Complementary developments in microscopy and mathematical modeling have been critical to our understanding of cardiac excitation–contraction coupling. Historically, limitations imposed by the spatial or temporal resolution of imaging methods have been addressed through careful mathematical interrogation. Similarly, limitations imposed by computational power have been addressed by imaging macroscopic function in large subcellular domains or in whole myocytes. As both imaging resolution and computational tractability have improved, the two approaches have nearly merged in terms of the scales that they can each be used to interrogate. With this review we will provide an overview of these advances and their contribution to understanding ventricular myocyte function, including exciting developments over the last decade. We specifically focus on experimental methods that have pushed back limits of either spatial or temporal resolution of nanoscale imaging , or have permitted high resolution imaging on large cellular volumes . We also review the progression of computational approaches used to integrate and interrogate these new experimental data sources, and comment on near-term advances that may unify understanding of the underlying biology. Finally, we comment on several outstanding questions in cardiac physiology that stand to benefit from a concerted and complementary application of these new experimental and computational methods. However, it is only rarely that studies of both types have been designed in a coordinated manner, with one providing essential information to the other. Experimental approaches have always provided the foundation of the field, but as our understanding has developed it has repeatedly required investigation of structures and function that were unobservable via available methods. At those points, mathematical approaches have often been applied to crystalize the prevailing arguments and assumptions, and to provide clear, quantitative, and actionable postulates that have sometimes reoriented the field. These contributions of the subdisciplines are depicted in As in many fields of physiology, experimental and 2+ release deep in the interior of the cell, thus verifying A F Huxley’s remarkable local stimulation experiments in skeletal muscle, which had pointed to their existence nearly a decade earlier , most aspects of the ECC mechanisms in cardiac and skeletal muscle remained to be established, but the fundamental membrane structures were more-or-less clear.Transmission electron microscopy (TEM) provided the earliest foundation for our understanding of nanoscale ECC in both cardiac ventricle and skeletal muscle. These studies were among the first applications of electron microscopy in biology and part of extensive structural characterizations performed by  earlier . Oxalateg tissue . Winegra2+ via fluorescent dyes (2+-induced Ca2+ release (CICR). Unless otherwise noted, we focus on the nanoscale structure-function relationships in ventricular tissue throughout this manuscript.Our basic concept of how these nanoscopic structures participated in cardiac ECC was introduced in the 1970s , well beent dyes . Still, 2+ that drives cardiac contraction is released from the cardiac SR at the terminal cisternae . In intact myocytes, cardiac ECC clearly required Ca2+ influx during the action potential (2+] around the SR in skinned (sarcolemma removed) preparations  was used to great effect for isolating, and reconstituting Ca2+ release function  the important sensitivities of macroscopic CICR to cytosolic Ca2+, ATP, and Mg2+. Prior EM observations  of these gigantic channels at the cytosolic face of the terminal cisternae suggested that they clustered together in an ensemble  during the action potential drives Ca2+ release from RyRs in the junctional SR. This conceptual model differed from skeletal muscle ECC, which occurred in very similar structures but was far less dependent on Ca2+ influx  clear observations that SR Ca2+ release was a graded phenomenon that depended on both the magnitude and velocity of triggering Ca2+ influx  the Ca2+ released by an RyR into its own local environment must diffuse away from the channel with approximately the same (or faster) kinetics than those with which the RyR channels close—a reasonable assertion based on available evidence, and one that has held the test of time; (2) when the number of RyRs at a release site is more than one (best estimates at the time suggested an average of ∼10), there must be a separate mechanism  to terminate the Ca2+ release. If such a mechanism does not exist, the local [Ca2+] that drives channel opening remains high and the various RyRs “trade off” in their opening over time, resulting in “latched” release. In part because of these clear postulates, one major direction that both experimental and computational efforts have pursued in the decades since has been to more accurately quantify the number of RyRs that participate at a release site. As Stern demonstrated, the interaction between this number and the local SR Ca2+ dynamics critically determines several micro- and macroscopic properties of cardiac ECC, notably including the mechanism of local Ca2+ release termination. With these fundamental functional characteristics becoming more clear, the terminology used to describe the structures involved in CICR also became more precise. The collection of RyRs at a single jSR terminal , together with that local jSR volume and its releasable Ca2+, became known as a Ca2+ release unit (CRU). Stern himself coined the term “couplon” to describe the CRU together with the local LTCCs that control Ca2+ release. Below we describe how super-resolution imaging has since added some subtlety to these definitions by identifying geometric subsets of RyRs within the CRU that likely behave semi-autonomously depending on conditions . Such subsets are often termed “clusters.”In essence, local control quantitatively formalizes the concept that individual Ca2+. However, it was clear that if local control was indeed the core mechanism of CICR, there were broad implications for how these nanoscopic local events were integrated over the whole cell, and how they both depended on and determined the macroscopic properties of cardiac ECC. With this seed, and parallel developments in both computing and imaging power, a second major direction for ensuing work has been to develop an integrated understanding of the interdependencies between locally controlled CICR and macroscopic cardiac ECC.In 1992, the simulations that 2+ release events (resulting from probabilistic opening of RyRs) should be observable in cardiac muscle. Interestingly, it was also at this time that the technology required to detect these elementary events had begun to become widely available. Laser scanning confocal microscopy (LSCM) critically enabled separation of in-focus and out-of-focus fluorescence, and built upon the basic concept for confocal optical sectioning developed and patented by Marvin Minsky in the late 1950s. At the time of Minsky’s patent, critical complementary technologies involving light collection, data processing and storage, and visualization were not yet available. Thus, it wasn’t until the late 1980s that commercial confocal microscopes began making their mark on biology, and could be applied to discerning these small localized Ca2+ release events. Only one year after Stern’s paper, Heping Cheng working with Jon Lederer and Mark Cannell, published the first measurements of these spontaneous Ca2+ release events that many had envisaged .By the early 1990s it was presumed that, given suitable optical conditions, local spontaneous Canvisaged . In that2+ fluorophores in conventional LSCM imaging volumes are typically not high. As a result, discernible sparks are often only those events that involve a relatively large regenerative Ca2+ release centered within the imaging volume. Many smaller or out-of-focus events simply contribute to the background fluorescence and noise. Recognizing this, experimentalists have since sought to observe and characterize these smaller events, and modelers have focused on understanding their contribution to triggered CICR and importance to macroscopic ECC. Ca2+ signals resulting from the opening of single RyRs were first suggested by Peter Lipp and Ernst Niggli, and named quarks for their presumed quantal underpinnings  myocytes, i.e., the quantitative relationship between LTCC-mediated sarcolemmal Ca2+ influx and RyR-mediated SR Ca2+ release, at the whole-cell level , and important for determining the threshold for spark termination and inter-CRU CICR (Ca2+ waves) at high SR Ca2+ load (2+ dynamics somewhat more measurable (2+ pool (jSR Ca2+ content) for determining spark termination. These characteristics appear to be the most fundamental for enabling reproducible CICR resulting from LTCC or spontaneous RyR opening. However, a range of dynamic factors also exert important physiological modulation of CICR. For example, voltage-dependent enhancement of unitary LTCC current potentiates CICR  . However, a range of evidence had also suggested that luminal regulation is a feature of RyR function, and may be crucial for both normal and pathologic RyR behavior (2+ threshold), even when the SR is not depleted  . As a remodelers . For thabehavior . Most redepleted . This ha2+ release. The Na+-Ca2+ exchanger (NCX), which predominantly functions in “forward” mode to extrude Ca2+ from the cell, reverses during the early repolarization of the action potential, due to the brief rise in cytosolic Na+ concentration and membrane depolarization. Various groups  from the RyR ensemble, and that this in turn destabilized the RyR closed conformation causing more frequent unitary RyR openings  most of the assertions in the original work have been subjected to intense scrutiny and counterarguments—see Eschenhagen’s excellent commentary for a comprehensive review  phosphorylation of the nearby S2814 site. It is now broadly accepted that CaMKII phosphorylation at S2814 is capable of conferring the majority of outcomes originally claimed by Marks for protein kinase A-mediated phosphorylation of S2808 . While the effects of regulation on other aspects of single RyR function need to be more comprehensively studied, this form of modulation has been a prominent finding in studies to date, and can be readily incorporated into modeling frameworks.In sum, the strong debates and vigorous effort that has ensued from Marks original studies has added a great deal to our understanding of regulatory influences in cardiac CICR. For the purposes of discussing how these regulatory effects impact structure-function relationships in CICR, it is true that the mechanisms generally converge upon either enhanced or reduced RyR Ca2+ release occurs at discrete sites , where cooperating RyRs generate Ca2+ sparks. Early estimates of CRU dimensions stemmed largely from 2D EM studies, using counts of RyR “feet” within the dyadic cleft  to determine local RyR interaction and release dynamics. The quantitative hierarchy of importance of structural versus RyR regulatory subtleties remains to be established, but it is clear that both are capable of markedly altering local control of CICR.The novel, imaging-driven insights into CRU morphology described above have inspired new directions in mathematical modeling over the past decade. Based on the realization that RyR clusters are of irregular shapes, dealized  and supedealized . These a2+ waves . These s2+ waves . Of cour2+ spark fidelity, and 2+ release is of reduced magnitude and slowed kinetics. Thus, by 2015, and thanks to the application of SMLM and structurally-defined modeling approaches, it was clear that CRU function (and even CRU definition) is critically dependent on the local arrangement of individual RyRs.The dSTORM studies highlighted above estimated a maximum occupancy of each imaged RyR cluster based on a crystalline, lattice arrangement of the RyR channels reported in earlier work . However2+ pool to determine the dynamic regime of Ca2+ release. That is, the relationship between RyR cluster size and the local jSR size, and thus Ca2+ content, is critically important. When SMLM-defined RyR locations are used to build computational structures, one must make important assumptions about these relationships  , but in ed by 2+ . Their w2+ diffusion and jSR Ca2+ refilling, which at this time remain poorly understood. This is noteworthy because these properties are central to aspects of macroscopic frequency dependence in cardiac ECC, and for the pathologic dynamics underlying Ca2+ waves, and electrical and mechanical alternans , is sufficient only for defining an area occupied by the channel. For insight into intra-cluster, real-time RyR location, the increased resolution of a technique like DNA-PAINT would be needed , but wit2+]), the frame time for recording Ca2+ release events is limited to approximately 2 ms with even the latest cMOS cameras. Thus, recorded Ca2+ signals are near the limit of Nyquist discrimination for single channel openings, and generally represent a complex integration of releases from multiple RyRs. One approach to addressing this issue was developed by the Lipp group (2+ released in previous frames (and its expected diffusion) to reveal only the newly released Ca2+ signal. It is hoped that this type of approach, together with the further development of camera technology , will shed further light into the true nature of RyR functional dynamics.Although much of this review has focused on developments in improving the spatial scale of imaging, it should be noted that the temporal scale of measurements is also a critical experimental limitation. While events such as RyR openings are quite brief . We thus expect that including the regulation of individual channels in mathematical models will be essential for fully describing CRU channel behavior, even as imaging techniques continue to advance. Perhaps more importantly, the manner in which RyR localization and regulation contribute to whole-cell function will likely require an entirely different level of integration across the spatial scales of cardiac ECC—from CRU to cell. In the following section we deal specifically with the challenges to developing modeling approaches that may be capable of that level of integration.The above discussion has indicated that there remain substantial limits in attaining adequate spatial and temporal resolution to discern CRU structure and function based on experiments alone. Surely, this is good news for mathematical modelers, but will modelers be side-lined when technology eventually enables imaging of individual dyadic proteins and their Caclusters , suggest2+ release events (sparks and spikes) exhibit a range of dynamic regimes, such as latched release and stochastic attrition, which depend critically on the number and proximity of RyR channels in a local CRU domain. These shifts in dynamics represent the direct influence of stochastic properties of individual channels on the local ensemble dynamics. Furthermore, as mentioned, modern imaging has made it abundantly clear that the number of RyRs present in many CRUs  spans a range that crosses these transitions in ensemble dynamics. Finally, spatial gradients in [Ca2+] resulting from continuum diffusion in both the cytosol and SR also clearly contribute to determining those dynamic transitions. In combination, these characteristics enforce several basic requirements for accurately modeling cardiac CICR. Specifically, the scales covered by the model should allow for the stochastic operation of single RyRs to interact with realistic variability in the size and spatial organization of RyR clusters, as well as diffusive transport of Ca2+ in the cytosol and SR. As we discuss below, these requirements impose a substantial computational burden when simulating CICR among multiple CRUs up to subcellular scales. Thus, while the local studies of As described above, the characteristics of unitary SR Ca3) region of an average cardiac myocyte (via SMLM). In principle, these datasets should be collected in similar cells. That is, for example, from the same species, same cardiac chamber, or otherwise similar in terms of any variable known to impart systematic differences in membrane ultrastructure or RyR localization. From these datasets it will be possible to construct representative distributions of the quantities (1–3), as well as many others, e.g., the volumetric density of jSR terminals that are not coupled to t-tubules. Of course, the characteristics of the membrane and RyR cluster geometries will not be spatially correlated because they will be collected in different cells. However, given sufficient data, some form of registration between the distributions should allow for assigning reasonable cluster sizes to individual jSR geometries. This approximation could be made considerably more precise with correlative SMLM-EM datasets. Still, to continue with our thought experiment; presuming these essential data (1–3 above) are available, it is in principle possible to construct a model with full geometric detail of all jSR terminals incorporating realistic RyR cluster sizes and inter-cluster distances for an intact myocyte. It is another matter entirely to determine if such a model could be simulated with fully stochastic RyR dynamics and local diffusive coupling.To discuss these challenges in concrete terms it first helps to construct the hypothetical “ideal” model. Given the recent advances in serial sectioning EM and SMLM described above, it seems plausible to expect that existing or near-term datasets could yield: (1) the spatial distributions and geometric sizes of all jSR terminals in an average cardiac myocyte [via serial sectioning scanning electron microscopy (SS-SEM)], (2) the total volume and regional volume densities of the network SR in an average cardiac myocyte (via SS-SEM), and (3) the variation in RyR cluster sizes and their spatial distribution in a representative  of 12–15 nm is typically required , which generally requires considerably finer temporal resolution. Of course, the scale of the problem can be reduced by employing finite element approaches involving larger mesh blocks  in regions where the transporter gating dynamics are less stiff and gradients are less severe. Still, this problem represents a substantial computational effort. To date, very large cardiac simulations involving both classical and more recent electrodiffusion models  that dictated the cleft [Ca2+] at any point in time. Thus, he was able to reduce the dyadic dynamics to a set of equations, the size of which was determined by the number of channels (both LTCCs and RyRs) assumed to be in each dyad. This in turn allowed grouping of the rate constants that applied at similar timescales to each channel species and permitted a 9-state (and even 4-state) model of each dyad. This strategy recapitulated the essential properties of local control, while markedly reducing computational cost. Unfortunately, this approach still assumes a single spatially homogeneous dyadic [Ca2+], which from the more recent super resolution studies described above seems unlikely to reflect the nanoscale reality.The computational challenges imposed by simulating detailed stochastic dynamics across the comparatively large volume of a myocyte have been appreciated for some time. Some of the earliest efforts to perform this type of multiscale integration were begun by Ray Winslow’s group at Hopkins, and Robert Hinch at Oxford. Initially, 2+ leak (which represents sub-spark release events such as quarks) in a whole cell model involving distributed dyads. These clever studies diffusively coupled their 20,000 jSR terminals to a common network SR and employed thorough mass conservation and reuptake-release balance. Their dyadic model utilizes a modified version of the well-known “sticky-cluster” model developed by Sobie  have used a variety of simplifications to maintain tractability. Notably, the proof-of-principle work from by Sobie . This fo2+ sensitivity from changes in RyR cluster morphology or other disease-associated structural changes can only be described as phenomenological. To fully simulate the interaction of these changes, which are generally observed simultaneously in disease, will provide a key foundation for understanding whether individual changes in dyadic structure and single channel function contribute to the pathology, or if either is in fact compensatory in nature. As mentioned above, the ability to integrate these effects over an entire cell will allow us to begin these interrogations. This can be considered one of the major goals of the new modeling frameworks that can now be developed based on state-of-the art SSEM and SMLM imaging.The above approximations are important first steps toward simulating whole-myocyte CICR while retaining the critical stochastic characteristics of RyR gating. However, their ability to simultaneously discriminate regulatory effects  that impact RyR CaIn 1960, Over the history of cardiac ECC there have been many instances in which technological or methodological advances have permitted step-changes in the progression of knowledge and shifts in the working paradigm. Rarely, however, has there been a period in which a range of major and complementary technical developments have occurred in such quick succession, as has been the case over the past decade. Like many others, we are eager to see what these developments yield for the field. We particularly expect data sources that are now becoming available to provide a foundation for reconciling multiple levels of investigation and postulates from past decades. We hope this, in turn, will provide new tools and approaches for understanding the relationship between dynamic nanoscale changes and whole-cell function, and new insight to mechanisms of disease that span these scales. Perhaps more than any other aspect, we expect this period to require integrated design of experiments and simulations to fully leverage the power of these new technologies and methods.WL and AE co-designed, wrote, edited, and developed figures for manuscript. HP-D wrote, edited, and developed figures for manuscript. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


OM (1 µM) slowed the kinetics of contractions and relaxations and evoked an oscillation between normal and reduced intracellular Ca2+ transients, action potential lengths and contractions in isolated canine cardiomyocytes. Echocardiographic studies and left ventricular pressure–volume analyses demonstrated concentration-dependent improvements in cardiac systolic function at OM concentrations of 600–1200 µg/kg in rats. Administration of OM at a concentration of 1200 µg/kg was associated with hypotension, while doses of 600–1200 µg/kg were associated with the following aspects of diastolic dysfunction: decreases in E/A ratio and the maximal rate of diastolic pressure decrement (dP/dtmin) and increases in isovolumic relaxation time, left atrial diameter, the isovolumic relaxation constant Tau, left ventricular end-diastolic pressure and the slope of the end-diastolic pressure–volume relationship. Moreover, OM 1200 µg/kg frequently evoked transient electromechanical alternans in the rat in vivo in which normal systoles were followed by smaller contractions (and T-wave amplitudes) without major differences on the QRS complexes. Besides improving systolic function, OM evoked diastolic dysfunction and pulsus alternans. The narrow therapeutic window for OM may necessitate the monitoring of additional clinical safety parameters in clinical application.Omecamtiv mecarbil (OM) is a promising novel drug for improving cardiac contractility. We tested the therapeutic range of OM and identified previously unrecognized side effects. The CaThe online version contains supplementary material available at 10.1007/s00395-021-00866-8. Stock solutions with concentrations of 1 and 10 mM were prepared in dimethyl sulfoxide (DMSO) and stored at 4 °C.A detailed description of the statistical methods used can be found in the supplementary material.2+ levels in the presence of 1 µM OM. For example, the force value in relaxing solution (pCa 9) was 1.1 ± 0.2 kN/m2, compared with 0 kN/m2 in the absence of OM  increased from 5.86 ± 0.02 to 6.42 ± 0.06  severalfold at low Ca2+ concentrations  at an OM concentration of 1 µM decreased irrespective of Ca2+ concentration  increased from 5.06 ± 047 to 10.40 ± 1.11 s and from 2.80 ± 0.21 to 8.75 ± 1.14 s at an OM concentration of 1 µM at low and submaximal Ca2+ levels, respectively  slowed down from 2.67 ± 0.38 to 18.51 ± 1.72 s and from 2.74 ± 0.34 to 12.00 ± 1.44 s at an OM concentration of 1 µM at submaximal and high Ca2+ levels, respectively  stiffness (Fpassive) increased from 0.88 ± 0.10 to 3.88 ± 0.44 kN/m2 and from 1.03 ± 0.12 to 3.25 ± 0.41 kN/m2 at submaximal and high Ca2+ levels, respectively, in response to OM 1 µM  OM (yielding a maximum dose of 1200 µg/kg BW) improved cardiac systolic function as revealed by echocardiography  increased from 9.2 ± 0.4 to 15.2 ± 0.7 ms and the maximal rate of diastolic pressure decrement (dP/dtmin) decreased from − 11,642 ± 603 to − 8096 ± 614 mmHg/s, indicating impairment of active relaxation. The LV end-diastolic pressure (LVEDP) increased from 8.6 ± 0.8 to 26.4 ± 1.2 mmHg and the end-diastolic P–V relationship (EDPVR) increased from 0.040 ± 0.001 to 0.098 ± 0.009 mmHg/µL, suggesting stiffening of the left ventricle.OM treatment was also associated with signs of diastolic dysfunction on echocardiography Fig. a: peak mmed Fig. b. The isP < 0.05) and diastolic  blood pressure at a cumulative dose of 1200 µg/kg BW. There was no effect on heart rate, irrespective of the development of hypotension   shed light on the initiation of these transient periods. They started gradually, either in parallel with an increase in heart rate Fig. b or elseans Fig. d. This pEchocardiography Fig. a–d and dP < 0.05) without an alteration in R-to-R duration: the value of the R–R distance was 137 ± 2 ms after the normal beats and 137 ± 1 ms after the consecutive altered beat at an OM dose of 1200 µg/kg BW  in parallel  decreased at the diminished contractions from 0.69 ± 0.04 to 0.41 ± 0.08  in which normal beats alternate with diminished cardiac contractions on a beat-to-beat basis, a feature similar to In recent clinical trials, OM was tested by pharmacokinetic titrations, resulting in a plasma concentration of 318 ± 129 ng/mL (representing 0.79 µM) . In the 2+ sensitization when OM was administered at the therapeutic concentration. This effect was paralleled by slower kinetics for both contraction and relaxation, and by elevated passive stiffness.In our in vitro experiments, cellular effects were studied at an OM concentration of 401 ng/mL (1 µM), which overlaps with the reported effective clinical serum concentrations of the drug , 25–28 Ww) were affected by the lowest dose of OM (200 µg/kg). It is important to note that improvement in EF (one of the major systolic parameters considered in human heart failure) was observed without a marked increase in stroke volume. This raises the question of whether improvement in EF alone should be considered as a favorable outcome in heart failure studies with OM. Here, we found that the major contributor to improved EF was the reduced diastolic volume of the LV chamber, which did not result in a marked improvement in LV systolic function but was paralleled by prominent diastolic dysfunction.Regarding our in vivo results, the positive effects of OM on the systolic function of the rat  occurred at a dosage of 600 µg/kg BW. Interestingly, some diastolic parameters  conditions. This is compatible with the finding that OM increased the passive stiffness of permeabilized human cardiac myocytes in an apparently Ca2+-independent mechanism, as well as slowing down relaxation after contraction. Our data suggest that the level of increased Ca2+ sensitivity may be extreme: both reduced diastolic length and increased cardiomyocyte passive stiffness occurred at very low (diastolic) Ca2+ concentrations.Cellular contractility, Caf humans , 23. OM pulsus alternans and T-wave alternans were not mentioned in the clinical studies.It was proposed in previous reports , 13 that2+ sensitivity of force production: i.e. low cytoplasmic Ca2+ concentrations are sufficient for both the initiation and maintenance of contraction. However, we also identified a marked reduction in the rate of force generation and relaxation under conditions where the Ca2+ concentration reached a steady state in permeabilized human cardiomyocytes, suggesting a previously unidentified action of OM on the Ca2+ regulation of the actin–myosin complex. It appears that both Ca2+-dependent activation and dissociation are much slower in the presence of OM. OM also increased the resting stiffness of cardiomyocytes. This can be explained by (1) a Ca2+-independent initiation of force production or (2) a direct interaction of OM with the contractile protein machinery, in addition to the myosin motor. These cellular data suggest an alternative mechanism for the clinically observed decrease in LV diameter: it may be the result of Ca2+ sensitization of contraction occurring at diastolic Ca2+ concentrations, similar to that evoked by the Ca2+-sensitizer EMD 57,033 [2+ transients at low concentrations. In contrast, OM affected the APD and T-wave morphology at higher concentrations. These latter, previously unidentified features can be explained by (1) altered ryanodine receptor (RyR) function and (2) slower dissociation of Ca2+ from the troponin complex.The increase in SET can be explained by the apparent increase in the CaD 57,033 . Additiopulsus alternans, in the rat. This was present in 23 of 30 rats at a high OM dose (1200 µg/kg). It occurred during periods of slightly elevated heart rates or higher stimulation frequencies in isolated canine LV cardiac myocytes (nine of 14 cells at a stimulation frequency of 5 Hz), suggesting that this feature is heart rate-dependent but seems to be independent of species. This alternating cardiac performance involved a mechanically normal systole with prolonged ejection, such that the left ventricle was unable to relax sufficiently to be filled before the following systole. This resulted in an incomplete systole, without major changes in QRS morphology or corrected QT interval, but with an alternating pattern of T waves. T-wave alternans has been described in heart failure patients, mostly during progression of the disease. It has been suggested that T-wave alternans can be responsible for the initiation of life-threatening arrhythmias [The second major novelty of our work is that OM evoked a dose- and apparently heart rate-dependent, transient, electromechanical alternation, a feature similar to OM dose 10 µg/kg. hythmias .2+ sensitization evoked by OM may also lead to an increased probability of ventricular arrhythmias. Baudenbacher et al. [2+ sensitization. Arrhythmias were evoked by the Ca2+ sensitizer EMD 57,033 and prevented by blebbistatin (a myofilament Ca2+ desensitizer). The same authors also proposed that Ca2+ sensitization provokes ventricular arrhythmias through Ca2+ transient alternans, which is exactly what we found with OM. Moreover, it was also shown by TnT mutations that alterations in Ca2+ sensitization may lead to changes in AP characteristics [2+ alternans leading to increased dispersion of excitability and refractoriness of myocytes, which may serve as a substrate for re-entry mechanisms and thus ventricular arrhythmias [In addition to T-wave alternans, the Caeristics . Indeed,hythmias , 19, 30.Use of oral OM treatment in patients with heart failure with reduced LV ejection fraction in the GALACTIC-HF study , showed When considering the limitations of our study, we must highlight the fact that the in vivo experiments were performed in rats, while the in vitro ones were performed in isolated canine and human cardiac myocytes. Caution is appropriate when extrapolating experimental data to possible human outcomes. It needs also to be noted that all the experimental data were obtained in healthy animals. It is known, however, that canine cardiac myocytes are good models of human electrophysiology. Finally, the results showed a severe drop in blood pressure values in response to OM treatment without increases in heart rate, which raises the possibility of an effect of anesthesia on the in vivo cardiovascular parameters (i.e. a blunted baroreflex may dispose the rats to develop hypotension).Supplementary file1 (PPTX 68 KB)Supplementary file2 (DOCX 40 KB)Below is the link to the electronic supplementary material.


One such essential entry pathway for calcium into the cell is the Ca2+ release-activated Ca2+ (CRAC) channel. It consists of the Ca2+ sensing protein, stromal interaction molecule 1 (STIM1) located in the endoplasmic reticulum (ER) and a Ca2+ ion channel Orai in the plasma membrane. The Orai channel family includes three homologues Orai1, Orai2 and Orai3. While Orai1 is the “classical” Ca2+ ion channel within the CRAC channel complex and plays a universal role in the human body, there is increasing evidence that Orai2 and Orai3 are important in specific physiological and pathophysiological processes. This makes them an attractive target in drug discovery, but requires a detailed understanding of the three Orai channels and, in particular, their differences. Orai channel activation is initiated via Ca2+ store depletion, which is sensed by STIM1 proteins, and induces their conformational change and oligomerization. Upon STIM1 coupling, Orai channels activate to allow Ca2+ permeation into the cell. While this activation mechanism is comparable among the isoforms, they differ by a number of functional and structural properties due to non-conserved regions in their sequences. In this review, we summarize the knowledge as well as open questions in our current understanding of the three isoforms in terms of their structure/function relationship, downstream signaling and physiology as well as pathophysiology.Ca In the quiescent state, the intracellular Ca2+ concentration is very low, in the range of 0.1 µM, whereas on the extracellular side and in intracellular organelles, such as the endoplasmic reticulum (ER) and the mitochondria, the Ca2+ levels are high, in the range of 1–2 mM. Activation of the cell, e.g., by receptor-ligand binding, triggers the activation of Ca2+ selective ion channels and cytosolic Ca2+ levels enhance as a consequence. Depending on the stimulus, Ca2+ signals develop in various patterns, ranging from transient to stable signals. This ensures the versatility of the Ca2+ ion in regulating a wide variety of biological processes, from the short term, such as secretion, to the long term, such as gene transcription or proliferation. This diversity of Ca2+ signaling mechanisms is further established by a toolbox of Ca2+ sensing, Ca2+ buffering, Ca2+ binding and Ca2+ transporting proteins, which can act in a cell type-specific manner [Cac manner ,4.2+ entry pathway into the cell is represented by the Ca2+ release-activated Ca2+ (CRAC) ion channel [2+ sensor STIM1 located in the ER and the Ca2+ ion channel Orai1 situated in the plasma membrane. Defective signaling mechanisms of the CRAC channel proteins  can lead to diseases such as severe combined immunodeficiency, thrombocytopenia, tubular aggregate myopathy, ectodermal dysplasia or cancer [A prominent Ca channel ,12,13,14r cancer ,15,16. Ar cancer ,18. The r cancer ,18. It ir cancer ,20. Whilr cancer ,23,24,25r cancer ,31,32,33To optimize available or to develop novel, target-specific therapeutic strategies, a detailed understanding of the CRAC channel components and their isoforms is required. In this review, we highlight the current knowledge on isoform-specific properties and physiological as well as pathophysiological roles of the three Orai homologues.2+ signals arise from Ca2+ influx via store-operated Ca2+ ion channels. They are controlled by the amount of Ca2+ in the intracellular Ca2+ stores, typically the ER. Their activation mechanism is considered to be a biphasic process in which depletion of the ER Ca2+ store is associated with Ca2+ influx from the extracellular side. The best characterized store-operated Ca2+ ion channel is the CRAC channel [In a wide variety of cell types, intracellular Ca channel ,34,35.2) and generate inositol 1,4,5-trisphosphate (IP3). IP3 binds to the IP3 receptors (IP3R) in the ER membrane, which releases Ca2+ from the ER lumen to the cytosol and activates the CRAC channel [In detail, receptor stimulation at the cell membrane leads to the activation of a signaling cascade that activates phospholipase C via a tyrosine kinase cascade or G proteins to hydrolyze phosphatidylinositol 4,5-bisphosphate  domains [2+ levels in the ER triggers the release of Ca2+ originally bound to STIM1. Subsequently, STIM1 proteins undergo a conformational change and oligomerize [2+ release-activated Ca2+-activating domain (CAD) region, couples to Orai [The STIM1 proteins located in the ER membrane sense ER luminal Ca domains ,39,40,41gomerize ,38,42,43gomerize ,46,47,48gomerize ,49,50,51gomerize ,52. An igomerize ,49,50,51 to Orai ,54, whil2+ influx in many cells, including immune cells, cardiomyocytes, airway, vascular smooth muscle cells, endothelial cells and melanocytes [Since the discovery of the three Orai proteins, Orai1 has been the most investigated. It is ubiquitously expressed and triggers together with STIM1 the “classical” store-operated Caanocytes ,23,24,25anocytes ,21,55. Ianocytes ,31,32,33anocytes ,31,32,33anocytes .Overall, it is important to understand the structure and function of the three Orai isoforms. In particular, their distinct structural and functional features, which will be reviewed in the following section, might arise as promising for target-specific therapeutic strategies.Structurally, all three Orai proteins consist of four TM domains flanked by the cytosolic N- and C-terminus and linked by two extracellular and one intracellular loop ,58,59 FA–D. DespThe cytosolic and extracellular regions show stronger differences in sequence. The N-terminus (aa 1–90) exhibits 34% sequence identity, while the C-terminus (aa 265–301) is about 46% comparable  Table 1Table 1. Drosophila melanogaster Orai, homology modeling strongly suggests that all human Orai homologues form hexameric complexes [Prior to the first reported Orai structure , Orai chomplexes ,71,72. Iomplexes ,74.2+ selective ion pore of the Orai channel [2+ accumulating region (CAR) [2+ ions are attracted by the CAR region and transferred to the selectivity filter [2+ permeation through the hydrophobic segment to the basic region. The role of the basic region in Ca2+ permeation is still controversial. Structural studies suggest that the residues with positively charged side chains are shielded by negatively charged chloride ions, and thus, either maintaining the closed state [2+ permeation [Within the hexameric complex, all six TM1 domains are located in the center and form the Ca channel ,68,69,70on (CAR) , which ion (CAR) ,75,76. Ay filter . STIM1 cy filter . This aled state  or allowrmeation . The posrmeation . Alternarmeation . Key resThe pore region is surrounded by a ring of TM2 and TM3 and at the periphery by TM4 regions connected to the helical C-termini  Figure E,F. Ther2+ (ARC) channel [4 (LTC4)-regulated Ca2+ (LRC) channels have been reported to form Orai1/3 heteromultimers, altough the ratio of Orai1:Orai3 subunits is currently elusive [Aside from homomeric Orai assemblies, Orai homologues have been reported to be heteromerized , which i channel . It cont channel ,87,88,89 channel . Further elusive .2+ ion channels, whereas the store-independent ARC channel is a pentameric assembly of Orai1 and Orai3 subunits. A number of isoform-specific differences in the sequence of human Orai isoforms exist. Among those, several ones have already been reported to lead to structural and/or functional differences, which will be described in detail below.Overall, the Orai subunits can form homo- or heteromeric, hexameric complexes to form store-operated Ca2+ selective currents develop, all with a reversal potential of +50 mV [All three Orai isoforms have in common that they can be activated by STIM1 upon store-depletion. As a result, strongly inwardly rectifying Caf +50 mV . Howeverf +50 mV .The activation of Orai channels involves in the initial step the direct interaction with STIM1. The indispensable prerequisites for their coupling are their C-termini ,93. MoreWe distinguish between isoform-specific differences which impact STIM1/Orai coupling, Orai activation or both .277AEF279   . It is r1–78, was restored upon the exchange of the loop2 by that of Orai3 (Orai1 ΔN1–78 Orai3-L2) [1–53) was abolished by inserting Orai1 loop2 in place of that of Orai3. In line with these studies, constitutive Orai1 mutants that lost their activity by truncation of the N-terminus also showed restored activity after the swap of Orai3 loop2, independent of STIM1 [We reported that an opening-permissive communication of the N-terminus and loop2 is essential for STIM1/Orai1 interaction and Orai1 activation . Using arai3-L2) . Similarof STIM1 . Using MWe recently showed that inter- and intra-subunit salt-bridge interactions at the cytosolic helical extensions of TM1, TM2 and TM3 are critical for STIM1 coupling as well as pore opening A,E. A siThe nexus region is in close proximity to the loop2-TM3 region, forming the hinge plate . Two con2+ currents to levels comparable to those of Orai2 and Orai3. In support, Orai2 and Orai3 chimeras containing the swapped Orai1 N-terminus showed a marked increase in currents [Moreover, the Orai N-terminus controls CRAC channel activation and biophysical characteristics in an isoform-specific manner, as reviewed in detail in Krizova et al. . Noteworcurrents .2+ selective currents, other common CRAC channel hallmarks include the fast Ca2+-dependent inactivation (FCDI) and the increase in IDVF versus I2+Ca [2+ influx into the cell. Interestingly, FCDI, which is typically recorded within the first 100 ms of a hyperpolarizing voltage step of 2 s [Aside from the store-operated activation of highly Caus ICa2+ ,95. FCDIp of 2 s ,84, occup of 2 s ,84,107. p of 2 s ,84,107. p of 2 s . Specifip of 2 s . Moreovep of 2 s . The extp of 2 s ,110. A p2+-containing solution to a divalent free Na+-containing solution, it is of note that the ratio of IDVF versus I2+Ca differs among the three isoforms. The reason for that likely underlies the distinct extents of FCDI. Indeed, the pronounced inactivation of STIM1-mediated Orai3 currents corresponds to an increased ratio of IDVF versus I2+Ca compared to Orai1 [Concerning the typical enhancements of CRAC channel currents when switching from a Cato Orai1 .The isoform-specific properties of the N-terminus are further crucial in the formation of ARC channels, which have been reported to form pentameric assemblies of three Orai1 and two Orai3 subunits. A chimeric approach revealed that the substitution of a single Orai3 N-terminus by that of Orai1 switched the channel into a more store-operated than an arachidonate-activated one .The activation signal of STIM1-Orai coupling at the periphery of the channel complex is finally transmitted to the pore region in the center. A series of GoF mutations, each located in one of the four transmembrane domains (TMs), suggested that pore opening involves structural alterations throughout all TM domains ,119,120.2+ entry  blocks STIM1-mediated Orai1, but not Orai3 currents (Reactive oxygen species (ROS) are essentials triggers in a variety of physiological and pathophysiological processes. They are generated outside as well as inside the cell and are produced by redox-active proteins. It has been reported that hydrogen peroxide  as well as its oxidomimetic mutation C195D were demonstrated to undergo inhibitory interactions with S239 located in TM4 . MutatioA set of compounds modulating Orai channels are currently available. Although none of those has been used in clinics in recent years several ones have reached phases of clinical trials. Most blockers have been reported to possess poor selectivity on Orai channels ,18.3+ and Gd3+, which selectively block Ca2+ ion channels at low concentrations. They lead to an equal and significant block of different Orai channel isoform currents [The most commonly used inhibitors are the trivalent lanthanides Lacurrents ,17,18.2-APB represents the most popular drug in the characterization of the structure/function relationship of CRAC channels ,67,126. 2+ entry in a comparable manner to 2-APB [Two derivatives DPB162-AE and DPB163-AE affected store-operated Cato 2-APB ,132. Botto 2-APB .2+ entry already at low concentrations. As the effects of these novel compounds were investigated on native SOC of breast cancer cells (MDA-MB-231) and HEK 293 cells, their effects on Orai1 and Orai3 individually remain of interest to determine potential isoform-specificity [Recently, a library of 2-APB derivatives has been synthesized containing substitutions at one of the phenyl rings of 2-APB. Among those, there are ones which inhibit store-operated Cacificity .Despite 2-APB showing an isoform-specific differences in its action on the three Orai homologues, it belongs to one of the most unselective blockers. Several other available blockers, such as Synta66, GSK7975A or RO2959, are rather selective to Orai channels, as they show almost no significant inhibitory effect on a variety of other cellular receptors, transporters or ion channels. Among those, RO2959 blocks STIM1-mediated Orai2 and Orai3 currents to a weaker extent than those of Orai1 . This maAltogether, isoform-specific differences have been discovered for several available pharmacological compounds F. The ch2+ signals are prominent for the versatility to regulate a variety of signal processes ranging from short to long term response. This is established by the spatiotemporal dynamics of calcium signaling patterns, including transient, oscillatory or robust ones [Cellular Caust ones ,139,140.2+ plateaus involved in sustained Ca2+ entry [2+ oscillations triggered by physiological receptor stimulation, whereas they are not involved in sustained Ca2+ entry. Indeed, various Orai isoform knockout cells showed that only Orai1-containing cells predominantly exhibited sustained Ca2+ entry. In contrast, KO cells with sustained expression of Orai2 or Orai3 either alone or together with other isoforms showed oscillatory Ca2+ level increases. Interestingly, a lower oscillation frequency was detected in cells containing only Orai2 or Orai3 alone compared with cells containing two or three isoforms. This isoform-specific behavior correlates with an enhanced STIM1 coupling to Orai2 and Orai3 compared to Orai1 in the resting state as well as with their isoform-specific FCDI, which is the highest for Orai3 [Orai1 is mainly indispensable for the development of Ca2+ entry ,141,142.or Orai3 .2+ levels can be translated into various output signals by diverse effector molecules. Among the latter, we focus in this section on the nuclear factor of activated T-cells (NFAT), typically found in the cytosol in the vicinity of the Ca2+ source, and responsible to convey the signal of Ca2+ influx to the nucleus to trigger gene transcription. This is essential for triggering the innate and adaptive immune cell response through the transcription of inflammatory mediators such as cytokines. The four members of the NFAT family, NFAT1–4, are kept inactive in the cytosol by phosphorylation. The movement of NFAT into the nucleus is triggered by local Ca2+ entry [2+-triggered NFAT movement into the cytosol was shown to be specific for Ca2+ influx via Orai1 and absent for Orai2 and Orai3 [2+ binding protein, calcineurin, a Ca2+-dependent phosphatase, and AKAP79, a prototypical A-kinase anchor protein. Mechanistically, Ca2+ entry leads to the attraction of calcineurin to calmodulin bound to Orai1 via the scaffolding protein AKAP79. AKAP79 binds to NFAT1 via its C-terminal leucine zipper motif, thus bringing it in close proximity to Orai1 upon Ca2+ entry  cells to the respective sites and induce their differentiation into effector TH cells. The latter proliferate and secrete cytokines. It has been demonstrated that CRAC channels in naïve T helper cells are more susceptible toward H2O2 than those of effector T helper (TH) lymphocytes. The reason for this is that Orai3, which is not redox sensitive, is upregulated in the effector TH cells  [Especially for estrogen receptor-expressing (ER+) breast cancer and non-small cell lung cancer (NSCLC), there is ample evidence that Orai3 functions as an attractive therapeutic target in these pathogenic cell types. In contrast to healthy breast tissue, estrogen receptor positive (ER+) breast tumorigenesis exhibit store-operated Caby Orai3 ,170  , but nots (T47D) . Overall2+ entry. Furthermore, Orai3 was identified to trigger the proliferation of lung adenocarcinoma cells. Similar to breast cancer cells, in lung cancer cells Orai3 expression has also been demonstrated to be regulated by ERα.Concerning lung cancer cells, in lung adenocarcinoma cells, a higher Orai3 expression B has bee2+ entry, though Orai3 is also expressed [In prostate cancer cells, the expression profile of Orai3 is still controversial. While Dubois et al.  demonstrxpressed . Holzmanxpressed  identifixpressed . Furtherxpressed ,161.Overall, Orai1 plays an important role in maintaining physiological processes in the human body as well as the individual cell. In contrast, Orai3 in particular is a critical determinant in cells to adapt to altered pathological conditions such as increased oxidative stress or tumorigenesis.Collectively, in this review, we have assembled the current knowledge on the structural and functional differences of the three Orai isoforms. Since Orai1 is the most universal ion channel and a variety of disease-related mutations are known, it has been the best characterized to date. However, there is increasing evidence that Orai2 and Orai3 are also of considerable importance, particularly in the development of disease. The universality of Orai1 makes therapeutic approaches difficult to dangerous, as this, in turn, could lead to immunosuppression and promote the development of diseases such as cancer. Hence, understanding the differences of Orai isoforms, both mechanistically as well as physiologically, is critical and may open new avenues for the development of potential future therapeutic strategies that selectively target one of the three homologues.2+ signaling as well as downstream signaling events, such as altered gene transcription, are differentially regulated by the three isoforms. The range of cellular Ca2+ signaling is massively expanded by the possibility of Orai isoform to heteromerize. Although Orai1 is a versatile ion channel in health and disease, there is increasing evidence that cells use a molecular switch mechanism that leads to the up- or downregulation of a particular Orai isoform to adapt to changing physiological or pathophysiological conditions. However, a set of questions still remains to be resolved.While the general mechanism of STIM1-mediated activation of the three Orai isoforms is comparable, they can be distinguished in their structural, functional and pharmacological properties. A number of differences in the structure/function relationship of Orai1 in particular compared to Orai3 have been elucidated, such as that of the cytosolic regions. Moreover, Orai1 and Orai3 differ in pH and redox sensitivity. There is evidence of isoform-specific pharmacological modulation of Orai channels. In addition, patterns of CaSeveral functional differences have been extensively characterized, particularly for Orai1 and Orai3. Less attention was paid to Orai2, which mimics some properties of Orai3  as well as some properties of Orai1 . While the isoform-specific roles of cytosolic segments are well understood, investigations of the role of the second extracellular loop (loop3), which largely differs in sequence identity among the three isoforms, is still pending. Additional efforts need to be made to understand the homo- and hetermomerization mechanisms of Orai subunits and key sites required for that.With respect to the structural resolutions, only those of dOrai channels in both the closed and open states are currently available. However, for an atomistic characterization of the three human Orai isoforms, their structural resolutions are still outstanding. This could further improve our understanding of the bulk of functional isoform-specific behaviors. The ultimate goal is to obtain structural resolutions of these homologues in complex with STIM1. This would also provide a structural understanding of STIM1/Orai interaction interfaces, obviously occurring in an isoform-specific manner at least for Orai C-termini ,99. Here2+-ATPase (SPCA2). Furthermore, however, Orai3 also plays a role in breast cancer cells and is expressed in excess there. It would need to be clarified whether SPCA2 can also regulate Orai3 or whether this is isoform-specific. Another modulatory protein, CRACR2A, has been reported to directly interact with STIM1 and Orai1 to impact their function [A number of accessory proteins have been identified in recent years to regulate the function of Orai1 channels. However, nothing is known for Orai2 and Orai3 in this regard. The identification of isoform-specific regulatory proteins may open further targets for selective therapeutic intervention, as may be possible for AKAP79 in gene transcription. For example, it is interesting to note that in breast cancer cells, Orai1 has been reported to be regulated by Secretory pathway Cafunction , while nfunction .2+ entry via Orai3, it remains to be determined how cytokine production in T-cells is possible, potentially via transcription factors, which can be specifically triggered by Orai3.Finally, a detailed understanding of the signaling cascades altering the expression of a certain Orai homologue in disease, as well-known for Orai3 in cancer, is still outstanding. For instance, in breast cancer cells, it has been shown that Orai3 upregulation is controlled by the ERα receptor. Such links might offer valuable targets to interfere with disease states. Furthermore, since NFAT1 mediated gene transcription has been recently reported to be impaired upon store-operated CaIn terms of therapeutic approaches, it remains crucial to develop not only selective strategies, but also methods of local drug delivery so that potential side effects can be circumvented. Recent advances in the field of nanotechnology have made it possible to deliver chemotherapeutic agents directly to the tumor site. This has significantly reduced the side effects and systemic toxicities related to these drugs ,185,186.


Our findings highlight ER Ca2+ as a master regulator of cholesterol metabolism and identify a mechanism by which CF may protect against CVD.Evidence suggests that caffeine (CF) reduces cardiovascular disease (CVD) risk. However, the mechanism by which this occurs has not yet been uncovered. Here, we investigated the effect of CF on the expression of two bona fide regulators of circulating low-density lipoprotein cholesterol (LDLc) levels; the proprotein convertase subtilisin/kexin type 9 (PCSK9) and the low-density lipoprotein receptor (LDLR). Following the observation that CF reduced circulating PCSK9 levels and increased hepatic LDLR expression, additional CF-derived analogs with increased potency for PCSK9 inhibition compared to CF itself were developed. The PCSK9-lowering effect of CF was subsequently confirmed in a cohort of healthy volunteers. Mechanistically, we demonstrate that CF increases hepatic endoplasmic reticulum (ER) Ca Caffeine may reduce cardiovascular disease risk, but the underlying mechanisms for these effects are incompletely understood. Here the authors report that caffeine inhibits the activation of the transcription factor SREBP2 to promote LDLc clearance through the PCSK9-LDLR axis. Recent advancements in therapies available for the management of dyslipidemia and CVD have led to the characterization of PCSK9 as a hepatocyte-secreted circulating factor capable of enhancing the degradation of cell-surface LDLR5. By extension, PCSK9 also reduces the ability of metabolically active tissues, such as the liver, to remove excess LDLc from the blood. Based on these seminal discoveries, anti-PCSK9 antibodies are now available to patients at high risk of CVD, yielding an unprecedented 60–70% reduction of LDLc levels6. Although efficacious, the high cost and/or need for subcutaneous administration of anti-PCSK9 antibodies poses a limit to their availability to patients worldwide7. Such circumstances warrant the need for additional studies examining the molecular mechanisms that modulate the expression and secretion of PCSK9 from hepatocytes in order to develop more cost-effective therapies.Increased levels of circulating low-density lipoprotein cholesterol (LDLc) are tightly linked to the development of cardiovascular disease (CVD). Despite the approval of several therapies that lower LDLc, many patients fail to reach their LDL lowering goal due to intolerance, adverse events, or simply the high cost of medications. An important regulator of LDLc is the sterol regulatory element-binding protein 2 (SREBP2), which is an endoplasmic reticulum (ER)-resident transcription factor. SREBP2 is activated by reductions in intracellular cholesterol and loss of ER Ca8. On the contrary, accumulating evidence now suggests that moderate to high levels of CF (>600 mg), consumed daily in the form of non-alcoholic beverages, are associated with a reduction in CVD risk9. Although biochemical studies have shown that CF increases intracellular Ca2+ levels and induces vasodilation of the vascular endothelium via release of nitric oxide11, a cellular process known to be cardioprotective12, molecular mechanisms supporting clinical evidence are currently lacking.Caffeine (CF) or 1,3,7 trimethylxanthine, is best known as a stimulant alkaloid of the central nervous system found in various plants and is commonly found in coffee and tea. The majority of published literature demonstrates that the average adult habitual caffeine drinker consumes between 400 and 600 mg of CF daily and organizations like Health Canada and the Food and Drug Administration conclude that such doses are not negatively associated with toxicity, cardiovascular effects, bone status, calcium imbalance, behavior, the incidence of cancer or effects on male fertilityIn the current study, we demonstrate that clinically relevant concentrations of caffeine suppress SREBP2 transcriptional activation in liver hepatocytes, thereby leading to a reduction of PCSK9 in both mice and humans. Using structure/activity relationships (SAR), we have also generated several xanthine derivatives with heightened antagonism against SREBP2 and PCSK9, compared to CF. Overall, these studies characterize the mechanism by which CF impacts the expression of genes well-known to mediate CVD risk.13, including HuH7 and HepG2 cells, as well as primary mouse- and human-hepatocytes , were treated with CF for 24 h and assessed for PCSK9 expression via immunoblots and real-time PCR , a pharmacological agent that depletes intracellular sterols. Like TG-treated cells, CF attenuated U18-induced PCSK9 expression  and subsequent induction of a downstream marker (phosphorylated acetyl CoA carboxylase [pACC]) in the livers of C57BL/6J mice  CF may increase ER Ca2+ levels, and (b) other agents known to increase ER Ca2+ levels may also block SREBP2 activation and PCSK9 expression. To test this hypothesis, we first examined cytosolic Ca2+ levels in CF-treated cells using the high-affinity fluorescent Ca2+ indicator, Fura-2-AM. Consistent with previous studies, CF significantly increased cytosolic Ca2+ levels in immortalized hepatocytes -based calreticulin chameleon Ca2+ sensor, which increases in fluorescence intensity upon Ca2+ binding22. The low-affinity Ca2+ indicator, Mag-Fluo-4, was also utilized for the direct assessment of ER Ca2+ and increases in fluorescence intensity upon Ca2+ binding24. The fluorescence intensity of cells treated with CF and control agents, TG and CDN, was assessed using a fluorescent spectrophotometer and visualized using a fluorescent microscope , ryanodine is known to facilitate ER Ca2+ loss by enhancing RyR-mediated Ca2+ transients; whereas high dose ryanodine (10 µM) is known to block RyR-mediated ER Ca2+ leakage26. The compound 2APB also blocks the exit of ER Ca2+ by antagonizing IP3Rs28. In contrast to these two agents that modulate ER Ca2+ release, CDN is an established allosteric activator of the SERCA pump and thus increases the entry of Ca2+ into the ER7. Consistent with our hypothesis, we observed that high-dose ryanodine, 2APB, and CDN, blocked SREBP2 and PCSK9 at the mRNA transcript level in the presence and absence of TG  treatment increased PCSK9 and SREBP2 expression , which were previously shown to increase ER Ca2+ levels27, also blocked PCSK9 secretion . In support of this notion, previous studies have demonstrated that (a) GRP78 is highly promiscuous in its client specificity31, capable of binding to one site every 36 amino acids of a randomly generated peptide32, (b) Ca2+ and ATP bind to GRP78 in a cooperative manner and that ATP is necessary for the peptide binding and folding abilities of this chaperone33, and (c) overexpression of GRP78 can attenuate the activation of SREBPs in response to ER stress14.GRP78 is among a number of Ca2+ levels enhance GRP78 peptide binding capacity, HuH7 cells were treated with either CDN, which increases ER Ca2+ levels, or TG which causes ER Ca2+ depletion. Following treatment, the interaction between GRP78 and SREBP2 was examined via immunoprecipitation of the former. By affecting ER Ca2+ levels, however, these agents also directly impact the expression and abundance of GRP78 compared to untreated cells. Therefore, assessment of the relative binding capacity GRP78 for SREBP2 between treatments required normalization of immunoprecipitations to equivalent GRP78 protein levels  were also treated with CF. Our findings demonstrate that siGRP78 treatment significantly increased the mRNA and secreted forms of PCSK9, as well as the mRNA levels of SREBP2 Fig. . Consist2+ depletion induces a compensatory unfolded protein response (UPR), we also postulated that CF may attenuate UPR marker expression by increasing ER Ca2+ levels. Upon assessment of PHHs treated with CF, we observed a reduction in mRNA transcript levels of ER stress markers GRP78 and the activating transcription factor 4  we observed a significant reduction of circulating PCSK9 and triglyceride levels Fig. . A time-udy Fig. . Immunohudy Fig. . Despite36. Treatment with alirocumab led to a significant increase in hepatic LDLR expression . Consistent with our observations in cultured hepatocytes and in mice, CF reduced plasma PCSK9 levels in healthy subjects by 25% (n = 12) and 21% (n = 8) at the 2- and 4-h time points, respectively 11. Although the aforementioned interactions do not directly support our observation, in which CF increased ER Ca2+ levels, CF is also known to block ER Ca2+ release via inhibition of the IP3-receptor41. In addition, CF was previously shown to bind to hepatic RyR and potentially block RyR-mediated Ca2+ release42. Given the broad range of targets known to interact with CF, the identification of exact molecular mechanisms pertaining to its protective effect on the vascular system is challenging. In support of the aforementioned protective effect of CF on CVD risk, CF has been shown to promote vasodilation of the vascular endothelium by means of stimulating Ca2+ release via CICR and leading to the activation of eNOS34. In vascular smooth muscle cells, following CICR-induced vasodilation, CF also increases intracellular Ca2+ levels by inducing non-selective cation channels at the cell surface43 and has been shown to block IP3Rs44. These studies, as well as those of others46, are consistent with our observations that CF increases intracellular Ca2+ levels and attenuates ER stress. Collectively, however, there exists a range of mechanisms by which CF affects intracellular Ca2+ levels, which tend to differ between tissue and/or cell types. In hepatocytes, our data strongly suggest that CF increases cytosolic and ER Ca2+ levels following a 24 h exposure.CF is known to exert its effect through a range of molecular targets, including the antagonism of adenosine receptors, GABA receptors, and phosphodiesterase enzymes, as well as inducing intracellular Ca2+ reserve, capable of extruding Ca2+ for signaling and/or excitatory purposes and removing excess cytosolic Ca2+ following periods of excitement. The Ca2+ sequestering capacity of the ER, which far exceeds that of the cytosol, depends on the abundance of ER-resident low-affinity/high capacity Ca2+-binding proteins47. The interaction of Ca2+ with these proteins also promotes chaperone function. During conditions of ER Ca2+ depletion, chaperones lose their folding capacity and misfolded polypeptides accumulate in the ER. The UPR, which increases the abundance of ER-resident chaperones, is then triggered in order to restore ER folding capacity and ER Ca2+ levels48. Conversely, increasing ER Ca2+ levels appears to have a net protective effect on ER homeostasis, given the observed reduction in UPR marker expression by agents that activate SERCA and increase ER Ca2+ influx or those capable of blocking leakage from either IP3R or RyR49. Consistent with this observation, we also found that CF protected against TG-induced ER stress in cultured hepatocytes and reduced the expression of a variety of ER chaperones in the livers of mice.The ER serves as an important and dynamic CaSREBP1 and SREBP250. Although the exact mechanism by which ER stress activates the SREBPs remains elusive15, previous studies have suggested and/or demonstrated that (a) ER stress can reduce the expression of INSIG1, a negative regulator of the SREBPs51, (b) ER stress-induced caspases can cleave and activate the SREBPs in a manner independent of conventional S1P activation in the Golgi and53, and (c) GRP78 can dissociate from the SCAP-SREBP complex thus liberating SREBP from the constraints of the ER54. We now report the finding that ER Ca2+ levels serve to fine-tune the peptide-binding capacity of GRP78, thereby affecting the ER retention of its binding partners, such as pre-mature SREBP2.In addition to UPR chaperones, ER stress is also known to promote the activation of the self-induced transcription factors that regulate the expression of fatty acid- and cholesterol-regulatory genes, namely de novo expression of PCSK9 and the LDLR, the observed induction of cell-surface LDLR in the face of SREBP2 inhibition likely occurs in response to the loss of circulating PCSK9 levels. Several studies have demonstrated that, with a half-life of five minutes55, PCSK9 expression closely follows that of SREBP256. In contrast, with a 144-fold increased half-life compared to PCSK9 (12 h)57, expression of the LDLR appears less dependent on de novo synthesis and more on factors that influence its stability at the cell surface, like circulating PCSK9. Because SREBP2 also induces the expression of HMGR, it is also possible that CF reduces circulating LDLc levels via inhibition of HMGR-mediated de novo cholesterol synthesis. It is well-established, however, that statins block HMGR, induce SREBP2 activity and increase circulating PCSK9 levels. In contrast to statins, our findings demonstrate that CF blocks both SREBP2 and PCSK9. Thus, with reduced circulating PCSK9 levels, it is unlikely that HMGR activity is increased in response to CF58.We also observed that CF induced the protein expression of the LDLR and increased LDLc uptake in cultured hepatocytes. Given that SREBP2 regulates 59. Developing our understanding of the regulatory mechanisms that modulate the expression and secretion of PCSK9 from hepatocytes may aid in the development of anti-PCSK9 therapies that are less costly than those that are currently available. Overall, results from our study support a model in which small molecules like CF, capable of increasing ER Ca2+ levels, can block the activation of SREBP2 by enhancing GRP78 chaperone function and binding capacity  per 1 ml complete medium containing plated cells. Human PCSK9 was overexpressed using the bicistronic pIRES-EGFP plasmid; calnexin using the mPA-GFP-N1 plasmid. To attenuate the expression of GRP78 and CD36, siGENOME smartpool siRNA was purchased from GE Dharmacon (M-008198-02 and L-010206-00-0005 respectively) and transfected using lipofectamine RNAiMAX as per the manufacturer’s protocol.HuH7 (kind gift from Dr. Nabil G. Seidah) and HepG2  cells were routinely grown in complete Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 100 U/ml of penicillin and streptomycin (Sigma-Aldrich). CF, ryanodine, 2 APB, CDN, theobromine, paraxanthine, 8-cyclopentyl-1,3-dimethylxanthine (8CD), 8-(3-Chlorostyryl) CF (8CC), PSB603, cyclopiazonic acid and U18666A were purchased from Tocris Bioscience. All cell treatments were carried out for 24 h unless otherwise stated. Cells were transfected with a cocktail consisting of plasmid DNA (1 µg), X-tremeGENE HP , and opti-MEM 2+ in Huh7 and HepG2 cells was measured using a high-affinity Ca2+ indicator, Fura-2-AM (Thermo Fisher Scientific). ER Ca2+ levels were assessed using the low-affinity Ca2+ indicator, Mag-Fluo-4, and via transfection of cells with D1ER. The D1ER plasmid encodes an ER-resident calcium binding protein linked to a fluorescent protein and increases in fluorescence intensity upon Ca2+ binding7. For assessment using indicators, cells were plated in black clear-bottom 96-well plates to a confluence of 70–75% and treated with Ca2+ modulating agents for 24 h (n = 6). Cells were then washed and incubated with Fura-2-AM (2 µM) or Mag-Fluo-4 (2 µM) for 45 min at 37 ˚C in HBSS containing 20 mM HEPES and 2% pluronic acid v/v (Thermo Fisher Scientific). Fluorescence intensity of intracellular Fura-2-AM was measured at two distinct wavelengths (ex 340/em 515 and ex 380/em 515), following three consecutive washes, to assess bound and unbound states using a SpectraMax GeminiEM fluorescent spectrophotometer . Fluorescence intensity of Mag-Fluo-4 was quantified at a single wavelength (ex 495/em 515). For assessment using D1ER, HuH7 cells were plated in black clear-bottom 96-well plates to a confluence of 70–75% and transfected (n = 6). Twenty-four hours later, cells were treated with Ca2+ modulating agents for an additional 24 h and quantified (ex 495/em 515) using a SpectraMax GeminiEM fluorescent spectrophotometer.Intracellular CaCells were washed in phosphate-buffered saline (PBS), lysed in 4X SDS-PAGE lysis buffer and separated on 7–10% polyacrylamide gels in denaturing conditions. Gels were transferred to nitrocellulose membranes using the BioRad mini trans-blot system, blocked in 5% skim milk in tris-buffered saline (TBS) for 1 h and incubated in primary antibody overnight for 16 h at 4 ˚C. Membranes were then exposed to horse radish peroxidase conjugated secondary antibodies and visualized using EZ-ECL chemiluminescent reagent (Froggabio). Band intensities were quantified using ImageJ software (BioRad) against membranes reprobed for housekeeping proteins. A complete list of antibodies used for immunoblot analysis is presented in Supplementary Table Cells were grown in 10 cm dishes were resuspended in ice cold non-denaturing immunoprecipitation buffer containing 20 mM Tris HCL, 137 mM NaCl, 1% NP-40, 2 mM EDTA, and protease inhibitors (Roche). Total cell protein was normalized using a protein assay (BioRad) and 1 mg of protein from each sample was incubated with 2 µg of capture antibody targeted against GRP78  and rotated on a platform for 24 h at 4 ˚C. Following incubation, samples were exposed to 100 µl of Protein G magnetic Surebeads (BioRad) for an additional 2 h on a rotating platform at 4 ˚C. Beads conjugated to the anti-GRP78 antibody were subsequently isolated and the remaining sample was collected and labeled “input” for use as controls. The magnetic beads underwent four consecutive washes using the non-denaturing IP buffer and resuspended and boiled in 100 µl of 4X SDS-PAGE sample buffer.Cells were plated in 4-well chamber slides and incubated in complete DMEM for 24 h and exposed to treatments 24 h later for an additional 24 h. Cells were fixed with 4% paraformaldehyde for 30 minutes and washed with either non-permeabilizing, or permeabilizing PBS containing 0.025% Triton-X. Cells were then blocked with 1% bovine serum albumin (BSA) for 30 min and stained with anti-GFP antibody for 1 h in PBS containing 1% BSA. Afterwards, cells were washed and incubated with Alexa 488 fluorescently labeled secondary antibodies (Thermo Fisher Scientific), as well as the DAPI nuclear stain. Slides were then mounted with permafluor and visualized using the EVOS FL color imaging system at either ×20 or ×40 magnification. A complete list of antibodies used for immunofluorescence staining is presented in Supplementary Table Following treatment, live cells were incubated in complete DMEM containing 5 µM Thioflavin-T  for 15 min. Cells were then fixed in 4% paraformaldehyde and mounted with permafluor. Fluorescent staining was visualized using the EVOS FL color system at either ×20 or ×40 magnification.Liver tissues were fixed in formaldehyde and subsequently embedded in paraffin for sectioning. Four µM thick sections underwent epitope retrieval and were subsequently stained with primary antibodies for 16 h at 4 ˚C. Slides were then exposed to biotin-conjugated secondary antibodies for 45 min and then streptavidin peroxidase for 10 min. Staining was visualized using the Nova Red HRP Substrate (Vector Laboratories). A complete list of antibodies used for immunohistochemical analysis is presented in Supplementary Table RNA purification/isolation was performed using RNeasy mini kits (Qiagen) and normalized to 2 µg RNA using a NanoDrop spectrophotometer. Samples were then reverse transcribed into cDNA using Superscript Vilo cDNA Synthesis kit (Thermo Fisher Scientific). Real-time PCR was executed with Fast SYBR Green (Thermo Fisher Scientific) using the ΔΔct method on the ViiA7 real-time PCR platform (Thermo Fisher Scientific). A complete list of primers used for PCR analysis is presented in Supplementary Table Secreted PCSK9 levels were assessed directly in cell culture medium of cells grown in FBS-free medium for 24 h or in the serum isolated from either mice or human subjects. Mouse PCSK9 was measured using the Quantikinine ELISA kit  and human PCSK9 using the PCSK9 Quantikinine ELISA kit . Serum ApoB levels were also quantified using ELISAs . Serum samples were diluted as per manufacturer’s instructions.Pcsk9−/− or Ampkβ1−/− mice on the C57BL/6J background and were performed in strict accordance with the McMaster University animal care guidelines and approved by the McMaster University ethics board. Mice were housed in 12:12 hour light:dark cycles and fed regular chow ad libitum . CF (25–100 mg/kg − 8 h) and CDN (50 mg/kg) treatments were administered via intraperitoneal injection unless specified otherwise. Primary mouse hepatocytes were isolated using a two-step process with EGTA  and collagenase  in 12-week-old male mice on the C57BL/6J background. Cells were then washed, centrifuged, and plated following isolation in cell strainers. Hepatosure 100-donor pooled primary human hepatocytes were purchased from Xenotech. Primary hepatocytes were regularly grown in William’s E medium  containing 10% fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin.All animal studies were carried out in 12-week-old, 8 h-fasted, male wild-type, Cells were seeded plated in black clear-bottom 96-well plates for 24 h and treated with experimental agents for an additional 24 h. During the last 5 h of treatment (19–24 h) cells were exposed to DiI-LDL (10 µg/ml) and then washed with two changes of pre-warmed (37 ˚C) HBSS containing 20 mM HEPES prior to analysis. The intracellular fluorescence intensity of DiI was then quantified using the SpectraMax GeminiEM fluorescent spectrophotometer .n = 8 males; n = 4 females) underwent fasting for 12 h prior to oral administration of 400 mg CF . Blood was collected prior to administration and at 2 and 4 h following administration. All subjects completed and signed consent forms and studies were approved by the Hamilton Integrated Research Ethics Board, project number 5805. The subjects were not compensated for their participation in the study.Healthy human subjects between the ages of 22 and 45  in to a solution of MeCN/H2O  were added 3,5-bis(trifluoromethyl)benzaldehyde , azobisisobutyronitrile , and n-bromosuccinimide . The reaction mixture was stirred at 30 °C for 3 h. A precipitate was filtered and rinsed ethyl acetate (2 × 5 mL) and methanol (3 × 5 mL) to obtain the crude product. Chromatography over silica gel using gradient elution with DCM and methanol offered MLWK-1812 phenyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione, 196.1 mg, 0.50 mmol, 40% yield) as a white solid. Mp: 329-330o C. 1H NMR : d (ppm) 14.38 , 8.76 , 8.24 , 3.53 , 3.28 . 13 C NMR DEPTQ : d (ppm) 154.32, 151.09, 148.14, 146.17, 131.38-130.81, 126.39, 125.41-120.76, q (J = 273.1 Hz), 123.31, 29.86, 27.84. FT-IR (cm-1): 3052.09, 2943.32, 2759.41, 1704.53, 1649.52, 1601.04. LCMS (ESI) m/z: 393.07807 calculated for ([M + H] + ); 393.07684 observed. The general procedure for the synthesis of MLRA-1812 was based on Bandyopadhyay et al.oC. 1H NMR : δ (ppm) 13.86 , 8.05–8.01 , 7.83-7.79 , 3.49 , 3.27 . FT-IR (cm-1): 3281.65, 3039.61, 1695.87, 1651.37, 1602.14. LCMS (ESI) m/z: 311.07504 calculated for ([M + H]+); 311.07481 observed. The general procedure for the synthesis of MLRA-1812 was based on Bandyopadhyay et al.60.A reaction procedure analogous to that described for MLRA-1812 was followed using 2,4,5-trifluorobenzaldehyde to yield MLRA-1820 -3,7-dihydro-1H-purine-2,6-dione, 117.9 mg, 0.38 mmol, 30% yield) as a light brown solid. Mp: 361–363 t-tests, while comparisons between multiple groups were compared using one-way ANOVAs with the Tukey HSD post-hoc test. The paired Student’s t-test was used to compared pre- and post-treatment values in human subjects. Differences between groups were considered significant at p < 0.05  and all values are expressed as mean ± s.d. Data from studies conducted in cell lines and mice are representative of at least three independent experiments.Statistical comparisons between two groups were conducted using unpaired Student’s Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary