However, the molecular detail of the switching mechanism and how the Ca2+ signal received at cardiac troponin C (cTnC) is communicated to cardiac troponin I (cTnI) are still elusive. To unravel the structural details of troponin switching, we performed ensemble Förster resonance energy transfer (FRET) measurements and molecular dynamic (MD) simulations of the cardiac troponin core domain complex. The distance distributions of forty five inter-residue pairs were obtained under Ca2+-free and saturating Ca2+ conditions from time-resolved FRET measurements. These distances were incorporated as restraints during the MD simulations of the cardiac troponin core domain. Compared to the Ca2+-saturated structure, the absence of regulatory Ca2+ perturbed the cTnC N-domain hydrophobic pocket which assumed a closed conformation. This event partially unfolded the cTnI regulatory region/switch. The absence of Ca2+, induced flexibility to the D/E linker and the cTnI inhibitory region, and rotated the cTnC N-domain with respect to rest of the troponin core domain. In the presence of saturating Ca2+ the above said phenomenon were absent. We postulate that the secondary structure perturbations experienced by the cTnI regulatory region held within the cTnC N-domain hydrophobic pocket, coupled with the rotation of the cTnC N-domain would control the cTnI mobile domain interaction with actin. Concomitantly the rotation of the cTnC N-domain and perturbation of the D/E linker rigidity would control the cTnI inhibitory region interaction with actin to effect muscle relaxation.Cardiac troponin (cTn) is the Ca The TF is a filamentous structure composed of the heterotrimeric troponin complex and tropomyosin bound to the double helical actin filament 2+-activated regulation of the muscle. The heterotrimeric troponin from the cardiac muscle is formed by three individual proteins: troponin C (cTnC), troponin I (cTnI) and cardiac troponin T (cTnT). cTnC is the Ca2+ binding protein and cTnI is the actin binding protein. At low levels of Ca2+, two regions of troponin I, the inhibitory region (cTnI-Ir) and the mobile domain (cTnI-Md), bind to actin to inhibit actomyosin ATPase and relax the cardiac muscle. The cTnT anchors the troponin complex on the actin filament and also interacts with tropomyosin. In the relaxed cardiac muscle, the cTnI−actin interactions act as a regulatory switch to prevent cross-bridge formation between actin and the myosin head through steric blocking of myosin-binding sites on actin via tropomyosin 2+ binds to cTnC and induces an interaction between the regulatory (switch) region (cTnI-Rr) of cTnI and a hydrophobic patch located in the N-domain of cTnC, thus leading to an open conformation in the N-domain of cTnC 2+-based thin filament regulation, the molecular details of how the Ca2+ signal received at cTnC is transmitted to cTnI are still not well understood.Striated muscle contraction is triggered by Ca2+ regulation and cardiac muscle functioning, it is important to elucidate the Ca2+-induced changes to the structure and the structural dynamics of the TF proteins, particularly the troponin complex. The cardiac troponin core domain structure at saturating Ca2+ was solved 2+-free state is available. Although the crystal structure of the cardiac troponin in Ca2+-bound state provides some detailed structural information of cTnI-cTnC interaction, a number of functionally important regions involved in Ca2+-regulation mechanism were absent from the structure. For example, the N-terminal extension (residue 1–32) of cTnI, which is an important region for protein kinase A phosphorylation, and the portions of the N- and C-domains of cTnT, which tethers troponin to the thin filament, were absent in the crystal structure. Structures of the inhibitory region (residues 137–148) and the mobile domain (residue 192–210) of cTnI, were not defined. The lack of such information on the structure and dynamics of these critical regions and no knowledge of the structure of the cardiac troponin complex in the Ca2+-free state pose a barrier in our understanding of cardiac troponin function. Based on the core domain structure of the cardiac troponin complex at saturating Ca2+, the authors proposed an activation model 2+ binding and dissociation. However, no experiment have supported such large anharmonic fluctuation, especially that of the cTnI-Rr. In an effort to understand dynamics of cardiac troponin in Ca2+ activation process, Tobacman's group has used deuteration studies to unravel the dynamics of the cardiac troponin at varying biochemical states 2+-saturated state. They made no attempt to unravel the structure of the troponin complex in the Ca2+-free state. There is no detailed structural model available where by the structures of the cardiac troponin complex in the Ca2+-free and Ca2+-saturated states have been compared. Till today there is no model that can shed light as to how the Ca2+ signal received at cTnC is propagated to cTnI. This knowledge is very necessary because the cTnI in concert with tropomyosin regulate myosin-actin interaction. There are two segments of cTnI that bind to actin, the troponin I inhibitory region and the troponin I mobile domain. The molecular details of the conformational rearrangement within cTnC that convey the Ca2+ signal to these regions of cTnI to bind or dissociate from actin is still an unknown.To fully understand Ca2+-saturated and Ca2+-free states. The recovered FRET mean distances and half-widths, together with 4 additional FRET distances from literature (two in the Ca2+-saturated state and two in the Ca2+-free state), were used as distance restraints and bond-energies while performing molecular dynamics (MD) simulations of the existing cardiac troponin complex 2+-saturated and Ca2+-free states. This approach helped gain insight into the differences in structure and dynamics of the cardiac troponin complex in the Ca2+-saturated and the Ca2+-free states. The results of our studies helped us unravel the structural pathway by which the Ca2+ signal received at cTnC is transmitted to cTnI.In this study, we take a new approach by using Förster resonance energy transfer (FRET) technique to measure distances within the cTn complex and come up with a model of the how the cardiac troponin functions. As with every structure determination technique there are advantages and drawbacks. NMR is a very good technique for structure determination but is generally limited by the size of the protein. Particularly with the size of troponin complex, the structural power of NMR is significantly limited. Generally, it is easy for NMR to work with each individual troponin subunit, but difficult to work with the whole troponin complex. Furthermore, NMR requires high concentration of proteins to get sufficient signals, which makes it more difficult to work with the troponin complex because of its low solubility and high tendency to precipitate at high concentration. Though FRET has lower resolution than NMR, it can be easily applied to large sized protein complexes and work with proteins at low concentration that is close to physiological conditions. Using FRET we were able to acquire some important structural information about the functionally important regions of the troponin complex, such as the N-terminal extension of cardiac troponin I, the mobile domain of cardiac troponin I and the C-terminal of cardiac troponin T, which were missing in crystal structure of cardiac troponin core domain. But just a bunch of distances determined by FRET will not mean much unless they can be translated to structural information. Therefore the acquired FRET distance and half-width information were applied as distance restraints and bond-energies in this study while we performed molecular dynamics simulations of the cardiac troponin complex in explicit solvent. Time-resolved FRET measurements were performed to acquire a total of 45 FRET distances between cTnI and cTnT and between cTnC and cTnI in the Ca2+-saturated troponin crystal structure. Intra-protein FRET distances of cTnI, from each of the residues 129 and 151 located at the two ends of the inhibitory region to each of the residues 5, 15, 30 and 43 on the N-terminal of cTnI were determined. These measurements were performed in the troponin complexes at both the Ca2+-free and Ca2+-saturated states. In total 45 pairs of inter-/intra-protein FRET distance distributions were recovered from these measurements (meaning that 45 distances were obtained in the Ca2+ free state and 45 in the Ca2+ saturated state). When restrained MD simulation of the cardiac troponin complex 2+-saturated state to be pushed to the Ca2+-free state. Also we performed free MD simulations expecting it to yield information on the dynamics of the cardiac troponin complex.In the crystal structure of the core domain cardiac troponin complex 0 (∼29 Å) of this donor- acceptor pair is compatible to the measured inter-protein distances in the range of 20–29 Å. For all other inter-protein distance measurements between cTnT and cTnI  was covalently attached to the single-cysteine residues of cTnI and the acceptor (DABM) to the single-cysteine residues in cTnT. For FRET measurements of the intra-protein distances between cTnI, the cTnI residues 129 and 150 were mutated to tryptophan, which served as the energy donor. The cTnI residues 5, 15, 30 and 43 of cTnI in the troponin complexes was mutated to cysteine and labeled with AEDANS which served as the FRET acceptor. To determine the distances between the N-terminal extension of cTnI and cTnC, we labeled each of the selected cTnI N-terminal residues with AEDANS, which served as the FRET donor, and labeled the residues of cTnC with the FRET acceptor, DDPM. Each selected donor and acceptor labeled proteins were reconstituted into a troponin complex and donor fluorescence intensity decays in the absence and presence of the acceptor were acquired at both Mg2+ (Ca2+-free) and at Ca2+-saturated states. The acquired fluorescence intensity decays were analyzed to derive FRET distance information in term of distance distributions 2+-free state (relaxed) to the Ca2+-saturated state (activated). All recovered mean distances and half-widths are given in In these FRET measurements, the distances between three cTnT residue  located in the C-terminus of cTnT and selected cTnI residues were measured using either AEDANS (5-(iodoacetamidoethyl) aminonaphthelene-1-sulfonic acid) or MIANS naphthalene-6-sulfonic acid) attached to the cTnI residues as FRET donors. DABM  or DDPM maleimide) attached to the cTnT residues served as FRET acceptors. During our FRET measurements, we found the distances between cTnI residue 131 and cTnT residues 276 and 288 were much shorter than others. To measure the short distances, the MIANS-DDPM pair was used because the Rand cTnI , the AED2+-saturated state 2+-free and Ca2+-saturated states, we carried out MD simulations. The cardiac troponin core domain was simulated in three different conditions. The first was restrained MD simulations. During the restrained MD simulations the experimentally determined FRET distances and half-widths were applied as distance restraints and bond energies. This approach helped us select the starting structure shown in The distances and half-widths generated from the FRET experiments were used to model the cardiac troponin complex. The crystal structure of the cardiac troponin core domain that was resolved in the Ca2+-free state) and opening (in Ca2+-saturated state) be observed 2+ concentrations were detected by experimentally monitoring the distance between the cTnC residues 13 and 51 in the presence of cTnI 2+-free (1YVO.pdb) and the Ca2+-saturated state structures (1YTZ.pdb). The distance increase was the result of Ca2+ binding to the skeletal troponin C which opened the hydrophobic pocket. Therefore, the above set of criteria used for validating the biologically relevant position of the N-terminal extension of cTnI in the cardiac troponin complex has sound experimental backing. After docking the NMR structure of the N-terminal extension of cTnI . The sites 3 and 4 on the C-domain bind to either Ca2+ or Mg2+. Under physiological conditions, it is likely that in the Ca2+ saturated state the C-domain is occupied by Ca2+, while in the Ca2+ free state, the C-domain binds Mg2+, but it may also binds to Ca2+ as suggested by literature 2+ binding affinity (10−7M). For clarity, we retained the Ca2+ in sites 2, 3 and 4 in the Ca2+ saturated state, and removed the Ca2+ from site 2 and renamed the Ca2+ to Mg2+ in sites 3 and 4 when modeling the Ca2+-free state.Simulation of the cardiac troponin complex  in the C2+-free state …(NE)Arg83, Tyr5(OH)…(NZ)Lys94, and Tyr5(OH)… (OE2)Glu94. These bonds helped the helix N stay in close proximity to the D/E linker and the hydrogen bonds between Arg83 and Glu94 via Tyr5 helped keep the D/E linker from experiencing any major anharmonic fluctuation. The closing of the cTnC N-domain hydrophobic pocket led to hydrogen bond formations between cTnC residues Gly30…Val72, Asp33…Ser69, and Ser37…Ser69. These bonds were formed between the defunct site 1 and site 2 of cTnC. It is interesting to note that similar hydrogen bonds also exist in the crystal structure of cardiac troponin core domain complex (1J1E.pdb) between the above said sites . There was also a hydrogen bond between the Gly91 on the D/E linker and Glu161 of cTnC.The hydrogen bonds found in this state may be classified into two categories, (i) Intra molecular hydrogen bonds and (ii) inter molecular hydrogen bonds. The list of these hydrogen bonds is given in 2+ the cTnI-Ir became more flexible and experienced anharmonic motion and moved close to the cTnC helix C. This resulted in hydrogen bonds between cTnC residues Met60, Asp62, Asp65 with cTnI residues Arg146, Thr144, Lys139 respectively. In the context of the thin filament the enhanced flexibility of the cTnI-Ir would serve to help it translocate towards actin rather than towards cTnC helix C. The cTnC residue Asp67 located on site 2 hydrogen bonded to cTnI His35 located on the N-terminal extension of cTnI. On the C-domain of cTnC, Glu135, Asp139 located on helix G, hydrogen bonded with cTnI residues Lys38 and lys37. Asn143 located on cTnC site 4, hydrogen bonded with cTnI residues Arg137, Gly138. Phe156 located on the cTnC H-helix hydrogen bonded with the Lys37 located on the N-terminal extension of cTnI.Upon analyzing the hydrogen bonds formed between cTnC and cTnI we observed that the unfolding of helix N enabled hydrogen bond formation between cTnC Asp2 and cTnI Lys37 located on the N-terminal of cTnI and between cTnC Asp3 and Lys 51 of cTnI. Also Glu32 located on site 1 of cTnC hydrogen bonded with Glu33 and Thr32 on the N-terminal extension of cTnI. As we traverse from the N-terminus of cTnC towards the C-terminus, we observed hydrogen bonds between the loop, which connects helices B and C, with the cTnI-Rr. These hydrogen bonds held the cTnI-Rr close to the cTn complex and did not let the cTnI-Rr drop off the cTnC N-domain hydrophobic pocket  complex where the cTnI-Rr falls away from the skeletal troponin C (1YVO.pdb)). Due to the absence of regulatory CaIn this state the cTnI residues Leu129-Asp135 formed a network of hydrogen bonds between cTnC-cTnI-cTnT. Looking at the network of hydrogen bonds in this region it led us to the conclusion that this is the region where the cTnI-Ir is tethered to the cTn complex. The hydrogen bond network may be seen in 2+-saturated state simulation of the cardiac troponin complex depicted in 2+ ions from the crystal structure. The Ca2+-saturated FRET distances and half-widths from The Ca2+-free state the helix N was totally unfolded). The helix A was folded from Lys21-Ile26 (in the Ca2+-free state the helix A was folded from Lys17-Ile26), the helix B was folded from Glu40-Leu48 , the helix C was folded from Pro54-Val64 (in the Ca2+-free state the helix C was folded from Pro54-Met60), the helix D was folded from Phe74-Met81 (in the Ca2+-free state it was folded from Phe74-Lys86), the helix E was folded from Glu94-Phe104 (in the Ca2+-free state it was folded between Ser98-Phe104), the helix F was folded from Leu114-Thr124, the helix G was folded from Glu130-Asp139 and the helix H was folded from Tyr150-Met157. During the simulations, the N-domain of cTnC maintained an open conformation. This was monitored by measuring the distance between cTnC residues 13 and 51  and the C-terminal end of cTnT was extended and positioned away from the cTn complex  and 11.1 ns (Ca2+-saturated state) were further simulated for 250 ns. This simulation was performed to understand the dynamics of the cardiac troponin complex in either biochemical states. These simulations are notated as simulations II.The troponin complex at the end of 9.5 ns . In the crystal structure (1J1E.pdb) the separation distance was 8.95 Å, whereas in the Ca2+-free state the separation distance increased to15.70 Å. This loss of Ca2+-induced structural change in the binding loop caused conformational rearrangement of helices B and C. These helices were nearly orthogonal to each other in the Ca2+-saturated state, whereas in the Ca2+-free state they were parallel. This conformational change has direct implication in the interaction between the cTnC hydrophobic patch and the regulatory region of cTnI because the rearrangement of helices B and C control the orientation of the cTnI-Rr held within the cTnC hydrophobic pocket (discussed under the structure of cardiac troponin I).Upon comparing the final distance restrained simulated structure of the Ca2+. The helix D in the Ca2+-free state assumed a helical conformation between residues 74–86, whereas in the Ca2+-saturated state the helix was confined to residue 74–81. In the Ca2+-saturated state the helix E was fully folded whereas in the Ca2+-free state the cTnC residues 94–97 of the helix E were unfolded. The Ca2+ based folding and unfolding of helix D is observed in the crystal structures of the cTnC N-domain in the Ca2+-free  in the Ca2+-free state, were seen arching towards the cTnI-Ir (138–144)  . We post138–144) . It is k2+ from the regulatory binding site of cTnC in the troponin complex not only led to a flexible D/E linker of cTnC, but also caused the N-domain of cTnC to rotate clockwise with respect to the C-domain by 40 degrees. The rotation commences from Asp87 of the central D/E linker and is most conspicuous in the angle of helix D. In the Ca2+-saturated state the cTnC helix D is nearly 70 degrees to the normal whereas and in the Ca2+-free state it dropped to 30 degrees. The rotated N-domain was stabilized by an intra-protein hydrogen bond network formed between helix N and helices D and E of cTnC  nor had it moved away from cTnC as suggested by the authors who solved the structure of cardiac troponin core domain 2+-free state the cTnI-Rr was dilated and had rotated clockwise with the whole N-domain to 80 degrees to the normal (was positioned at 1 and 7 O'clock) and in the Ca2+-saturated state it retained it secondary structure and was oriented at 120 degrees to the normal (between 11 and 5 O'clock). It seemed like the cTnI-Rr undergoes a swivel like motion. To understand what could cause the dilation we ran an electrostatic surface analysis of the cTnC N-domain and observed that the reorientation of helix B caused a gap in the hydrophobic pocket within which the cTnI-Rr is held  because tropomyosin is farther away in the thin filament scenario, and the C-terminal of cTnT would be less unfolded in the Ca2+-free state and positioned closer to the cTn complex  because tropomyosin would be closer to the cTnT in the Ca2+-free state.The cTnT C-terminal residues Asp271-Lys288 experienced unfolding in both the Ca2+-induced anharmonic motion of the residues 202–223 at the N-terminus of cTnT was observed in the superimposed images. When Ca2+ was absent, the structure had moved away from the Ca2+-saturated state by ∼70 degrees  and open the cTnC N-domain hydrophobic pocket. This would cause the cTnI-Rr held within the cTnC N-domain hydrophobic pocket to regain its secondary structure and undergo a change in orientation. This would retract the cTnI-Md from actin. Concomitantly the refolding of helix E would make rigid the D/E linker region and retract the cTnI-Ir towards the troponin complex, thereby initiating cardiac muscle contraction.With all these pieces of information, we propose a sequence of events that would lead to cardiac muscle relaxation from the activated state. The loss of regulatory Ca2+ fluctuations cause the cardiac troponin complex to take two separate pathways which result in either muscle contraction or muscle relaxation. We used FRET distances and half-widths to model the cardiac troponin complex in the Ca2+-free and Ca2+-saturated states. This approach helped us to compare the structures in either biochemical states and come up with a model of how the Ca2+ signal received at cTnC is transferred to cTnI.To summarize, the CaThe wild-type recombinant cTnC was over-expressed in E. coli strain BL21 (DE3) cells and purified as previously described Two pairs of sulfhydryl donor-acceptor probes were used for FRET measurements between cTnI and cTnT: (a) AEDANS-DABMI  and (b) MIANS-DDPM. The second pair of probes was used to measure two distances, TnI 131 to TnT 276 and TnI 131 to TnT 278, and the first pair was used to measure all other distances. For distance measurements between cTnC  and cTnI  the AEDANS-DDPM pair was used. The procedure for sulfhydryl labeling of the proteins have been previously described 2+ regulation of the actin-activated S1-ATPase activity assay was carried out as described previously 2+ regulatory activity were negligible.Reconstitution of cTn with cTnC, donor-labeled cTnI, and acceptor-labeled cTnT was carried out in 6 M urea at a molar ratio of TnC∶TnI∶TnT = 1.5∶1.0∶1.2, following a previous procedure 2, 0.15M KCl, 1 mM DTT, and 1 mM EGTA(ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid)) in the presence and absence of 3 mM CaCl2. The emission spectra were corrected for variation of the detector system with wavelength. The donor quantum yields were determined by the comparative method using quinine sulfate as the standard 0, the Förster critical distance at which the efficiency of energy transfer is 0.5. In this calculation, a value of ⅔ was used for the orientation factor κ2.The steady-state fluorescence measurements were made at 20°C on an ISS PC1 photon-counting spectrofluorometer using a band pass of 3 nm on both the excitation and emission monochromators with samples (2 µM protein) in the working buffer (30 mM Mops (3-(N-mopholino)propanesulfonic acid) at pH 7.0, 5 mM MgCl2. The fluorescence intensity decays were measured in the time domain with a time-correlated single photon-counting lifetime spectrometer. The samples containing AEDANS as donor were excited with 348 nm LED light and the emission was isolated with a 410 nm cut-off filter, while the samples containing tryptophan as donor were excited with 295 nm LED light the emission was isolated with a 340 nm interference filter. The intensity decay of the donor from the donor-alone and the donor-acceptor samples were each collected into 1024 channels of a multichannel analyzer under identical conditions. The decays were analyzed by an exponential model. The distance-dependent donor decay of a donor-acceptor pair separated by a given distance r is given byDi is the decay time of the donor in the absence of acceptor, αDi is the associated amplitude, and R0 is the Förster distance. The observed decay for an ensemble of donor-acceptor pairs is given by0 was experimentally determined for each set of experimental conditions. The reduced chi-squares ratio (Time-resolved fluorescence measurements were carried out at 20°C with samples (2 µM protein) in the working buffer or in the buffer containing 3 mM CaClThe coordinates of the cTn complex (PDB ID-1J1E) α's of the participating residues during EM, SA and MD simulations as previously described 2+ in site 2 of cTnC. In our simulations structure displayed in In preparation for MD simulations the protein was placed in a triclinic box filled with 71739 SPC water molecules and set to physiological pH. The salt concentration was set at 0.15M NaCl to mimic experimental salt concentrations and sodium counter ions were added to maintain electroneutrality. The MD simulations were carried out on Cheaha cluster at the UAB IT Research Computing, University of Alabama at Birmingham, Alabama, USA. The GROMOS96 43a1 force field embedded in GROMACS During the restrained MD simulations, during the initial 200 ps, SA was performed on the N-terminal extension of cTnI. During the SA the temperature of the whole system was maintained at 300 K with the exception of the 40 amino acids that comprises the N-terminal extension of cTnI (1–40 amino acids). The temperature of the N-terminal extension of cTnI was increased in increments of 50 K from 300 K to 500 K and lowered to 300 K over a period of 200 ps. During the simulations the neighborhood list was updated every 10 steps and the bonds were not constrained during EM, SA and MD simulations. The electrostatic interactions were computed using PME . The nonbonded interactions were treated with a cut-off commencing at 10 Å and reaching zero at 14 Å. The simulations were performed under constant temperature and pressure  www.pymol.org) and CCP4MG package The structures were viewed using Rasmol Figure S1The opening and closing of the cTnC N-domain hydrophobic pocket in simulations II. The distance between the cTnC residues 13 and 51 are plotted as a function of time. The Ca2+ saturated and Ca2+-free states are colored red and blue respectively.(TIF)Click here for additional data file.Figure S2The distance between cTnC residues 13 and 51C monitored over 250 ns of simulations. Monitoring the distance between the two cTnC residues 13 and 51 helped reveal the cTnC N-domain hydrophobic pocket fluctuations between open and closed states. After allowing the initial 25 ns for equilibration, based on the minimum distance between the cTnC residues 13 and 51, we can say the time the Ca2+ saturated state structure spent in the closed state was from 67–70 ns, 81–111 ns and from 124–136 ns which is ∼18% of the simulation time. Likewise the time the Ca2+-free state spent in the open state was from 79–109 ns, 114–122 ns, 127–137 ns, which is ∼19.2% of the simulation time.(TIF)Click here for additional data file.Figure S3RMSF of the cTn complex simulated for 250 n after the distance restraints were released (simulations II). The root mean square fluctuation of the cardiac troponin complex was calculated over 250 ns. In the graph the C-alphas from 1–161 pertain to cTnC, 162–249 pertain to cTnT, 250–442 pertain to cTnI. In the Ca2+-saturated state fluctuations of more than 4 Å are observed from C-alpha 235–249. These pertain to the residues in the C-terminal end of cTnT helix H2 (residues 274–288). In the same state, the C-alphas 264–280 (correspond to residues 14–30 of cTnI) correspond to the N-terminal extension of cTnI. Towards the end of the x-axis we can see that the C-terminal end of cTnI (pertains to the cTnI-Md) experiences fluctuations in both the biochemical states. In the Ca2+-saturated state the C-alphas from 412–442 (corresponding to cTnI-Md residues 161–191) experience fluctuation, whereas in the Mg2+ (Ca2+ free) state the C-alphas from 425–442 experience fluctuation (correspond to cTnI-Md residues 174–191).(TIF)Click here for additional data file.Figure S4The opening and closing of the cTnC N-domain hydrophobic pocket (simulations III). The cTn complex was simulated in the absence of any distance restraints. The distance between the cTnC residues 13 and 51 was plotted as a function of time. The Ca2+-saturated and Ca2+-free systems are colored burgundy and sky blue.(TIF)Click here for additional data file.Figure S5RMSD of the protein in simulation III. The cTn complex was simulated without distance restraints. The RMSD of the cTn complex was plotted as a function of time. The Ca2+-saturated and Ca2+-free systems are colored burgundy and sky blue.(TIF)Click here for additional data file.Figure S6RMSF of the protein in simulation III. The root mean square fluctuations of the cardiac troponin complex were calculated for the cTn complex which was simulated for 150 ns without any restraints. In the graph the C-alphas from 1–161 pertain to cTnC, 162–249 pertain to cTnT, 250–442 pertain to cTnI. Fluctuations of more than 3 Å are observed in the N-terminal helix H1 of cTnT in both the Mg2+ (Ca2+-free) and Ca2+-saturated states. The C-alphas 162–177 in the graph pertain to cTnT residues 202–217 in the crystal structure). Fluctuations are observed at the N-terminal extension of cTnI . Towards the end of the x-axis fluctuations are observed in the C-terminal end of cTnI . These pertain to residues 164–191 of the cTnI-Md that are experiencing fluctuations in both the biochemical states.(TIF)Click here for additional data file.Figure S72+-free and Ca2+-saturated states after 150 ns of simulations.Structure of the cTn complex in the Ca (a) Depicts the structure of the cTn complex in the Ca2+-free state after 150 ns of simulations. (b) The structure of the cTn complex in the Ca2+ saturated state after 150 ns of simulations. The cTnC N-domain helices have not unfolded because no distance restraints were in place.(TIF)Click here for additional data file.File S1(DOCX)Click here for additional data file.


There is increasing evidence that astrocytes are able to discriminate between different Ca2+-linked stimuli, as the efficiency of some Ca2+ dependent processes – notably release of gliotransmitters – depends on the stimulus that initiates the Ca2+ signal. The spatiotemporal complexity of Ca2+ signals is substantial, and we here tested the hypothesis that variation in the kinetics of Ca2+ responses could offer a means of selectively engaging downstream targets, if agonists exhibited a “signature shape” in evoked Ca2+ response. To test this, astrocytes were exposed to three different receptor agonists  and the resultant Ca2+ signals were analysed for systematic differences in kinetics that depended on the initiating stimulus. We found substantial heterogeneity between cells in the time course of Ca2+ responses, but the variation did not correlate with the type or concentration of the stimulus. Using a simple metric to quantify the extent of difference between populations, it was found that the variation between agonists was insufficient to allow signal discrimination. We conclude that the time course of global intracellular Ca2+ signals does not offer the cells a means for distinguishing between different neurotransmitters.Astrocytes express a wide range of receptors for neurotransmitters and hormones that are coupled to increases in intracellular Ca Once triggered, increases in Ca2+ concentration activate downstream effectors such as transcription factors, kinases and exocytotic machinery + concentration, reactive gliosis in response to injury and maintenance of the blood brain barrier Astrocytes are non-excitable cells of the central nervous system, which nevertheless express a great many neurotransmitter and hormone receptors 2+ signalling may take is the subject of much debate. One question raised by the number of receptors coupled to Ca2+ elevation in astrocytes is how selectivity of function can be maintained. The appropriate physiological response of a cell in situ to glutamate (a fast excitatory transmitter) may be expected to differ from its response to ATP (a diffusely acting inhibitory transmitter), or histamine (a neurotransmitter and inflammatory messenger) for example, and yet all of these agonists have been shown to trigger Ca2+ signals in glia The form that a putative computational code in astrocyte Ca2+ signals differs with the receptor type that initiates it. Such spatially-restricted signalling is certainly known for Ca2+ microdomains that locally trigger exocytosis, but many transmitters are capable of triggering “global” Ca2+ signals that spread throughout the cell and even between cells via gap junctions. Another possibility that has been explored is that the interval between repetitive Ca2+ oscillations generated by a stimulus may encode information regarding agonist concentration 2+ response could carry information about the stimulus that triggered it – that the kinetics of cytosolic Ca2+ concentration could vary depending on the nature of the initiating stimulus.One possibility is that the spatial range of Ca2+ signalling apparatus can generate Ca2+ oscillations of strikingly different duration and range 2+ dynamics – to encode information about the type of agonist and/or its concentration into the timing of a global Ca2+ response, even within a single cell type 2+ signalling apparatus, could all generate diversity in the dynamics of cytosolic Ca2+ concentration. For example, in the case of glutamate, different temporal profiles of Ca2+ response may be generated by Ca2+ influx through GluA2-deficient AMPA receptors versus InsP3-dependent release of Ca2+ from internal stores after activation of mGluRs. Additionally, despite mGluR1, P2Y and H1 receptors all being coupled to Gq proteins, activation of the different receptors may generate changes in InsP3 concentration (and associated Ca2+ release) with differing kinetics, due to variation in desensitization kinetics, receptor density, or the strength of coupling to Gq proteins between the three receptor types. Co-expression of different receptor classes, such as Gq-coupled H1 and Gs-coupled H2 receptors may also generate different Ca2+ kinetics than expression of only a single receptor class, if crosstalk between the signalling cascades alters the activity of some of the many components of the Ca2+ signalling apparatus. Such mechanisms of signal diversification could allow astrocytes exposed to transient concentrations of neurotransmitters in situ to encode information about the nature of the stimulus into the kinetics of the provoked response.Such variation in kinetics is observed between different cell types, where the expression of different levels of the channels, pumps and buffers of the cellular Ca2+ signals that they generate. We examined the long-term response of the cells to tonic application of glutamate, ATP and histamine at various concentrations, but also used a high-throughput imaging technique to analyse the short-term Ca2+ response of naïve cells to the agonists. We compared astrocytes cultured from both cerebellum and cortex, to determine whether the dynamics of the Ca2+ signal elicited by the agonists varied in predictable ways. We found that although Ca2+ responses showed substantial heterogeneity in kinetics, the time course of the Ca2+ signal did not vary systematically between agonists, suggesting that the cells could not exploit this variability to discriminate between stimuli.In this study, we sought to test the concept that astroglial cells could discriminate between different agonists, at different concentrations, by systematic variation in the time course of the global Ca2+ indicator fluo-4, and then exposed to ATP, glutamate and histamine for 20 min at a concentration of 100 µM. All three agonists evoked Ca2+ signals in both cerebellar and cortical astrocytes , the P2Y agonist 2-[Methylthio]adenosine-5′-trihydrogen diphosphate (2-MeS-ADP), and the H1 agonist 2-pyridylethylamine dihydrochloride (Pyri di), but very few cells responded to the mGluR1 agonist (S)-3,5-Dihydroxyphenylglycine (DHPG), or the H2 agonist dimaprit dihydrochloride (Dim di). Cortical astrocytes gave similar responses  and classified the responses into four broad categories  against each of the kinetic parameters examined  can be compared to known overlap of the idealized data.Carrying out such an analysis on simulated data (random data sets sampled from a Gaussian distribution) shows that progressively increasing the difference between population means by multiples of the standard deviation (SD) increases the value of D . When th2+ spikes, but they did not correlate with mixing time, suggesting they arose from spontaneous activity rather than mechanical artefacts , the population responses were skewed and largely overlapping, regardless of stimulus  at three different concentrations . The population response did not appear to differ significantly with agonist concentration, beyond a tendency for longer latencies to be lost at higher concentrations .A final comparison was made between cerebellar and cortical astrocytes, stimulated with ATP, glutamate and histamine ; Table 52+ kinetics would be less than the variation between agonists across the whole population (if the hypothesis was correct). It is possible, however, that the converse is true – that variation in kinetics between agonists may occur in each cell, but the differences between cells overwhelms the trend. This possibility is harder to test, as it requires repetitive addition of agonists to the same cell population; consequently, the cells will not be naïve for the later additions , and the protocol is less amenable to high-throughput screening. Nevertheless, we exposed astrocytes to 100 µM ATP, histamine and glutamate for 1 min sequentially with 7 min recovery period between additions, and then analyzed the response kinetics of those cells that responded to all three stimuli  can exhibit kinetically complex Ca2+ signals, such as repetitive oscillations in Ca2+ concentration from basal levels, sinusoidal oscillations on a raised baseline, short bursts of oscillations, or prolonged high amplitude plateaux of Ca2+2+ dynamics can arise from several sources, but a major cause is the inherent stochasticity of the mechanisms underlying global Ca2+ signal generation.In this study we tested the hypothesis that the spatiotemporal complexity of Ca2+ signals arise from the amplification of elementary release events generated by the opening of a small number of clustered ER Ca2+ channels. It is the best known example of how the random behaviour of individual channel openings can be amplified to a whole cell scale, most notably in the variation in interspike intervals during baseline oscillations 2+ release channels, buffers and clearance pumps can give rise to dramatic changes in Ca2+ dynamics. This variability in the balance of release and extrusion mechanisms has been shown to result in intercellular heterogeneity of response to agonists in genetically identical cell populations 2+ handling proteins is sufficient to account for large scale changes in global signal dynamics Global Ca3 and Ca2+ between adjacent cells. A study by Venance et al. 2+-dependent release of gliotransmitters such as ATP can initiate paracrine (or autocrine) signalling in cultured astrocytes In the case of astrocytes, however, there are additional sources of complexity. Gap-junctional coupling between cells can provide a route for diffusion of InsP2+ signal towards certain kinetic profiles. However, the evidence accumulated in this study suggests that the substantial heterogeneity in the kinetics of Ca2+ responses does not correlate with the initiating stimulus during either short- or long-term stimulation.In principle, different neurotransmitters and hormones could engage these various sources of kinetic heterogeneity to varying degrees, and so bias the Ca2+); for example, glutamate in cerebellar astrocytes showed single spikes or sustained responses whereas ATP showed bursts of spikes or repetitive spikes. However, these trends were not robust; all classes of response were observed to differing degrees for all transmitters. No class could be taken to be characteristic (or predictive) of stimulation with a particular agonist. A similar lack of distinctiveness was observed when the initial response of cells was analysed – heterogeneity existed in the range of parameter values, but did not vary in a systematic manner with the type or concentration of stimulus  a measure of the difference between the populations was needed.One ramification of the population responses we observed is that the kinetic parameters do not have a Gaussian distribution, which means that the use of parametric tests for assessing changes in mean parameter values for small sample sizes – a not uncommon practice – is inappropriate, and may give rise to spurious conclusions. Use of non-parametric tests may protect against this problem, but to answer the specific question raised by our hypothesis of DKL) is a metric derived from information theory and used in communications applications to assess signal degradation between a reference signal and the measured output signal. If signals are identical, the DKL is zero, but as the signals diverge, DKL increases, in principle to infinity. Application of this measure to biological data has been carried out D were small (mean ≈0.2) and indistinguishable from control differences (mean = 0.15±0.1 SD). Although some comparisons returned higher values of D (maximum 0.69), this would be predicted due to chance, and there was no obvious pattern to suggest that a given kinetic parameter consistently showed large differences between agonists or concentrations  would be need to be reliably identified from the Ca2+ kinetics resulting from a single exposure to the agonist. This would require response distributions with negligible overlap and D values that approach 1. In an idealised example there is still considerable overlap at D values as high as 0.87  Act 1986, and were approved by the Babraham Research Campus Animal Welfare, Experimentation, and Ethics Committee (Certificate of Designation PCD 80/4804).2+-and Mg2+-free Hank's HEPES-buffered saline solution , 22 mM glucose, 20 mM HEPES, 100 units.ml−1 penicillin, 100 µg.ml−1 streptomycin, 0.1 mM L-serine, 0.5 mM L-glutamine and 1 mM sodium pyruvate (pH adjusted to 7.2 with NaOH). Meninges and blood vessels were carefully removed using fine forceps. The dissected tissue was finely chopped and dissociated enzymatically for 45 minutes at 37°C in dissection buffer supplemented with 15 µg/ml papain. Cerebella and cortical tissue was subsequently mechanically dissociated using repeated trituration through fire-polished Pasteur pipettes in the presence of 20 mg/ml DNase in medium containing DMEM , 4500 mg.l−1 glucose, 4 mM L-glutamine, 110 mg L-pyruvate, 10% foetal bovine serum, 50 µg/ml L-proline, 100 units.ml−1 penicillin, and 100 µg/ml streptomycin. Cells were plated at a density of 5×104 cells/ml (cerebellar astrocytes) or 8×104 cells/ml  on poly-l-lysine coated coverslips for long-term stimulation experiments or 96 well imaging plates  for high throughput experiments. Medium was changed 24 hours after plating, and every 3–4 days thereafter.Neonatal (P2) rat pups were sacrificed by cervical dislocation and decapitation. Whole brains were removed under sterile conditions. Using a dissection microscope, the cerebellum and cerebral cortices were removed and transferred to separate vessels containing ice cold dissection buffer composed of: CaCerebellar and cortical astrocytes were fixed in PBS containing 2% paraformaldehyde and 0.05% glutaraldehyde for 20 minutes at room temperature. Cells were washed several times with phosphate buffered saline (PBS) before being permeabilized in PBS containing 0.2% triton X-100 for 15 minutes. Blocking of non-specific binding sites was achieved by incubation in PBS containing 0.1% triton-X-100, 10% normal goat serum and 1% bovine serum albumin. Cells were incubated with primary antibody  in this buffer for 1 hour at room temperature. After further washing, cells were incubated with secondary antibody (goat anti-rabbit IgG) conjugated to Alexa Fluor 488 indicator  for a further hour. After a final wash, coverslips were mounted onto slides with Vectashield containing the DNA counterstain 4′-6′ diamidion-2-phenylindole .Cells were imaged on a point-scanning confocal microscope (Zeiss LSM 510 META) with 1.40 na, 63×oil objective. Indicators were excited at 488 nm (Alexa Fluor 488) and 405 nm (DAPI) and emission detected in bandwidths of 505–550 nm (Alexa Fluor 488) 420–480 nm (DAPI).2+ indicator fluo-4 AM  for 30 min in buffer containing: 135 mM NaCl, 3 mM KCl, 10 mM HEPES, 15 mM D-glucose, 2 mM MgSO4 and 2 mM CaCl2. Cells were then left to de-esterify accumulated fluo-4 AM for a further 30 min before transferring to the stage of an Olympus IX70 inverted microscope . Loaded cells were excited at 450/20 nm with an exposure time of 100 ms, imaged at a frame rate of 2 Hz, and fluorescence emission (535/50 nm) was detected with an ORCA-ER camera . Camera and shutter  were controlled by Wasabi software . Agonists were added by manual displacement of bath solution with excess buffer containing agonist at the indicated concentration.For long-term stimulation experiments, astrocytes were loaded with 1 µM of the CaFor high throughput imaging, a BD Pathway 855 High-Content Cell Analyser  was used . For these experiments, cells were co-loaded with hoechst 33342 and fluo-4 AM. Fluo-4 fluorescence  was imaged continuously at a frame rate of 8 Hz, and a single image of Hoescht staining  was acquired at the end of the sequence. Solution exchange was automated, with a 1∶10 dilution of stock solution into each well with an addition time of 1 s, followed by a re-dispensing mixing step (at 20 µl/s), giving a total mixing time of 2 to 3 s.t divided by mean intensity for 0–20 s before addition of agonist (F/F0).Regions of interest centred on the cell nucleus were defined manually for long-term stimulation experiments, or by segmentation based on the Hoescht image (using AttoVision software) for high-throughput experiments. Fluorescence intensity was expressed as the ratio of fluorescence at time 2+ signal that reaches the nucleus) if F/F0 increased >1.045 fold (at least 3 SD above the pre-stimulation mean). Peak amplitude is the largest fold-change in fluorescence, rise time is the time taken to increase from 10% to 90% of peak, latency is the time taken from addition of agonist to 10% of peak, and area under curve (AUC) is the integral of fluorescence intensity from 10% of peak to the end of the experiment. Cells were excluded from further analysis if Ca2+ signals occurred before addition of agonist.Kinetic parameters were measured with bespoke software . Cells were defined as responders , burst of spikes , repetitive spikes , and sustained response . This protocol successfully sorted 2216 cells into the four classes from a total population of 2247 (31 cells were not classified = 0.14%).For spike counting, traces were smoothed by averaging 50 adjacent time points (6.25 s) and subtracting the result from the original time course. The spike threshold was set at 0.4, with a minimum width of 5 s. Mean interspike interval (ISI) was also determined. For classification of responses to long-term stimulation, the following criteria were used: D) between two normalized histograms A and B, with n bins:To assess how different two populations are, we used a simple subtraction method to quantify the difference  to 1 (non-overlapping populations).D varies with the extent of overlap, we used idealized surrogate data sets. Two normalized random Gaussian distributions (standard deviation = 1) were generated using Origin . The difference between population means was then systematically increased, and D was calculated for each difference .


Cells were isolated from anesthetized 3-month-old male and female Fischer 344 rats, paced at 4 Hz (37°C), exposed to simulated ischemia (20 mins) and reperfused. Cell shortening (edge detector) and intracellular Ca2+ (fura-2) were measured simultaneously. Cell viability was assessed with Trypan blue. Ischemia reduced peak contractions and increased Ca2+ levels equally in myocytes from both sexes. However, contraction amplitudes were reduced in reperfusion in male myocytes, while contractions recovered to exceed control levels in females . Only 60% of male myocytes excluded trypan blue dye after ischemia and reperfusion, while all female cardiomyocytes excluded the dye (p<0.05). Parallel experiments were conducted in myocytes from ∼24-month-old female rats or 5–6-month-old rats that had an ovariectomy at 3–4 weeks of age. Beneficial effects of female sex on myocyte viability and contractile dysfunction in reperfusion were abolished in cells from 24-month-old females. Aged female myocytes also exhibited elevated intracellular Ca2+ and alternans in ischemia. Cells from ovariectomized rats displayed increased Ca2+ transients and spontaneous activity in ischemia compared to sham-operated controls. None of the myocytes from ovariectomized rats were viable after 15 minutes of ischemia, while 75% of sham cells remained viable at end of reperfusion (p<0.05). These findings demonstrate that cardiomyocytes from young adult females are more resistant to ischemia and reperfusion injury than cells from males. Age and OVX abolish these beneficial effects and induce Ca2+ dysregulation at the level of the cardiomyocyte. Thus, beneficial effects of estrogen in ischemia and reperfusion are mediated, in part, by effects on cardiomyocytes.Sex differences in responses to myocardial ischemia have been described, but whether cardiomyocyte function is influenced by sex in the setting of ischemia and reperfusion has not been elucidated. This study compared contractions and intracellular Ca There are important sex-related differences in the pathophysiology of many cardiovascular diseases, including ischemic heart disease 2+ loading, such as β-adrenergic stimulation or increased external Ca2++-Ca2+ exchanger 2-adrenergic receptors Studies in animal models also have provided evidence for sex differences in responses to myocardial ischemia and reperfusion. Experiments in intact hearts from rats and mice have shown that young adult females exhibit better recovery of contractile function and fewer arrhythmias in reperfusion than age-matched males 2+ levels, along with post-ischemic contractile dysfunction (stunning) and reduced viability in reperfusion 2+ and cell viability were compared throughout ischemia and reperfusion in ventricular myocytes from 3-month-old rats of both sexes. Some studies also used myocytes from aged (∼24-month-old) female rats and myocytes from 5–6-month-old female rats who had undergone a bilateral OVX at 3–4 weeks of age. Our results demonstrated that individual ventricular myocytes from young adult females were significantly more resistant to ischemia and reperfusion injury than cells from age-matched males. This female advantage was abolished by either advanced age or by removal of ovarian estrogen through OVX. These data indicate that beneficial actions of estrogen in myocardial ischemia are mediated, in part, by actions on the myocytes themselves.We have developed a model of ischemia and reperfusion injury in ventricular myocytes isolated from the hearts of male animals For full details of Methods, please refer to Protocols were approved by the Dalhousie University Committee on Laboratory Animals (No. 10-029) and followed Canadian Council on Animal Care Guide to the Care and Use of Experimental Animals . Sodium pentobarbital anesthesia was used and all efforts were made to reduce suffering.2+ buffer (200 µM) followed by nominally Ca2+ free buffer plus collagenase and dispase II (37°C). After digestion, the ventricles were minced, stored in a high K+ substrate-enriched buffer and filtered before use. OVX was confirmed by uterine atrophy.Myocytes were isolated from 3 month old male and female Fischer 344 rats, ∼24 month old females and from 5–6 month old females after OVX or sham operation at 3–4 weeks. Ventricular myocytes were isolated as described 3, 0.9 NaH2PO4, 4 KCl, 0.5 MgSO4, 1.8 CaCl2, 5.5 glucose; pH 7.4, 95% O2, 5% CO2). Cells were paced at 4 Hz with trains of 20 (3 ms) pulses followed by a 2.5 s delay. The pacing frequency of 4 Hz was chosen to be near the physiological frequency in the rat. The 2.5 s pauses were incorporated in the protocol to observe spontaneous activity if it occurred. Control recordings were made for 15 minutes in normal Tyrode’s solution, then cells were exposed ischemic Tyrode’s solution for 20 minutes  2, 10% CO2 was directed over the experimental chamber to reduce the pO2 as described previously Myocytes were loaded with fura-2 AM as described 2+ transients were recorded simultaneously as described 2+ levels were recorded with a DeltaRam fluorescence recording system , Birmingham, NJ) and data were acquired with Felix32 software (PTI). Fura-2 was excited at 340 and 380 nm and emission was measured at 510 nm . Unloaded cell shortening was measured (120 Hz) with a video edge detector  and CCD camera . The ratio of fluorescence at 340 and 380 nm was converted to Ca2+ concentration as described previously Contractions and Ca2+ transients once these responses had reached steady state. The incidence of spontaneous activity (beats that occurred during the 2.5 s pause) was recorded in each experiment. Alternans  were quantified with an alternans ratio  Data were analyzed with Clampfit 8.2 software (Molecular Devices). The last three responses in the 20 pulse train were averaged to quantify contractions and Ca2+ transients recorded from male and female myocytes after 15 minutes of pacing at 4 Hz, just prior to exposure to simulated ischemia and reperfusion. Contractions were normalized to cell length, as we found that cells from females were smaller than cells from males .Selected physical characteristics of young adult male and female rats were compared as shown in om males . Peak conditions . Systoli2+ transients were compared throughout ischemia and reperfusion in myocytes from young adult male and female rats. 2+ transients (top) and contractions (bottom) recorded from a male myocyte at selected time points throughout an experiment. 2+ transients recorded from male myocytes during ischemia and reperfusion compared to responses in time controls. Data were normalized to values recorded after 15 minutes of pacing at 4 Hz to facilitate comparisons between groups. Contractions were essentially abolished by ischemia but recovered with an overshoot immediately upon reperfusion  and reduced viability in reperfusion.To determine whether responses of individual cardiomyocytes to myocardial ischemia were influenced by the sex of the animal, contractions and underlying Caerfusion . Howevererfusion . In contperiment . Ischemierfusion . Reperfuerfusion . There w2+ transients (top) and contractions (bottom) in ventricular myocytes from female rats. Mean data show that contractions decreased significantly in ischemia, but recovered upon reperfusion and exceeded time controls throughout reperfusion . However, ischemia did induce mechanical and Ca2+ transient alternans in myocytes from aged female rats, but not in cell from the younger female animals .To determine whether OVX would augment ischemia and reperfusion injury at the cellular level, responses of cardiomyocytes from sham-operated and OVX female rats were compared throughout ischemia and reperfusion. Physical characteristics of the sham-operated and OVX rats used in this study are shown in erfusion . OVX als2+ transients also increased in ischemia in OVX cells, but not in sham controls  that has been well documented in aging male hearts 2+ sequestration and disrupt intracellular Ca2+ regulation as shown in this study and previously Aging also resulted in diastolic Ca2+ transients, which were larger than sham even prior to ischemia as shown in the present study and previously 2+ transients and contractions, and this was followed by the uptake of trypan blue dye within 15 minutes of exposure to ischemia. The larger Ca2+ transients and spontaneous Ca2+ release in observed in OVX cells in ischemia suggests that SR Ca2+ load is much higher in OVX myocytes than in sham controls. Indeed, we and others have shown that SR Ca2+ load is elevated by OVX even in the absence of ischemia 2+ overload in ischemia and leads to spontaneous release of SR Ca2+ and ultimately a loss of cell viability.Previous studies in rats have shown that serum estradiol levels decline by more than 60% between the ages of 3 mos and 14 mos 2+ dysregulation is a major mechanism that contributes to the increased sensitivity of OVX hearts to ischemia and reperfusion injury 2+ dysregulation in OVX myocytes may help explain the increased propensity for reperfusion arrhythmias observed in aromatase knockout mice, which exhibit both estrogen suppression and testosterone elevation Our results indicate that intracellular CaIn the present study, ovaries were removed at 3–4 weeks of age but the rats were 5–6 months of age when we examined responses to ischemia and reperfusion. This resulted in an OVX model characterized by long term estrogen withdrawal and the lack of exposure of the heart to normal pubertal systemic estrogen modeling. It is important to note that this model differs from the more gradual reduction in ovarian steroids that would be observed during menopause. This model also may result in hearts that are particularly susceptible to ischemia and reperfusion damage. Additional experiments with other time frames for estrogen deprivation could be explored in the future to address these issues. It is also important to note that the present study examined only acute responses of cardiomyocytes to ischemia and reperfusion. The impact of ovarian estrogen suppression on other factors that may contribute to chronic post-ischemic heart failure also would be interesting to evaluate.We also found that both aging and OVX caused increased a large increase in body weight when compared to younger female rats. This increase in body weight would be expected to increase systemic volume load and this could promote cardiomyocyte hypertrophy. Indeed, increased volume load may contribute to the increase in cardiomyocyte length we observed in the aged group. However, cardiomyocyte length was not affected by OVX, which suggests that increased volume load does not lead to cellular hypertrophy in OVX hearts, at least over the time frame of our investigation. It is likely that an increase in adipose tissue contributes to the increase in body weight observed in aged and OVX animals, so adipose tissue is a potential non-gonadal source of estrogen in our study 2+ transients, and cell length recorded prior to exposure to ischemia and reperfusion also were examined in this study. Basal contractions and Ca2+ transients were larger in myocytes from OVX rats compared to sham-operated controls, as we and others have previously reported 2+ transients also did not decline with age in female rats and aged myocytes were hypertrophied, as reported in our previous study 2+ transients were similar in amplitude in myocytes from 3 month-old male and female rats, when cells were paced at 4 Hz in 1.8 mM Ca2+ buffer. Previous studies that have reported smaller contractions and Ca2+ transients in female rat cells when compared to males used slower pacing frequencies and/or lower external Ca2+ concentrations 2+ loading is reduced by low pacing frequencies and/or reduced external Ca2+ concentrations.Peak contractions, Ca2+ homeostasis in individual cardiac myocytes exposed to simulated ischemia. Intracellular Ca2+ dysregulation plays an important role in cardiovascular diseases, so our data indicate that beneficial actions of estrogen in the setting of myocardial ischemia are mediated, at least in part, by actions on Ca2+ handling mechanisms in the myocytes themselves. Sex hormone-regulated pathways within cardiac myocytes may be a fruitful area to explore for the identification of new targets for the treatment of ischemic heart disease in men and women.In summary, the results of our study show that resistance to myocardial ischemia and reperfusion injury is present in individual ventricular myocytes from young female animals. Our study also showed that this was abolished by either advanced age or by long term removal of ovarian estrogen. Aging and OVX both caused dramatic changes in intracellular CaAppendix S1Supplemental materials and methods.(DOCX)Click here for additional data file.


As well as activating ATP-consuming processes, cytosolic Ca2+ increases may also potentiate mitochondrial ATP synthesis. Until recently, the ability to study the role of mitochondrial Ca2+ transport in glucose-stimulated insulin secretion has been hindered by the absence of suitable approaches either to suppress Ca2+ uptake into these organelles, or to examine the impact on β-cell excitability. Here, we have combined patch-clamp electrophysiology with simultaneous real-time imaging of compartmentalised changes in Ca2+ and ATP/ADP ratio in single primary mouse β-cells, using recombinant targeted  as well as entrapped intracellular (Fura-Red), probes. Through shRNA-mediated silencing we show that the recently-identified mitochondrial Ca2+ uniporter, MCU, is required for depolarisation-induced mitochondrial Ca2+ increases, and for a sustained increase in cytosolic ATP/ADP ratio. By contrast, silencing of the mitochondrial Na+-Ca2+ exchanger NCLX affected the kinetics of glucose-induced changes in, but not steady state values of, cytosolic ATP/ADP. Exposure to gluco-lipotoxic conditions delayed both mitochondrial Ca2+ uptake and cytosolic ATP/ADP ratio increases without affecting the expression of either gene. Mitochondrial Ca2+ accumulation, mediated by MCU and modulated by NCLX, is thus required for normal glucose sensing by pancreatic β-cells, and becomes defective in conditions mimicking the diabetic milieu.Glucose induces insulin release from pancreatic β-cells by stimulating ATP synthesis, membrane depolarisation and Ca In particular, Ca2+ entry into the cytosol, triggered by elevated ATP, is expected to enhance ATP hydrolysis, for example by activating granule exocytosis 2+ ATPases which pump the cation out of the cytosol 2+-induced drop in ATP is then predicted to open KATP channels, thereby arresting Ca2+ influx 2+ has been suggested to induce repolarisation of the plasma membrane by opening Ca2+-activated K+ channels 1Fo ATPase The interplay between cytosolic Ca2+ uptake have remained unclear in any mammalian cell type. However, two reports in 2011 identified a Ca2+-selective mitochondrial uniporter, MCU, encoded by the Ccdc109a gene 2+ sensing subunit MICU1 2+ transporting entity. Conversely, mitochondrial Ca2+ efflux was proposed to be mediated by the Na+-Ca2+ exchanger NCLX 2+ transport in the β-cell, and may thus modulate insulin secretion, is currently unknown.Until very recently, the molecular entities responsible for catalysing mitochondrial Ca2+ transfer across the mitochondrial membrane in β-cells and (b) the impact of these changes on cytosolic ATP dynamics and electrical excitability. To these ends, we have deployed a recently-developed, molecularly-addressed GFP-based recombinant probe for mitochondrial Ca2+ ([Ca2+]mit), 2mt8RP 2+ probe (Fura Red) allowing us to image [Ca2+]cyt simultaneously with [Ca2+]mit in individual primary mouse β-cells. These measurements have been combined with perforated patch electrophysiology to allow plasma membrane potential (Vm) to be recorded or controlled without perturbing cellular composition or metabolism 2+]cyt via voltage-gated Ca2+ channels 2+]cyt and [Ca2+]mit in real time. In parallel, the novel ATP sensor Percevalcyt). These combined approaches have allowed us to characterise the roles of MCU and NCLX as regulators of mitochondrial ATP synthesis in the β-cell.In the present study, we have sought to explore (a) the molecular mechanisms responsible for Ca2+]cyt and/or [Ca2+]mit might influence glucose-induced increases in [ATP/ADP]cyt. The latter parameter was therefore imaged in single mouse β-cells expressing the GFP-based probe Percevalcyt  and an increase in input resistance, followed by depolarisation of the plasma membrane and a [Ca2+]cyt rise, as expected. This was closely followed by a drop in [ATP/ADP]cyt (iii). The 33±4% drop (“trough” in cyt quickly recovered and displayed a steady further increase (iv). The increase was not associated with any significant decrease in [Ca2+]cyt, and thus was not likely to reflect a lowering demand for Ca2+ extrusion or other ATP-consuming processes. Furthermore, setting Vm to −70mV via the patch pipette, thus closing voltage-gated Ca2+ channels, led to a prompt decrease in [Ca2+]cyt (v). The application of the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) resulted in an abrupt decrease of [ATP/ADP]cyt, as expected (vi), and an elevation of [Ca2+]cyt, presumably due to a compromise in Ca2+ pumping across the plasma and ER membranes.β-Cells maintained at low (3 mM) glucose exhibited a resting V/ADP]cyt , iii. Thincrease , iv. TheCa2+]cyt , v. The expected , vi, andcyt elevation in β-cells in two distinct phases  using the patch pipette in voltage-clamp mode  and Ca2+-dependent (the one that follows) Ca2+ entry.An increase in glucose from 3 mM to 17 mM resulted in a rapid elevation of [ATP/ADP]evel ii, . Notablycyt iii,  Remarkabased iv, . These ePerceval were associated with the closure of ATP-sensitive K+ channels, as expected. This seemed an important question since fluctuations in “global” cytosolic ATP/ADP differ in some circumstances from those immediately beneath the plasma membrane, as recorded with a targeted luciferase-based probe We next sought to determine whether the apparent increases in cytosolic ATP/ADP ratio reported with m. These pulses were too small to trigger any voltage-gated Ca2+ conductance and therefore had no effect on Ca2+ entry. The addition of 17 mM glucose decreased Im during the Ca2+-independent phase of [ATP/ADP]cyt increase  m thus was found to decrease from the initial value of 0.43±0.09 nS/pF to 0.09±0.02 nS/pF (n = 12) during the Ca2+-independent phase. A strong and significant correlation  between the elevation of [ATP/ADP]cyt as recorded with Perceval, and the closure of KATP changes as measured above,  and [Ca2+]cyt and [Ca2+]mit were stable (i). Exposure to 17 mM glucose led to an increase in [Ca2+]cyt (ii) which was followed later by an increase in [Ca2+]mit, presumably reflecting Ca2+ uniporter-mediated uptake (iii). [Ca2+]cyt and [Ca2+]mit reached their maximal amplitudes 47±6 s and 134±25 s, respectively, after the onset of glucose-induced electrical activity  plays a role in the regulation of the [ATP/ADP]cyt increase that follows an initial and small Ca2+-induced drop. To test this possibility directly we therefore reduced the expression of the recently-identified mitochondrial Ca2+ uniporter, MCU 2+ entry into mitochondria, whilst the imposed cytosolic Ca2+ increases were unaffected  phase of the [ATP/ADP]cyt increase, i.e. the [ATP/ADP]cyt recovery, was significantly impaired in the β-cells where MCU expression was reduced  independently of a Ca2+ increase, we explored the glucose-induced changes in this parameter prior to [Ca2+]cyt elevation using tetramethyl rhodamine, ethyl ester (TMRE). The resting Ψm (measured as −127±4 mV in control vs −133±5 mV in MCU− cells) and the kinetics of the glucose-induced change  and studied the impact on the dynamics of [Ca2+]cyt and [Ca2+]mit in response to Vm manipulation. FFA+ cells displayed slower dynamics of [Ca2+]mit increase (+ β-cells. This effect was not likely to be caused by changes in resting Ψm (−135±4 mV in control vs −137±4 mV in FFA+ cells) or the kinetics of the glucose-induced change in Ψm (homeobox-1 (Pdx1), in contrast, was significantly reduced by the chronic glucolipotoxicity, in line with earlier observations To this end, we cultured primary mouse β-cells under glucolipotoxic conditions  These studies also represent the first use of the novel ATP/ADP probe 2+ increases are essential for the sustained (second) phase of [ATP/ADP]cyt increase in response to high glucose. Interestingly, we show . Future studies are required to address this question and the role of NCLX in the β-cell.Recent studies 2+ concentration. In the second phase, the elevation of cytosolic Ca2+ concentration leads to a gradual increase in mitochondrial Ca2+ (cyt (first phase) was not affected by the MCU silencing whereas the second phase of [ATP/ADP]cyt increase was essentially eliminated.Overall, our data support a two-phase model , in whicial Ca2+ . This, i2+ accumulation may be essential for increases in cytosolic ATP/ADP in response to the sugar. However, this earlier study relied on the over-expression in the mitochondrial matrix of a high affinity (and high capacity) calcium-binding protein, S100G. Whether the presence of this protein within the mitochondrial matrix may interfere with normal mitochondrial function  is unclear.A recent study 2+ accumulation, catalysed by MCU, is revealed here to be essential for the second phase of glucose-induced ATP synthesis by glucose. What may be the consequences for electrical activity and insulin secretion? Increases in ATP are believed to be involved in both “KATP-dependent” and “KATP-independent” regulation of exocytosis by glucose 2+ accumulation in the regulation of plasma membrane electrical activity  action on secretory granule movement or fusion, perhaps powered by ATP increases Mitochondrial Caactivity  suggesti2+ transport into mitochondria .Female CD1 mice were sacrificed by cervical dislocation as approved by the United Kingdom Home Office (HO) Animal Scientific Procedures Act, 1986 and designated as “Schedule 1” procedure. Animals were maintained under HO Licence PPL 70/7349 (Holder Dr I Leclerc), which received local ethical committee approval, and all participants received approved local training at Imperial College. Pancreatic islets were isolated by collagenase digestion +-palmitate and 17 mM glucose for 72 h. Palmitate was prepared as a 150 mM stock in ethanol; the working solution also contained 0.67% fatty-acid free BSA (Sigma). Control medium contained, respectively, 0.67% FFA-free BSA and 0.17% ethanol.Chronic glucolipotoxicity was modelled by culturing the cells in medium containing 0.5 mM Na6 infectious units/ml. Cells infected with the GFP+ control particles (sc-108084) at the same titre displayed a multiplicity of infection of two, 36 hours after infection. Particles delivering non-target shRNA (sc-108080) were used as a negative control.MCU was silenced in primary β-cells by 24h incubation with shRNA-bearing lentiviral particles , at 1×10EcoRI, then extension using T4 DNA-polymerase and finally by restriction with HindIII to liberate the insert. The HindIII/blunt insert was cloned into pShuttleCMV previously digested with EcoRV and HindIII.cDNA encoding Perceval c oxidase (COX) while the targeting efficiency was improved by using two tandem repeats of the addressing sequence cDNA encoding 2mt8-ratiometric pericam (2mt8RP) was kindly provided by Prof Tullio Pozzan (University of Padua). “Mt8” refers to the first 36 amino acids of subunit VIII of human cytochrome Ppia), and FFA treatment effect as fold change with 95% confidence intervals was calculated using the ΔΔCT method on 7500 Software .RNA was purified from islet samples using Trizol. RNA was quantified by Nanodrop spectrophotometer then reverse transcribed using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems). mRNA abundance was quantified by qPCR using Sybr Green PCR Master Mix (Applied Biosystems) on a 7500 Fast Real-time PCR machine. Expression of each gene was normalised to cyclophilin A  respectively. 2mt8RP, Fura-Red and Indo-1 were used at single excitation and emission wavelengths. Either dye was dissolved in DMSO (4mM) containing 4% F127-Pluronic. Cells were loaded with Fura-Red by incubation with 4μM of the dye in the extracellular solution for 30 min. Imaging experiments were performed on an Olympus IX-71 microscope with UPlanFL N ×40 magnification objective. For acquisition, an F-View-II camera and MT-20 excitation system equipped with a Hg/Xe arc lamp were used, under control of CelÎR software (Olympus). Excitation/emission wavelengths were (nm): 410/535 (2mt8RP), 490/630 (Fura-Red), 490/535 . Images were acquired at a frequency of 0.2 Hz with typical excitation times of 10 ms. The acquisition of the fluorescence and electrophysiological data was synchronized using TTL pulses. Imaging data was background-subtracted, analysed and presented as F/F0  and F0/F . Whole cells were selected as regions of interest (ROI) to minimize the effect of the cell drift. For cell clusters, only the patched cell was included in the ROI. Every [Ca2+] recording was subjected to the dynamic range control by applying, at the end of the trace, solutions containing 10 μM ionomycin: “Ca2+-free” (0.5 mM EGTA), “Ca2+-max” (5 mM Ca2+). For the [ATP/ADP]cyt recordings the dynamic range was controlled by high glucose (maximum after >30 min of exposure) and 2µM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone .Simultaneous imaging of  and [ATP/ADP]cyt, in response to a glucose step from 3 to 17 mM. To monitor the input resistance, the protocol included 10-ms injections of repolarising 10-pA current applied every 20s. The parameters of the current injections were chosen to minimise their effect on the glucose-induced electrical activity. To control Vm and impose electrical stimulations, the mode was periodically switched to voltage-clamp m was held at the value of −70 mV, with 0.5 Hz +5/−10 mV pulses to monitor the KATP conductance . The pipette tip was dipped into pipette solution, and then back-filled with the same solution containing 0.17 mg/ml amphotericin B. Series resistance and cell capacitance were compensated automatically by the acquisition software. Recordings, triggered by the TTL pulse, were started in current-clamp mode, and the depolarization of the plasma membrane was monitored simultaneously with cyt increase and the decrease in Gm were closely associated in time. Gm was calculated from Im traces Click here for additional data file.


Due to their self-catalytic properties, small RNAs with bulge bases are hypothesized to be primordial molecules which could form elementary translation systems. Using molecular dynamics simulations, we study the binding propensity of small RNAs by calculating the free energy barrier corresponding to the looped out conformations of bulge bases, which presumably act as the binding sites for ligands in these small RNAs. We find that base flipping kinetics can proceed at atmospheric pressure but with a very small propensity. Furthermore, the free energy barrier associated with base flipping depends on the stacking with neighboring bases. Next, we studied the base flipping kinetics with pressure. We find that the free energy associated with base looping out increases monotonically as the pressure is increased. Furthermore, we calculate the mean first-passage time of conformational looping out of the bulge base using the diffusion of reaction coordinate associated with the base flipping on the underlying free energy surface. We find that the mean first-passage time associated with bulge looping out increases slowly upon increasing pressures  RNA molecules are very diverse both structurally and functionally RNA is hypothesized as a primordial molecule due to its ability to form efficient catalysts and its similarity to DNA, where it can act as a molecular information machinery −1 as compared to the uracil bulge The active site in the small tRNA-like molecules, that is believed to bind to aa-AMP, consists of AG/AG bulge in the stem Related is the question of the effect of RNA hydration on RNA kinetics. For binding to proceed, the bulge base has to flip out of the local helical plane, and so has to overcome both the bending rigidity and solvation energy barrier to solvate in water. The solvation of different substances in water is a widely studied problem 5′GGGGAGG3′-5′CCCCCC3′, and (ii) with a single adenosine bulge in sequence 5′CCCCACC3′-5′GGGGGG3′, and (iii) an AA-bulge in 5′GGGGAGG3′-5′CCACCCC3′. Next,we study the effect of pressure on the bulge base looping out for sequences (i) and (ii) with a single A-bulge. The organization of the paper is as follows. In section “In this paper, we first compare the propensity of bulge base looping out of a double strand RNA (dsRNA) for three different sequences: (i) with a single adenosine bulge in sequence 2 (5′-GGGGAGG-3′/5′-CCCCCC-3′) (2 (5′GGGGAGG3′-5′CCCCACC3′) embedded between (GC)2 (The energy minimized starting structures of three different RNAs (i) with a single A-bulge embedded between (GC)CCCC-3′) , (ii) wiC-3′) 2  were creAfter the position restrained simulations, unrestrained Molecular Dynamics (MD) was carried out at four different values of the pressures −1.deg−2 were distributed uniformly along the reaction coordinate To quantify the relative propensity of binding of RNA at different pressures, we chose the dihedral angle ′N1) see  as the r−1, suggesting that although −1) is sufficient enough for the bulge to get displaced of the free energy minimum configuration. Due to a large free energy barrier (−1) a complete looped out conformation of single A-bulge is less favorable and hence consistent with experimental fact that RNA with single A-bulge does not show appreciable aminoacylation (unpublished work). In −1). Moreover, the secondary minimum as seen in the the case of single A-bulge  is absent. Hence, the rate of base flipping would be enhanced for AA-bulge compared to single A-bulge base in structure I.In ucture I , we find−1, suggesting that the propensity of single A-bulge base looping out from the local helical plane would decrease upon increasing pressure and so the binding propensity of incoming ligands.We next studied the effect of pressure on the kinetics of bulge bases in structure I and structure II. −1. Comparing In order to calculate the effective rate n by see  where  aAs we have seen in the sections above, the base flipping kinetics changes as the pressure is increased – namely, the free energy barrier for the bulge base to flip out increases with pressure. Moreover, the transient barrier which presumably is due to the base triplet formation of the bulge base with the neighboring bases disappears at pressures We first looked at whether the observed change in the pressure dependence of the kinetics is a result of ordering of water around RNA. To this effect, we calculated the OOO-bond angle To quantify the ordering/disordering of water molecules around Since, we only consider a thin hydration shell, the expression for ensemble average Since we did not see any sudden change in the ordering of hydration shell around RNA that might lead to the base flipping barrier observed at  to  see . Moreove2 in 5′GGGGAGG3′-5′CCCCCC3′ (structure I), and embedded between (CG)2 in 5′CCCCACC3′-5′GGGGGG3′ (structure II), and AA-bulge embedded between (GC)2 in 5′GGGGAGG3′-5′CCACCCC3′ (structure III). Specifically, we calculate the free energy barrier associated with the base looping out from the local helical plane of the RNAs. We find that a single A-bulge base embedded between (GC)2 has a much larger free energy barrier of complete looping out compared to both AA-bulge embedded between (GC)2, and A-bulge embedded between (CG)2. Among the three structures studied here, the free energy of looping out of A-bulge is minimum for A-bulge base embedded between (CG)2. Our results indicate the importance of stacking interactions with neighboring bases in determining the rate of base flipping. It further suggests that certain structural features of the bulge bases may facilitate the binding of small molecules to small RNAs. Usually, an A-bulge base with weaker neighbor stacking will have faster rate of flipping as compared to AA-bulge with the same neighbor stacking. We have studied only a few simple cases here. A complete underlying physical picture could only come from looking at various structural motifs and identifying the structural features that may increase or decrease the binding propensity of small RNAs.In summary, in this paper we have investigated the effect of neighbor stacking and pressure on the kinetics of an Adenosine bulge base embedded between (GC)Motivated by the fact that near the thermal vents the pressure is very high, we next studied the effect of pressure on the kinetics of A-bulge base for two configurations. We find that, upon increasing the pressure, the propensity or likelihood of base flipping decreases. At pressure The effect of hydration, namely the increased hydration level of the bulge bases led to slowing down of the bulge flipping kinetics. It implies that the structural changes in water and the RNA hydration at high pressures may be relevant to self-catalytic properties of small RNAs. Indeed, water exhibits many anomalous behavior


DMPK gene which induce the mis-splicing to fetal/neonatal isoforms of many transcripts, including those involved in cellular Ca2+ homeostasis. Here we monitored the splicing of three genes encoding for Ca2+ transporters and channels  during maturation of primary DM1 muscle cells in parallel with the functionality of the Excitation-Contraction (EC) coupling machinery. At 15 days of differentiation, fetal isoforms of SERCA1 and CACN1S mRNA were significantly higher in DM1 myotubes compared to controls. Parallel functional studies showed that the cytosolic Ca2+ response to depolarization in DM1 myotubes did not increase during the progression of differentiation, in contrast to control myotubes. While we observed no differences in the size of intracellular Ca2+ stores, DM1 myotubes showed significantly reduced RyR1 protein levels, uncoupling between the segregated ER/SR Ca2+ store and the voltage-induced Ca2+ release machinery, parallel with induction of endoplasmic reticulum (ER) stress markers. In conclusion, our data suggest that perturbed Ca2+ homeostasis, via activation of ER stress, contributes to muscle degeneration in DM1 muscle cells likely representing a premature senescence phenotype.The pathogenesis of Myotonic Dystrophy type 1 (DM1) is linked to unstable CTG repeats in the  Myotonic dystrophy  is the most prevalent form of adult muscular dystrophy with a frequency of 1 in 8,000 individuals worldwide , has a sDifferent histopathological changes occur in the DM1 muscle, including predominance and atrophy of type I fibers, augmented incidence of ring fibers and variability in fiber size, increased number of central nuclei and sarcoplasmic masses . Other sin situ hybridization (RNA-FISH), sequester two splicing regulators implied in the development of the great majority of DM1 symptoms. The misregulated alternative splicing, or spliceopathy, is linked both to increased levels of CUG binding protein 1 (CUG-BP1)  is maintained three to four orders of magnitude lower than the intra-SR/ER [Ca2+] [In skeletal muscle cells excitation-contraction coupling (ECC) and regulation of intracellular CaR [Ca2+] .n expansion on the splicing ; SERCA1 ; CACNAIS gene  were performed as previously described  and GRP78 , compared to the expression of β-actin , were quantified by Sybergreen RT-PCR using the ABI PRISM7000 sequence detection system.escribed . Total P2+ measurementsCa. T10-15 myotubes were seeded on glass coverslips, and loaded with the fluorescent Ca2+ sensitive probe Fura2-AM . [Ca2+] was assessed by calculating the ratio of fluorescent intensities measured at 340 nm and 380 nm excitation and 520 nm emission on Olympus IX81 inverted fluorescence microscope as previously described  fetal variant was found only in the adult DM1 muscle samples, but not in the pathological DM1 myotubes. This difference could be due to the absence of innervation in our aneural muscle culture model, which otherwise leads to the consequent physiological adaptation of mature muscle fibers, together with RNA instability and the presence of other RyR1 splicing variants, such as RyR1 ASI(+), in adult muscle.The splicing pattern of the SERCA1, RyR1 and Cap < 0.01) of relative controls. Importantly, we found no changes in the expression or subcellular distribution of SERCA2 and Cav1.1  and CHOP  as compared to controls. Western blot analysis showed that CHOP levels were increased about twofold in DM1 myotubes as compared to controls at both T10 and T15  and C/EBP homologous protein (CHOP) by quantitative RT-PCR. As shown in  and T15 B,C, whil and T15 B,C. In onchanged A,C, we fnchanged A,B.These results confirmed that ER stress is already induced in early phases of differentiation in DM1 myotubes, and that the marked induction of the proapoptotic CHOP might be responsible for triggering cell death at T15 of differentiation, associated with the general derangement of ER/SR structure. 2+ homeostasis and ER stress during maturation of DM1 muscle cells. We have chosen time points of 10 and 15 days of differentiation for the analysis, corresponding to the commencement of the previously observed apoptotic features (T10), and when the majority of myotubes displays features of full blown apoptosis (T15) ..2+ releain vitro muscle maturation. In the same cellular model (T10 and T15 DM1 myotubes) we previously described a splicing unbalance of MBNL1 and Insulin Receptor and nuclear accumulation of CUG-containing DMPK mutant transcripts (foci) [DMPK gene plays a role in determining the splicing unbalance of all analyzed genes.We found that both fetal SERCA1b Δex22 and fetal Cav1.1 Δex29 were significantly increased in T15 DM1 myotubes as compared to controls, where the proportion of adult isoforms of SERCA (ex22) and Cav1.1 (ex29) increased with the progression of the s (foci) , stronglin vitro muscle maturation were missing. The splicing pattern of SERCA1-ex22 and Cav1.1-ex29 has a similar developmental window [C-terminal region [2+ transient in adult muscle [2+ transients, that may be further amplified by local Ca2+-dependent activation of RyR1 within the triad junction. Increased ex29 skipping and enhanced Cav1.1 gating may explain the augmented nifedipine-sensitive Ca2+ influx in DM1 myotubes [2+ homeostasis (RyR1 and SERCA1 mis-splicing) may lead to a chronic Ca2+ overload already observed in DM1 myotubes [Aberrant splicing of RyR1, SERCA1 and Cav1.1, has been well documented in adult DM1 muscle ,19,29, bl window ,19. Recel window . No funcl region  in COS-1myotubes . The commyotubes ,26, and myotubes .2+ from the ER/SR store and could be the consequence of the accumulation of excess Ca2+ in the microdomain and consequent activation of Ca2+-dependent proteases [2+ related proteins were inconclusive, suggesting, from one side, a decrease in the total amount of SERCA protein in cultured DM1 myotubes [et al. reported a trend towards increased protein levels of SERCA1 and 2 in the ER fraction of DM1 mouse, but not of the RyR1 [vs. Ca2+-dependent phosphorylation and ELISA [2+ homeostasis in myotonic dystrophy could be determined by the failure to complete the developmental switch to a specific set of alternatively spliced genes, resulting in insufficient Ca2+ release from the ER/SR due to the reduction of functional RyR1 protein levels. It has been previously reported that increased ROS levels can trigger increased opening probability of the RyR [2+ signals . Taken  the RyR , which mnals See .2+ transfer to the mitochondria [2+ binding proteins calsequestrin and calnexin remained unchanged in DM1 myotubes. Moreover, the overall structure of the ER/SR has not changed drastically as judged by the distribution of SERCA on the immunofluorescence images. Thus, the missing accumulation of grp78 protein is most likely the result of increased turnover of the protein, probably mediated by the ER-associated degradation system [The long-term consequence of deregulated protein synthesis can be linked to apoptotic events through the observed ER stress response. The kinetics of cellular responses to ER stress can typically be divided in two phases. First, adaptation  aims to re-establish normal protein folding in the ER by up-regulating key chaperones such as grp78, and to provide the energetic basis for cell survival by up-regulating mitochondrial function . In the chondria , or by ichondria . Here, wn system . Our finn system  in relatn system . Most imn system . Data ren system  and BIN1n system .2+ handling, coupled to deregulated Ca2+ homeostasis and excitation-contraction coupling, leads to muscle weakness by ER-stress and consequent apoptosis.In summary, our data suggest a general pathologic mechanism in DM1 muscle, in which mis-splicing of pre-mRNAs of genes involved in muscle Ca