Here, we show that apoptotic response of AML cells to the synergistically acting polyphenols curcumin (CUR) and carnosic acid (CA), combined at low, non-cytotoxic doses of each compound was mediated solely by disruption of cellular Ca2+ homeostasis. Specifically, activation of caspase cascade in CUR+CA-treated AML cells resulted from sustained elevation of cytosolic Ca2+ (Ca2+cyt) and was not preceded by endoplasmic reticulum stress or mitochondrial damage. The CUR+CA-induced Ca2+cyt rise did not involve excessive influx of extracellular Ca2+ but, rather, occurred due to massive Ca2+ release from intracellular stores concomitant with inhibition of Ca2+cyt extrusion through the plasma membrane. Notably, the CUR+CA combination did not alter Ca2+ homeostasis and viability in non-neoplastic hematopoietic cells, suggesting its cancer-selective action. Most importantly, co-administration of CUR and CA to AML-bearing mice markedly attenuated disease progression in two animal models. Collectively, our results provide the mechanistic and translational basis for further characterization of this combination as a prototype of novel Ca2+-targeted pharmacological tools for the treatment of AML.Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by extremely heterogeneous molecular and biologic abnormalities that hamper the development of effective targeted treatment modalities. While AML cells are highly sensitive to cytotoxic Ca Acute myeloid leukemia (AML) is one of the most aggressive hematologic malignancies in adults, with 5-year relative survival rates of 17–19% ), relaps2+ tightly controls diverse processes, ranging from enhanced proliferation and survival in response to transient elevation of cytosolic Ca2+ ([Ca2+] cyt) to apoptosis triggered by long-term [Ca2+] cyt rise cyt [2+ homeostasis in this cancer cell type are currently available.Cellular Cacyt rise . Consequgnancies –10. Feasl trials . AML cel [2+]cyt –15, but 2+ homeostasis in neoplastic cells, though the exact mechanisms underlying polyphenol-mediated Ca2+ changes remain poorly understood. Different polyphenols have been reported to suppress AML in cell-based and murine models cyt was expressed as the mean Fluo-3 fluorescence intensity.For evaluation of steady state cyt rise after the addition of thapsigargin followed by [Ca2+]cyt decline toward the baseline.For evaluation of endoplasmic reticulum Ca2+cyt-extruding function of the plasma membrane Ca2+ ATP-ase (PMCA) was assessed as described cyt decline.The Caescribed  with modhibitor) . SamplesExperiments were carried out in the Ben-Gurion University SPF animal facility in accordance with the protocol (IL-35-05-2012) approved by the University Committee for the Ethical Care and Use of Animals in Experiments.4 syngeneic C1498 AML cells in 100 μl PBS. Seven days later, animals were randomly assigned to four groups (6 mice/group) and were started on the following supplements mixed with the standard diet (ad libitum): Group 1 (vehicle) - 2.05% (w/w) methylcellulose (Sigma); Group 2 – 0.05% (w/w) CUR + 2% (w/w) methylcellulose; Group 3 – 2% (w/w) CA-rich rosemary extract (RE) + 0.05% (w/w) methylcellulose; Group 4 – 0.05% (w/w) CUR + 2% (w/w) CA-rich RE. Animal appearance and behavior were then assessed daily and the body weight was measured 3 days per week. Moribund mice were humanely sacrificed and overall animal survival was estimated by the Kaplan-Meier analysis, as described previously [For the systemic AML model , twenty-eviously .6 HL60 cells in 200 μl PBS. Seven days later, mice were randomly assigned to four groups (6 mice/group) and were started on the following treatments : Group 1 (vehicle) – 0.1% DMSO in PBS; Group 2 – 0.5 mg CUR in DMSO/PBS; Group 3 – 0.5 mg CA-rich RE in DMSO/PBS; Group 4 – 0.5 mg CUR + 0.5 mg CA-rich RE in DMSO/PBS. Mice were monitored for another 12 days. Animal appearance and behavior were assessed daily and the body weight was measured every 3 days. At the end of the experiment, all mice were humanely sacrificed and necropsied. Abdominal tumors were excised, weighed and then fixed in 10% neutral buffered formalin and embedded in paraffin.For the peritoneal AML tumor model , twenty-In Situ Apoptosis Detection Kit  according to the manufacturer's protocol. Slides were counterstained with hematoxylin for visualization of the nuclei. An average percentage of apoptotic cells was calculated after scoring a total of 200 cells in each of 17 random fields at 400× magnification.For the TUNEL assay, tissue sections were deparaffinized and apoptosis was assessed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using ApopTag Peroxidase t test. Animal survival data were statistically evaluated using a log-rank (Mantel-Cox) test. The statistical analysis was performed with GraphPad Prism 6.0 Program . Data are presented as the mean ± SD. P < 0.05 was considered statistically significant.The significance of the differences between the means of various subgroups was assessed by unpaired two-tailed Student's


At the cellular level cardiac hypertrophy causes remodelling, leading to changes in ionic channel, pump and exchanger densities and kinetics.Previous studies have focused on quantifying changes in channels, pumps and exchangers without quantitatively linking these changes with emergent cellular scale functionality.Two biophysical cardiac cell models were created, parameterized and validated and are able to simulate electrophysiology and calcium dynamics in myocytes from control sham operated rats and aortic‐banded rats exhibiting diastolic dysfunction.2+ channel and sarco/endoplasmic reticulum Ca2+ATPase as the principal regulators of systolic and diastolic Ca2+, respectively.The contribution of each ionic pathway to the calcium kinetics was calculated, identifying the L‐type Ca2+, through changes in expression of key Ca2+ modelling protein densities, is drastically reduced following the aortic banding procedure; however the cells are able to compensate Ca2+ homeostasis in an efficient way to minimize systolic dysfunction.Results show that the ability to dynamically change systolic Ca2+ transient. However, the relative roles of the underlying subcellular processes and the positive or negative impact of each remodelling mechanism are not fully understood. Biophysical cardiac cell models were created to simulate electrophysiology and calcium dynamics in myocytes from control rats (SHAM) and aortic‐banded rats exhibiting diastolic dysfunction. The model parameters and framework were validated and the fitted parameters demonstrated to be unique for explaining our experimental data. The contribution of each ionic pathway to the calcium kinetics was calculated, identifying the L‐type Ca2+ channel (LCC) and the sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) as the principal regulators of systolic and diastolic Ca2+, respectively. In the aortic banding model, the sensitivity of systolic Ca2+ to LCC density and diastolic Ca2+ to SERCA density decreased by 16‐fold and increased by 23%, respectively, relative to the SHAM model. The energy cost of ionic homeostasis is maintained across the two models. The models predict that changes in ionic pathway densities in compensated aortic banding rats maintain Ca2+ function and efficiency. The ability to dynamically alter systolic function is significantly diminished, while the capacity to maintain diastolic Ca2+ is moderately increased.Elevated left ventricular afterload leads to myocardial hypertrophy, diastolic dysfunction, cellular remodelling and compromised calcium dynamics. At the cellular scale this remodelling of the ionic channels, pumps and exchangers gives rise to changes in the Ca At the cellular level cardiac hypertrophy causes remodelling, leading to changes in ionic channel, pump and exchanger densities and kinetics.Previous studies have focused on quantifying changes in channels, pumps and exchangers without quantitatively linking these changes with emergent cellular scale functionality.Two biophysical cardiac cell models were created, parameterized and validated and are able to simulate electrophysiology and calcium dynamics in myocytes from control sham operated rats and aortic‐banded rats exhibiting diastolic dysfunction.2+ channel and sarco/endoplasmic reticulum Ca2+ATPase as the principal regulators of systolic and diastolic Ca2+, respectively.The contribution of each ionic pathway to the calcium kinetics was calculated, identifying the L‐type Ca2+, through changes in expression of key Ca2+ modelling protein densities, is drastically reduced following the aortic banding procedure; however the cells are able to compensate Ca2+ homeostasis in an efficient way to minimize systolic dysfunction.Results show that the ability to dynamically change systolic Ca ABaortic bandingAPaction potentialampCaCalcium amplitudeCaB2+background CaDCa2+ concentrationdiastolic CaHFheart failureLCC2+ channelL‐type CaNCX+/Ca2+ exchangerNaPCa2+ concentrationpeak CaPMCa2+‐ATPaseplasma membrane CaRyRryanodine receptorSERCA2+‐ATPasesarco/endoplasmic reticulum CaSRsarcoplasmic reticulumet al. et al. Heart failure (HF) due to left ventricular pressure overload following aortic stenosis or hypertension, remains a serious public health problem, associated with high morbidity and mortality Drazner, . It is c2+ is fundamentally important for transmitting this electrical timing signal into mechanic contraction. During each heartbeat, an electrical wave of depolarization spreads rapidly through the myocardium inducing an intracellular Ca2+ influx through the voltage dependent L‐type Ca2+ channels (LCCs)  in the sarcoplasmic reticulum (SR) , driving Ca2+ back to the SR and the Na2+/Ca2+ exchanger (NCX) and the sarcolemma Ca2+‐ATPase (PMCa) extruding Ca2+ out of the cell.In the heart, the role of Caet al. et al. 2+ channels, exchangers and pumps during the development of cardiac hypertrophy in rats. In particular, previous studies have reported the L‐type Ca2+ channel flux  or detrimental (decompensatory) for maintaining cardiac function. This is essential for identifying the proteins that derive detrimental mechanisms and manipulate them to maintain their regular levels.The use of experimental animal models of left ventricular pressure overload, such as the aortic banding (AB) procedure  at the expenses of systolic Ca2+.To address these issues, this study adopts a computational modelling based approach to quantify the relative role of each of the key changes in protein density and kinetics in cardiac myocyte Cae et al. , conduct. et al. . Røe et et al. (+/Ca2+ exchanger (NCX), Ca2+ pump (PMCa), sarcoplasmic reticulum Ca2+‐ATPase (SERCA), L‐type channel (LCC) and background Ca2+ channel (CaB). NCX and PMCa densities were constrained by Ca2+ transients induced by caffeine exposure following 1 and 6 Hz pacing. These results were then combined with Ca2+ transient measurements at 1 and 6 Hz to fit SERCA to the decay of the field stimulated Ca2+ transients at 1 and 6 Hz. The Ca2+ influx into the cell from the LCCs was fitted using the current–voltage relationship curve. The background Ca2+ current was modified to reproduce the peak of the Ca2+ transients at 1 and 6 Hz. The remaining Na+ and K+ currents were constrained using literature data to achieve a prolonged action potential duration commonly found in both human heart failure and in animal models of cardiac hypertrophy. The effect of EGTA in patched pipettes was modelled by setting [EGTA] = 0 mm and 0.6 mm in Ca2+ transient and AP experiment simulations, respectively.The model re‐fitting procedure is described in detail in the Appendix. Briefly, using the rat specific framework developed by Gattoni et al. , we para2+ dynamics. In three test models we introduced the AB NCX, LCC or SERCA model with all other parameters kept at the SHAM values . In three additional models we introduced pairs of LCC, NCX and SERCA AB models into the SHAM rat model  and finally we introduced NCX, LCC and SERCA AB models into the SHAM model (LCC–SERCA–NCX). We created two further models. The first started with the SHAM model and included SERCA, NCX and LCC AB models with the changes in K+ and Na+ channels from the AB model (LCC–SERCA–NCX–K–Na). The second was the same as the previous model but also included the background Ca2+ current parameters from the parameters used in the AB model. These nine models were simulated at 1 and 6 Hz giving a total of 18 simulated Ca2+ and AP transients. For each of these 18 models we performed graded changes in 17 of the main protein parameters regulating the electrophysiology currents: gSERCA: conductivity of the SERCA pump; KSERCA: half‐inactivation constant of the SERCA pump; JL: permeability of the L‐type channel; JR: permeability of the RyR; gCaB: conductivity of the background Ca2+ current; gSRl: conductivity of the SR leak; gt: conductivity of the transient outward potassium current; gNCX: conductivity of the Na+/Ca2+ exchanger; gpCa: conductivity of the Ca2+ pump; gBK: conductivity of the background K+ current; gBNa: conductivity of the background Na+ current; gK1: conductivity of the inward potassium current; gNa: conductivity of the Na+ current; gss: conductivity of the steady state potassium current; NaKmax: conductivity of the Na+/K+ exchanger; BCMDN: calmodulin concentration; and BTRPN: troponin concentration. Each parameter x was perturbed in the range log10(xnew) ∈ , where xnew is the parameter value used for a generic simulation and xBase is the fitted parameter value (in this way −1 corresponds to 10% of the control parameter and 1 corresponds to a 10‐fold increase). All developed models, representing 5814 parameter combinations were run to their limit cycle. For each model we identified how many of experimental phenotypes , DCa, relaxation time at 50% decay (RT50) and time to peak Ca2+ (Tpeak)) the model matched and we ranked models based on this number (1–4). A parameter combination is defined ‘viable’ if the considered simulated phenotype value falls within a 20% range of the experimental phenotype value .To test the resulting parameters for both the SHAM and AB rat models we performed a comprehensive model validation study. A schematic representation of the validation process is shown in Fig. 50 and Tpeak, were calculated for 11 protein densities: gSERCA, KSERCA, gCaB, gt, gK1, gNa, gss, VL , KL (concentration at LCC inactivation), JL, and gNCX. The sensitivity values were calculated with a 1% change in the protein density for both the SHAM and AB models’ proteins at 6 Hz, using the equation:p1 is the new parameter (increased by 1% from the fitted parameter), p0 is the fitted parameter, P(p1) is the tested phenotype value evaluated in the new parameter p1, and P(p0) is the tested phenotype value evaluated in the fitted parameter p0.The sensitivity of four calcium transient phenotypes, PCa, DCa, RT2+ and AP traces for the SHAM and AB models at 1 and 6 Hz. At 1 Hz, simulated DCa and RT50 in the AB model were reduced by 59% and 7%, respectively, compared with the DCa and RT50 simulated values in the SHAM model and consistent with a decrease of 42% and 17%, respectively, observed experimentally. At the same frequency, simulated PCa was increased by 3% although experimental measurement revealed a 7% decrease. Simulated SR Ca2+ content in the AB model at 1 Hz was increased by 28% compared with the simulated value in the SHAM case and compatible with increased SERCA activity in both experimental and simulated measurements.Fig. 50 in the SHAM model remained unchanged in the AB model with a value of 63 ms, falling within mean experimental measurements of 63 ± 6.4 and 57 ± 7 ms in the SHAM and AB cases, respectively. At the same frequency, simulated PCa in the AB model was decreased by 30% with respect to the simulated value in SHAM model, comparable to a 40% decrease in the experimental data. Simulated SR Ca2+ content in the AB model at 6 Hz was increased by 61% compared with the simulated value in the SHAM model, due to increased SERCA activity. Key Ca2+ characteristics are summarized in Table At 6 Hz, simulated DCa in the AB model decreased by 68%, consistent with the experimentally observed 67% decrease. Simulated RT2+ extrusion through SERCA, NCX and PCA by evaluating their simulated integrals during one cycle. Results in the SHAM case reveal a ratio of 86.6%, 10% and 3.4%, respectively, at 1 Hz, and 94%, 5% and 1%, respectively, at 6 Hz. Results in the AB case reveal a proportion of 92%, 6.3% and 1.7%, respectively, at 1 Hz and 96.4%, 2.8% and 0.8%, respectively, at 6 Hz. Prolonged duration of the electrical AP is shown in Fig. 90) increased by 66%, comparable to an increase of 50% in the experimental data. At the higher pacing frequency of 6 Hz, repolarization was faster compared with the same model at 1 Hz and we predicted APD90 to be increased by 50%, comparable with a 50% increase in the experimental data.We predicted the proportion of Ca2+ pump conductivity gPCaε and a decrease in the background current conductivity gCaBε. The resulting simulated PCa, DCa, RT50, Tpeak and Calcium amplitude (Caamp) values for those four parameter combinations produce very similar results, as shown in Table 2+ conductivity parameter gCaB = 0.6×10−8falls within the acceptable range.Our model validation tables are represented in Figs 2+ kinetics are associated with a change in the energetic cost of maintaining ionic homeostasis, we calculated the ATP consumption over one cardiac cycle. For each ion transporter, taking into account its specific stoichiometry , we calculated the ATP consumption per cardiac cycle as the integral of the rate of ATP consumption over each cardiac cycle.To determine if the changes in Cam per beat respectively in the SHAM and AB cases at 6 Hz. ATP consumption by SERCA, Na+/K+ and PMCa at 6 Hz shows values of 24.1, 4.15 and 2.9 μm, respectively, in the SHAM case and 27.4, 2.92 and 2.23 μm, respectively, in the AB case.Comparison of ATP consumption per cardiac cycle showed no relevant changes, with total values of 31.15 and 32.55 μTo estimate the importance of different proteins during the progression from SHAM to AB and to distinguish between compensatory and decompensatory mechanisms, a sensitivity analysis was performed. The sensitivity values are shown in Table + current parameter changes on all the studied Ca2+ phenotypes. In Table JL (LCC permeability) leads to a 5.7% increase in PCa, 5.8% increase in DCa, 7.6% increase in RT50 and 7.7% increase in Tpeak. Conversely, a 1% increase in gt (Ito conductance) leads to a 3.3% decrease in PCa, 3.6% decrease in DCa, 4.5% decrease in RT50 and 3.8% decrease in Tpeak.The sensitivity analysis for the SHAM model at 6 Hz in Fig. Tpeak in the AB model. A 1% increase in all tested parameters leads to less than 0.5% change in PCa and no change in Tpeak. A dominant role of gSERCA is visible on DCa where a 1% increase in this parameters produces a 1.6% decrease in DCa. JL and gSERCA have major effects on RT50 where a 1% increase in both those parameters leads to a 1.5% decrease in RT50.In the AB model at 6 Hz in Fig. 2+ transient and AP. Starting from the AB model, we performed a 10% backward linear change in all parameters (towards the SHAM case) and a 10%, 20% and 50% linear change in all parameters forward towards the AB case. In Fig. To observe the joint effect of all altered subcellular proteins during the transition from SHAM to the final AB state, in Fig. et al. Alterations of excitation–contraction coupling, resulting from altered proteins in calcium homeostasis, are thought to play a critical role in heart failure  suggesting that the decrease in Ca2+‐ATPase mRNA levels may be a marker of the transition from compensatory hypertrophy to failure in rats.We simulated an increase in SERCA activity following AB, concordant with the experimental measurements in this work. Many studies have reported decreased SERCA activity or gene expression in hypertrophy and HF   and transient outward K+ (gt) conductivities are important in the SHAM model in that increased JL leads to increased PCa, DCa, RT50 and Tpeak while increased gt leads to decreased PCa, DCa, RT50 and Tpeak. The magnitude of this sensitivity is drastically reduced following AB. The ability to dynamically change systolic Ca2+ is an important property of cardiac myocytes in so much that it enables regulation of the strength of cardiac contraction and thus plays a role in the regulation of blood supply throughout the body across a wide range of different conditions, including exercise. In this context, reduced magnitude of the sensitivity following AB is a clear sign of diminishing function, in the sense that the system is less responsive to changes in the AB state compared with the SHAM state. Despite numerous investigations, the role of L‐type Ca2+ channels in the pathogenesis of cardiac hypertrophy and HF remains controversial  while decompensatory effects will be discordant in the SHAM and AB cases . In this way, we assume that there is a protein direction of change at SHAM that will improve function. However, if the sensitivity is discordant then this means that this given protein is having the opposite effect on Ca2+ function in AB compared with SHAM animals, thereby indicating a transition where changes in a protein that were initially beneficial are now detrimental. In our sensitivity analysis, all the SERCA, inward rectifying K+ current, steady state K+ current and transient outward K+ current parameters show decompensatory effects on peak Ca2+ while the L‐type Ca2+ current, the inward Na+ current and the transient outward K+ current parameters show decompensatory effects on diastolic Ca2+. Focusing on the magnitude of the sensitivity, the L‐type Ca2+ channel and the transient outward K+ channel are the most significant pathways regulating PCa while SERCA is the most significant pathway regulating DCa. The transient outward K+ channel shows decompensatory effects on both PCa and DCa while the L‐type Ca2+ current reveals a compensatory effect on PCa and a decompensatory effect on DCa. SERCA changes reveal a compensatory effect on DCa and a decompensatory effect on PCa. Overall, results following AB reveal the major compensatory effect of SERCA on DCa at the expenses of PCa, highlighting the trend of the system to reduce DCa in order to maintain a physiological Ca2+ balance as a response to AB , the inwardly rectifying K+ current (IK1) and the steady state K+ current (Iss), all playing a significant role in rat repolarization , diastolic Ca2+ (DCa), relaxation time of the Ca2+ transient at 50% decay (RT50) and time to peak of the Ca2+ transient (Tpeak). For each box in the tables we have performed 19 different simulations . The box is coloured in grey if at least one of those 19 simulations falls inside a 20% variability of the metric AB experimental range. For example, if we focus on the top left table in Fig. gSERCA (row) and in at least one of these 19 simulations we have found a PCa value falling inside a 20% variability of the PCa metric AB experimental range. Trivially, all the boxes for the AB model will be grey, as we already know that there is at least one PCa value falling inside a 20% variability of the PCa metric AB experimental range and that corresponds with the parameter value chosen in our AB model. It must be noted that, when considering more than one Ca2+ phenotype in Figs 2+ handling at both 1 Hz and 6 Hz.We have developed a validation method by exploring nine model variants , obtained through permutation of parameters. For each simulation we have evaluated four metrics: peak Ca2+ pump conductivity gPCaε and a decrease in the background current conductivity gCaBε. In Table 50, Tpeak and Caamp values for those four parameter combinations.Among the 5814 combinations tested in the validation process only four parameter combinations are able to match all four phenotypes at both pacing frequencies and correspond to an increase in Caet al.  and transient receptor potential (TRP) channels might influence both systolic and diastolic calcium concentrations, and we did not have sufficient data to constrain these potential contributors to Ca2+ regulation. However, recent studies have shown that SOCE only appears in about 30% of hypertrophied rat myocytes and are absent from adult cardiomyocytes  decrease in peak Ca2+ at the more physiological 6 Hz is captured and the model replicates the overall Ca2+ transient shape, as shown in Fig. The model was fitted to representative traces. In Table et al. . The project has also received funding from the European Union's Horizon 2020 research and innovation programme  under grant agreement No 647714, South‐Eastern Norway Regional Health Authority, Anders Jahre's Fund for the Promotion of Science, the Norwegian Institute of Public Health, Oslo University Hospital Ullevål, and the University of Oslo. The authors acknowledge financial support from the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy's and St Thomas’ NHS Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust.


Adenosine can be released by a number of different mechanisms that include Ca2+ dependent processes such as the exocytosis of ATP. During sustained pathological network activity, ischemia and hypoxia the extracellular concentration of calcium ions (Ca2+) markedly falls, inhibiting exocytosis and potentially reducing adenosine release. However it has been observed that reducing extracellular Ca2+ can induce paradoxical neural activity and can also increase adenosine release. Here we have investigated adenosine signaling and release mechanisms that occur when extracellular Ca2+ is removed. Using electrophysiology and microelectrode biosensor measurements we have found that adenosine is directly released into the extracellular space by the removal of extracellular Ca2+ and controls the induced neural activity via A1 receptor-mediated membrane potential hyperpolarization. Following Ca2+ removal, adenosine is released via equilibrative nucleoside transporters (ENTs), which when blocked leads to hyper-excitation. We propose that sustained action potential firing following Ca2+ removal leads to hydrolysis of ATP and a build-up of intracellular adenosine which then effluxes into the extracellular space via ENTs.Neural circuit activity increases the release of the purine neuromodulator adenosine into the extracellular space leading to A It is unclear what happens to adenosine signaling under these conditions, but since some of the adenosine release mechanisms are Ca2+ dependent, it would be predicted that adenosine release would also fall. However, it has been observed that removing extracellular Ca2+ actually enhances the amount of adenosine released during ischemia : 127 NaCl, 1.9 KCl, 8 MgCl2, 0.5 CaCl2, 1.2 KH2PO4, 26 NaHCO3, 10 D-glucose (pH 7.4 when bubbled with 95% O2 and 5% CO2). Slices were stored at 34°C for 1–6 h in standard aCSF . This study was carried out in accordance with the recommendations of the U.K. Animals (Scientific Procedures) Act (1986). All experiments were approved by the local Animal Welfare and Ethics Board at the University of Warwick (AWERB).Sagittal slices of hippocampus (300–400 μm) were prepared from male Sprague-Dawley rats, at postnatal days 18–30. In accordance with the U.K. Animals (Scientific Procedures) Act (1986) rats were killed by cervical dislocation and decapitated. Hippocampal slices were cut with a Microm HM 650V Microslicer in cold (2–4°C) high Mg2+ aCSF consisted of standard aCSF with no Ca2+, 1 mM Mg2+ and 1 mM EGTA. For the experiments where high K+ aCSF was puffed onto area CA1 in the hippocampus, the high K+ aCSF had the same composition as zero Ca2+ aCSF but the total K+ concentration was elevated to 10–25 mM with KCl.A slice was transferred to the recording chamber, submerged in aCSF and perfused at 6 ml/min (32°C). For extracellular recording, an aCSF-filled microelectrode was placed on the surface of stratum pyramidale in CA1. Extracellular recordings were made using a differential model 3000 amplifier . Signals were filtered at 3 kHz and digitized on line (10 kHz) with a Micro CED (Mark 2) interface controlled by Spike software . Standard cylindrical microelectrode biosensors  were inserted into the slice in CA1  consist of enzymes trapped within a matrix around a Pt or Pt/Ir (90/10) wire  and a CCD camera . Whole-cell current clamp recordings were made from single or pairs of pyramidal cells in area CA1 of the hippocampus using patch pipettes (5–10 MΩ) manufactured from thick walled glass  and containing (mM): potassium gluconate 135, NaCl 7, HEPES 10, EGTA 0.5, phosphocreatine 10, MgATP 2, NaGTP 0.3 and biocytin 1 mg ml−1 . Voltage recordings were obtained using an Axon Multiclamp 700B amplifier  and digitized at 20 kHz. Data acquisition and analysis was performed using Pclamp 10 (Molecular Devices). If the series resistance exceeded 14 mΩ than recordings were aborted, the mean series resistance was 11.2 ± 0.2 mΩ (n = 30). The standard I-V relationship was obtained by injecting step currents starting between −400 pA and −300 pA, incrementing by 100 pA until a regular firing pattern was induced. A plot of step current against average voltage response (measured at steady state after the activation of IH) around the resting potential was used to measure the input resistance . ATP was purchased from Roche . All substances were dissolved in distilled water except NBTI which was dissolved in DMSO .2+, we first characterized the neural activity produced when extracellular Ca2+ was removed. We investigated early changes in activity (0–1 h) following perfusion with zero-Ca2+ aCSF , unlike some previous studies where activity was measured 1–2 h after Ca2+ removal  with the firing of single action potentials at ~4–8 Hz . When the extracellular Ca2+ was washed back in, even after prolonged periods of activity (up to an hour), most pyramidal cells had resting membrane potentials (−71 ± 3 vs. −67 ± 7 mV) and IV relationships (steady state input resistance 121 ± 15 vs. 115 ± 13 mΩ) which were not significantly different to that observed at the start of the recording. Thus the removal of extracellular Ca2+ did not induce irreversible changes in pyramidal cell properties. Some recordings were lost before the return of extracellular Ca2+, data from these cells is included in our analysis.Before assessing any adenosine release following the removal of extracellular Caz Figure . This waz Figure . The prez Figure , cell 2 z Figure . This paz Figure . These d1 receptors plays a role in controlling the activity induced by Ca2+ removal, the A1 receptor antagonist 8CPT (2 μM) was applied to slices. Application of 8CPT markedly depolarized the membrane potential  and produced a significant increase in the action potential firing rate  biosensors to directly measure any changes in the extracellular concentration of adenosine, we first tested whether changing the ionic environment had effects on the biosensor sensitivity to adenosine. Changing from standard aCSF to zero Cae Figure  even wit2+-free aCSF a large current was induced on ADO biosensors  which was absent on null sensors and thus was due to the detection of purines . This current had a number of distinct phases, which were particularly evident when the ADO biosensor traces were deconvolved and the phases were separated: there was an initial increase in baseline current , spontaneous purine-release events were superimposed on a sustained elevated extracellular purine concentration in the majority of slices  there was no consistent difference between the rise of the signals on ADO and INO biosensors (mean rise 52 ± 10 vs. 55 ± 8 s) suggesting adenosine is metabolized before detection. In Figure 1 receptors on activity  confirms that adenosine is released (since inosine is inactive) and then metabolized before biosensor detection.When release-events were detected on both ADO and INO biosensors, we compared the rise of the signals to see if we could separate components produced by adenosine and by its break-down product inosine. If adenosine is released into the extracellular space and then metabolized to inosine, a signal directly produced by adenosine would be expected to be faster than a signal produced by inosine, as it will be slowed by the intervening metabolism step. In this case the signal on the ADO biosensor would be faster than that on the INO biosensor. In contrast, if adenosine is metabolized to inosine before detection then the rise-time of events on ADO and INO biosensors will be similar as they are both detecting inosine. However, this is further complicated as the rise of biosensor signals also depends on the distance between the purine release site and the biosensor, with the rise slowed as the distance increases  during established Ca2+ free activity and it reliably evoked a current on the ADO biosensor . Since the periodic depolarizations that are observed are probably due to waves of extracellular K+  onto CA1 at least 30 min after removal of Ca2+. This reliably elicited large currents on the ADO biosensor  had no effect on the biosensor baseline current. This data is consistent with the spontaneous purine release events arising from periodic enhanced activity.We hypothesize that these spontaneous increases in extracellular purine concentration are unlikely to arise from the frequent short depolarizations and associated action potential firing which is homogeneous and is therefore more likely to produce the tonic increase in adenosine concentration. Instead we propose they arise from the occasional periods of enhanced activity and rapid depolarization blocks that we observed with the whole cell patch clamp recordings. We have shown that periods of depolarization, with enhanced firing, can be localized or can spread across the tissue Figure  which isy Figure , this sh2+? It could arise from either the release of ATP and its subsequent extracellular metabolism or the direct release of adenosine. The release of ATP by exocytosis would seem unlikely as there is no extracellular Ca2+ and no currents were observed on ATP biosensors , and second by applying NBTI and dipyridamole after removal of extracellular Ca2+, during phase 3 . In slices pre-incubated with NBTI and dipyridamole the initial two phases of adenosine release persisted but the later sustained current and spontaneous events (phase 3) were either greatly reduced or abolished . In a further six slices, NBTI and dipyridamole were applied after Ca2+ removal during phase 3 and they reduced the amplitude of both the sustained current  and the spontaneous release-events  would have dissipated. Application of NBTI and dipyridamole depolarized CA1 pyramidal cells  greater than the amplitude of the short spontaneous baseline depolarizations 3.5 ± 0.5 mV) and increased their firing rate and some cells went into depolarization block . Thus inhibition of ENTs enhances the depolarization of CA1 pyramidal cells, increases their firing rate and pushes some cells into depolarization block. This is the opposite of what occurs under quiescent conditions when extracellular Ca2+ is present. Here the block of ENTs in the hippocampus leads to the accumulation of extracellular adenosine, activation of A1 receptors and the inhibition of transmitter release  there were periodic spontaneous adenosine release events which were superimposed on the sustained extracellular concentration of adenosine (and its metabolites). Our data suggest that these spontaneous release events arise following strong activity probably induced by extracellular K+ waves : (1) in some slices these release-events were localized and only measured on one biosensor and in other slices the events were measured on both biosensors. This is consistent with the activity measured from whole-cell patch clamp recordings, where large depolarizations and depolarization blocks, could be either localized or could be synchronized across cells; (2) Bikson et al. (+. These events could be mimicked by puffing high K+ onto CA1 (in Ca2+ free conditions) and this produced robust adenosine release; (3) application of 8CPT, to block A1 receptors, enhanced firing and also produced robust adenosine release and finally; (4) long lasting membrane potential hyperpolarizations often followed strong bursts of activity  than the decay of the hyperpolarization (decay ~45 s) but this inconsistently can be explained by the fact that biosensors detect both adenosine and its metabolites, with the slow decay the result of diffusion of purines out of the slice . This could potentially be tested by applying a cocktail of K+ channel blockers, since blocking of A1 receptors leads to marked depolarization, increased firing and in many cells depolarization block. These bursts of increased neural activity probably represent escape from adenosine suppression control. These may occur because the waves of increased extracellular K+ concentration weaken the hyperpolarizing effect of GIRK activation (as the outward K+ gradient will be reduced or reversed).Once Can et al.  showed, Experiments were designed by MJW and MJER. Experiments were carried out by MJW and RD. Data was analyzed by MJW and MJER. Article was written by MJW and MJER.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.


During EC coupling, the mammalian DHPR embraces functional duality, as voltage sensor and l-type Ca2+ channel. Although its unique role as voltage sensor for conformational EC coupling is firmly established, the conventional function as Ca2+ channel is still enigmatic. Here we show that Ca2+ influx via DHPR is not necessary for muscle performance by generating a knock-in mouse where DHPR-mediated Ca2+ influx is eliminated. Homozygous knock-in mice display SR Ca2+ release, locomotor activity, motor coordination, muscle strength and susceptibility to fatigue comparable to wild-type controls, without any compensatory regulation of multiple key proteins of the EC coupling machinery and Ca2+ homeostasis. These findings support the hypothesis that the DHPR-mediated Ca2+ influx in mammalian skeletal muscle is an evolutionary remnant.Skeletal muscle excitation–contraction (EC) coupling is initiated by sarcolemmal depolarization, which is translated into a conformational change of the dihydropyridine receptor (DHPR), which in turn activates sarcoplasmic reticulum (SR) Ca 2+ channel. Here the authors show that mice where Ca2+ influx through the DHPR is eliminated display no difference in skeletal muscle function, suggesting that the Ca2+ influx through this channel is vestigial.In mammalian skeletal muscle, the DHPR functions as a voltage sensor to trigger muscle contraction and as a Ca This sudden increase in intracellular Ca2+ concentration triggers Ca2+-sensitive Ca2+ release channels or ryanodine receptors (RyR) to open and release massive amounts of Ca2+ ions from sarcoplasmic reticulum (SR) stores into the cytoplasm, sufficient to finally induce muscle contraction6. This phylogenetically ancient EC-coupling mechanism, which is fully dependent on Ca2+-induced Ca2+ release (CICR), is still extant in skeletal muscles of invertebrates and protochordates12, and interestingly, in all cardiac and smooth muscles up to the evolutionary most derived vertebrates13.Excitation–contraction (EC) coupling in skeletal muscle can be understood only in an evolutionary context. The phylogenetic earliest mechanism involves muscle membrane depolarization, which triggers pore openings of the voltage-gated sarcolemmal 2+ influx-independent, inter-channel protein–protein interaction between the DHPR and skeletal-muscle-specific RyR type-1 (RyR1)14. Upon this physical interaction, the voltage-induced conformational change of the DHPR is transmitted to RyR1, which subsequently opens, releases large amounts of Ca2+ from the SR stores and ultimately leads to muscle contraction16. This phylogenetically most derived skeletal-muscle-specific mechanism of depolarization-induced Ca2+ release combines the advantage of accelerated EC coupling efficacy along with lower energy consumption due to reduced Ca2+ extrusion across the sarcolemma17. A prerequisite for this Ca2+ -independent physical DHPR–RyR1 interaction was a transition in the ultrastructural positioning of the DHPRs from random clustering around the RyR (the protochordate/cardiac-type) to an arrangement into tetrads, i.e., four DHPRs positioned strictly adjacent to the homotetrameric RyR118.However, during evolution to vertebrates, novel skeletal-muscle-specific DHPR and RyR isoforms emerged, giving rise to a new and unique mechanism of EC coupling: Vertebrate skeletal muscle EC coupling is based on a Ca2+ channels19. However, the physiological significance of this DHPR Ca2+ current, which is certainly not (immediately) required for EC coupling14, is still unresolved despite being under investigation since nearly half a century19. So far, one can only speculate that this DHPR Ca2+ inward current, might contribute in the form of excitation-coupled Ca2+ entry (ECCE)22 and have a crucial role in Ca2+ homeostasis, e.g., by SR store filling, or is essential for skeletal muscle development via regulation of acetylcholine receptor pre-patterning and formation of neuromuscular junctions24, or has a negative inotropic effect29, which is crucial for overall muscle health and fibre integrity29, or finally, is just an evolutionary remnant of the ancient CICR stage30. In other words, it is unclear whether the skeletal muscle DHPR Ca2+ current is exaptational or vestigial. However, to date it was not possible to answer any of these questions due to the unavailability of an appropriate animal model system with a fully occluded skeletal muscle DHPR.Interestingly, in addition to their role in conformational EC coupling, mammalian skeletal muscle DHPRs retained their ancient function as Ca31. This mutation leads to reduced Ca2+ pore-binding affinity and thus changes the Ca2+ selectivity of the DHPR by allowing flow of monovalent cations instead of Ca2+33. The E1014K mouse displays reduced muscle endurance, decreased muscle fibre size and a shift in fibre-type distribution towards fast fibres31. The authors concluded that Ca2+ permeation and/or binding via/to the DHPR pore plays an important modulatory role in muscle performance31. However, a drawback of this interpretation is the negligence of the presence of a persisting permeation of monovalent cations through the mutant EK channel34, which might, for instance, affect the membrane potential during muscle activity32.Recently, a mouse model carrying a mutation in the DHPR selectivity filter (E1014K) was generated2+-conducting DHPRs in their skeletal muscles35. Consequently, we engineered the selectivity-filter-adjacent point mutation N617D, previously identified to be responsible for DHPR non-conductivity in zebrafish fast, glycolytic muscle2 into the mouse genome. Electrophysiological investigations on cultured skeletal muscle primary myotubes and adult fibres from the resultant homozygous mutant mouse with non-conducting (nc) DHPR showed complete block of DHPR Ca2+ influx but unaltered intracellular SR Ca2+ release. Notably, and in contrast to the E1014K model, the ncDHPR pore does not show permeation of monovalent cations36.With the intention to create a mouse model with a fully occluded skeletal muscle DHPR, we used a DHPR pore-blocking mechanism refined by evolution. Previously, we found that zebrafish and all other euteleost fishes express non-CancDHPR mouse model to explore the specific role of the DHPR-mediated Ca2+ current in skeletal muscle via a range of muscle performance tests on whole animals and isolated muscles, as well as at the cellular and molecular level with biophysical, immunohistochemical, biochemical and quantitative PCR (qPCR) techniques. Surprisingly, these experiments did not point to any differences between ncDHPR mice and their wild-type (wt) littermates, irrespective of whether mice were young or aged, or the isolated muscles were fast- or slow-twitch type. Overall, our results strongly support the hypothesis that the Ca2+ influx through the mammalian skeletal muscle DHPR is irrelevant for muscle performance and consequently can be considered as an evolutionary remnant  of the phylogenetic stages of early chordates, where DHPR Ca2+ influx was the exclusive trigger for RyR activation in skeletal-muscle EC coupling.Here we use the 2+ influx in skeletal muscle, we generated a non-Ca2+-conducting (nc) DHPR knock-in mouse. Mutation N617D in pore loop II, previously identified to be responsible for the complete loss of Ca2+ conductance in zebrafish DHPR α1S-b was introduced into the murine CACNA1S gene  compared  with wt myotubes (Imax = −4.87 ± 0.31 pA pF−1)  between ncDHPR and wt myotubes , but again no inward Ca2+ current in ncDHPR myotubes (Imax = −0.07 ± 0.04 pA pF−1)  of elevated temperature on intracellular Ca2+ release, neither in wt nor ncDHPR myotubes  compared  with wt fibres (Imax = −19.1 ± 2.62 nA)  voltage-dependence of peak Ca2+ release flux  Fig. . Howeverbes Fig. , pointinnse Fig.  with indlux Fig.  and Ca2+ity Fig.  between 2+ influx in SR store filling, Fura-FF-AM loaded adult interosseous muscle fibres were challenged with RyR agonist 4-CmC (4-chloro-m-cresol) under conditions that inhibited Ca2+ uptake by sarco/endoplasmic reticulum Ca2+-ATPase(SERCA)   between ncDHPR and wt fibres, indicating identical steady state SR Ca2+ loading in both genotypes.To assess the importance of DHPR Caid) Fig. . SERCA b2+ influx in ncDHPR mice did not cause embryonic lethality and animals had normal posture and gait, and could not be visually distinguished from wt littermates. No difference  in male  was indistinguishable  between both genotypes.The absence of DHPR Caale Fig.  and femaale Fig.  body weiize Fig. , inset wP > 0.05, unpaired t-test) between ncDHPR and wt mice.To identify possible muscle-type-specific differences we chose extensor digitorum longus (EDL) muscle, which primarily consists of fast-twitch fibres and soleus (SOL) muscle, which is more a slow-twitch muscle compared with EDL. Basic physical parameters such as wet weight, length and diameter of SOL and EDL muscles, isolated from young  in the fibre-type composition, neither in the slower-twitch SOL muscle nor the fast-twitch EDL  in the fibre cross-sectional area (CSA) of SOL and EDL muscles were observed between both genotypes  mice. Home-cage ambulatory activity recorded over 60 h was indistinguishable  between ncDHPR and wt mice (average age: 4.0 months). Both the genotypes displayed normal behaviour, with higher activity during the dark cycle and lower activity during the light cycle  were challenged with a voluntary activity wheel experiment for 21 days. Consistent with above results, the cumulative average distance covered by ncDHPR mice (19.29 ± 6.31 km) was similar  to that of wt control mice (21.28 ± 3.71 km)  Fig. .Fig. 5Vo2+ influx on skeletal muscle functioning in intact muscles independent of other parameters and under high frequency stimulations38, we compared ex vivo contractile properties, viz. force and fatigability of SOL and EDL muscles isolated from ncDHPR and wt mice. For this part of the study, young  and aged  mice were used to examine putative age-related accumulative degenerative effects on muscle performance due to the loss of DHPR Ca2+ influx. On being subjected to force-frequency tests, SOL and EDL muscles did not show any differences  in maximum twitch force between young ncDHPR and wt mice . Furthermore, the maximum twitch force observed in both muscle types of aged mice was indistinguishable  between ncDHPR and wt . The tetanic force exhibited by SOL and EDL at increasing stimulation frequencies  in young  or aged  ncDHPR and wt mice.To test the impact of the loss of DHPR Ca38, the implications of the loss of DHPR Ca2+ influx in the ncDHPR mouse might become evident only under extreme fatiguing conditions. Hence, we subjected isolated SOL and EDL muscles to fatigue-inducing repetitive high-frequency tetanic stimulations  between muscles isolated from young ncDHPR and wt mice . Likewise, there was no significant difference in fatigability between muscle derived from aged ncDHPR and wt mice . Akin to the physiological muscle parameters, kinetic parameters of single twitches such as time to peak, time to half-relaxation and twitch duration were similar  in muscles from ncDHPR and wt, regardless of whether isolated from young or aged mice  in young ncDHPR  and wt  mice. Next, we compared muscle strength of all four limbs37 by letting the mice hang upside down on a cross-wired grid for a maximum of 600 s. The latency to fall from the inverted grid was indistinguishable  between young ncDHPR and wt control mice . Mice that achieved the maximum hanging time were also similar for both genotypes (14 ncDHPR and 12 wt mice). Likewise, no significant difference  in performance was observed between aged ncDHPR and wt mice . None of the aged mice reached the maximum hanging time of 600 s. Finally, we performed a highly specific grip strength test to exclude other aspects of behaviour and coordination37, and exclusively measure the maximal muscle strength of forelimbs. In this test, the mouse was allowed to grasp onto a trapezoidal steel grip and after a gentle pull, the maximal force (in g) exerted before it releases the grip was recorded. Consistent with results obtained with other tests, young and aged ncDHPR mice displayed similar  muscle strength to their age-matched control wt siblings  time such as the age-matched wt control mice  between young ncDHPR and wt mice . Consistently, aged ncDHPR and wt mice displayed equal performance . Next, we designed a prolonged Rotarod protocol (600 s) at constant speed (20 rpm for young and 15 rpm for aged mice) to access endurance. Latency to fall displayed by young ncDHPR mice was comparable  to their wt counter mates . Likewise, endurance level was indistinguishable  between aged mice of both genotypes .Ex vivo measurements on isolated SOL and EDL muscles showed no alterations in fatigability in the absence of DHPR-mediated Caice Fig. . LikewisncDHPR mice39. Interestingly, young mice of both genotypes accomplished the entire 0.9 km run with similar efficiency. The total average resting time cumulated during the 1-h task, as well as the cumulative number of rests at the end of the task were indistinguishable  between both genotypes  competence to their age-matched wt siblings  in the cumulative number of rests between aged ncDHPR and wt mice. Altogether, results from all the above experiments indicate that ncDHPR mice exhibit equivalent muscle performance to their wt siblings, which convincingly points to the insignificance of DHPR Ca2+ influx for skeletal muscle function.Resistance to fatigue is an optimal, comprehensive measure of muscle performance and one of the most powerful tools for analysing fatigue resistance is a treadmill task. An acute exhaustive treadmill protocol was used to measure the enforced maximum endurance limit of pes Fig. . Finallypes Fig. , aged mings Fig. . The com2+ influx has no obvious impact on general physiological parameters and whole animal muscle performance, neither in young nor aged ncDHPR mice. Apparently, the question arose whether up- or downregulation of any of the key triadic proteins responsible for either influx or efflux of Ca2+ ions compensates for the lack of DHPR-mediated Ca2+ influx in the ncDHPR mice. To address this issue, we first opted for qPCR analysis to quantify the messenger RNA levels transcribed from a selected panel of genes encoding for proteins involved in EC coupling and Ca2+ homeostasis, with β-actin (ACTB) and eukaryotic translation elongation factor 1 alpha 2 (EEF1A2) as reference genes. Total RNA was isolated from skeletal muscle of mice at different developmental stages viz. neonatal mice and adult SOL and EDL muscles. Starting with the main players of EC coupling, DHPR and RyR1, we found no differences  in transcript levels between neonatal ncDHPR and wt mice  with that recorded from wt myotubes , STIM1, SERCA1, PMCA1, NCX (isoforms NCX1 and NCX3) and CSQ (isoforms CSQ1 and CSQ2) were unaltered in skeletal muscles of neonatal ncDHPR mice compared  with wt mice  at the transcriptional level of all the above mentioned proteins in adult SOL or EDL muscles between both genotypes with EEF1A2 as reference gene  as internal reference, indicated comparable protein levels  between both genotypes  muscles derived from adult pes Fig.  and 12. 2+ influx is irrelevant for proper skeletal muscle functioning.Overall, the surprising outcome of our study from the plethora of in vitro, ex vivo and whole-animal tests underlines the fact that DHPR Ca29 that DHPR Ca2+ influx has a negative regulatory effect on skeletal muscle contraction by inhibition of the Ca2+-sensitive adenylyl cyclase (AC) isoforms, AC5 and AC6. Apparently, our knock-in ncDHPR mouse provides the perfect system to re-investigate this hypothesis. As intracellular SR Ca2+ release was unaltered in ncDHPR mice  with wt mice  in expression levels of AC5 and AC6 in adult SOL and EDL muscles between both genotypes  from control wt muscle  between ncDHPR and wt diaphragm muscles , which expresses a non-Ca2+-conducting (nc) DHPR in skeletal muscle. Despite more than 40 years of research, the role of this Ca2+ influx in mammalian skeletal muscle which occurs during conformational EC coupling is still obscure and controversial19. Theoretically, there are three possible roles for this enigmatic DHPR Ca2+ influx: (i) it still marginally contributes to skeletal muscle EC coupling via a rudimentary CICR effect; (ii) it is important as an exaptational current, i.e., during evolution it underwent a change in function from the crucial initiator of CICR to a Ca2+ current that serves another function now; or lastly, (iii) it is just a vestigial current, viz., an evolutionary remnant Ca2+ current from phylogenetically ancestral CICR stages12 but without any functional importance.In this study we investigated the physiological consequences of the elimination of skeletal muscle inward Ca2+ current still contributes to EC coupling via rudimentary CICR, because it is small, slowly activating and considerably lags behind the activation of SR Ca2+ release41. Moreover, previous biophysical experiments on skeletal muscles ex vivo, muscle fibres and myotubes from frogs to rodents, explicitly showed that pharmacological blocking of the DHPR or elimination of extracellular Ca2+ did not obstruct EC coupling45 and, consequently, it is widely accepted that the DHPR Ca2+ influx is not an (immediate) prerequisite for the contractile activation30.Nevertheless, contemplating these three options it seems very unlikely that this DHPR Ca2+ influx  also exist, because it is apparently regulated in different ways. For example, the accessory DHPRα2δ-1 subunit functions as major determinant of the characteristic slow l-type Ca2+ current kinetics47 and the DHPRγ1 acts similar to an endogenous Ca2+ antagonist that reduces Ca2+ entry under conditions promoting modest membrane depolarization48. DHPR Ca2+ influx is also massively ( ~ 8-fold) enhanced in developing fetal mouse skeletal muscle due to the expression of an alternatively spliced DHPRα1S subunit50 and even more substantially amplified ( ~ 30-fold) by retrograde RyR1-DHPR coupling52. Hence, the probability of this regulated skeletal muscle DHPR Ca2+ influx to function as an exaptational Ca2+ current cannot be ruled out. In this case its character would have completely changed, from a crucial EC coupling signal transmitter to a supporter of muscle cell Ca2+ homeostasis , tightly linked to the EC coupling process.However, substantial arguments against a complete and long-term insignificance of the DHPR Ca2+ conductivity in mammalian skeletal muscle, we engineered point mutation N617D from pore loop II of zebrafish DHPRα1S-b, identified to be responsible for the complete lack of Ca2+ conductivity in fast, glycolytic muscle2, into the murine gene CACNA1S  might induce gross physiological effects such as myotonia or paralysis. However, very surprisingly, our results clearly demonstrate that the absence of the physiological DHPR Ca2+ influx in ncDHPR mice is irrelevant for SR store filling  such as TRPC, STIM and Orai53, because it shows that despite the lack of ECCE in the ncDHPR mouse muscle the SOCE machinery is not upregulated to compensate for the loss of ECCE. However, analyses of mRNA or protein levels of these major players in EC coupling and Ca2+ homeostasis do not necessarily equate to their activity levels since a number of these proteins are posttranslationally regulated. Still, the evidence that these key proteins remain uninfluenced by the lack of DHPR Ca2+ influx is very high.Eventually, to gain deeper and clearer insight into the role of DHPR Cacle Fig. . Survivaing Fig. . DHPR Caice Fig. –12. This2+ entry pathway20 via the skeletal muscle DHPR22, activated by repetitive or prolonged depolarizations and to be essential for store repletion53. The complete lack of Ca2+ influx in ncDHPR mouse myotubes even after 2-s prolonged depolarizations in the presence of the 1,4-dihydropyridine (DHP) agonist BayK864436, presents convincing evidence that skeletal muscle of the ncDHPR mouse does not require ECCE for its proper functioning.ECCE was described as store-independent Ca2+ current is vestigial seems strongly and clearly confirmed by all our findings, earlier studies on frog26, crab27 and rat28 skeletal muscles pointed to positive inotropic effects of different l-type Ca2+ channel blockers. Recently, analogous inotropic effects were described by ex vivo Ca2+ channel-blocking experiments on mouse diaphragm muscle29. It was hypothesized that DHPR Ca2+ influx attenuates skeletal muscle contraction via inhibition of Ca2+-sensitive AC isoforms, AC5 and AC6, which in turn leads to reduced intracellular cAMP levels, known to potentiate muscle contraction mainly through PKA-dependent RyR1 phosphorylation and thus preserving muscle fibre integrity29. With our ncDHPR mouse model we re-examined this apparently paradoxical Ca2+ antagonist effect on muscle contraction. As demonstrated explicitly in the Results section, perfusion of isolated diaphragms of ncDHPR mice with two chemically distinct organic l-type Ca2+ channel blockers, the DHP nifedipine and the phenylalkylamine (PAA) verapamil, surprisingly and in stark contrast to the hypothesis of Menezes-Rodrigues et al.29, induced identical positive inotropic effects in wt and ncDHPR mice  apparently paradoxical Ca2+ antagonist effect on muscle contraction. The most likely reason that we can speculate is that the antagonist concentrations (10 µM nifedipine and 50 µM verapamil) used in all these ex vivo experiments are ~ 6,000- and 25,000-fold, respectively, beyond the Kd-values observed for skeletal muscle membrane binding55 and thus might bind and interact non-specifically to low-affinity binding sites of other muscle proteins besides its specific high-affinity binding pocket on the DHPRα1S56. It is quite possible that such undefined low-affinity protein–DHP/PAA complexes are responsible for the observed amplification of skeletal muscle contraction.Although the hypothesis that the DHPR Ca2+-conducting DHPR31. With this mouse model, it has been proposed that Ca2+ permeation through (or just Ca2+ binding to) the DHPR has a significant impact on muscle function. In contrast to the ncDHPR mouse model, the E1014K mouse displayed decreased SR Ca2+ store refilling, increased fatigue, decreased muscle fibre size, increased type-IIb fibre count31 and an obese phenotype57. The most probable reason for this disparity might be different biophysical consequences due to the implementation of distinct DHPR pore mutations. In the ncDHPR mouse, the N617D pore mutation adds a negative charge in close vicinity of the selectivity filter glutamate in repeat II of the DHPRα1S, which induces an aberrant high-affinity Ca2+ binding to the pore36. This results in complete occlusion of the pore for all ions under physiological conditions. Conversely, in the E1014K mouse model, the E to K charge conversion in repeat III leads to impairment of the Ca2+ selectivity filter, enabling only low-affinity (mM) Ca2+ binding to the pore33. Thus, the E1014K-DHPR does not allow Ca2+ permeation but shows a strong conductivity for monovalent cations32, which consequently turns it into a junctionally targeted, slowly activating, non-inactivating Na+ channel34.Furthermore, our results and conclusions are also incongruent with a study on another mouse model (E1014K mouse) with a non-Ca2+ pore-binding (as in the case of wt or N617D-DHPR) and destabilized by low-affinity Ca2+ pore-binding (as in the case of E1014K-DHPR). Only this option allows discussing the claim of the study on the E1014K model that Ca2+ permeation through and/or binding to the DHPR pore has an important modulatory role in muscle performance31. As the ncDHPR mouse does not show any of the aberrant muscle phenotypes found with the E1014K model, we can undoubtedly exclude the importance of DHPR Ca2+ permeation for skeletal muscle performance. However, as Ca2+ binds firmly to the N617D DHPR pore36 in contrast to the E1014K channel, a putative crucial sole Ca2+ binding effect to the DHPR to sustain skeletal muscle function cannot be excluded.It is quite possible that this E1014K-specific leakiness for monovalent cations causes the observed alterations in muscle performance and is responsible for the differences between the two mouse models. Alternatively , a proper conformation might be required for fine-tuning the DHPR signalling function, which is stabilized by high-affinity Ca2+ current is fully dispensable in mammalian skeletal muscle and is most likely a non-functional, tolerated evolutionary remnant  from the ancestral Ca2+-influx-dependent skeletal muscle EC coupling found in early chordates10. The final switch-off of this vestigial skeletal muscle DHPR Ca2+ influx took place only in the phylogenetically most derived clade among the vertebrates—the euteleost fishes3.We are aware of the innate dilemma of a study, which intends to describe the non-existence of a phenotype and thus, despite an exhaustive scope of experiments, the fact that a stone was left unturned cannot be excluded. Nevertheless, all our results strongly suggest that the DHPR-mediated Ca1S non conducting via introduction of point mutation N to D into exon 13, simultaneously with a silent mutation for restriction enzyme site PflF1 for RFLP test, was deliberated by our lab. Finally, the ncDHPR mouse on C57BL/6NTac background was generated at the Institut Clinique de la Souris—ICS .The strategy for transforming the mouse DHPRαad libitum. At least 24 h before the start of the experiments mice were housed individually and acclimatized to the behavioural facility. All experiments were performed on mutant and gender- and age-matched wt mice. Mice were organized into two age groups, “young” between 3 and 7 months and “aged” between 14 and 22 months old. Only male mice from both age groups were used in this study, unless indicated otherwise.Animal care and all experimental procedures were conducted in strict accordance with the guidelines of the European Union Directive 2010/63/EU and approved by the Austrian Ministry of Science (BMWF-5.031/0001-II/3b/2012). Mice were housed on a 12/12-h light/dark schedule, with access to food and water 258. l-type Ca2+ currents simultaneously with intracellular SR Ca2+ release using 0.2 mM Fluo-4 in the patch pipette solution were recorded from these cultured myotubes, as described59. Myosin-II blocker N-benzyl-p-toluene sulphonamide  was continuously present in the bath solution.Myoblasts from skeletal muscles of new born up to 5 days old pups of wt, heterozygous or homozygous for non-conducting DHPRs were isolated and plated on 35-mm ECL-coated (Merck Millipore) culture dishes in Dulbecco’s modified Eagle’s medium in a 37 °C incubator with 5% CO60. Briefly, single muscle fibres were enzymatically isolated from interosseous muscles of young mice, voltage-clamped on a two-microelectrode setup and dialysed with an artificial intracellular solution containing 0.2 mM Fura-2 and 15 mM EGTA, to allow parallel fluorescence recordings of Ca2+ currents and SR Ca2+ release. The high intracellular EGTA concentration dominates Ca2+ binding in these experiments and facilitates the calculation of the time course of the Ca2+ release flux from the SR61. Depolarization-induced fluorescence ratio signals (380 and 360 nm excitation) were subjected to a removal analysis using a Ca2+ distribution model fit approach60. The results were used to estimate the voltage-dependent Ca2+ release flux and converted to SR Ca2+ permeability by correcting for putative SR Ca2+ depletion during the depolarizing voltage steps60.Measurements on voltage-clamped muscle fibres and signal analysis were performed as described62 were loaded with 5 µM of the ratiometric low-affinity Ca2+ indicator dye Fura-FF-AM in HEPES-buffered standard Krebs–Ringer solution for 30 min at room temperature, washed and equilibrated with Ringer solution containing 100 µM BTS for another 30 min to supress contractions. The bath solution was then replaced with Ca2+-free Ringer solution to eliminate the influence of external Ca2+. Target cells were superfused using Ca2+ releasing solution, i.e., Ca2+-free Ringer solution containing 500 µM of the RyR agonist 4-CmC to trigger Ca2+ release and 30 µM CPA to inhibit SERCA Ca2+ sequestration. A period of 20-s application of the Ca2+ releasing solution was always bracketed with 20-s superfusion with Ca2+-free Ringer solution  based on an inverted microscope  equipped for epifluorescence (Fluar ×40/1.3 oil-immersion objective)63.Primary-cultured interosseous muscle fibresion Fig. . For Ca2ncDHPR and wt mice were immunostained overnight at 4 °C with antibodies against fibre-type-specific myosin heavy chain (MHC) isoforms. Primary antibodies against MHC isoforms I (BS-D5), IIa (SC-71), IIb (BF-F3) and IIx (6H1)  at a dilution of 1:2,000, except 6H1, which was 1:200, were used. Secondary staining was performed for 1 h at room temperature (RT) with anti-mouse IgG Alexa 594 or anti-mouse IgM Alexa 594 . Images were taken under a fluorescence microscope (Olympus BX53) with a ×20 objective lens. The number of antibody-stained fibres were counted and normalized to the total number of muscle fibres per field64. Saved images were additionally used for calculating the fibre CSA with Metamorph software .Eight-micrometre-thick cryosections of snap-frozen SOL and EDL muscles from 2 to 3 months old ncDHPR mice65 and mounted vertically between a force transducer  and static clamp in an organ bath equipped with platinum electrodes and under continuous perfusion with carbogen-(95% O2 + 5% CO2) saturated Krebs–Ringer solution. Optimal muscle stretch was determined by applying single twitches at supramaximal voltage (25 V for 1 ms) and set at the length that generated maximal force. After 10 min of equilibration, EDL and SOL muscles were subjected to different force frequency  and repetitive tetanic fatiguing  protocols38  mounted on top of every home cage.For voluntary activity wheel test, mice were individually habituated for 2 days before the start of the experiment in cages furnished with activity wheels . The mice had continuous free access to the wheels, which could be revolved in either direction. Activity wheels were equipped with photoelectric sensors interfaced to a computerized system that automatically recorded the distance travelled every 5 min continuously for 21 days. Up to six mice were run in parallel and for each genotype the distances travelled per day were calculated and plotted as averaged, cumulative values.ncDHPR and wt mice.Three standard tests were used to compare the motor coordination and muscle strength between ncDHPR or wt mice from the young group were used. The mouse was placed on one end of a 120-cm long beam resting 50 cm above a padded surface. An enclosed safety box was mounted at the other end of the beam. After three pre-training trails, the time needed to traverse the beam and reach the safety box was recorded for every mouse.For the beam walk test, ncDHPR and wt mice.For the wire hang test, a custom-designed device consisting of a cross-wired grid, positioned between two poles 35 cm apart and 55 cm above a padded surface was constructed. The mouse was placed in the centre of the grid and shaken gently three times, to ensure firm gripping to the wire (1.7 mm in diameter) with all four paws. The grid was then immediately turned upside down and the latency to fall was recorded. Three trials were done with every mouse, with a recovery interval of some minutes, and only the highest score was used for analysis. The maximum hanging time was set to 600 s and the mouse that did not fall during this period was removed and given the maximum score. Body weight was recorded at the end of the experiments and the average weight was similar between ncDHPR and wt mice.Maximal muscle strength of the forelimbs was measured using a grip strength test (Bioseb). The mouse was gently lowered by the tail and allowed to grip a metal bar (1.3 mm in diameter) with the front paws only. Keeping the torso horizontal, a slow gradual pull was exerted in backward direction until the grip was released. The force applied to the bar by the animal at the time of the release is recorded by the sensor as the maximum force reached. Three measurements separated by 30-s inter-trial intervals were collected from each animal and the maximum value was used for statistical analysis. Mice were weighed at the end of the testing session and there was no difference in average weight between ncDHPR and wt mice.Two standard methods were used to compare fatigability and endurance between ncDHPR and wt mice were placed on the rotarod at an initial speed of 4 rpm for 25 s. After the warmup phase, the rotarod was set to accelerate steadily from 4 to 40 rpm for 300 s. Each mouse was subjected to three trials with 15 min inter-trial intervals. For rotarod endurance task, mice were placed on the rod rotating at a constant speed, 20 rpm for young and 15 rpm for aged mice for 600 s. Latency to fall was recorded and only the highest score among the 3 trails was considered for analysis.Rotarod tests at accelerating and constant speeds were used to assess muscle fatigue and endurance. Mice were familiarized and trained on a four-lane rotarod (TSE Systems) by running them for 5 min per day for 3 days at a speed of 4 rpm. For testing the fatigability, young and aged −1 at a 15° inclination. Acute exhaustion protocols following an initial 5-min warm up at 5 cm min−1 were designed for young and aged mice. In case of young mice, band speed was increased 2 cm s−1 every minute till 27 cm s−1 and then maintained at the maximum speed for an additional 45 min. The entire treadmill task was 0.9 km in 1 h. For aged mice, a milder protocol with a speed increment of 1 cm s−1 every minute up to 17 cm s−1 was used. The entire run was 0.55 km distance in 1 h. To encourage continuous forward running, the front end of the treadmill was covered with a black cloth and electric stimulus at the back end was set at 0.2 mA. The number of rests and body weight was recorded for each mouse. Finally, the average cumulative resting time collapsed into 5 min bins, was plotted against the duration of the task. In addition, average number of cumulative rests during the entire task was calculated.Treadmill test was performed to assess the degree of fatigability under rigorous conditions. Mice were run on a five-lane treadmill with electric stimulus grid (Bioseb). Three days before the experiment, mice were acclimatized and trained on the treadmill by running them for 5 min per day at a speed of 9 cm s2+ homeostasis was assessed by comparative TaqMan quantitative reverse-transcriptase PCR (qRT-PCR). Total RNA was extracted from skeletal muscle of neonatal pups (up to 2 days old) and adult (7 months old) EDL and SOL muscles using RNeasy Mini Kit (Qiagen). Following RNAse-free DNAse treatment, first-strand complementary DNA synthesis was performed using random primers and M-MLV reverse transcriptase (Promega). For every gene, flanking primer pairs and 5′ FAM (6-carboxyfluorescein) and 3′ BHQ1 (Black Hole Quencher)-labeled TaqMan probe spanning exon–exon boundaries, were designed and purchased from Eurofins, Germany (Supplementary Table CT method with two endogenous reference genes—ACTB (β-actin) and one of the most stable mammalian skeletal muscle housekeeping gene, EEF1A2 66.Putative transcriptional regulation of key triadic proteins involved in EC coupling and CancDHPR and wt mice were excised, homogenized and lysed in sample buffer containing 250 mM Tris-HCl (pH 8.5), 2% lithium dodecyl sulfate, 10% glycerol and 0.5 mM EDTA and centrifuged at RT, 16,000×g for 5 min. Protein concentration measurements and separation by SDS-polyacrylamide gel electrophoresis on 3–8% Tris-Acetate or 4–12% Bis-Tris NuPAGE gels (Thermo Fisher Scientific) was performed as described previously67. Immunostaining was performed with different primary antibodies followed by appropriate secondary antibodies  with the central tendon facing up and the rib end towards the bottom and kept under continuous perfusion with carbogen (95% O2 + 5% CO2) saturated Krebs–Ringer solution. Optimal muscle stretch was determined by applying a single twitch at supramaximal voltage (25 V for 2 ms). After 10 min of equilibration under continuous stimulation , nifedipine (10 µM) or verapamil (50 µM) was added to the bath solution to investigate the effect of l-type Ca2+ channel blockers on muscle contraction29. Nifedipine was washed out after 25 min and stimulation continued for another 15 min, whereas verapamil was washed out after 40 min with further stimulation for 20 min. At the end of each experiment, muscle wet weight, diameter and length were measured. All recordings were performed at room temperature ( ~ 26 °C). Baseline force was calculated by taking the average twitch peak force of the initial 6 min before adding the drug. Finally, the average peak twitch force, collapsed into 2-min bins and expressed as percentage of baseline force, was plotted against the duration of the experiment.Three- to 5-mm strips with rib overhangs were dissected from diaphragm muscle isolated from euthanized 3–6 months old 64; hence, power calculations were not necessary. Animals were arbitrarily allocated to the specific groups , but without the use of an explicit randomization procedure. Experiments did not require blinding and thus were not performed under blinded conditions. In our study, no data points, samples or mice were excluded from analysis. Based on our previous publications59, all statistical analyses are considered appropriate. n-Values represent the number of mice, muscles, muscle fibres or myotubes, as specified. Variance is similar between comparison groups. All data are expressed as means ± s.e.m. and statistical analyses were conducted using unpaired Student’s t-test. P < 0.05 was considered statistically significant.Sample sizes of mice or tissue are based on previous publicationsAll relevant data are available from the first/corresponding author on reasonable request.Supplementary InformationPeer Review file


Moreover, extracellular Ca2+ levels impact on distinct processes in mammalian reproduction. The calcium-sensing receptor (CaSR) translates changes in extracellular Ca2+-concentrations into cellular reactions. This review summarizes current knowledge on the expression of CaSR and its putative functions in reproductive organs. CaSR was detected in placental cells mediating materno-fetal Ca2+-transport such as the murine intraplacental yolk sac (IPYS) and the human syncytiotrophoblast. As shown in casr knock-out mice, ablation of CaSR downregulates transplacental Ca2+-transport. Receptor expression was reported in human and rat ovarian surface epithelial (ROSE) cells, where CaSR activation stimulates cell proliferation. In follicles of various species a role of CaSR activation in oocyte maturation was suggested. Based on studies in avian follicles, the activation of CaSR expressed in granulosa cells may support the survival of follicles after their selection. CaSR in rat and equine sperms was functionally linked to sperm motility and sperm capacitation. Implantation involves complex interactions between the blastocyst and the uterine epithelium. During early pregnancy, CaSR expression at the implantation site as well as in decidual cells indicates that CaSR is important for blastocyst implantation and decidualization in the rat uterus. Localization of CaSR in human extravillous cytotrophoblasts suggests a role of CaSR in placentation. Overall, evidence for functional involvement of CaSR in physiologic mammalian reproductive processes exists. Moreover, several studies reported altered expression of CaSR in cells of reproductive tissues under pathologic conditions. However, in many tissues we still lack knowledge on physiological ligands activating CaSR, CaSR-linked G-proteins, activated intracellular signaling pathway, and functional relevance of CaSR activation. Clearly, more work is required in the future to decode the complex physiologic and pathophysiologic relationship of CaSR and the mammalian reproductive system.Active placental transport of maternal serum calcium (Ca Moreover, it is modulated by various physiological stimuli including extracellular pH, L-aromatic amino acids, and ionic strength. Different ligand binding sites can stabilize distinct conformational changes, which results in “ligand-biased signaling.” As a consequence, CaSR was shown to interact with the heterotrimeric G proteins Gq/11, Gi/o, G12/13, and Gs and is able to target all major intracellular signaling pathways. CaSR-mediated activation of Gq/11 results in stimulation of phospholipase C (PLC), Cai/o can inhibit adenylyl cyclase (AC). On the other hand, it can activate mitogen-activated protein kinases (MAPK) such as ERK1/2 and JNK. This can lead to transactivation of the epidermal growth factor receptor (EGFR). Activation of G12/13 modulates several pathways. This can lead to migration via rho-mediated actin polymerization and membrane ruffling or induce cell differentiation. It can also target tyrosine kinases, protein phosphatases, or activate certain AC isoforms. CaSR-coupling to Gs also activates ACs. Furthermore, CaSR activation can stimulate PLA, phosphatidylinositol 3-kinase (PI-3K) and PI-4K. Overall, major consequences of CaSR activation in cells are CaCaSR is, however, promiscuous in ligand binding; it can be stimulated by other divalent  activity, which stimulates expression of Ca2+-channels with appropriate response elements. And finally, CaSR can increase K+-channel activity, and as a consequence increase membrane potential and a Ca2+-driving force. Many intracellular signaling pathways, in turn, are sensitive to cytosolic Ca2+-rises, and as a consequence, cellular processes such as proliferation or migration can result from CaSR activation  or as a tumor suppressor gene  , mainly activation of MAPK pathways  has been investigated so far, probably because MAPK activation is of great importance in reproduction  the proliferation of the stem cells (spermatogonia) resulting in spermatocytes, (2) two meiotic divisions that give rise to haploid spermatids, and (3) spermiogenesis, the differentiation into mature sperms , while a CaSR agonist (AC-265347) inhibited PY similarly to CaIn isolated equine sperms, CaSR protein was located predominantly in the head of the sperm, with lower expression seen along the tail. Proteins detected with an anti-CaSR antibody by western blotting had a molecular weight of 100 and 75 kDa . It was concluded that CaSR activation had a pro-apoptotic impact on germ cells in diabetic rats and overall participated in diabetes-induced testicular damage.Diabetes, being an increasing problem worldwideWhile CaSR expression in normal rat Leydig cells could not be shown  receptor present on the surface of murine cells inducing Ca2+ release from ryanodine receptor-gated intracellular Ca2+ stores  is characterized by hyperandrogenism, oligo- and/or anovulation, and polycystic ovarian morphology. PCOS is associated with insulin resistance, hyperinsulinemia and central obesity. Excessive proliferation of theca cells is often associated with PCOS; this is a major cause of infertility due to ovarian hyperandrogenism  first proliferate and then enter the first meiotic division, which is halted in a prophase state of meiosis I until sexual maturity. This first meiotic arrest is characterized by a large nucleus called the germinal vesicle  from the pituitary gland, which stimulates the release of the oocyte from the ovary (ovulation). Upon that stimulus, meiosis I continues and is terminated shortly before ovulation. Oocytes at metaphase I (MI) stage are defined as oocytes with no GV (intact nucleus) and no polar body . For transition of the unfertilized egg to a developing embryo, egg activation is initiated by alterations in Ca2+ concentrations in the surroundings. Induced proliferation of these cells due to high Ca2+ concentrations may be of physiological relevance during the healing of the ruptured surface following each ovulation, when neighboring cells are locally exposed to ovarian follicular fluid. At least in pig ovarian follicular fluid, Ca2+ concentrations of around 2.34 mmol/L have been measured  cells. There, inositol triphosphate production increased after stimulation with Gd3+ and Ca2+. Expression of an interfering mutant CaSR inhibited the proliferative response to elevated extracellular Ca2+. CaSR agonists induced tyrosine phosphorylation, ERK activation and proliferation. Expression of interfering mutants for Ras, Raf, and MKK1 indicated that proliferation of ROSE cells in response to increased CaExpression of CaSR in the ovary was first described in human surface epithelial cells Figure . ProteinOvarian surface epithelial cells are the cell type primarily responsible for malignant ovarian carcinoma, which is the most lethal female genital malignancy. McNeil et al.  observed2+ in mobilization of intracellular Ca2+ during oocyte maturation, activation, and fertilization  and was not observed at suboptimal external Ca2+(0.5 mM). However, variations of Ca2+ between 0.5 and 4 mM Ca2+ in the absence of the agonist did not stimulate in vitro oocyte maturation. In oocytes treated with NPS R-467, CaSR immunostaining increased at the plasma membrane, while it was reduced in the cytosol. Finally, treatment of oocytes as well as cumulus cells with CaSR agonist resulted in an increased activity (phosphorylation) of MAPK (ERK) in these cells . Increasing extracellular Ca2+ resulted in a biphasic response of the cells; an initial inhibitory effect on apoptosis was followed by a delayed phase of increased apoptosis. As the initial inhibitory effect of the Ca2+ on apoptosis was mimicked by applying the CaSR agonists Mg2+ and Gd3+, an involvement of CaSR in inhibition of apoptosis was suggested  as well as the ions Ca2+ and Mg2+. The authors suggested that activated CaSR may play a role in securing the survival of avian follicles after their selection . Current data support a role of CaSR in maturation (completion of first meiosis) of oocytes and a contribution of the MAPK ERK signaling pathway. However, the interplay of CaSR with other molecules that maintain oocytes in the meiotic arrest and those that initiate meiotic resumption (such as LH) , to house the growing conceptus and to expulse the fetus during delivery . If communication between the embryo and the endometrium fails, then implantation fails. This is an important cause of infertility. There are estimations that only 50–60% of all conceptions advance beyond 20 weeks of gestation and from the pregnancies that are lost, 75% represent a failure of implantation. Failed implantation is also a major limiting factor in assisted reproduction. To treat disorders such as infertility, which increases worldwide due to an increasing toxic environment, but also diseases such as obesity, it is important to understand the molecular mechanisms of implantation and placentation  placenta formation, two distinct populations of trophoblast cells, the villous and the extravillous trophoblast, which exhibit distinct functions, become evident. The expanding chorionic villi of the placenta, which mediate materno-fetal transport of nutrients are covered by a multinucleated layer of syncytiotrophoblast (STB). This cell layer is maintained by proliferation and fusion of the underlying villous cytotrophoblasts. Extravillous trophoblasts, in contrast, invade the uterus and interact with maternal cells. These cells are important for proper placentation.invasive as seen in humans, most primates, dog, cat mouse, rat, or rabbit meaning that the conceptus will break through the surface epithel of the maternal uterus and invades the underlying stroma or can be non-invasive , integrating the uterine epithelium in the placenta. The depths of invasion as well as the degree of proximity between maternal and fetal circulation can vary largely among species. In humans and guinea pigs, the conceptus invades the stroma so deeply that the uterine surface epithelium is restored over it . Other species , may invade the stroma only partially and project into the uterine lumen (eccentric implantation). This can result in a contact to other sites of the uterine lumen and additional placental development . While in some species the maternal tissue remains relatively intact , meaning that trophoblast cells contact maternal capillary endothelium, in other species, trophoblast cells also invade the maternal endothelium and ultimately bath in maternal blood . These types of placentas are classified as haemochorial, which means that all maternal tissue layers are removed and the chorion  bathes directly in maternal blood. But even structures of murine and human placentas differ to some extent. Whereas, the term human placenta is monochorial, which means that one continuous trophoblast cell layer, the STB, separates maternal and fetal blood circulation, the murine placenta is trichorial. Moreover, in rodent placenta, but not in human, other primate, or ruminant placentas the primitive yolk sac, which in all species participates in nutrient exchange between the fetal and maternal circulations before the formation of the placenta, is incorporated into the placenta and turns into the intraplacental yolk sac (IPYS). The IPYS is positioned between fetal vessels and maternal blood spaces, well situated for exchange of substances between mother and fetus. It is a bilayered membrane, where smaller parietal or cuboidal cells on a thick basement membrane (Reichert's membrane) overlie maternal blood spaces and vessels, while tall columnar cells on the visceral or endothelial side overly the fetal vessels. Between these layers is the sinus of Duval, which communicates with the yolk sac cavity and the uterine lumen . Current knowledge on placental Ca2+ transfer as well as regulation of maternal and fetal Ca2+ homeostasis have been reviewed in several recent publications  to stimulate Ca2+ secretion into milk when the Ca2+ supply is adequate  by immunization of mice with intact parathyroid cells. Two of these antibodies (G11 and B6) interfered with the Ca2+-sensing mechanism of parathyroid cells .The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.