed previously  compounds (m activity in relation to MCU activity in cardiac cell mitochondria.A leucine zipper EF hand-containing trans-membrane protein (LETM1) found in non-mammalian cells is thought to be a molecular component of CHEm . Knockdochondria . Alternachondria  support + efflux  or that ompounds , and to ompounds . The preOF1-ATPsynthase/ase directionality is governed by ΔΨm and its “reversal potential” EREV -ATPase, which in turn is dependent on the concentration of the reactants ATP/ADP, and H+  with a decrease in ΔΨm at an IMM gradient of approximately 0.35 ΔpHm units  whereby the H+ leak is compensated by augmented H+ pumping by complex V; this resulted in slow mCa2+ influx (“Ca2+ leak”) that could be blocked by Ru360, which indicates the influx likely occurred via MCU. But if compensatory H+ pumping is blocked by OMN, the matrix becomes acidic, the ΔpHm gradient falls lower, and slow mCa2+ efflux occurs via CHEm thus masking the slow mCa2+ influx  can result in maintained NADH levels despite the DNP-induced H+ leak. In the presence of OMN, however, NADH was gradually oxidized  and curve fitting for approximating conversion to ΔΨm  was sufficient to compensate for the continued inward influx of H+ mediated by DNP thus restoring the ΔpHm gradient, but not the pmf, and thus preventing activation of CHEm. Ru360 blocked this additional uptake of mCa2+ by the MCU so that [Ca2+]e did not continue to fall.The net Can to ΔΨm ). This i2+ influx under our experimental conditions occurs primarily via the MCU. The efflux of Ca2+ via the CHEm is slow so we expect the re-uptake of Ca2+ via the MCU also would be slow. Although the JCHE flux equation alone predicted that slow mCa2+ influx could have occurred via CHEm this is unsustainable if [H+]m < [H+]e. It is likely that voltage-dependent transport of net Ca2+ inward is mostly responsible if there is at least a partially maintained ΔΨm  despite mCa2+ extrusion via CHEm. Interestingly, under the condition of a fully polarized ΔΨm (no DNP and no OMN)  and Ca2+ chemical gradient.We predict that the major conduit for both fast and slow mCa no OMN) –D, m became lowered due to CHEm. Because inhibiting the MCU with Ru360 caused a robust increase in [Ca2+]e, this indicated the Ca2+ effluxed via CHEm re-enters via the MCU unless this pathway is blocked. Under the unique condition of collapsed ΔΨm (100 μM DNP) and ΔpHm gradients, the secondary, slow uptake of mCa2+ is absent , which indeed occurs if there is remaining ΔΨm. This means that net Ca2+ efflux due to CHEm  ROS, adenine nucleotide levels, and other factors are believed to contribute to mPTP formation during IR injury. But in our study we did not utilize IR to induce increases in Ca2+ and ROS or decreases in pHm or ΔΨm; (2) Altering just the driving force for protons across the IMM using DNP or external pH to exchange Ca2+ ion for H+ ions is not compatible for a mechanism to cause or prevent formation of mPTP but it is for inducing mCHE activity; (3) Transient mPTP formation is controversial and based largely on the utility of calcein or other small particles to mark mitochondrial release of small molecules with free flowing ions such as Ca2+  CsA to H2O2 ; (4) CsAormation . Some in2-induced effects attributed to CHEm. CsA ceased all apparent CHEm activity after adding CaCl2 when pHe was 6.9 or 7.15, as assessed by measurements of extra-matrix [Ca2+]e, pHm, and ΔΨm  through a Ru360-sensitive pathway if H+ pumping counteracts a H+ leak; however, there is net slow mCa2+ efflux that overrides ΔΨm-mediated Ca2+ influx that is activated via CHEm if there is a high ΔpHm gradient. In cardiac mitochondria, the rapid and slow mode of uptake of mCa2+ appears to be dependent primarily on the trans-membrane [Ca2+] and ΔΨm gradients if outward H+ pumping counteracts inward H+ entry. In contrast, slow extrusion of mCa2+ by CHEm appears to be dependent primarily on the [ΔH+]m gradient induced by H+ influx/leak by DNP or by an acidic pHe. Importantly, if NCEm and NHEm are inactivated, blocking complex V might prevent delayed Ca2+ overload and instead stimulate Ca2+ extrusion via CHEm if there is an inward H+ leak. In intact cells, this can also serve to preserve TCA cycle-generated ATP, i.e., substrate level phosphorylation. Such passive homeostatic balance of Δ[Ca2+]m may occur during cardiac injury when there is mCa2+ loading accompanied by declines in NADH redox state, pHm and Ψm. We conclude that the differences in the rate and magnitude of mCa2+ influx/efflux in partially depolarized mitochondria, in the presence or absence of FOF1-ATPase activity, can be ascribed to the underlying changes in pmf components, ΔpHm and ΔΨm, after rapid mCa2+ loading.This study furnishes new insights into the bioenergetic and dynamic mechanisms in cardiac cell mitochondria of delayed, slow mCaDS proposed the study and its initial design. JH conducted most experiments, carried out initial statistical analysis, constructed initial figures, and participated in design, interpretation and writing. AG, AB, CB, CN, and MB conducted supporting experiments. AC, W-MK, and RD participated in theoretical interpretation of the results and text editing. DS and AC supervised the team in subsequent experimental designs, interpretation of results, and manuscript construction and writing.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.


Mentoring plays a pivotal role in workplace-based learning, especially in the medical realm. Organising a formal mentoring programme can be labor and time intensive and generally impractical in resource constrained medical schools with limited numbers of mentors. Hence, informal mentoring offers a valuable alternative, but will be more likely to be effective when mentors and protégés share similar views. It is therefore important to gain more insight into factors influencing perceptions of informal mentoring. This study aims to explore mentors and protégés’ perceptions of informal mentoring and how these vary (or not) with gender, age and the duration of the relationship.acceptance, counselling, friendship, parenting, psychological support, role modelling and sociability.We administered an Informal Mentor Role Instrument (IMRI) to medical practitioners and academics from Egypt, Pakistan and Saudi Arabia. The questionnaire was developed for the study from other validated instruments. It contained 39 items grouped into 7 domains: A total of 103 mentors and 91 protégés completed the IMRI. Mentors had a better appreciation for the interpersonal aspects of informal mentoring than protégés, especially regarding acceptance, counselling and friendship. Moreover, being older and engaged in a longer mentoring relationship contributed to more positive perceptions of interpersonal aspects of mentoring, regardless of one’s role (mentor or protégé).It can be concluded that the expectations of mentors and protégés differed regarding the content and aim of the interpersonal characteristics of their mentoring relationship. We recommend mentors and protégés to more explicitly exchange their expectations of the informal mentoring relationship, as typically practiced in formal mentoring. Additionally, in our study, seniority and lasting relationships seem crucial for good informal mentoring. It appears beneficial to foster lasting informal mentoring relationships and to give more guidance to younger mentors.Supplementary information 10.1186/s12909-019-1791-8. Mentoring is a relationship between a mentor  and protégé  that aims to guide personal and professional development over time –4. Numerchoice. The two types of mentoring are complimentary, not mutually exclusive. Informal mentoring provides a valuable alternative due to the spontaneous nature of the relationship [Mentoring can be either formal (i.e. institution-instigated) or informal (i.e. occurring spontaneously). Formal mentoring is essentially a task or an assignment and is typically perceived as such, whereas the informal relationship is a tionship . Yet thetionship . In additionship  Moreovertionship .Conflicts may occur when a mentor assumes competing roles as a guide and an evaluator for the same cohort of protégés or when protégés betray trust, damage the mentor’s reputation, rely heavily on their mentor, ignore the mentor or are ungrateful to them. Unsuccessful informal mentoring may be attributed to poor implementation and discrepancies in protégé-mentor perceptions. That said, it is important to learn more about the factors that influence perceptions of informal mentoring , 19, 20.Research on mentoring to date has been directed in large part towards formal mentoring that is embedded within a formal curriculum. Largely unexplored, however, are the factors that enhance initiation, perception and preservation of these informal relationships by physicians or faculty . Scant aInformal mentoring relationships are fluid in nature and rest on implicit and diverse expectations between mentor and protégé. Sometimes one of the partners does not even acknowledge being in a mentoring relationship. In order for mentors and protégés to benefit the most from the relationship, awareness of their distinct, yet complementary roles, is key.personal, interpersonal and professional level [Perceptions of informal mentoring in the medical realm may be mediated by several factors that operate at the al level . At the al level , 22. At al level . As roleal level , 23, 24.ideal mentor and varying expectations from the relationship [Although mentors have traditionally paired with protégés who were 8–15 years younger of age, there is a growing tendency towards similar-age (peer-to-peer) or even reverse-age mentoring. We postulate that age can impact the mentoring experience because junior and senior protégés have different perceptions of what is the tionship . Also, oGender may also moderate the mentoring experience. Expectations and behaviours of mentors and protégés have been shown to differ between sexes. Males, for instance, were more likely to serve as mentors and mainly provided career-related advice , while tspeed mentoring programme in which protégés each met with six mentors for ten minutes per pairing [The duration of the informal mentoring relationship may impact on mentoring experiences. It can vary from a few months up to a lifetime. An example of a short-lived mentoring experience was reported by Cellini and colleagues who organised a  pairing . Three mThe continuation of a relationship depends on open communication, availability, mutual respect, clear goal setting, role modelling, the mentor’s coaching skills, and the presence of a protégé portfolio , 30, 31.age, gender and the duration of the relationship. To this end, the following two research questions will be addressed: (1) Do mentors and protégés differ in their perceptions of the interpersonal aspects of informal mentoring? (2) How do mentors and protégés’ perceptions of informal mentoring vary with their age, gender and the duration of the relationship?To recap, the present study investigates the dynamics of informal mentoring by measuring the differences in perceptions between mentors and protégés and how these perceptions vary (or not) with We targeted medical practitioners, academic staff members and graduate and doctoral students in governmental institutions in Egypt, Pakistan and Saudi Arabia. We asked voluntary participants if they were presently or recently engaged in an informal mentoring relationship and whether they fulfilled the role of mentor or protégé in this relationship.We searched the literature for instruments that measure perceptions of mentoring relationships , 32–37. All items were rated on a 5-point Likert scale, ranging from 1 (strongly disagree) to 5 (strongly agree), the higher scores representing more favourable perceptions. We assessed the reliability of the IMRI by calculating Cronbach’s alpha for each of the seven subscales. The alpha per subscale ranged between .90 and .98 for protégés and between .51 and .89 for mentors , age (in years), gender , and the duration of the current or most recent informal mentoring relationship (in months). To make the IMRI applicable to both mentors and protégés, we changed the original wording of the items by eliminating all mentions of ‘my mentor’, ‘my protégé’, ‘me’ or ‘you’ and replacing them with ‘the protégé’ or ‘the mentor’, as appropriate. Other adjustments were the replacement of all references to ‘school’ and ‘education’ by ‘work in the medical education field’ and the rephrasing of items to be written in the positive. We produced both an English and an Arabic paper version of the questionnaire to make it intelligible to all participants. The English questionnaire was also available in online format.We administered the Arabic paper version in person to attendees of departmental meetings, workshops and seminars at the medical college of Zagazig University, Egypt, and collected their responses on the spot. The English version was distributed both online and in paper format to graduate students and faculty members at the medical college of Riphah University in Pakistan and to faculty members at the college of medicine, University of Dammam, Saudi Arabia. We informed participants of the study’s purpose verbally while distributing the paper version or, in the case of the online version, through a short standard paragraph posted at the beginning of the survey.After having explained the rationale of the research, the volunteered participants were asked to sign an electronic consent form. Then the survey started with the items that asked for participants’ background information. Participants were consequently asked if they had any past experience with informal mentoring, which was defined as follows: ‘Informal mentoring is based on natural personal matching and mutual interests between a junior and a senior person. Its ultimate outcome is professional development and considerable impact and satisfaction for both.’ Participants were instructed to enter ‘0’ and to stop completing the questionnaire if they had never had an informal mentoring experience. Ethical clearance was obtained to administer the IMRI English & Arabic versions of the used questionnaire  and compared these average group scores. We addressed the second research question by investigating the relation of participants’ age and the duration of the current or most recent informal mentorship with the domain scores, which yielded the continuous variables of Age (in years) and Duration (in months). Since we expected the distribution of Duration to be considerably skewed, we also used the log10-transformed version of Duration in the analysis, which we labelled ‘LgDur’. We considered participants’ gender as a potential confounder, and therefore included Gender  in the analysis as a control variable. Moreover, as the relationship between a subscale and Age and Duration could vary in accordance with participants’ role of mentor or protégé, we included the variable ‘Role’ in the analysis as a grouping variable . In a final step, we obtained descriptive statistics of the above-mentioned variables and investigated the factors explaining the variation in each domain score in a multiple regression analysis, using the domain score as dependent variable and Age, LgDur, Role, Gender and the interactions RoleLgDur (= Role×LgDur) and RoleAge (= Role×Age) as independent variables. For ease of interpretation and to improve numerical stability in the regression analysis, we centred the continuous variables of Age and LgDur. That is, we subtracted the variable’s average value from the original value, as in Age centred = Age – average (Age). In the stepwise regression analysis, we first entered Age, LgDur, Role and Gender into the model. After that, we used a backward procedure to investigate the interactions which we retained only if found to contribute significantly to the explanation of the variance in the dependent variable. We used the standard regression coefficient (src) as an indicator of effect size, applying Cohen’s rule of thumb that values of 0.1, 0.3 and 0.5 indicate a small, moderate, and large effect, respectively. Results were considered statistically significant when A total of 226 participants completed the questionnaire, 180 of which were from Egypt, 32 from Pakistan and 14 from Saudi Arabia. From these participants, 25 were excluded because they failed to indicate which role they fulfilled (protégé or mentor) in their most recent mentoring relationship. Another six participants were excluded because they did not answer 7 or more of the 39 items in total, leaving 194 participants whose data formed the input to our analysis. Following the guideline that you need minimum 50 + 8 x ‘the number of independent variables’ participants per variable in your statistical model, our sample size of 194 respondents adequate to test statistical models with four independent variables.b of the intercept represents the domain score of a male protégé  of average age 1(37.9 years) whose current or most recent mentoring relationship lasted 12.9 months which corresponds to the average LgDur (1.11) or 101.11.As anticipated, the distribution of the variable Duration demonstrated unacceptably high values of skewness and kurtosis 4.90, 36.21), while the logarithmically transformed version of Duration, LgDur, exhibited considerably better values  that were within the acceptable range. Tables 1, while sc, which varied from .23 to .27, indicating effects of moderate size. Hence, compared to protégés, mentors had more positive perceptions of three out of seven domains. As to our second research question about the influence of age, gender and the duration of the mentoring relationship on perceptions, we found moderate effects (sc .25 and .29) of age for Counselling and Parenting, with perception scores increasing by 0.020 and 0.027, respectively, with each additional year. Furthermore, we found that perception scores for all subscales increased significantly as relationships lasted longer, the regression coefficients b showing increments of 0.34 to 0.52 each time Duration was multiplied by a factor of 10. Corresponding effect sizes were small to moderate, as evidenced by the sc’s which ranged from 0.15 to 0.28. Controlling for gender, we found it to be a confounder only in the domain of Friendship, since female participants on average had a 0.262 higher score for Friendship than their male peers, the sc of 0.15 signalling a small effect. Finally, the non-significance of the interactions ‘Role Age’ and ‘RoleLgDur’ confirmed that being older and engaged in a longer mentoring relationship contributed to more positive perceptions of interpersonal aspects of mentoring, regardless of one’s role (mentor or protégé).With respect to our first research question as to whether mentors and protégés have different perceptions of mentoring, we found that mentors on average had significantly higher perception scores for Counselling, Friendship and Acceptance (increases of 0.4 to 0.5) than protégés, the corresponding acceptance, counselling and friendship, which may be because mentors were only connected with the protégés they liked [The present study examined how participants’ role (mentors’ vs protégés), their age, gender and the duration of the relationship influenced perceptions of informal mentoring relationships. Among a heterogeneous cohort of informal mentors and protégés, we found significant differences in perceptions of mentorship domains based on roles. Further, we found that age might be contributing to such perceptions across both roles. In terms of roles, mentors had more positive perceptions in the domains of ey liked , 39. Thicounselling and parenting. Younger mentors reported less favourable perceptions of informal mentoring, which can possibly be attributed to an exaggerated sense of their importance and narcissistic personality traits. At the start of their career, the less experienced mentors believed they had the right to unlimited success and may take advantage of others [As for age, there was a positive correlation between age and mentors and protégés’ perceptions in the domains of f others .The notion that age is of significance to career choice, counselling and professional development has previously been reported by studies on mentoring in the medical context , 5, 23. Mentorship could present itself in different patterns. Among these patterns, the hierarchical relationship between senior and junior members represents the commonly encountered model . Culturafriendship and role modelling. This seems plausible, since mentoring dyads are more likely to become friends when the relationship lasts longer. The reverse may also be the case: when partners are united in friendship and protégés perceive their mentors as role models, the relationship may persist. A ‘friend’ and a ‘role model’ can be the personal qualities partners see in the other in the early stage of identification, when mentors and protégés first connect with each other [With respect to duration, our study advocated that longer relationships were associated with more positive perceptions of different aspects of informal mentoring, notably ch other , 48.Another determinant of mentoring perceptions, albeit to a minor extent, was gender. A meta-analysis was conducted by O’Brien et al., 2010 to address mentoring relationships from the perspective of gender differences between mentor- and protégé. According to this study, males described giving more advice on career development than female mentors. Equally, female mentors conveyed providing more psychosocial support than male mentors. There were no gender differences for protégés regarding career development, However, male protégés stated obtaining less psychosocial support than female protégés .friendship into the informal mentoring relationship. Other female-specific features emphasised in the literature are that female protégées expect their informal mentors to be their counsellors, coaches, teachers and in some occasions even their sponsors [Women were more inclined to infuse a sense of sponsors . Moreovesponsors , which dsponsors , sometimsponsors . Finallysponsors , women gsponsors .homophily’ and ‘liking attraction’ [. Homophily or similarity attraction is the tendency of mentors and protégés to engage with those who share the same core values, attitudes and characteristics. Similarity breeds connection, a principle that governs relationships of all time, including marriage, friendship and work. Without a baseline understanding and similarity between mentors and protégés, mentoring relationships are likely to fail [liking, means that people are drawn to those who seem to like them [trust between two individuals [The above results should be interpreted in the context of two phenomena in interpersonal relationships that have been of great interest to social scientists since the 1950s: ‘raction’ , 55 . Ho to fail , 39. Theike them . Interesividuals .pragmatic, albeit sophisticated, transactional beneficial relationship that is based on give and take. By suggesting a couple of theoretical implications, this study made a humble attempt to contribute to this understanding and to offer more insight into the motivations that drive mentor-protégé dyads. The study recalls that mentoring relationships do not exist in a vacuum. We therefore must acknowledge and further examine their context to unearth the personal, interpersonal and circumstantial variables that can make or break these relationships.As was highlighted before, Social Exchange Theory  is oftenHowever, some limitations need addressing. First, the general pattern of informal mentoring relationships that emerged was restricted by the variables we selected, as is the case in most quantitative studies. Moreover, we did not match mentors’ perceptions with those of their partner protégés, and we certainly invite future research to make up for this shortcoming. The large standard deviation of the mean scores on the IMRI subscales underlines the importance of future studies investigating mentor-protégé pairs, so they reflect on the same mentoring relationship. Furthermore, the IMRI was, although based on items from internationally published instruments, not fully validated in the countries of this study. A future validation study is recommended. Finally, we solely focused on perceptions of the informal mentoring relationship, without exploring whether favourable perceptions were also associated with better outcomes, such as a better receptivity to feedback by protégés . PreviouMentoring can be one of the most fulfilling and transformative relationships we experience at work, but effectiveness cannot be taken for granted. Mentors had a better appreciation for the interpersonal aspects of informal mentoring than protégés. Moreover, perceptions of both mentors and protégés improved with age and the duration of the mentoring relationship in our study. We therefore recommend nurturing lasting informal mentoring relationships and to give more guidance to younger mentors and protégés.Additional file 1: English and arabic versions of the used questionnaire. (DOCX 31 kb)


R- and S-enantiomers. Its inhibitory potency towards nine cytochromes P450 (CYP) was studied to evaluate the drug–drug interactions between the enantiomers. Enzyme inhibition was evaluated using specific CYP substrates in human liver microsomes. With CYP3A, both enantiomers exhibited reversible and time-dependent inhibition. S-AML was a stronger reversible inhibitor of midazolam hydroxylation: the Ki values of S- and R-AML were 8.95 µM, 14.85 µM, respectively. Computational docking confirmed that the enantiomers interact differently with CYP3A: the binding free energy of S-AML in the active site was greater than that for R-AML . Conversely, R-AML exhibited more potent time-dependent inhibition of CYP3A activity  than S-AML . R-AML was also a significantly more potent inhibitor of CYP2C9 (Ki 12.11 µM/S-AML 21.45 µM) and CYP2C19  is available as a racemate, i.e., a mixture of  R- and S-enantiomers  have been reported, many of which are associated with reduced substrate metabolism. Several studies have demonstrated the clinical significance of CYP2C9 and CYP2C19 polymorphisms  [The single point assay was done as follows: at 37 °C . Subsequprotocol . To findtion [I] ,43.Docking studies were carried out using the Molecular Operating Environment (MOE) software package  ,41,43,44


LMNA) have been associated to diverse pathological phenotypes with skeletal muscles and the heart being the most affected systems. When affected, the heart develops a wide array of phenotypes spanning from dilated cardiomyopathy with conduction defects to arrhythmogenic right ventricular cardiomyopathy. The surprising large number of cardiac phenotypes reflects the equally large number of specific mutations identified in the LMNA gene. In this review, we underlie how mutations in LMNA can impact the activity and the spatial/temporal organization of signaling mediators and transcription factors. We analyzed the ever-increasing amount of findings collected in LmnaH222P/H222P mice whose cardiomyopathy resemble the most important features of the disease in humans and a number of key evidences from other experimental models. With this mini review, we attempt to combine the newest insights regarding both the pathogenic effects of LMNA mutations in terms of signaling abnormalities and cardiac laminopathies.Nuclear lamin A/C are crucial components of the intricate protein mesh that underlies the inner nuclear membrane and confers mainly nuclear and cytosolic rigidity. However, throughout the years a number of other key physiological processes have been associated with lamins such as modulation of both genes expression and the activity of signaling mediators. To further solidify its importance in cell physiology, mutations in the lamin A/C gene ( LMNA, cytogenetic location: 1q22) thanks to alternative RNA splicing events   , underliy (ARVC) . StrikinLMNA have a very aggressive and fast clinical course that could culminate with sudden cardiac death from malignant ventricular arrhythmias and end-stage heart failure occurring at earlier ages compared to other familial cardiomyopathies. Current therapies involve implantable pacemakers and defibrillators to manage arrhythmia and conduction defects in order to prevent sudden cardiac death  causing muscular dystrophy and DCM , an important mediator of fibrosis and extracellular matrix deposition (Lamins are well-known molecular scaffolds that tether at the nuclear envelope mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (ERK) 1/2 and its downstream transcription factor c-Fos, a major regulator of important cellular processes such as cell proliferation, death, survival, and differentiation . Mutatioairments , 2012a longation , 2017. I22P mice . All the22P mice . The mecposition , and itsposition . Of noteposition .H222P/H222P mice. Thus, pharmacological compounds able to correct defective actin dynamics could be used as new therapeutic approach to improve left ventricular dysfunction in LMNA-mediated cardiomyopathy  together with left ventricular dilation markers in ventricular tissue of Lmna2P/H222P , 2018. D22P mice . So far,22P mice  and like22P mice . Strikin22P mice .H222P/H222P mice also showed reduced expression of WNT and β-catenin  overexpression, with activation of caspase-8/caspase-3 and caspase-9-dependent release of cytochrome c from mitochondria .We identified in members of an Italian family a novel heterozygous lamin A/C variant consisting of a in-frame duplication of 21 nucleotides in the exon 2 of the MNA gene . This mu∗4 . Both mutations generated a premature termination codon before the NLS. When exogenously expressed in HEK293 or HL-1 cardiomyocytes both mutants mislocalized and accumulated within the endoplasmic reticulum (ER). Of note, Lmna R321X ER-accumulation led to unfolded protein response (UPR), an adaptive mechanism involving a complex transcriptional program that induces the hyperphosphorylation of PERK, a kinase localized on the ER membrane . Of note, partial restoration of CX43 activity in Lmna D243Gfs∗4-expressing cells by lithium improved cell-to-cell signal propagation suggesting CX43 as potential pharmaceutical target for this form of cardiolaminopathy.When we functionally characterized the second nonsense poptosis . The facmonomers . While p2+ dynamics. On one hand, Lmna R321X ER accumulation led to an abnormal Ca2+ handling by the cell; ER Ca2+ content was significantly reduced as well as the Ca2+-mediated agonist responses in both the cytosol and the nucleus. On the other hand, Lmna D243Gfs∗4 expression affected the regular Ca2+ oscillatory pattern of cardiomyocytes through a CX-43 dysfunction. Thus, the effect of LMNA mutations on Ca2+ changes might represent a new and under-investigated event in the pathogenesis of LMNA-mediated cardiomyopathies. Of note, 2+ sensitizer able to increase muscular Ca2+ sensitivity without increasing cytosolic Ca2+ levels, ameliorated systolic dysfunction, reduced cardiac interstitial fibrosis and modulated the expression of genes involved in cardiac remodeling when administrated in LmnaH222P/H222P mice. Nuclear and cytosolic Ca2+ changes can activate Ca2+-dependent PKC isoforms either resident within the nucleus or translocated from the cytosol, respectively. Interestingly, lamin A has at least two consensus sites for PKC and others for kinases that can be modulated by Ca2+ (During the functional characterization of both truncated mutants we uncovered critical changes in intracellular Ca by Ca2+ .LMNA-mediated cardiomyopathies will help cardiologists to design more precise and effective therapeutic protocols.Overall, we believe that the exact dissection of all the cellular pathogenic mechanisms induced by mutations in lamin A/C gene will pave the road toward personalized treatment for these cardiomyopathies. Patients carrying different mutations with the same cardiac phenotype should not be therapeutically approached with the same strategy. For instance, cardiac fibrosis is not only induced by apoptosis but also by abnormal CX43 expression triggers due to enhanced activity of non-excitable cardiac fibroblasts . Of noteAG conceived the idea of this article. AG and MC wrote and edited the manuscript. Both GP and MS reviewed and critically edited the content 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. The reviewer DB and handling Editor declared their shared affiliation at the time of the review.


Frequently, breast cancer cells modulate their calcium signaling pathways to optimize growth. Unique calcium pathways in breast cancer cells could serve as a way to target tumorigenic cells without affecting normal tissue. Resveratrol has previously been shown to activate calcium signaling pathways. We use cell viability, single-cell calcium microscopy, and RT-PCR assays to determine the activity and mechanism of three different 4′-esterified resveratrol derivatives. We demonstrate that two of the derivatives reduce cell viability more effectively than resveratrol in MDA-MB-231 human breast cancer cells. The derivatives also activate similar pro-apoptotic calcium signaling pathways. In particular, the pivalated and butyrated resveratrol derivatives are intriguing putative chemotherapeutics because they are more effective at decreasing cell viability in vitro and inhibiting the plasma membrane Ca Breast cancer is the most common form of cancer in American women; in fact, one in eight women will be diagnosed with breast cancer during her lifetime . Breast 2+]i) is increased by an influx of calcium from the high calcium environments of the extracellular matrix (~1 mM) or the endoplasmic reticulum (ER) (~1 mM). Additionally, the [Ca2+]i can be increased by decreasing calcium efflux from the cytosol i [2+]i signaling.Specifically, RES has been shown to activate apoptosis in tumor cells via increases in [Ca2+]i . However2+]i . Therefo2+]i . We testOur interest in RES derivatives as potential chemotherapeutics and their pathways stems from the ability of RES to decrease cell viability in various cancer cell lines ,19,20. W2+ signaling [2+]i normally seen after treatment with TG  in several cancer cell lines [2+]i, we needed to determine whether the derivatives increased [Ca2+]i or acted by a different mechanism to decrease cell viability. Thus, we utilized live-cell microscopy to detect changes in relative [Ca2+]i after RES derivative addition. Treatment with 150 μM BuRV, IsoRV, and PIV induced increases in relative [Ca2+]i in MDA-MB-231 cells (2+]i were quantified by finding the area under the curve (AUC) after treatment with RES derivatives in MDA-MB-231 cells (2+]i that was significantly larger (by 21.66%) than the increase in relative [Ca2+]i induced by RES. In addition to analyzing the AUC for each compound, we determined the relative [Ca2+]i at the end of each assay. PIV also had an endpoint calcium concentration that was 46.03% (p < 0.05) higher than RES. This gives further evidence that PIV has a more pronounced effect on [Ca2+]i than RES.RES induces increases in cytosolic calcium concentration  and/or by activation of G-proteins, presumably through RES binding to a member of the G-protein coupled receptor (GPCR) family i to Ca2+ coming from stores within the cell. We pretreated cells with 5 μM thapsigargin (TG), a sarcoendoplasmic reticular Ca2+-ATPase (SERCA) inhibitor (SERCA is responsible for pumping Ca2+ from the cytosol to the ER), 5 min prior to the addition of 150 μM RES, BuRV, IsoRV, or PIV. With SERCA inhibited by TG, any increase in [Ca2+]i after RES derivative treatment could be attributed to PMCA inhibition, as Ca2+ flows from the ER and accumulates in the cytosol with impaired exit to the extracellular space. In our assay, we calculated the AUC after treatment with RES derivatives to quantify the PMCA inhibition activity of each derivative.To test the inhibitory activity of RES derivatives on PMCA, we utilized a protocol established by Samad et al. . Briefly2+]i following SERCA inhibition, indicating that these RES derivatives act as PMCA inhibitors in MDA-MB-231 cells  than increases in [Ca2+]i induced by RES after SERCA inhibition. These results suggest that, relative to RES, BuRV and PIV act as more potent PMCA inhibitors in MDA-MB-231 cells. The proportional decrease in cell viability induced by BuRV and PIV indicates that PMCA inhibition may play a role in the RES derivative anticancer effect.Treatment with 150 μM BuRV, IsoRV, and PIV induced significant increases in relative [Ca31 cells . In MDA-2+ source(s) to increase [Ca2+]i, we measured changes in [Ca2+]i after treating MDA-MB-231 cells with RES, followed by RES derivatives 6 min later  pathway. PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol triphosphate (IP3), which binds to the IP3 receptor (IP3R), a calcium channel that opens when IP3 binds to release ER Ca2+ i induced by RES derivatives is dependent on PLC. We compared the AUC after treatment with RES or derivatives to cells that were pretreated with U-73122 prior to treatment with RES or derivatives  for 10 min prior to the addition of RES derivatives. We compared the AUC after treatment with RES or derivatives to cells that were pretreated with 2-APB prior to treatment with RES or derivatives , only RES-treated cells have cell viability restored when pretreated with BAPTA-AM. The ability of RES derivatives to induce changes in breast cancer cell viability, apparently independent of calcium signaling, may indicate that the 10 μM BAPTA-AM was insufficient to block the increase in [Ca2+]i increases (as determined by the AUC) significantly larger than those induced by RES, which correlates with the increased effect of PIV on cell viability. Our PMCA inhibition assay revealed that RES as well as all RES derivatives under consideration inhibited PMCA in MDA-MB-231 cells, while only BuRV and PIV were found to be more potent inhibitors of PMCA than RES. Our IP3R and PLC inhibition assays also indicated that the RES and IsoRV-induced increases in [Ca2+]i show some level of dependency upon IP3R stimulation, but not PLC stimulation. Overall, our data indicates that, much like RES, ER-dependent Ca2+ release is utilized by RES derivatives to increase [Ca2+]i. Although decreases in cell viability induced by RES derivatives were not attenuated by BAPTA-AM treatment, RES derivatives activate an increase in [Ca2+]i. The experiments we conducted examining how Ca2+ accumulates in the cytosol provide further information on how RES and RES derivatives may affect [Ca2+]i and cell viability through the same pathway. However, it is unlikely that the molecular mechanism for RES and its derivatives is solely explained by calcium signaling. RES is a multifunctional small molecule that has been shown to activate several proapoptotic pathways i in MDA-MB-231 cell lines. TNBCs have uniquely modified calcium signaling pathways bis[N-[2-(acetyloxy)methoxyl]-2-oxoethyl]-bis[(acetyloxy)methyl] ester (BAPTA-AM-Acetoxymethyl ester) (15551) (BAPTA-AM), and trans-resveratrol (70675) (RES) were purchased from Cayman Chemical . U-73122 (J62898) and 2-Aminoethyl diphenylborinate (A16606) (2-APB) were purchased from Alfa Aesar . 3--2,5-diphenyltetrazolium bromide (AC158990050) (MTT) was purchased from Acros Organics . 4′-Butyrate resveratrol (BuRV), 4′-Isobutyrate resveratrol (IsoRV) and 4′-Pivalate resveratrol (PIV) were synthesized by the Andrus lab in the Department of Chemistry and Biochemistry at Brigham Young University i were measured using Fura-2, a Ca2+-binding dual-excitation fluorophore that excites at 340 nm when bound to Ca2+ and at 380 nm when not bound to Ca2+ i following treatment with RES derivatives. For experiments studying plasma membrane Ca2+-ATPase (PMCA) inhibition, fresh Ringer’s solution was removed and replaced with a Ca2+-free buffer, Ca2+- and Mg2+-free Hank’s Balanced Salt Solution (HBSS).Fura-2 loading and experimental protocols performed were similar to those performed by Peterson et al. . Cells w3R) inhibition, cells were incubated in inhibitors  for 10 min prior to the addition of RES derivatives.Following Fura-2 loading, cells were imaged with an Olympus IX51 inverted microscope. Treatments were added manually in 100-μL volumes. All RES and RES derivatives were in 1.5% DMSO in Ringer’s solution, with the exception of experiments studying PMCA inhibition in which HBSS was used in place of Ringer’s solution as a vehicle. Pretreatments of 2-APB were also in 1.5% DMSO in Ringer’s solution, while TG and U-73122 pretreatments used Ringer’s solution alone as a vehicle. In all experiments, cells were imaged for 1 min prior to the addition of treatment to establish a baseline. In experiments studying PMCA inhibition, cells were incubated in inhibitor (TG) for 5 min prior to RES derivative addition. In experiments studying phospholipase C (PLC) and inositol triphosphate receptor . The calcium traces were quantified by determining the area under the curve (AUC) or total calcium response after treatment addition using GraphPad Prism 7 .Images acquired were analyzed using CellSens software  were identified representing one cell each. At least 10 ROIs per experiment were chosen and analyzed in order to measure changes in relative [CaTM SMARTpool siRNA. Transfection media was prepared according to the manufacturer’s instructions and contained 8% FBS. The siRNAs used in this study were those for p53  and the non-targeting control siRNA . All experiments had mRNA knockdown >70% at 6 h post-siRNA  with Bonferroni correction. Statistically significant differences between RES or a derivative treatment and the vehicle treatment are indicated by * if


What may be surprising is that the identification of the molecules involved in mitochondrial Ca2+ transport occurred only in the last decade for both the influx  and the efflux  pathways. In this review, we will focus on the description of the amazing molecular complexity of the MCU complex, highlighting the numerous functional implications of the tissue-specific expression of the variants of the channel pore components (MCU/MCUb) and of the associated proteins .In the last few decades, a large body of experimental evidence has highlighted the complex role for mitochondria in eukaryotic cells: they are not only the site of aerobic metabolism (thus providing most of the ATP supply for endergonic processes) but also a crucial checkpoint of cell death processes (both necrosis and apoptosis) and autophagy. For this purpose, mitochondria must receive and decode the wide variety of physiological and pathological stimuli impacting on the cell. The “old” notion that mitochondria possess a sophisticated machinery for accumulating and releasing Ca Indeed, Ca2+ ions are responsible for decoding a variety of extracellular and intracellular stimuli, which, in animals, range from endocrine secretion to gene expression, muscle contraction, and synaptic transmission5. The efficacy of Ca2+ as a signalling molecule relies mainly on the maintenance of a steep Ca2+ gradient between the concentration in the extracellular (few mM) and intracellular (∼100 nM) environments. This >10,000-fold difference ensures that even very small changes in intracellular Ca2+ concentration ([Ca2+]i) are effective in regulating the numerous Ca2+-sensitive proteins of the cell, such as catalytic enzymes, channels, and transcription factors6. The maintenance of a low [Ca2+]i is tightly controlled by the presence of pumps and transporters both at the plasma membrane7 and at the membrane of organelles accumulating large amounts of Ca2+ 9. The endoplasmic/sarcoplasmic reticulum (ER or SR in striated muscle) is undoubtedly the main intracellular Ca2+ store; nevertheless, other cellular compartments actively participate in modulating [Ca2+]i: first of all mitochondria but also the Golgi apparatus10, endosomes, and lysosomes11.Ca2+ handling was first demonstrated in the 1960’s, when their ability to actively accumulate Ca2+ was evaluated in different cellularex vivo models14. This occurred even before the advent of the chemiosmotic theory15, which provided the thermodynamic basis for Ca2+ entry into mitochondria. In addition, the role for mitochondria in cell function has gradually expanded, and its initial identification as the powerhouse for cellular energy supply was subsequently integrated into a more complex activity that includes the regulation of cell death, metabolism, and signalling pathways18. Notably, many mitochondrial functions are directly regulated by the level of Ca2+ ions inside the organelles19. The control of mitochondrial Ca2+ concentration ([Ca2+]mt) is thus of primary relevance for cell physiology, and emerging evidence converges on the concept that its dysregulation is of utmost importance in the establishment of pathological conditions.The role for mitochondria in Ca2+ uptake, the mitochondrial Ca2+ uniporter (MCU) complex 21, and, one year after, by the cloning and molecular characterisation of the gene coding for the MCU23, the entry pathway was characterised at the molecular level and the route for the genetic manipulation of mitochondrial Ca2+ uptake was finally open.Since the first reports of mitochondrial Cain silico screenings23 identified theCCDC109a gene product as the long-sought pore-forming unit of the MCU channel mediating mitochondrial Ca2+ uptake in mammalian cells. Purified MCU protein reconstitution in lipid bilayers revealed a Ca2+ channel activity with the electrophysiological features of the hypothetical MCU20. In addition, it was clearly shown that MCU downregulation in mammalian cells inhibits mitochondrial Ca2+ uptake while its overexpression increases mitochondrial Ca2+ accumulation, thus proving that it isbona fide the channel mediating Ca2+ uptake in mitochondria23.In 2011, two independent24. A single MCU moleculeper se is thus not able to form the channel but should organise in oligomers, and MCU was initially proposed to arrange itself into tetramers25. This hypothesis was very recently confirmed by coherent high-resolution cryo-EM data from four independent groups on different MCU orthologs28. These seminal studies finally unveiled the structure and arrangement of MCU protomers within the complex and the exact position of the channel selectivity filter. The 3D reconstruction of EM data unambiguously revealed that MCU forms tetramers with a nonobvious symmetry: the transmembrane domain displays a fourfold symmetry, while the N-terminal domain (NTD) on the matrix side shows a twofold symmetry axis. These findings confuted the pentameric MCU architecture that was previously proposed based on magnetic resonance and negative staining EM data fromCaenorhabditis elegans MCU-ΔNTD protein, in which a significant portion of the NTD was removed to facilitate the analysis29. In addition, the MCU DIME motif, which contains the two critical acidic residues directly involved in the cation coordination, has now been shown to reside at the beginning of––and thus integral to––the second transmembrane helix (TMH), and not in the loop connecting the two transmembrane helices, as previously suggested29.The sequence of MCU consists of two transmembrane α-helix domains spanning the inner mitochondrial membrane (IMM), the second of which contains the critical DIME motif responsible for the channel selectivity filter, linked by a short loop toward the mitochondrial intermembrane space (IMS), while both the C- and the N-termini face the mitochondrial matrixet al. showed, MCU is not the only pore-forming unit of the oligomer, but it associates with the protein encoded by the MCU paralog geneCCDC109b, which was thus named MCUb25. The characterisation of MCUb demonstrated that it acts as a negative regulator of MCU activity both in lipid bilayer experiments and when overexpressed in mammalian cells25 , pointed to a more subtle and less obvious involvement of mitochondrial Ca2+ uptake in organ metabolism and organism development. This issue has been elegantly addressed by recent studies implementing tissue-specific modulation of MCU expression (by ablation/downregulation or overexpression) in adult tissues. For example, cardiac-specific tamoxifen-inducibleMCU deletion in adult mice, differently from the germline genetic ablation ofMCU, which did not protect from ischemic-reperfusion injury34, clearly protects from ischemia-reperfusion heart damage, abrogates the contractile responsiveness to β-adrenergic stimulation responsible for the so-called “fight-or-flight” response, and reduces heart bioenergetics reserve capacity, even though it does not induce phenotypic abnormalities either in basal conditions or after cardiac overload and does not alter cardiomyocytes’ resting [Ca2+]mt39. These results suggest that MCU may be dispensable for cardiac homeostasis in basal conditions, while it appears to play a major role in cardiac metabolic flexibility during acute stress. Similarly to what has been reported for the heart tissue, germline deletion ofMCU did not protect from hypoxic/ischemic brain injury40. However, brains from conditional neuronal-specific inducibleMCU-deleted mice as well as primary cortical neurons silenced forMCU show a significant reduction of the hypoxic/ischemic damage and decreased cell death without impairment of neuronal mitochondria metabolism41. Once more, these findings point to the existence of a strong drive for organs and tissues to respond to chronicMCU ablation by establishing adaptive metabolic shunts in order to cope with/bypass impaired mitochondrial Ca2+ signalling. Such adaptation to mitochondria dysfunction has also been described in the cardiac-specific–/–Tfam mouse, the murine model for human cardiomyopathies with mtDNA depletion42, in which the alteration in mitochondrial metabolism due to oxidative phosphorylation (OXPHOS) deficiency induces a secondary upregulation of MCU protein and a concomitant downregulation ofNCLX transcription43. The resulting increased mitochondrial Ca2+ uptake and reduced Ca2+ efflux in–/–Tfam cardiac mitochondria, although inducing an elevated and potentially damaging [Ca2+]mt, appear somehow functional to ensure an efficient Ca2+-dependent mitochondrial respiration that otherwise would be compromised by the pathology41.After the molecular identification of the MCU pore components and auxiliary factors, the path for the genetic manipulation of mitochondrial Ca2+ uptake machinery should be evaluated with caution, and the potential induction of unpredicted phenotypes, as a consequence of adaptive/maladaptive responses, should be taken into account. This is particularly relevant whenin vivo models are considered.Overall, these findings lead us to the following consideration: experimental systems featuring long-term genetic manipulation of the mitochondrial Ca2+ signalling in the maintenance of organ and organism homeostasis. The decrease or enhancement of [Ca2+]mt by AAV-mediated MCU silencing or overexpression, respectively, demonstrated that mitochondrial Ca2+ uptake regulates myofibre trophism through the modulation of the activity of the insulin growth factor-1/Akt pathway and the transcription of the peroxisome proliferator-activated receptor gamma coactivator 1 alpha 4 gene (PGC1α4). In particular, the overexpression of MCU in both adult and neonatal muscles induces significant fibre hypertrophy, which is not accompanied by alterations in mitochondrial membrane potential or other metabolic parameters (such as glycogen content and succinate dehydrogenase activity)44. On the contrary, MCU downregulation leads to marked fibre atrophy as demonstrated by reduction of fibre size, inhibition of Akt activity, reduction ofPGC1α4 transcripts, and impaired activation of the pyruvate dehydrogenase complex44, in agreement with the data from the–/–MCU mice34  has been fundamental to uncover the metabolic rewiring occurring at the level of skeletal muscle cell but also, importantly, systemically45. In line with previous data44, the muscle of–/–skMCU mice shows decreased performance and smaller fibre size compared to that of control mice45. Moreover, it shows a shift toward fast fibre type and an enhanced preference for fatty acid oxidative metabolism45. Interestingly, the ablation ofMCU in skeletal muscle also impinges on other tissues, as shown by the increased catabolic response in both liver and adipose tissue of–/–skMCU animals45. This clearly points out how the mitochondrial metabolic adaptation in response to altered Ca2+ dynamics (i.e.MCU deletion) within the muscle tissue may have major systemic impact on different tissues and, importantly, on the modulation of global metabolism.Moreover, the recent description of a mouse model bearing constitutive skeletal muscle-specific deletion of MCU  mostly due to breathing failure for the underdevelopment of the respiration coordination centres (cerebellum and Purkinje cells)49. The few surviving knockout mice display muscle impairment and ataxia48, a phenotype very similar to that described in children with aMICU1 mutation50. The analysis of–/–MICU1 mitochondria revealed increased matrix [Ca2+]mt, increased sensitivity to permeability transition pore (PTP) opening and cell death, and reduced production of ATP, similar to what was reported inMICU1-silenced hepatocytes and Hela cells47. Overall, these data clearly highlight the relevance of MICU1 gatekeeping function in the control of mitochondrial Ca2+ homeostasis and metabolism. Nevertheless, an additional intriguing feature of MICU1 action has recently emerged, which consists of its ability to confer MCU selectivity for Ca2+ over Mn2+. Indeed, the DIME motif in the MCU pore subunit appears to be unableper se to discriminate between Ca2+ and Mn2+ in the absence of MICU151. However, when present, MICU1 prevents Mn2+ entry in mitochondria, thus ensuring the stringent Ca2+ selectivity of MCU51. In this scenario, the proper stoichiometry of MICU1 is thus crucial to avoid mitochondrial Mn2+ uptake and to prevent Mn2+ toxicity. This concept may have relevant implications for human pathologies, especially for those diseases caused by MICU1 deficiency53 but also for Parkinson’s disease, as already suggested51.MICU1 was the first regulator of mitochondrial Ca54, which share 41% and 34% identity with MICU1, respectively, but display a broad and non-overlapping expression pattern compared to MICU155. In particular, MICU2 was shown to bind covalently to MICU1 through disulphide bridges, and the resulting dimer endows the genuine MCU gatekeeper activity56. In this scenario, cytosolic Ca2+ elevation causes Ca2+ binding to the EF-hand domains of the MICU1–MICU2 dimer, allowing MICU1 to function as a positive regulator of MCU activity, thus efficiently promoting Ca2+ entry into the organelle56  and MICU3 (EFHA2)e56 see.54. Along this line, its role as a positive regulator of mitochondrial Ca2+ uptake has been demonstrated recently55. Indeed, the expression of MICU3 in Hela cells, which normally do not express it, showed that it interacts with MICU1, but not with MICU2, forming obligatory dimers55  owing to the MICU1–MICU2 gatekeeper action, as already discussed, becomes extremely efficient upon [Ca2+]i elevation. Indeed, Ca2+ binding to the MICU1–MICU2 regulatory complex converts it from an inhibitory regulator (ensuring gatekeeping) at basal [Ca2+]i to an activator, strongly enhancing the flux through the channel. The threshold for MCU activation is directly dependent on the Ca2+ affinity of MICUs’ EF hands (which range from 300 nM to 1.3 µM), since the cooperative Ca2+ binding to these domains induces a conformational change in the MICU1–MICU2 dimer, which acts as a molecular switch relieving MCU inhibition57.By the interaction of monomers with non-overlapping functions, the MICU proteins confer an important property of the mitochondrial Ca2+ crosstalk between single inositol trisphosphate receptor (IP3R) and MCU channels58. Indeed, the presence of MICU2 has been shown to decrease the Ca2+ affinity of the MICU1 gatekeeper, thus elevating the threshold for MCU opening58. In the context of the intracellular environment, this increased threshold for mitochondrial Ca2+ uptake translates into the need for a shorter distance between the ER Ca2+-releasing channel (IP3R) and the mitochondrial Ca2+ uptake channel (MCU)58.Despite the role for MICU2 in the regulation of MCU gatekeeping and activation being controversial still, an intriguing hypothesis is that it has evolved to spatially restrict the Ca2+ uptake rate. The relative proportion of the MICU proteins, in particular, has been demonstrated as the basis of the tissue-specific differences in cellular Ca2+ dynamics59. Indeed, the different ratio of MICU1:MCU and MICU1:MICU2 proteins in the liver, the heart, and skeletal muscle has been correlated with the distinct mitochondrial Ca2+ handling of these three tissues59. The level of MICU1 expression, which is much lower in cardiac tissue (while MCU and MICU2 are present at similar levels) compared to liver, has been suggested to determine the lower maximal Ca2+ uptake capacity and less-steep Ca2+ dependence that characterise cardiac mitochondria compared to liver mitochondria, as electrophysiological data32 and intracellular Ca2+ measurements59 revealed. The low amount of MICU1 in cardiac mitochondria is instrumental to reduce mitochondrial Ca2+ uptake upon cytosolic Ca2+ elevation, allowing the decoding of the repetitive cytosolic Ca2+ spikes of the beating heart into a graded increase of matrix Ca2+. This integration of frequency fluctuations is crucial to prevent mitochondrial Ca2+ overload, which will be otherwise detrimental to the cardiac myocytes. By contrast, the relatively high MICU1 level and its cooperative activation of MCU in hepatocytes ensure the punctual and massive mitochondrial Ca2+ increase at every single cytosolic Ca2+ spike59, which is functional to liver oxidative metabolism.In line with this, in the last few years, the stoichiometry of MCU components has emerged as a crucial feature of the modulation of mitochondrial Ca60  and the matrix (EMRE)62. However, this hypothesis was subsequently questioned by other groups, whose data supported an opposite topology for the EMRE protein, with the short N-terminus exposed to the matrix while its acidic C-terminus faces the IMS64. In this conformation, EMRE action would be that of supporting MCU Ca2+ transport activity by interacting with the first TMH of MCU within the IMM and to interact also with MICU1 at the IMS via its C-terminus poly-aspartate tail, thus ensuring MICU1 binding to the channel and gatekeeping activity63.To add further complexity to the study of MCU channel regulation, other molecules have been shown to interact with MCU in recent years. Indeed, the quantitative mass spectrometry analysis of the MCU-interacting proteins revealed the presence of a critical component of the MCU complex, the essential MCU regulator (EMRE) see, a 10 kD65, or by expression of proteolytic-resistant EMRE constructs62, leads to uncontrolled MCU channel opening causing mitochondrial Ca2+ leakage and mitochondrial Ca2+ overload, resulting in neuronal cell death66. The existence of a tight control of EMRE protein levels has also been demonstratedin vivo in the–/–MICU1 mice, where EMRE is reduced in a genetic condition characterised by increased [Ca2+]mt basal levels48. These findings clearly underline a negative regulatory mechanism that keeps EMRE expression in check in order to cope with changes in MCU activity48.Recently, the control of EMRE protein levels has emerged as a crucial aspect of MCU complex regulation. Indeed, a correct amount of EMRE is fundamental to guarantee the exact stoichiometry of the different MCU components. Alteration of EMRE protein levels by ablation of the AAA-proteases responsible for its degradation68, despite the original proteomic analysis of the MCU interactors failed to recover it61, is the MCU regulator 1 (MCUR1). MCUR1 is a coiled-coil-containing protein encoded by theCCDC90A gene initially identified by a RNAi screen searching for a mitochondrial membrane protein involved in MCU homeostasis67, whose precise function in the regulation of MCU activity and mitochondrial metabolism is still debated. Indeed, MCUR1 has been shown to be required for MCU-dependent Ca2+ uptake, since its knockdown dampens mitochondrial Ca2+ entry while its overexpression enhances it67. However, a direct action of MCUR1 on MCU activity was questioned by the finding that its silencing leads to a significant loss of mitochondrial membrane potential (ΔΨm), whichper se can account for the decreased mitochondrial Ca2+ uptake69. In this context, MCUR1 has been suggested to act primarily as a cytochrome c oxidase (COX) assembly factor69, and this concept would also be supported by the fact that a MCUR1 orthologue is present in budding yeast70, which lack MCU, and is required for yeast COX activity and survival in non-fermentable medium69. Nevertheless, the patch clamp data of Ca2+ currents from voltage-clamped mitoplasts point to an effective role for the MCUR1 protein on MCU channel conductance, independently of ΔΨm71. In line with this, experiments inDrosophila cells, which are resistant to Ca2+-induced mitochondrial permeability transition (MTP)72 and in which no MCUR1 homologs are found, show increased sensitivity to Ca2+-dependent PTP opening when heterologous human MCUR1 is expressed, which is not accompanied by alterations in the rate of mitochondrial Ca2+ uptake72. Conversely, MCUR1 knockdown in mammalian cells renders them resistant to Ca2+ overload, providing protection from cell death72. This led the authors to conclude that MCUR1 regulates the Ca2+ threshold for the MPT and may act as a molecular bridge connecting the MCU channel to the MTP complex. Whether this hypothesis holds true still has to be tested.Another protein that has been associated with the MCU complexMCUR1 in endothelial and cardiac tissuesin vivo by mouse conditional knockout models68 points to an involvement of MCUR1 in the assembly and function of the MCU channel. In particular, MCUR1 binding to MCU and EMRE appears to be necessary for MCU oligomerisation and stability, since–/–MCUR1 cells will display a significantly decreased amount of MCU-containing high-molecular-weight complexes and reduced rate of mitochondrial Ca2+ uptake68. This, as expected, impinges on cellular energetics, as it is reflected by the dramatic reduction of ATP levels and consequent activation of the pro-survival autophagy pathway in cells depleted of MCUR172. Whether the pleiotropic roles for MCUR1 in the regulation of mitochondrial Ca2+ uptake, OXPHOS efficiency, and the apparent discordant phenotypes of MCUR1-depleted cells depend on the intrinsic transcriptional and metabolic differences of different cell types or on the species-specific features of the mitochondrial Ca2+ machinery is still an open question, and further investigation is needed to clarify it.More recently, the genetic deletion of2+ uptake machinery has been revealed to be far more complex than that resulting from just considering the cooperative actions of the different MCU components and their possible combinations. Indeed, the activity of the MCU channel relies on a multifaceted integration of regulatory mechanisms acting on both the MCU pore subunits and its co-regulators. Moreover, these mechanisms have been shown to occur at multiple levels––transcriptional, post-transcriptional, and post-translational––and their systematic identification has started to be accomplished only in recent years.The regulation of the mitochondrial Ca2+ signalling through the involvement of calmodulin (CaM) and the activation of CaM-kinase (CaMK)73. It has been shown that the immediate-early gene Npas4, a neuronal transcription factor with neuroprotective action against excitotoxicity74, acts directly downstream of the NMDAR signalling and of CaMK to modulateMCU transcription73. In another context, chromatin immunoprecipitation and promoter reporter analyses revealed that the Ca2+-regulated transcription factor cyclic adenosine monophosphate response element-binding protein (CREB) directly binds the MCU promoter and stimulates its transcriptional activity in chicken DT40 lymphocyte cells75. The CREB-mediated activity-dependent modulation of MCU expression in response to intracellular Ca2+ mobilisation via IP3R and store-operated Ca2+ entry (SOCE) has also been proven to be crucial to ensure a prompt metabolic flexibility and cell survival in lymphocytes75.In neurons, for example, MCU transcription appears to be under the control of activity-dependent cytosolic Ca76, where a strong inverse correlation between MCU expression level and the abundance of microRNA (miR)-25 has been reported in both tumour cell lines and colon adenocarcinoma samples76. Indeed, MCU expression is found to be upregulated in both colon and prostate cancer cells that show a reduced level of miR-25, and the overexpression of miR-25 in these cells downregulates MCU and increases cell sensitivity to apoptosis76. Similarly, in breast cancer, the downregulation of miR-340 is correlated with increased MCU expression in highly metastatic cells, while MCU targeting by miR-340 blocks the metabolic shift from OXPHOS to aerobic glycolysis that would otherwise favour cell migration and invasiveness77. In the context of pulmonary arterial hypertension (PAH), in which vascular cells are hyper-proliferative and apoptosis resistant, exhibiting a cancer-like phenotype, the decreased MCU function that underlies the key phenotypic features of PAH  has been ascribed to the downregulation of MCU expression by both miR-25 and miR-13878. The overexpression of MCU or the blocking of the MCU-targeting miRs in PAH cells indeed restores Ca2+ dynamics, mitochondrial metabolism, and cell proliferation to physiological levels78. MiR-25 has also been shown to target and downregulate MCU in the heart, where it is suggested to protect myocytes from oxidative damage by reducing [Ca2+]mt levels79. Another two muscle-specific miRNAs (myomiR), miR-1 and miR-206, have been implicated in the regulation of MCU expression in muscle80, of which miR-1 plays a major role in the context of cardiac development in both mice and humans. In mice, miR-1 upregulation is crucial during the first few postnatal weeks for the modulation of MCU activity in order to prevent massive [Ca2+]mt elevation that would otherwise occur, since mitochondria distribution undergoes heavy remodelling at this time point and organelles are placed in close contact with the SR Ca2+-releasing channels80.The existence of post-transcriptional regulation of MCU has been initially described in cancer2+-dependent tyrosine kinase Pyk2 has been shown to directly interact and phosphorylate MCU in cardiac cells in response to α-adrenergic stimulation81. The MCU phosphorylation by Pyk2 has been proposed to promote its oligomerisation and to enhance mitochondrial Ca2+ entry, thus stimulating mitochondrial metabolism and favouring cytosolic Ca2+ clearance in cardiac cells81. In addition, the atomic resolution data of the N-terminus of MCU (residues 72–189) allowed the description of the exact structure of the MCU matrix domain82. This domain was shown to acquire a β-grasp-like fold and to bind Mg2+ and Ca2+ through a MCU-regulating acidic patch (MRAP)82. The disruption of cation binding via mutation of the MRAP sequence impairs MCU oligomerisation, reduces basal [Ca2+]mt levels, and leads to significant dampening of mitochondrial Ca2+ uptake after agonist stimulation, highlighting the role of divalent cations in the regulation of MCU activity81. This has been proposed as a possible feedback mechanism of MCU regulation aimed at preventing excessive mitochondrial Ca2+ entry in conditions of elevated [Ca2+]i.Post-translational modifications of MCU proteins have also been described. Indeed, the Ca83. One of these modifications, S-glutathionylation, was recently shown to occur in MCU protein as the result of oxidation at the evolutionarily conserved Cys-9784. The S-glutathionylation has been demonstrated to promote a MCU conformational change, which increases oligomerisation84. In human pulmonary microvascular endothelial cells (HPMVECs), this enhances MCU activity and promotes increased mitochondrial Ca2+ entry both in basal conditions and upon agonist challenge, causing cellular bioenergetics crisis and higher sensitivity to cell death84.Finally, a wide range of chemical modifications have been reported to target the thiol moiety of Cys residues in many proteins, enabling biological switching of structure and reactivity oxidation2+ entry in mitochondria has made a great step forward in the last few years, and new possibilities for effective modulation of MCU activity have been revealed. In this line, very recently, a systematic orthologous interspecies chemical screen based on the combination of an optimised yeast cell system and a mammalian mitochondria-based NCC library drug screen picked out and validated the first MCU-specific inhibitor molecule to be described: mitoxantrone85. Mitoxantrone has been utilised already in clinical practice for its antineoplastic action against non-Hodgkin’s lymphomas and acute myeloid leukaemia86; however, the anti-tumour properties of this drug appear to rely on a different molecular moiety with respect to its anti-MCU activity, thus opening up the possibility for the chemical engineering of new lead compounds to specifically target MCU function. This is of outmost relevance for the design of novel potential therapeutic approaches to pathologies in which mitochondrial Ca2+ signalling dysfunction is involved88, including those characterised by primary MCU dysfunction due to mutations in its components53.Overall, the understanding of the mechanisms controlling Ca2+ uptake regulation.In conclusion, despite many of the molecular mechanisms refining MCU activity in the cell having been described, of which the most relevant are presented in this review, a number of other possibilities for the modulation of uniporter function may exist , and additional efforts are needed from the scientific community to fully unravel the complexity of mitochondrial Ca2+ transport machinery. Most of the components of the MCU complex have been identified, and several cellular andin vivo models have been generated that helped to define the physiological relevance of mitochondrial Ca2+ uptake. Still, many issues remain obscure and need to be investigated. The entire spectrum of molecular mechanisms by which different cell types finely tune mitochondrial Ca2+ uptake appears to be related not only to the metabolic needs but also to the breadth of cellular activities modulated by organelle Ca2+ levels. We believe that the complete picture will emerge when at least three conceptual mechanisms are fully investigated, i.e. gene expression (the MCU and MICU1 isoforms show significant tissue-specific differences in expression), alternative splicing (as in the case of MICU1.1), and post-transcriptional and post-translational regulation . We then need to explore the functional interplay between mitochondrial Ca2+ transport and other ion fluxes, such as Na+ and K+, for which complexity and partial redundancy of transport mechanisms have been described but molecular definition still lags behind. Unravelling this cross-talk will greatly enhance our insight into the regulation of mitochondria, these fascinating organelles with pleiotropic effects on a cell’s life and death.In the last few years, tremendous advances have been made in defining the molecular identity of the mitochondrial Ca2+]i, intracellular Ca2+ concentration; [Ca2+]mt, mitochondrial Ca2+ concentration; CaM, calmodulin; CaMK, calmodulin kinase; CREB, cyclic adenosine monophosphate response element-binding protein; COX, cytochrome c oxidase; EMRE, essential mitochondrial Ca2+ uniporter regulator; ER, endoplasmic reticulum; IMM, inner mitochondrial membrane; IMS, intermembrane space; IP3R, inositol trisphosphate receptor; MCU, mitochondrial Ca2+ uniporter; MCUR1, mitochondrial Ca2+ uniporter regulator 1; MICU, mitochondrial Ca2+ uptake protein; miR, microRNA; MRAP, mitochondrial Ca2+ uniporter-regulating acidic patch; MTP, mitochondrial permeability transition; OXPHOS, oxidative phosphorylation; PAH, pulmonary arterial hypertension; PGC1α4, peroxisome proliferator-activated receptor gamma coactivator 1 alpha 4; PTP, permeability transition pore; SR, sarcoplasmic reticulum; TMH, transmembrane helix[CaGiorgia Pallafacchina wrote the original draft, Sofia Zanin drew the figures, and Rosario Rizzuto revised the manuscript.