Composite materials are widely used in many engineering applications and fields of technology. One of the main defects, which occur in fiber-reinforced composite materials, is delamination. It manifests itself in the separation of layers of material and the damaged structure once subjected to mechanical loads degrades further. Delamination results in lower stiffness and the decrease of structure’s carry load capability. Its early detection is one of the tasks of non-invasive structural health monitoring of layered composite materials. This publication discusses a new method for delamination detection in fiber-reinforced composite materials. The approach is based on analysis of energy signal, calculated with Teager–Kaiser energy operator, and comparison of change of the weighted instantaneous frequency for measurement points located in- and outside of delamination area. First, applicability of the developed method was tested using simple models of vibration signals, reflecting considered phenomena. Next, the authors’ weighted instantaneous frequency was applied for detection of deamination using signals obtained from FEM simulated response of the cantilever beam. Finally, the methods effectiveness were tested involving real experimental signals collected by the laser Doppler vibrometer (LVD) sensor measuring vibrations of the delaminated glass-epoxy specimens. Fiber-reinforced polymer (FRP) composite materials have found application in a wide range of engineering structures—from parts of vehicles, through structures like pedestrian bridges to wind turbine blades. The main cause of their popularity is very good strength to mass ratio.Both, during manufacturing as well as use, FRP structures are subjected to a number of degradation processes, such as cracks or delamination. Degradation can result from loading the structure with force over accepted level, e.g., as a result of impulse, or due to violating construction’s fatigue strength on cyclic load. According to present state of technology, the origination of a defect does not preclude further use of a structure. However, further operation of an object, in accordance with a given level of safety, implies the need for early detection, identification, and localization of the fault as well as controlling fault development. Detection of an early phase of a fault allows to change operation conditions or to plan maintenance and repairs ,2.Delamination is the separation of part of a material of FRP which causes local change of its stiffness. Further loading of a structure, will effect, in the growth of delamination area or, in an extreme case, its rapid development causing destruction or irreversible loss of operation ability of a structure, e.g., like in the case of helicopter rotor blade delamination which took place in Israel in 2009 . Due to Those methods are based on observations of different physical phenomena such as: ultrasonic waves ,5, acousDue to economical and practical reasons, the methods based on observation and analysis of vibrations have gained big popularity, mainly in structural health monitoring applications. It is caused by the fact that the vibration signal can be easily measured by relatively cheap equipment . There iLiterature presents many techniques, which are dedicated to dealing with vibrational response of a structure and aim for detection and/or identification of the structural damages, such as delamination. A great number of methods are based on inverse problem analysis ,20. For In , the autAnother group of methods identify delamination by the use of so-called damage indicators. The most popular damage indicators are based on time domain signals or modal characteristics extracted from the vibrational signal. The example of such an indicator is the probabilistic delamination indicator (PDI) presented in . PDI is The methods based on the use of damage indicators are significantly simpler and less time consuming then the inverse analysis methods, but there are still areas that need to be improved. Many indicators exhibit sensitivity problems, need a reference state, and do not provide the possibility of detecting false alarms, reducing their reliability.Intelligent signal processing-based algorithms can be pointed as the next group of delamination identification methods. An interesting example of signal processing-based identification is presented in . The autAs the delamination is a very serious damage and can result in catastrophic failure, there is a very high concern in the scientific community in finding the effective methods allowing for its identification. The papers mentioned above represent only a small fraction of the published literature resources in the field, showing the diversity of scientific tools adopted for delamination identification purposes. In the more broad discussion on the vibration-based delamination assessment methods, their potential and classification are a job for a self-contained review article rather than for the introductory part of the research paper; a general conclusion coming from at least few review papers ,30,31 caIn the context of the above, the proposed method should not be considered as a stand-alone solution, being a perfect remedy, but as one of possible assisting methods in the vibration-based SHM system. Nonlinear dynamics of breathing deformation, observed in the finite element model of the vibrating specimen as well as in experimental tests, points to the need of selecting proper signal processing and analysis methods allowing to detect this phenomena.The presented approach, is based on the Teager–Kaiser energy operator (TKEO). The analysis of the time domain signal using the Teager–Kaiser energy operator enables observation of transient disturbances of the signal’s instantaneous frequency (IF) . Such diIn contrast to many present vibration-based methods, the original TKEO-based delamination detection method does not require knowledge of dynamic behavior of the structure in reference, i.e., healthy state, nor referring to the numerical model. In the considered case of breathing delamination, comparison of instantaneous frequency of vibration signals recorded in measurement points located within and outside delamination allows to create a useful failure indicator.The paper is organized in the following order: Present-day publications ,34 as weTo reflect nonlinear dynamic behavior of the structure with breathing delamination and to perform preliminary analysis, the dedicated numerical model of a vibration signal was created. The model must be considered simplified because it is not reflecting any particular breathing delamination of any specific structure. Additionally, it assumes that change of instantaneous frequency takes place in a contiguous way without rapid transitions.For the preliminary tests, it was assumed that half-periods of the created signal will differ by 2 Hz and instantaneous frequency will continuously vary from 9 to 11 Hz and back. In the first approach, indicated as Model 1, the numerical model was created by combining half-periods of two single harmonic signals with different frequencies using the cubic spline interpolation function St , smoothlTo build the numerical signal, the upper half-period of 9 Hz waveform and the bottom half-period of 11 Hz waveform were joined without interval. Due to different curvature of half-periods, the connection point was smoothed with spline interpolation. Due to spline smoothing, the zero-crossing point is shifted from the original zero-crossing of 9 and 11 Hz waveforms. In the The Model 2 ) and instantaneous frequency (Equation (5)) of the analyzed signal:In the considered case of the nonlinear signal with half-period frequency variation, the Hilbert transform demodulation does not allow to properly detect changes occurring in the signal. According to this approach, the investigated signal has weak amplitude and frequency modulations. The spectrum of the obtained instantaneous frequency signal reveals existence of the 9.95 Hz component as well as the contribution of its higher harmonics.The modeled discrepancy of frequency is not correctly represented in the instantaneous frequency . The HilThe Teager–Kaiser energy operator is a differential operator and was presented in  while itTeager–Kaiser energy In , the autFor a certain class of AM-FM signals, described as (Equation (8)):ion (9)) :(9)ETKt=Extending this concept, the TKEO (Equation (6)) can be applied for determination of the envelope (Equation (10)) and instantaneous frequency (Equation (11)):The demodulation procedure based on Equations (10) and (11) is known as the energy separation algorithm (ESA) and was presented in ,48. DiscIt is important to emphasize here, that because in the general case, the base formula (9) allows for determination of approximate value of Teager–Kaiser energy, Equations (10) and (11) are accurate for harmonic signals only. In the case of signals with AM and FM modulations, both for CESA and DESA, the additional high frequency components arise in related Teager–Kaiser energies. In consequence, some additional conditions have to be imposed on the signal to minimize discrepancy, such as : the anaSeveral DESA algorithms exist  and althOne must be aware of the limitations of this approach. The vibration signal coming from breathing delamination is not an AM-FM class signal and does not fulfill the conditions allowing for proper demodulation. As a result, it is not possible to fully correctly estimate changes occurring in the signal. The value of the instantaneous frequency is overestimated. Additionally, as  discusseComparison of analyzed methods clearly show, that only the one based on TKEO demodulation allowed for estimation of modeled changes of IF and revealed phenomena on interest. The spectrum analysis as well as Hilbert demodulation did not make it possible to identify variation of instantaneous frequency.To define applicability of the described Teager–Kaiser weighted instantaneous frequency (Equation (14)), a number of test cases were investigated. Both steady-state and decaying signals (Equation (15)) were analyzed:The reason for performing such tests was the higher ease of FE modeling of the structure with breathing delamination producing decaying signals, which were later used as input data for analysis of the method effectiveness in the case of the cantilever beam with delamination . HoweverSignals with amplitude decaying factor from 1 (no decaying) to 0.1 (fastest decaying) were analyzed to identify whether decaying increases estimation error. For each considered case, the investigated signal was limited to the time range from the beginning till the moment when the signal’s absolute envelope reached the value of 0.2. Statistical parameters for each case, including the signal’s duration, are presented in The quality of the instantaneous frequency estimation was strongly related to the time length of the signal taken to the analysis. For the signals with low values of ux  and uy . Besides, the CF boundary conditions scheme was applied to the whole delaminated beam, meaning that the beam was fixed in one end. The standard surface-to-surface contact interaction introduced between two faces of the delamination, allowed neither penetration nor separation among the sublaminate structures.The finite element modeling for simulation of a delaminated beam behavior was performed with the use of the commercial finite element software ANSYS. The delaminated beam was composed of four blocks, as it is presented in Four-nodal plane elements with a plane strain condition defined in the width direction were used. This is a rather rarely used practice in analysis of beams upon out of plane loading, but this approach was verified by the comparison of the eigenvalues obtained from the discussed model with the eigenvalues of full 3D model. There were no meaningful frequency differences for the first three modes for considered geometry. The use of four-nodal plane elements allows to reduce time cost of computation in comparison to use of 3D brick elements. The additional asset is, that those elements allow to create more realistic conditions at the delamination tips than standard beam elements.As delamination splits part of the beam into two distinct segments, which are connected in the delamination tips and can come into interaction during the vibrations, the additional boundary conditions, called contact conditions ,53,54, hTo enforce maintaining the constraints in contact modeling, the penalty method was used with contact status defined as it is shown in The value of contact stiffness The number of equilibrium equations in this method, for most of the cases, is much smaller compared to other methods and the time of the computation can be shortened.For the purpose of demonstrating the breathing delamination effect, transient analysis were performed with the initial condition imposed as the displacement field. Those conditions recreated deformation of the beam, caused due to application of static, perpendicular force in the beam tip. Delamination of the length The numerical FE model described in To obtain the band-limited signal, low-pass filtration was applied in order to remove influence of higher frequencies. As it can be seen in It can be observed that the deviation of For validation purposes, the signals were also analyzed with the Hilbert transform demodulation approach. In contrast to the TKEO-based method, which allowed to identify variation of 2 and epoxy resin ARALDITE LY3505  with hardener XB 3403 , by the use of vacuum-bag method. During the hand lay-up of the fabric, three 0.0001 m thick Teflon inserts of different dimensions were placed between the 6th and 7th layer of the composite. Four individual test specimens of nominal dimension 0.18 m × 0.02 m × 0.0014 m were cut from the laminate after resin solidification and the inserts were removed after cutting out specimens. The inserts allowed to create a partially separated layer with controlled length and edges. The created delamination span through the whole width of the beams. Throughout vibration tests, specimens were clamped along one of the tips with 0.03 m distance and active length was equal to 0.15 m. The center of delamination was located at 0.075 m from the free end, i.e., in the center of the active part of the specimen. During the experiment, four specimens were tested: one without delamination  of basis weight 163 g/mThe schematic diagram is presented in The measurements were conducted in two steps. In the first one, all specimens were excited with pseudo-random noise signal and modal analysis was performed in the range  Hz. This band covered the first five bending modes of the specimens. The results are presented in It must be pointed out, that in the case of the first two modes, the differences between modal frequencies of the individual specimens can be related to its weight and width variation, as well as influence of mounting errors. First modal frequencies might not be an objective criterion for technical state assessment.In the second step, the specimens were excited with the harmonic signal with the frequency equal to the identified first modal frequency. During the test of a single specimen, the Polytec vibrometer recorded signals from measurement points: M1 (center of specimen) and M2 (free end of specimen—out of delaminated area). For each measurement point, the vibrometer recorded the simultaneously time domain vibration signal of specimen and reference vibration of mounting clamp. The sampling frequency was equal to 12,800 Hz. Duration of the recorded waveforms was 5.12 s.Because fluctuation of instantaneous frequency, discussed in the TSA displacement signals from measurement points M1 and M2 of the successive specimens as well as reference signal, measured by the sensor located on mounting clamp, were processed with the use of the DESA-2 algorithm to obtain The weighted instantaneous frequency of reference signal was oscillating close to the assumed frequency of the excitation signal as it is presented in For comparison of the presented signals and evaluation of delamination, statistical parameters can be used such as: standard deviation (Equation (17)), coefficient of variation (Equation (18)), or values span (Equation (19)) of the weighted instantaneous frequency Values of those parameters computed for all the samples are presented in S and coefficient of variation V. Change of standard deviation between samples with increasing size of delamination is presented in For the performed measurements, the most informative parameters were standard deviation fw caused by opening and closing of breathing delamination. Based on the measures like standard deviation, it is possible to build a failure indicator.The experimental work confirmed periodic changes of weighted instantaneous frequency numerical models were created, reflecting breathing delamination phenomena in vibration signal;the conducted experimental tests confirmed half-period variation of instantaneous frequency resulting from breathing delamination;the weighted instantaneous frequency a failure indicator and a diagnostic method were proposed for detection of breathing delamination type failure.The paper discussed the application of the original Teager–Kaiser weighted instantaneous frequency The main findings of the conducted research can be summarized as:the use of the Teager–Kaiser operator for the construction of the new weighted instantaneous frequency parameter gives much better prospects for delamination detection than in the case of use of instantaneous frequency obtained using demodulation with the Hilbert transform;standard deviation of the method is efficient for the analysis of the real-world vibrational signals, when proper signal processing is applied.The performed research allowed to identify several limitations of the method, e.g., that the presence of noise in the signal causes that the calculation of The main benefit of The proposed approach can be useful for monitoring of simple beam-like structures e.g., composite bridge decks. However, the structure’s boundary conditions must allow for delamination breathing. This occurs for at least some of the boundary condition schemes, such as CF (clamped-free) or CC (clamped-clamped). As it was shown, in the case of a simple cantilever beam, the first mode was sufficient for delamination detection. In the case of more complicated structures, the proper excitation must be implemented to force breathing delamination phenomena to occur in all suspected components of the structure. In such a situation, multimodal testing can be necessary.The experimental research presented in the paper was limited to the case of unidirectional arrangement of reinforcing layers in the specimens. In order to verify the method’s effectiveness for other stacking sequences, future research work will cover testing of specimens with various orientation of layers and the ratio of the stiffness of the separated part (upper sublaminate in The definition of the applicability scope of the method to other types of failures and development of the algorithm, allowing for identification of size of delamination, is an open issue and will be the subject of future work.


Here, we expanded our existing computational models of Ca2+ signalling in heart to include MT-dependent mechanotransduction through X-ROS. Informed by new focused experimental tests to properly constrain our model, we quantify the role of X-ROS on excitation-contraction coupling in healthy and pathological conditions. This approach allowed for a mechanistic investigation that revealed new insights into X-ROS signalling in disease including changes in MT network density and post-translational modifications (PTMs), elevated NOX2 expression, altered Ca2+ release dynamics (i.e. Ca2+ sparks and Ca2+ waves), how NOX2 is activated by and responds to stretch, and finally the degree to which normalizing X-ROS can prevent Ca2+-dependent arrhythmias.Microtubule (MT) mechanotransduction links diastolic stretch to generation of NADPH oxidase 2 (NOX2)-dependent reactive oxygen species (ROS), signals we term X-ROS. While stretch-elicited X-ROS primes intracellular calcium (Ca The study conformed to the principles and regulations of The Journal of Physiology (ad libitum and were exposed to a 12 h light-dark cycle. Mice were deeply anaesthetized with isoflurane anaesthesia (3%), delivered by a veterinary grade vaporizer. Euthanasia was by rapid exsanguination secondary to the removal of the heart.Animal care and procedures were approved and performed in accordance with the standards set forth by the University of Maryland, Baltimore, Institutional Animal Care and Use Committee  and the ysiology . WT . Experiments were performed at room temperature (25°C).Adult, male mice (8–16 weeks) were anaesthetized by isoflurane vapour, followed by the excision of the heart and enzymatic isolation of ventricular cardiomyocytes (VCMs) as previously described . Cardiomα-tubulin antibody  and phalloidin conjugated with Alexa Fluor 633 (Thermo Fisher Scientific), followed by a 2 hour incubation in secondary anti-mouse antibody conjugated with Alexa Fluor 488 (Thermo Fisher Scientific). VCMs were imaged using a Nikon A1R inverted confocal microscope with a ×60/1.4 NA oil-immersion objective .Microtubule and sarcomere structure were profiled in cells fixed in paraformaldehyde (4%) for 15 min, followed by 10 min permeabilization with Triton X-100 (0.1%). SuperBlock™ phosphate-buffered saline (PBS) was used for blocking procedure and antibody dilution . The cells were incubated overnight (4°C) with mouse α-tubulin and its detyrosinated form , as described  stretch (All experiments were performed in custom-fabricated cell chambers mounted on an inverted microscope. The diastolic stretch was performed as previously described (μM of Fluo-4-acetoxymethyl (AM) ester (Thermo Fisher Scientific) and 0.01% Pluronic F127  followed by an additional 10 min for de-esterification. Cells were scanned using a 488 nm laser in a Nikon A1R inverted confocal microscope with a ×40/1.3 NA oil-immersion objective. The acquisition was performed in confocal line-scan mode at 1.84 ms per line. Automated analysis of line-scan images for Ca2+ spark location and properties were performed using ImageJ  with SparkMaster plugin  quench’ technique as we previously described . Here myocytes were loaded with 5 μM Fura-2 AM (Thermo Fisher Scientific) and 0.01% Pluronic F127 (see above) for 15 min followed by 10 min for de-esterification. Cells were imaged using an IX70 inverted fluorescence microscope  with a ×40/1.1 NA water-immersion objective. The Fura-2 was excited at its isobestic point using a 360 nm light from the DG-4 illumination system  and the emission was captured with a sCMOS camera  each second. Prior to stretch trial, the perfusion with normal Tyrode solution was switched to a Mn-Tyrode solution to establish the basal Mn permeability (30 s). Cells were then stretched for 60 s with a sinusoidal waveform  as described previously  for 5 min, followed by a second trial of Mn2+ quenching and 2 Hz cyclic stretch.To determine the relative magnitude of Cat test (for two groups) or one/two-way ANOVA (for more than two groups) was used for normally distributed data , full duration, half maximum (FDHM) and amplitude, 2+ spark frequency, post hoc test for multiple comparisons  and SPSS Statistics v 26 ).All the data are presented as mean value ± standard error of the means (SEM) unless otherwise noted. Following a test for normality, either Student’s unpaired ted data , 3 and 72+ signalling in heart utilized here are based on published work by our group  and ion current gating variables. These ODEs are then coupled to two discrete-state, continuous-time Markov chains representing the stochastic gating of the LCC and RyR2. See 2+ diffusion utilized consists of four reaction-diffusion style, partial differential equations which govern the diffusion of free cytosolic Ca2+, Ca2+ bound to Fluo-4, free SR Ca2+ and Ca2+ bound to Fluo-5N over the simulated region. This spatial region was designed to mimic a transverse confocal linescan of a ventricular cardiomyocyte. Within this spatial domain, Ca2+ release units (CRUs) are randomly distributed along the transverse direction with a mean inter-CRU distance of 0.6 μm. Details regarding channel gating were identical between the compartmental and spatial formulations. See The computational models of CaBased on results from our group and others, we have focused modelling MT-dependent MCT via three primary components that activate during sarcomere stretch: (1) mechanical strain within MTs activates NOX2; (2) NOX2 generates X-ROS signals; and (3) X-ROS signals sensitize the activation of RyR2 and SACs  is governed by:k  is a rate constant with units of s−1  which is given by:ρX is an ‘X-ROS scalar’ formed by the relative densities of MTs and NOX2  such that ρX = ρMTρNOX2, ρXo represents a basal oxidization factor, and πXA is the probability of NOX2 being activated (in state ‘A’ from above). Each RyR2’s gating is determined by the following two-state, continuous-time Markov chain represented by the following transition-state diagram,k12 is the basal RyR2 opening rate in μMη− s−1, Φ is a luminal regulation function  current was added to the existing computational model and was constrained by Ca2+ influx measurements shown here , and ECa is the Nernst reversal potential for Ca2+.Evidence suggests that sarcolemmal stretch-activated channels  contribute to the dynamic regulation of Cahere see . This Ca2+ signalling in the heart. First, we quantified the alterations in MT network associated with disease (i.e. MDX), then we characterized the amount of Ca2+ that enters a VCM during stretch, and finally how Ca2+ spark properties are altered during stretch in both healthy and diseased hearts. The computational model was constrained and simulations were performed to assess the ability of the model to make predictions regarding MT-driven changes in Ca2+ signalling.Having established the formulation for our computational model, we next performed a focused series of experimental tests to expand our understanding of three main areas of stretch-dependent Caα-tubulin  quench technique to assess the magnitude of Ca2+ influx in Fura-2-loaded VCMs both at rest and with stretch imposed by our single myocyte stretch device (see μm). Immediately upon application of Mn2+ containing perfusate, cation influx was observed via quenching of Fura-2  and spatial extent  of Ca2+ sparks are insensitive to stretch, the temporal extent  is significantly increased  demonstrated that the rate of X-ROS generation is maximal immediately upon stretch and rapidly decays with either the immediate return to resting length or if the stretch is maintained (βMT) that activates NOX2 to initiate a burst of ROS. If the stretch is sustained , this behaviour was translated into a change in RyR2 gating .First, our analysis of both model- and experiment-derived sparks showed that the model also identified FDHM as a spark parameter sensitive to stretch  and 5B. mdx model of murine DMD, we linked the disease-dependent increase in MT density, the abundance of glu-tubulin and increased NOX2 expression to the deleterious increase in Ca2+ sparks and arrhythmogenic Ca2+ waves. As an empirical test of our model we sought to account for this behaviour by altering model parameters a priori based on our published and new experimental results.A major pathological sequelae of Duchenne muscular dystrophy (DMD) is cardiomyopathy and arrhythmia. In the vs. WT  in our model as conservative estimates for their influence on X-ROS in MDX. We also included alterations in SERCA and NCX activity associated with MDX animals  is responsible for translating the mechanical energy of stretch to activate NOX2. However, within the cell, MT strain is determined by glu-tubulin-enriched MTs in concert with intermediate filaments (IF) and other cytolinker proteins, which also exhibit increased expression in disease. Furthermore, evidence suggests that MT and other cytoskeletal changes regulate the mechanical properties (i.e. stiffness) of the cytoskeleton to regulate MCT  and SR Ca2+ (by ~50 μM) was a parsimonious explanation for the sustained level of spark activity seen here, we remain intrigued that this level of Ca2+ could also activate nNOS-dependent NO signals (Our experiments, and computational model, also revealed MCT through X-ROS as a regulator of mechano-sensitive sarcolemmal Ca disease  regulatiin vitro approaches as shown here (see In summary, we presented a new computational model integrating MT-dependent MCT and cardiomyocyte ECC through X-ROS. In conjunction with advanced here see , this mo


Macadamia nut can induce fatal allergic reactions and changes in dietary habits will raise their consumption in industrialised countries. Until now diagnosis of macadamia nut allergy by sIgE solely relies on the macadamia nut extract, but single components are lacking.Macadamia nut proteins recognised by IgE from 2 macadamia nut extract positive sera were identified by mass spectrometry . Sensitisation to macadamia nut extract and heterologously expressed isoform VLAP-2–3 was evaluated in 82 nut allergic (NA) and 27 tolerant (NT) patients  comprehending 10 macadamia nut allergic (MA) and 18 explicitly reported macadamia nut tolerant patients (MT), using line blots. Co-sensitisation to additional VLAP isoforms and other vicilins was evaluated in 8 MA, 12 MT and 14 NA patients sensitised to VLAP-2–3. Functional properties were determined by indirect basophil activation.Even though proteins recognised by IgE were identified as VLAP-2–1, 2–2 and 2–3, only peptides specifically belonging to VLAP-2–3 were detected by mass spectrometry. The macadamia nut extract was recognised by 33% of NA patients (27/82) including 3 MA patients and 26% of NT patients . Similarly, 29% of NA (24/82) patients showed partly strong sIgE-binding to VLAP-2–3 including 3 MA patients with systemic reactions to macadamia nut. Contrary, VLAP-2–3 was recognised by only 2 NT (1 MT) patients (7%) with very low sIgE titres. Simultaneous recognition of the isoforms VLAP-2–1 and 2–2 was observed in all patients positive for VLAP-2–3 with partly reduced sIgE titres in 59% of these patients. Additionally, all three VLAP isoforms were able to repeatedly induce BAT reactivity upon sensitisation with a MA serum.VLAP proteins are the first described macadamia nut components with serological and functional allergenic properties and they are associated with systemic reactions to macadamia nut. Macadamia nut belongs to the group of tree nuts being able to induce even fatal allergic reactions , 2. EvenE. coli [For identifying novel macadamia nut allergens, proteins of crude macadamia nut extract were separated by 2D gel electrophoresis and subsequently used for western blotting. Proteins recognised by sIgE from two commercially purchased sera were analysed by mass spectrometry (MS) and proteins with known amino acid sequences were heterologously expressed in E. coli . For serMacadamia integrifolia proteins).Particularly proteins with a molecular mass between 53 and 67 kDa and an isoelectric point (pI) between 6.3 and 8.7 were strongly bound by sIgE from 2 macadamia nut sensitised sera , recognising both the macadamia nut extract and VLAP-2–3, showed increased sIgE titres against VLAP-2–3 (23 to 105 RU) compared with the extract (17 to 70 RU). Reversed sensitisation patterns were observed for the 3 MT patients positive for macadamia nut extract (3 to 18 RU) whose sIgE titres to VLAP-2–3 were lower compared with the extract. Their sIgE titres against VLAP-2–3 were either very low (5 RU) or even undetectable. While all 3 MA patients positive for VLAP-2–3 experienced moderate to severe symptoms, the other 7 MA patients except MA-7 suffered from rather mild to moderate symptoms , MT patients recognised VLAPs with almost negligible sIgE titres, indicating the potential of VLAPs to discriminate between MA and MT patients positive for the macadamia nut extract. Moreover, MA patients sensitised to VLAPs experienced moderate to severe symptoms upon ingestion, highlighting the potential of VLAPs as potential markers for systemic reactions to macadamia nut. Accordingly, the recombinant vicilin from walnut, Jug r 2, has been described as a marker for severe allergic reactions in patients without pollen-related sensitisation , 7. MoreIn conclusion, VLAPs from macadamia nut appear to be supportive in identifying patients with systemic reactions to macadamia nut and should be incorporated in component-resolved diagnostics of macadamia nut allergy.Additional file 1. Detailed description of methods. Detailed description of the MS analysis and the indirect basophil activation test related to the description within the manuscript.Additional file 2. Detailed and blinded information on the patient population. The data include detailed and individual information for the sera used within this manuscript.Additional file 3. Enclosed proteins detected by 2D gel electrophoresis and mass spectrometry analysis. The macadamia nut extract was separated by IEF and SDS-PAGE. Spots corresponding to sIgE-binding were analysed by mass spectrometry using peptide mass fingerprinting. The table shows the identified proteins in this fraction and MS/MS results are displayed in green.Additional file 4. Amino acid sequence alignment of the different VLAP isoforms. Amino acid sequence alignment of the different VLAP isoforms with highlighted amino acid substitutions; Cyan: VLAP-2-1 ≠ VLAP-2-2 and VLAP-2-3; Red: VLAP-2-2 ≠ VLAP-2-1 and VLAP-2-3 Green: VLAP-2-3 ≠ VLAP-2-1 and VLAP-2-2.


Obesity is a major risk factor for diabetes and cardiovascular diseases such as hypertension, heart failure, and stroke. Impaired endothelial function occurs in the earliest stages of obesity and underlies vascular alterations that give rise to cardiovascular disease. However, the mechanisms that link weight gain to endothelial dysfunction are ill-defined. Increasing evidence suggests that endothelial cells are not a population of uniform cells but are highly heterogeneous and are organized as a communicating multicellular network that controls vascular function.To investigate the hypothesis that disrupted endothelial heterogeneity and network-level organization contribute to impaired vascular reactivity in obesity.To study obesity-related vascular function without complications associated with diabetes, a state of prediabetic obesity was induced in rats. Small artery diameter recordings confirmed nitric-oxide mediated vasodilator responses were dependent on increases in endothelial calcium levels and were impaired in obese animals. Single-photon imaging revealed a linear relationship between blood vessel relaxation and population-wide calcium responses. Obesity did not alter the slope of this relationship, but impaired calcium responses in the endothelial cell network. The network comprised structural and functional components. The structural architecture, a hexagonal lattice network of connected cells, was unchanged in obesity. The functional network contained sub-populations of clustered specialized agonist-sensing cells from which signals were communicated through the network. In obesity there were fewer but larger clusters of sensory cells and communication path lengths between clusters increased. Communication between neighboring cells was unaltered in obesity. Altered network organization resulted in impaired, population-level calcium signaling and deficient endothelial control of vascular tone.The distribution of cells in the endothelial network is critical in determining overall vascular response. Altered cell heterogeneity and arrangement in obesity decreases endothelial function and provides a novel framework for understanding compromised endothelial function in cardiovascular disease. •The endothelium is an assembly of exquisitely organized cells in a highly-refined network that is essential to function.•In obesity, the network is disorganized into fewer, larger sensory clusters with increased path lengths between clusters•2+ responsesThe disorganized endothelial network resulted in deficient encoding of vasoactive stimuli to population level Ca•Changes in the network organization of endothelial cells contributes to obesity-related vascular dysfunction. Each ofImpaired endothelium-dependent function is also observed in various animal models of obesity, including obese Zucker ,12 and J2+ is a second messenger that relays signals from numerous endothelial cell membrane receptors to effector proteins that govern vascular function [2+ levels control endothelial derived hyperpolarizing factor, and regulate the synthesis and release of vasoactive mediators such as NO  or can grow, by recruitment of neighboring channels, into Ca2+ waves that propagate within cells or between cells. Studies of human arteries highlight a deficit in subcellular and whole-cell endothelial Ca2+ signals in obesity [Intracellular Cafunction . For exareviewed ). Ca2+ s obesity ,27, but  obesity  or absen2+ dynamics [Our recent work shows that endothelial control of vascular function is driven by cellular heterogeneity and large-scale, multicellular network Cadynamics . Even widynamics . Distincdynamics . These fdynamics  and funcdynamics  in endotdynamics ,44, and dynamics . The rol2+ dynamics in large populations of endothelial cells in intact blood vessels. We show that agonist-evoked, endothelium-dependent vasodilation arises from network-level interactions that occur among a heterogeneous endothelial cell population, and that this network-level control is impaired in prediabetic obesity. Obesity alters the network, leading to a reduction in the size of the agonist-sensitive cell population, an increase in the clustering of sensory cells, and an increase in the communication distance between cell clusters. These changes in endothelial cell network dynamics impairs the collective endothelial response and provides a new framework for understanding vascular dysfunction in obesity.Given the importance of endothelial function to the health problems in obesity and the absence of information on the pathophysiological correlate of endothelial cell heterogeneity, we investigated networked Ca2All data underpinning this study is available from the authors upon reasonable request. An expanded Material and Methods section can be found in the Supplemental Materials.2.1All animal care and experimental procedures were conducted in accordance with relevant guidelines and regulations, with ethical approval of the University of Strathclyde Local Ethical Review Panel and were fully licensed by the UK Home Office regulations  Act 1986, UK) under Personal and Project License authority. Studies are reported in compliance with the ARRIVE guidelines . Twenty-2.22+ imaging of en face artery preparations with and without simultaneous assessment of vascular tone. First-order mesenteric vessels were selected as a widely used model of vascular and endothelial function since there is a wealth of published background information to aid experimental design and data interpretation. For pressure myograph experiments, we used the open source pressure myograph system, VasoTracker [2+ imaging experiments, arteries were opened longitudinally, and endothelial cells were preferentially loaded with the Ca2+ indicator, Cal-520/AM (5 μM). Network-level Ca2+ signaling was imaged using a high (0.8) numerical aperture 16× objective (~0.8 μm2 field of view). This 16× objective also permitted quantification of vascular contractility in opened arteries using edge-detection algorithms [2+ signaling was imaged using a high (1.4) numerical aperture 100× objective. Network-level and subcellular Ca2+ signaling was assessed using custom Python software [2+ responses were evoked by agonists or photolysis of caged inositol triphosphate (IP3). Images were acquired at either 10 Hz (agonist- or caged IP3-evoked activity) or 20 Hz (spontaneous activity).First-order mesenteric arteries from SD- and HFD-fed rats were studied using pressure myography, and high-resolution single photon CaoTracker  and recogorithms . Subcellsoftware ,51. Ca2+2.3t-tests, independent 2-sample t-tests (with Welch's correction as appropriate), ordinary or repeated measures two-way ANOVA with multiple comparisons as appropriate and as indicated in the respective figure or table legend. All statistical tests were two-sided. A p value of <0.05 was considered statistically significant.Summary data are presented in text as mean ± standard error of the mean (SEM), and graphically as mean ± SEM or individual data points with the mean indicated. Graphically, paired data points are indicated by connecting lines. Concentration-response data were computed according to a three-parameter dose–response model. Data were analyzed using paired 33.13 muscarinic receptor antagonist, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide , or the NO synthase inhibitor, L-NG-Nitro arginine methyl ester , each applied intraluminally (+ channel blockers TRAM34 (1 μM) and apamin  in SD and HFD groups. These results suggest that the major mechanism underlying relaxation is endothelial NO production.Rats on a high-fat diet developed obesity, without complications associated with diabetes . To examine endothelial function in this model of obesity , we first assessed endothelium-dependent vasodilation to acetylcholine (ACh). ACh evoked concentration-dependent relaxations of established tone  in mesenteric arteries from SD and HFD rats A. In eacThe muscarinic-receptor mediated, NO-dependent relaxations were impaired in mesenteric arteries from the HFD group D. Howeve2+-dependent process [2+, we measured endothelial Ca2+ levels and vasoactivity before and after block of NO production or buffering endothelial Ca2+. Inhibiting NO synthesis using L-NAME (100 μM) enhanced PE-evoked contractions and inhibited ACh-evoked relaxations but had no effect on endothelial Ca2+ levels in SD or HFD groups . Preventing endothelial Ca2+ changes, by buffering Ca2+ with BAPTA, reduced basal intracellular Ca2+ levels, potentiated PE-induced contractions and inhibited ACh-induced relaxations in SD- and HFD-fed groups . The latter results demonstrate that endothelial Ca2+ signaling is required for endothelium-dependent relaxations.Endothelial production of NO is generally considered to be a Ca process . To dete3.22+ signaling and endothelium-dependent relaxation, we examined population-wide Ca2+ activity using high spatiotemporal resolution, wide-field single photon imaging (50–100 cells). We used a full ACh concentration series (1 nM–10 μM) to activate the cells while Ca2+ activity was recorded. The Ca2+ responses obtained in these experiments were normalized to maximal responses induced by ionomycin (1 μM), which were statistically similar in SD and HFD  than in SD (~80%).To examine the precise relationship between endothelial Ca and HFD A, inset,2+ response in obesity, we first determined the source of endothelial Ca2+ signals in our experimental model. To do this, the effects of several pharmacological interventions on various endothelial Ca2+ signaling parameters  were examined , consistent with previous studies demonstrating the role of the M3 receptor in the endothelial response to ACh [2+ (Ca2+-free PSS with 1 mM EGTA included in PSS), endothelial cells remained responsive to ACh, although the rise in cytoplasmic Ca2+ concentration was not sustained . This result suggests that the initial response to ACh involved Ca2+ release from the internal store and that Ca2+ influx is required for the sustained phase. ACh-evoked responses were significantly reduced by the phospholipase C inhibitor, U73312 (2 μM), when compared to its inactive analogue U73343 (2 μM). Compared to control responses (no drug) and in contrast to U73312 (2 μM), U73343 (2 μM), did not reduce steady-state Ca2+ responses or the number of active cells although the first peak was reduced. Two IP3 receptor (IP3R) inhibitors, 2-aminoethoxydiphenyl borate  and caffeine , did not reduce ACh-evoked endothelial Ca2+ activity. These results demonstrate that ACh evokes a Ca2+ signalling cascade consisting of: 1) IP3-mediated Ca2+ release from the internal store; and 2) Ca2+ influx via store-operated Ca2+ entry [2+ entry is dependent on that of the preceding Ca2+ release event, these results suggest that the impaired response in obesity arises from either reduced Ca2+ release from activated IP3 receptors or a reduction in the ability of IP3 to generate Ca2+ release.To explore the mechanisms underlying the impaired Cae to ACh . In the  , each i2+ entry ,57. As t3-mediated Ca2+ release in obesity, dynamic concentration-dependent activity from individual endothelial cells in intact blood vessels was examined. Spatial Ca2+ activity maps, generated from ΔF/F0 datasets show heterogeneity in the response of individual endothelial cells to ACh  of Ca2+ increases evoked by ACh were significantly impaired in the HFD when compared to the SD controls . This finding rules out the possibility that the reduced ACh-evoked endothelial Ca2+ activity in the HFD group arises from an impaired ability of IP3 to evoke Ca2+ release.One possible explanation for these results is that IP3.32+ signaling, and that an inability of IP3 to evoke Ca2+ release does not explain the findings. Intracellular communication is critical in determining coordinated endothelial responses. Interactions among endothelial cells permit Ca2+ signals in discrete clusters of cells to coalesce into networked signaling patterns of activity that drive tissue-level responses [2+ responses in obesity because of either or both of: 1) alterations in the spatial distribution of endothelial sensitivity to ACh; and 2) altered communication between endothelial cells.The results presented so far suggest that obesity impairs vascular responses by reducing endothelial Caesponses ,59. Thus3-mediated Ca2+ activity occurring between neighboring endothelial cells (2) were imaged and regions of interest were generated for each cell visualized . In both experimental groups, endothelial cells were arranged in a lattice network and each cell possessed an average of six immediate neighbors . This type of analysis provides information on how cells encode information at the population level and how they interact, yielding insights into network communication mechanisms. In SD and HFD-fed animals, the mean pairwise correlation coefficients significantly exceeded chance levels . However, the extent of pairwise synchronicity was statistically similar in the two groups . This result indicates that the extent of communication between activated cells is unaltered in obesity. In support of this conclusion, Ca2+ wave propagation initiated by focal release of IP3 was similar in SD and HFD . This finding also suggests that communication among cells is unaltered in obesity. Thus, whilst intercellular communication is unaltered, the density of ACh-sensitive cells is reduced, and the clustering of agonist-responding cells is increased in obesity.We next examined endothelial network communication by determining the functional connectivity of adjacent cells. In this analysis, a pairwise cross-correlation analysis was used to assess signal similarity between all neighboring endothelial cell pairs . In contrast to ACh-evoked Ca2+ activity, basal (unstimulated) Ca2+ events are mostly subcellular waves and the initiation site of individual events can be readily identified . However, there was no difference in the extent of endothelial Ca2+-event-MEP coupling in SD- and HFD-fed rats . Thus, it is unlikely that alterations in myoendothelial coupling contributes to the vascular dysfunction in obesity observed in the present study.An alternative route by which the endothelial network may be compromised is via changes in local signaling events. In many vascular diseases (e.g. hypertension ), dysfunchannels ,61, whicsee also ,32). Imp42+ responses. Functionally, the network deficit manifested as impaired vasodilator responses. Abnormal vasodilator function occurred despite a lack of effect on intercellular Ca2+ signal propagation or pairwise synchrony between adjacent endothelial cells, suggesting a spatial network disruption rather than failure in network communication. Together, these findings: 1) highlight the role of population-level interactions in governing endothelial function, 2) offer functional insight into the endothelial cell heterogeneity seen in many genetic studies; and 3) provide support for a new cellular heterogeneity and organization hypothesis of endothelial dysfunction in cardiovascular disease.Obesity, a disease characterized by excess body fat, is associated with type 2 diabetes, metabolic syndrome and cardiovascular diseases such as hypertension and stroke. Impaired endothelial function is a hallmark of obesity-related cardiovascular diseases , but mec2+ activity in individual cells at the population level (hundreds to thousands of cells) reveals spatial modules, or clusters of sensory cells, that detect activators and recruit agonist-insensitive cells to drive network activity. Recruitment of agonist-insensitive cells occurs via the transmission and propagation of Ca2+ signals and increases the overall population of active cells contributing to the vascular response. Here, we show that the size of the sensory cell population is reduced in obesity and there is an increase in the clustering of agonist-responsive cells, i.e., in obesity there are fewer but larger modules. Increased clustering of sensory cells results in activated cells communicating to a greater extent with other agonist-activated cells. Since endothelial Ca2+ signals decay with transmission distance [2+ responses and so impair endothelial vasodilator responses.Endothelial cells are interlaced with one another to form a regular mesh  network in which each cell has ~6 adjacent neighbors. This structural architecture is unaltered by obesity. However, recent application of network theory to endothelial cell populations has started to uncover how rich functionality emerges from these structural connections and how network connectivity permits multiple physiological processes to be controlled simultaneously . Observidistance , the incWhile widely acknowledged to exist (e.g. ), the fu2+-dependent dilator mechanisms differ also within specific vascular beds and depend on blood vessel calibre [2+ signaling at MEPs appears important for the relaxation of smooth muscle via endothelium-derived hyperpolarization in small mesenteric arteries [2+-event-IEL hole coupling in SD- and HFD-fed rats. These findings suggest that alteration in myoendothelial coupling is unlikely to contribute to the vascular dysfunction in obesity observed in the present study. Notwithstanding, the activation of the vasodilator pathway NO or endothelial-derived hyperpolarization may depend on the nature [2+ signaling modalities engaged by the vasodilator stimulus. Here, we demonstrate a role for endothelial cell heterogeneity in the agonist-evoked, NO-mediated relaxation of preconstricted first-order mesenteric arteries. Future studies will determine how cell heterogeneity plays a role in other endothelial vasodilator pathways.Endothelial cells in different vascular beds are also heterogeneous and endowed with specific functions suited to the organs they vascularize . There i calibre . In thisarteries , but note nature  or extene nature  of the v2+ responses, the relationship between population-level endothelial Ca2+ signaling and vasodilator function is linear and injective. Precisely how endothelial microcircuits interact and coordinate to control vascular reactivity via tissue-level endothelial Ca2+ signaling is an important question, particularly as the mapping of endothelial Ca2+ levels to vascular relaxation is unaltered in obesity. Cooperativity among cells is essential for generating coherent population-level responses and necessitates communication among endothelial cells. Such communication is achieved in endothelial cell networks via the transmission of IP3 or Ca2+ or both, through intracellular gap junctions that connect neighboring cells [2+ signals of neighboring cells was similar in control and obesity, suggesting comparable functional connectivity. Second, evoked Ca2+ wave propagation was similar in control and obesity. Consistent with a cell heterogeneity hypothesis of endothelial dysfunction, these findings suggest that the mechanisms underlying endothelial cell cooperativity are largely unaltered in obesity.In the present study, despite the unambiguous existence of local heterogeneity in cell organization and Cang cells ,82. Two 2+ signaling components, such as the various IP3R isoforms [2+ entry such as STIM and ORAI [2+ signals, is also heterogeneous across the endothelium [2+ signaling patterns in obese animals [Our finding that the density of agonist-sensitive endothelial cells is decreased, while clustering of agonist-responsive cells is increased, demonstrates that altered endothelial cell heterogeneity and network organization are responsible for deficient vascular function in obesity. Recent single-cell RNA sequencing studies of whole aorta have also identified distinct endothelial cell subpopulations . These sisoforms , or compand ORAI  may eachothelium . This ob animals ,17.Molecular and functional diversity in endothelial cells may arise from local microenvironmental factors. In support, transplanted endothelial cells gain structural phenotypes and gene expression patterns associated with new host tissue ,91 and eIn conclusion, the present results show that the endothelium is a collection of exquisitely organized cells, and that this organization governs endothelial function. Impaired vascular function in obesity arises from alterations in endothelial cell heterogeneity, specifically the altered distribution of sensory cells, and results in deficient network-level function (vasodilation). As the mechanisms disrupting endothelial network function are not yet clear, significant work remains to address a heterogeneity model of vasomotor dysfunction. Future studies will investigate drivers of altered endothelial heterogeneity and network dysfunction in obesity and its related vascular diseases. As collective cell behavior also controls the regeneration of injured endothelium , the conJGM & CW developed the concept. NMNA & SD developed the animal model. CW, XZ, MM, MDL, CB & HRH performed the experiments, and analyzed the data. CW, XZ, MDL, CB, HRH, & JGM interpreted the data. CW & JGM drafted the manuscript. All authors revised and edited the manuscript and approved the final version of the manuscript.None.


Heart failure is often associated with impaired cardiac relaxation. Little, however, is known about how diastolic intracellular Ca2+ concentration is regulated. This article first discusses the reasons for this ignorance before reviewing the basic mechanisms that control diastolic intracellular Ca2+ concentration. It then considers how the control of systolic and diastolic intracellular Ca2+ concentration is intimately connected. Finally, it discusses the changes that occur in heart failure and how these may result in heart failure with preserved versus reduced ejection fraction.Normal cardiac function requires that intracellular Ca So then I could tell themWhere the wind goes…But where the wind comes fromNobody knows.—AA Milne, “Wind on the Hill”1 the focus of this article is not on the extensively studied mechanisms that deliver calcium ions to the myofilaments and thereby produce systole. Rather, we review the much less well understood removal of Ca2+. Specifically, we will consider how diastolic intracellular Ca2+ concentration ([Ca2+]i) is controlled and how it changes in disease.In keeping with the above quotation from the collection of poems for children by A.A. Milne,2+]i results from the need for the myofilaments to be deactivated to allow ventricular filling. There may, however, be reasons for ensuring that diastolic [Ca2+]i is not too low as, the lower it is, to reach a given systolic level, more Ca2+ must be added to and removed from the cytoplasm on each beat. This will increase energy expenditure, and since Ca2+ cycling accounts for about 30% of the energy consumption of the myocardium,2 this may be a significant factor in requiring that diastolic [Ca2+]i is not too low.An upper limit for diastolic [Ca2+]i, the more Ca2+ must be added to produce a given increase in [Ca2+]i. This is because, at low [Ca2+]i, the cytoplasmic Ca2+ buffers become less saturated and their ability to absorb Ca2+ increases. Conversely, as [Ca2+]i increases, buffering power will decrease so a given increase in [Ca2+]i will require a smaller increase in total Ca2+ . In other words, by altering the level of saturation of buffers, diastolic [Ca2+]i determines the amplitude of the systolic transient produced by a given rise of total Ca2+ and therefore alterations of diastolic [Ca2+]i change the inotropic response. (2) A further consideration is that force depends steeply on [Ca2+]i so that, starting from an elevated diastolic [Ca2+]i, a smaller increase in [Ca2+]i will be required to produce the same change of force compared with at a normal diastolic [Ca2+]i.4 Therefore, an increase in diastolic [Ca2+]i will increase the level of developed force produced by a given systolic rise of [Ca2+]i.There are 2 factors. (1) The lower the diastolic i is increased by increasing the size of the L-type Ca current8 or the amount of Ca stored in the SR.10 The latter is determined by the balance of cellular Ca2+ fluxes. For example, increasing SERCA activity or decreasing Ca efflux on NCX will increase SR Ca2+ content. The decay of the Ca transient is largely due to SERCA-mediated reuptake into the SR with, particularly in larger species, significant contributions from NCX.4 The rate of this decay would be expected to affect end-diastolic [Ca2+]i since, all other things being equal, a faster decay will mean that [Ca2+]i is reduced to a lower level by the time of the next beat, resulting in a lower end-diastolic [Ca2+]i.The pathways that underlie cardiac calcium cycling are well understood6 Figure ; the ind2+]i. (1) Problems of indicator calibration make it much easier to measure changes than absolute levels of [Ca2+]i. This is a particular issue when comparing measurements between cells or animals. (2) When nonratiometric, Ca2+-sensitive, fluorescent indicators are used, the records are often normalized to the diastolic or resting fluorescence,11 making it difficult to measure diastolic [Ca2+]i. (3) In experiments using the whole cell version of the patch clamp, diffusion of Ca2+ and Ca2+ buffers into or out of the pipette may contribute to regulation of [Ca2+]i. Indeed, one of the uses of the whole-cell technique is to control the cytoplasmic ionic concentrations. (4) The major issue may be that, particularly in smaller animals, most experimental work studying Ca2+ cycling in cardiac tissues has used rates of stimulation considerably below normal heart rates. While the fact that ion currents and [Ca2+]i have reached steady state values helps dissect the fluxes responsible for the systolic Ca transient, it establishes an artificial situation. As discussed below, end-diastolic [Ca2+]i represents a balance between many Ca2+-handling mechanisms. In contrast, in a quiescent myocyte, the resting level of [Ca2+]i is determined entirely by the fluxes of Ca2+ across the sarcolemma13 because, in the steady state, there can be no net flux into or out of organelles. Such a net flux would result in a continuous change of organelle Ca2+ content—a situation incompatible with a steady state. At low rates of stimulation, [Ca2+]i will be identical to the resting level seen in the unstimulated case. These frequency-dependent effects are illustrated by making the RyR leaky with caffeine  P2+]i as being separate. From this viewpoint, diastolic [Ca2+]i is controlled at a certain level, and the mechanisms discussed above determine the magnitude of the systolic rise. We think that this is incorrect; the regulation of diastolic and systolic [Ca2+]i is inextricably linked.15 This has been demonstrated recently by investigating the effects of interfering with SR function on diastolic and systolic [Ca2+]i. Consistent with previous data,16 making the RyR leaky with caffeine decreased the amplitude of the systolic Ca2+ transient by decreasing SR Ca2+ content. This was accompanied by an increase in diastolic [Ca2+]i such that the average level of [Ca2+]i over the cycle was unaffected  and, therefore, a decrease in the energy to pump Ca2+ out of the cell  efflux through the RyR, will increase diastolic and decrease systolic [Ca2+]i. For example, in the presence of a normal SR, β-adrenergic stimulation increases systolic but has no effect on diastolic [Ca2+]i. In contrast, when the RyR is leaky, β-stimulation increases diastolic [Ca2+]i.14The average Ca+i is detl Figure . There i2+]i which activate NCX21 and thereby produce arrhythmogenic delayed afterdepolarizations.22 However, the resulting Ca2+ efflux will help maintain Ca2+ flux balance and thereby keep diastolic [Ca2+]i lower than would otherwise be the case. Under these conditions, addition of caffeine to empty the SR and thereby remove waves and their associated efflux results in a steady level of [Ca2+]i, which is greater than the minimum seen in the presence of Ca2+ waves.23Similar considerations also apply to conditions of calcium overload where waves of calcium release from the SR occur. Much attention has been directed to the detrimental effects of these increases in [Ca2+]i alone.26 That work showed that potentiation of RyR opening had no effect on systolic [Ca2+]i in the steady state. In those earlier experiments, the decrease of SR Ca content was exactly compensated for by an increase in fractional release from the SR so the amplitude of the Ca transient and the accompanying Ca2+ efflux were unaltered. Ca2+ flux balance could, therefore, be maintained at constant diastolic [Ca2+]i. In the more recent work,14 the degree of potentiation of the RyR was greater (higher concentrations of caffeine used), and, therefore, the SR Ca content fell to such a low level that even if it is all released, the systolic Ca transient is smaller than control. The consequent decrease of systolic Ca2+ efflux results in systolic efflux being less than influx, thereby loading the cell with Ca2+ and increasing diastolic [Ca2+]i until the increase of diastolic efflux returns the cell to Ca2+ flux balance.These consequences of flux balance are a generalization of those previously described for changes of systolic [Ca2+ fluxes before discussing how their integration leads to control of diastolic and systolic [Ca2+]i.In the context of the above general considerations, we will review important aspects of the underlying Ca2+]i potentially depend as much on the Ca2+ buffering properties of the cell as on the fluxes of total Ca2+.3 In quiescent cells (or at low pacing rates), an increase in buffering is not expected to change diastolic [Ca2+]i, since free [Ca2+]i (and not the Ca2+ bound to buffers) determines efflux, and this must balance influx, which is constant. At higher pacing rates, because an increase in buffering slows the rate of change of [Ca2+]i, the Ca transient cannot decay back to baseline and end-diastolic [Ca2+]i will rise. Accordingly, experimentally increasing the cytoplasmic buffering power slows the rate of decay of [Ca2+]i27 and elevates [Ca2+]i and force in diastole.28 An increase in diastolic [Ca2+]i in hypertrophic cardiomyopathy resulting from some troponin T mutations has been attributed to this mechanism29 and may contribute to contractile impairment at increased heart rates in this condition.The changes of [Ca2+]i will decay, and, all other things being equal, the further diastolic [Ca2+]i will fall before the next beat and, therefore, the lower will be end-diastolic [Ca2+]i. Experimentally decreasing SERCA activity can (see above) increase diastolic [Ca2+]i30 and pressure30 as a consequence of the slowing of the decay of the Ca2+ transient. The increased diastolic [Ca2+]i will compensate for the decreased systolic efflux resulting from the smaller Ca2+ transient thereby maintaining Ca2+ flux balance. It should, however, be noted that acute inhibition of SERCA has been reported to increase [Na+]i,31 and this can elevate diastolic [Ca2+]i via NCX. The origin of this increase in [Na+]i is unclear. One possibility is that the decreased amplitude of the Ca2+ transient will have decreased inactivation of the L-type Ca current, thereby increasing Ca2+ entry and thence efflux on NCX, leading to loading of the cell with Na+. Given that Na+ entry on NCX is a major component of Na+ entry into the cell,32 this will elevate [Na+]i. In another study, knockout of SERCA also elevated [Na+]i.33 These knockout mice have increased L-type Ca current, possibly to compensate for the lack of SERCA. This increased Ca2+ influx will need to be balanced by increased efflux on NCX. The consequent increase in Na+ influx may, therefore, account for the elevation of [Na+]i.The greater the activity of SERCA, the faster systolic i until the increase in diastolic Ca2+ efflux restores total efflux to equal influx. Leaky RyRs also slow the rate constant of decay of the systolic Ca transient.35 Under normal conditions, Ca release from the SR occurs more or less synchronously, a few milliseconds after the start of depolarization, in response to the rise of [Ca2+]i produced by the L-type current. Release from clusters of RyRs can be seen as calcium sparks.11 In contrast, after myocardial infarction, Ca sparks are observed on the falling phase of the systolic Ca transient.15 Increasing RyR phosphorylation and opening by overexpression of CaMKII -δC also leads to the appearance of delayed calcium sparks, which will interfere with the decay of [Ca2+]i and relaxation.35 Such late sparks have also been suggested to be a more general phenomenon particularly when the initial release of Ca2+ from the SR is depressed.36 A study in hypothyroid mice has linked the appearance of late sparks to impaired systolic and diastolic function.37As mentioned in a previous section, making the RyR leaky can decrease SR Ca content and thence the amplitude of the systolic Ca transient and systolic Ca+ entering to pump 1 Ca2+ out of the cell. This stoichiometry generates an electric current,38 and NCX activity is sensitive not only to the Na+ and Ca2+ concentration gradients but also to membrane potential; hyperpolarization increases and depolarization decreases net Ca2+ efflux. Depending on the ionic gradients and membrane potential, NCX can reverse direction with net Ca2+ influx coupled to Na+ efflux (reverse mode). At a normal resting potential, NCX works in the forward direction with Ca2+ efflux roughly proportional to [Ca2+]i.17 It should, however, also be noted that NCX is allosterically regulated by [Ca2+]i, thus limiting Ca2+ efflux at low [Ca2+]i.39 For an extensive review of NCX, see the article by Blaustein and Lederer.40NCX uses the energy provided by 3 Na+]i decreases the driving force available for NCX to remove Ca2+ from the cell and thereby increases developed force and the underlying systolic Ca transient. In rabbit ventricular myocytes, inhibition of the Na-K pump increases both [Na+]i and diastolic [Ca2+]i.41 However, at least with moderate increases in [Na+]i, there is often no increase in diastolic [Ca2+]i43 or developed force/cell length.46 While this may result partly from the low sensitivity of force and some Ca2+ indicators to [Ca2+]i, it may also be explained as follows . At first, this will be largely provided by an increase in systolic [Ca2+]i as a result of the increase in SR Ca content. Only with further reduction of NCX, perhaps because there is a limit to how much SR Ca content and thence systolic [Ca2+]i can increase, will diastolic [Ca2+]i increase appreciably.An increase in [Na2+ efflux is less well established.47 It has been argued that the PMCA is irrelevant to the control of bulk cytoplasmic [Ca2+]i and, instead, has a signaling function by controlling [Ca2+]i in microdomains near caveolae.48 Work from the Bers Laboratory finds that the contribution of the PMCA to Ca2+ removal in a variety of species is typically <10% of that of NCX.49 We find a larger contribution; inhibiting NCX with Ni2+ leaves 25% to 33% of the Ca2+ removal from the cell functional in rat.50 A concern with the use of Ni2+ is that it may not completely inhibit NCX, but similar results are seen when NCX is stopped by removal of Na+ ions.52 The NCX-independent Ca2+ efflux is abolished by the nonspecific PMCA inhibitor carboxyeosin.54 A substantial role for PMCA is also suggested by work on myocytes isolated from NCX knockout mice. These animals live normally, and their ventricular myocytes have normal Ca2+ transients. There is no change of PMCA expression, and the myocytes maintain Ca2+ flux balance by decreasing Ca influx through the L-type Ca current to 20%—a level at which PMCA alone can presumably balance it.56 This suggests that PMCA makes a contribution equivalent to 25% of that of NCX in the wild type. One caveat is that, as in other studies, the rate of Ca2+ removal from the cell was assessed from the rate of fall of the caffeine-evoked rise of [Ca2+]i. The available data do not provide caffeine exposures of sufficient duration to obtain accurate measurements,56 and further work is required to establish the role of PMCA in the regulation of diastolic [Ca2+]i.In addition to NCX, the myocyte also expresses a PMCA whose contribution to Ca2+ uptake and release from mitochondria could affect diastolic [Ca2+]i. As we have recently reviewed,6 there are conflicting reports in the literature with only some studies finding evidence in favor of beat-to-beat movements of Ca2+ into and out of mitochondria. On balance, at least in adult ventricular myocytes, while changes of mitochondrial [Ca2+] can be observed at slow rates of stimulation,57 they disappear at higher rates questioning their importance in regulating diastolic [Ca2+]i.57In principle, Ca2+ entry during the action potential is the L-type Ca current.58 In some regions of the heart, particularly in nodal tissues, there are also contributions from the T-type Ca channel.59 The stoichiometry of NCX means that it can also contribute to Ca2+ influx during depolarization, but, under normal conditions, this is much smaller than that through the L-type channel.60 In heart failure, the increase in [Na+]i will increase influx through NCX,61 and it is possible that the magnitude of Ca2+ influx through NCX may have been underestimated due to making measurements at slow rates where [Na+]i is decreased.The major route for Ca2+]i of maneuvers that alter the L-type Ca current. Inspection of most data shows little effect on diastolic levels,63 but the majority of experiments were performed at slow rates or used the whole-cell patch clamp technique. We found that decreasing the L-type Ca current with cadmium in cells where diastolic [Ca2+]i was elevated reduced diastolic [Ca2+]i.14 From first principles, one would expect 2 opposing effects.64 (1) Increased L-type Ca current will increase Ca2+ influx per unit time thereby requiring an increased average [Ca2+]i to balance it. Depending on the conditions, this may be achieved by increased systolic or diastolic [Ca2+]i. (2) The increase in L-type current will increase Ca2+ release from the SR, increasing systolic Ca, thereby contributing to the elevated average without the need to increase diastolic. This latter effect, however, is limited as it is impossible to release >100% of SR content. It should also be noted that, at least under some conditions, increasing L-type Ca current does not increase SR content.64 Additionally, the increase in systolic [Ca2+]i will increase the time taken for [Ca2+]i to decay back to baseline, increasing the tendency for diastolic [Ca2+]i to rise at shorter pacing intervals (higher heart rates). Quantitative considerations will determine whether the increase in systolic efflux is sufficient to balance the increase in influx or, alternatively, whether elevated diastolic [Ca2+]i occurs.Many studies have investigated the effects on systolic [Ca2+]i is of the order of 100 nmol/L indicating that some kind of background Ca2+ entry pathway must exist to balance Ca2+ efflux on NCX. Such a pathway accounts for the fact that, even in a quiescent cell, after being emptied with caffeine, the SR can be refilled by a mechanism that requires extracellular Ca2+.65 As mentioned above, Ca2+ waves can occur in cells held at a fixed membrane potential,23 again indicating an influx pathway to balance efflux on NCX during the waves. The magnitude of this influx is roughly proportional to external Ca2+ concentration in the range ≤5 mmol/L.23 Subsequent work, examining the effects on resting [Ca2+]i of abruptly removing external Ca2+, provided an estimate for the background Ca2+ influx of the order of 2 to 6 µmol/L per s in rat ventricular myocytes.66 A recent study estimated Ca2+ influx from measurements of average [Ca2+]i (see above) and found a value of about 4 µmol/L per s.14 These values compare to an entry on each action potential via the L-type Ca current of the order of 5 to 10 µmol/L. Therefore, at normal heart rates (in a rat) of 5 s−1, the background influx will be of the order of 10% of that carried by the L-type current. As regards the mechanism of this influx, one study identified a Ca2+ entry mechanism that increased on hyperpolarization of the surface membrane and was blocked by the relatively nonspecific agent gadolinium (Gd3+).67 It is, therefore, important to consider the identity of this flux.In the absence of stimulation, resting i, which can be blocked by Gd3+, and has been attributed to Ca2+ entry via TRPC channels.77 Similar results were found for the experimental myopathy produced by infusion of isoproterenol.78 Further evidence suggesting a role for TRP channels in contributing to setting diastolic [Ca2+]i comes from the observation that knocking out both TRPC1 and TRPC4 in mice decreased diastolic [Ca2+]i.79 The recent synthesis of specific antagonists of TRPC channels80 and an agonist81 should make it possible to study the role of these channels more precisely.Transient receptor potential (TRP) channels are also sensitive to Gd2+ into the cell activated by emptying the SR, so called store-operated Ca2+ entry, and in the HL-1 cell line, this has been suggested to contribute to resting [Ca2+]i.82 One issue is that much of the evidence for a role of store-operated channels in cardiac tissue comes from work on cultured or neonatal cells,84 and these may not be representative of adult myocytes. Some recent articles have, however, reported store-operated Ca2+ entry into adult mouse ventricular myocytes85 with the fluxes being inhibited by Gd3+.87 In many tissues, store-operated calcium entry is produced by a combination of the endoplasmic reticulum Ca2+ sensor STIM1  and the surface membrane channel Orai1 (see the article by Qiu and Lewis88 for review). Overexpression of STIM1 in mouse heart increases diastolic [Ca2+]i, as a result of increased Ca2+ entry into the cell and increased leak from the SR.89 It should, however, be noted that STIM1 has also been reported to interact with PLN and thereby control SERCA.90TRP channels have also been implicated in the influx of Ca+ in addition to Ca2+ and, by altering [Na+]i, could affect [Ca2+]i indirectly via NCX. All in all, it is clear that more work is required to characterize the contribution of TRPs, connexins, and as yet unidentified mechanisms to the background Ca2+ influxFinally, some TRP channels and connexins transport Na2+]i in ventricular trabeculae91 and isolated myocytes.95 It is important to note that the Ca2+ indicators used to measure [Ca2+]i buffer [Ca2+]i to some degree and potentially exaggerate the effects of increased frequency. It would be useful to repeat these experiments using as low concentrations of Ca indicators as possible. Of course the fact that, even in the absence of indicators, increasing stimulation rate increases diastolic force96 and decreases cell length92 means that excessive buffering cannot account for all the effects.Increasing the rate of stimulation increases diastolic [Ca2+]i  The increase in diastolic [Ca2+]i can occur abruptly on increase in rate,94 much faster than the presumed increase in at least global [Na+]i. (2) In the NCX knockout mouse (where changes of [Na+]i would not be expected to increase [Ca2+]i), the effects of rate on diastolic [Ca2+]i are similar to those in wild type.55 As discussed above, and in Figure 2+ influx to be balanced by increased efflux and, therefore, elevated average [Ca2+]i. Why does diastolic [Ca2+]i increase at higher rates? It might be thought that balance could occur simply by the increased frequency of Ca transients resulting in more systolic efflux. This, however, ignores that (1) the Ca transient cannot decay back to equilibrium before the next beat, resulting in end-diastolic [Ca2+]i rising and (2) there is less time for NCX Ca removal from the cell per beat. Finally, in species with a negative force-frequency relationship (and humans with heart failure100), the systolic Ca transient decreases at higher rates presumably reducing the systolic efflux per beat. Both Na-dependent and independent mechanisms may contribute. The increase in Ca2+ entry at higher rates demands increased Ca2+ efflux for flux balance. This will require an increase in average [Ca2+]i. The elevated [Na+]i will decrease NCX activity thereby requiring a greater increase in average [Ca2+]i.There are at least 2 possible explanations for the frequency-dependent increase in diastolic [Cai Figure . One is 2+]i will be determined by the net effects of β-adrenergic stimulation on Ca entry via the L-type current and on the shape and size of the Ca transient, which determine NCX removal. β-Adrenergic stimulation increases influx via ICa-L, which is partly balanced by a larger Ca transient amplitude and, therefore, greater systolic efflux via NCX. This latter effect is curtailed, however, by the accelerated decay of the Ca transient resulting from greater SERCA activity.101 Accordingly, in rat ventricular myocytes, studied at constant rate, β-adrenergic stimulation increases diastolic [Ca2+]i.94 It should also be noted that β-adrenergic stimulation phosphorylates phospholemman, increasing Na-K pump activity, decreasing [Na+]i, and thereby increasing the driving force for NCX-mediated Ca2+ efflux. This will decrease the level of average [Ca2+]i required to balance the increased Ca2+ influx.Physiologically, changes of β-adrenergic stimulation are the main cause of changes of heart rates. The effects of β-adrenergic stimulation will, therefore, be a combination of those of rate, discussed above, as well as direct effects. The latter include an increase in both Ca entry through the L-type Ca current and of SERCA activity (see above—via phosphorylation of PLN). The expected effects of these have been discussed earlier. In brief, the level of diastolic i, would be expected to decrease Ca2+ buffering and accelerate the decay of the Ca transient. Conversely, phosphorylation of PLN will increase affinity and increase buffering and should slow the decay. Experiments on mouse ventricular myocytes found that these effects were balanced such that there was no net effect on Ca2+ buffering.101 Further work is required to investigate the effects of β-adrenergic stimulation in a wider range of conditions.The Ca transient and accompanying NCX removal could also be affected by changes in Ca2+]i. Before turning to the changes of calcium cycling that occur in heart failure, it is important to set these in a clinical context.The previous sections have reviewed the fundamental mechanisms that regulate diastolic [Ca2+]i at the beginning of diastole, leading to dissociation of the thick and thin filaments. In the subsequent, passive phases of diastole, the pressure gradient distends the ventricle.102 While this phase depends heavily on passive properties of the myocardium including wall thickness and fibrosis, it is also determined by diastolic [Ca2+]i by setting the baseline myofilament activation and thus tension. Both active and passive processes require the heart to be sufficiently relaxed and compliant to fill with blood. During exercise, heart rate rises and diastolic interval decreases. Here, LV filling is maintained by increasing transmitral flow via an increase in pressure gradient. In the healthy heart, this gradient is generated by enhanced elastic recoil to reduce LV pressure in early diastole, without significantly changing left atrium pressure.103Ventricular filling in diastole relies on both a compliant ventricle and a pressure gradient between the left atrium and left ventricle (LV). In the early phase of diastole, active ventricular relaxation helps to generate this gradient by actively sucking blood into the ventricle via elastic recoil. This active phase is myocyte dependent and relies on the rapid decline in i (see below), increased extracellular collagen,106 increased myofilament crossbridge interactions due to metabolic changes independent of Ca2+,108 and loss of elastic recoil (due to failure of elastic compression in systole).109Diastolic dysfunction is frequently observed alongside systolic dysfunction in heart failure with reduced ejection fraction (HFrEF). There are multiple mechanisms for slowed relaxation in HFrEF, including abnormalities in Ca110). The increase in chamber stiffness and slowed relaxation observed in HFpEF causes a rise in LV filling pressures, which, when sufficiently high, results in the heart failure syndrome.111 It is increasingly clear that HFpEF is not a condition of diastolic dysfunction alone and some impairment in systolic function is present at rest and becomes more prominent during exercise.112 This systolic impairment may further exacerbate diastolic dysfunction because contractile impairment modifies the restoring forces that drive early diastolic recoil.113 Additionally, HFpEF is associated with a constellation of comorbidities such as diabetes mellitus, obesity, hypertension, aging, and kidney disease. Consequently, HFpEF is accompanied by systemic changes, including inflammation and endothelial dysfunction, tissue fibrosis, microvascular dysfunction and ischemia, and multiorgan impairment such as renal failure and sarcopenia. These contribute both to the diastolic impairment and the overall clinical phenotype.110 In spite of its complexity, the inherent defects underlying diastolic dysfunction can be broadly grouped into 2 classes: (1) external to the cardiac myocyte and (2) resulting from impaired myocyte function.Importantly, however, about half of patients with heart failure have diastolic dysfunction but a normal ejection fraction or heart failure with preserved ejection fraction ,126 which is also present in isolated myocytes.127 However, a significant proportion of HFpEF patients do not have LV hypertrophy, and severity of hypertrophy does not closely correlate with diastolic dysfunction.128 Instead, the bulk of this increase in resting tension can be explained at the sarcomere.Dysfunctional relaxation and higher passive stiffness in HFpEF is present at the level of the cardiac myocyte.130 Its properties can be directly modified by phosphorylation 131 and oxidative modification via disulphide bonds132 and S-glutathionylation.133 As such, in addition to changes in its expression, posttranslational modifications in titin allow for dynamic changes in cellular and diastolic stiffness, which are implicated in the pathophysiology of HFpEF. Finally and intriguingly, the titin N2BA isoform exhibits a small [Ca2+]i-dependent increase in stiffness.135 Although this may add further importance to the role of diastolic Ca2+ in diastolic dysfunction, the significance of this finding in vivo has not yet been established.The giant molecular spring titin, which spans the Z disk to M band, is a major determinant of passive tension by providing recoil in early diastole and resistance to stretch in late diastole.2+]i (see subsequent sections) and their sensitivity to Ca2+. Increased myofilament Ca2+ sensitivity secondary to hypophosphorylation of cardiac troponin I has been reported in HFpEF.136 Furthermore, abnormally high myofilament Ca sensitivity also contributes to the diastolic dysfunction observed in hypertrophic cardiomyopathy caused by sarcomeric gene mutations.138 Accordingly, the increase in resting tension in HFpEF myocytes has been linked with low PKG (protein kinase G) levels, which may impair relaxation by reducing phosphorylation of titin, cardiac troponin I, and PLN.140 A role for defective CaMKII phosphorylation of titin has also been proposed.131 In conclusion, although other factors may contribute to impaired diastolic function, it is important to consider the role of abnormalities in Ca2+ signaling.At the sarcomere level, there is also evidence implicating the actin-myosin filaments in HFpEF. Relaxation of these depends on both diastolic [Ca2+]i change in heart failure? We will first consider data from animal and human studies where systolic function is also impaired before moving on to HFpEF.Does diastolic i is concerned, measurements on ventricular strips from patients with heart failure found increases in diastolic force and [Ca2+]i, which were most obvious at higher stimulation frequencies.141 Ca transients in cells isolated from patients had a smaller amplitude and also slowed decay,42 which would be expected to increase diastolic [Ca2+]i. A subsequent study found little elevation of diastolic [Ca2+]i or force96 but pointed out that the lack of sensitivity of the Ca2+ indicator used may have made it hard to resolve changes of diastolic [Ca2+]i. Experiments on myocytes from patients with heart failure, using more sensitive fluorescent indicators (fluo-3 and fura-red), demonstrated an increase in diastolic [Ca2+]i with increasing rate,142 but no control data were available. Increasing the rate of stimulation increased diastolic force in ventricular muscle strips from patients with heart failure but not controls.143 In a rabbit model of aortic insufficiency/restriction, the amplitude of the systolic Ca transient decreased to about 70% of control with no change of diastolic [Ca2+]i.144 It should, however, be noted that the experiments were performed at a slow rate (0.5 Hz). Another study on rabbit myocytes found that pressure and volume overload–induced heart failure increased diastolic [Ca2+]i,145 and, in contrast to much of the other work discussed here, this was unaffected by stimulation frequency. Finally, a study of right side heart failure  showed a tendency to increased diastolic [Ca2+]i, particularly at elevated stimulation frequencies.146 Further complication is added by reports of a decrease in diastolic [Ca2+]i in a sheep tachypacing model of heart failure, albeit studied at low stimulation rates.147 Interestingly, this was accompanied by a decrease in the L-type Ca current, which would be expected to decrease average [Ca2+]i, perhaps contributing to the decrease in diastolic [Ca2+]i. Unchanged diastolic [Ca2+]i was found in ventricular myocytes from tachypaced dogs, but this also used low stimulation rates and whole-cell patch clamp.148 Decreased diastolic [Ca2+]i has also been found in a ferret aortic banding model of hypertrophy, again at low stimulation rates.149The decreased systolic Ca transient in heart failure may result in large part from a decrease in SR Ca2+]i.150 In another study on rats, using lipopolysaccharide administration, septic cardiomyopathy slowed the decay of [Ca2+]i.151 This was suggested to result from decreased activity of NCX and PMCA. This is surprising because these sarcolemmal transporters make only a small contribution (compared with SERCA) to the decay of the systolic Ca transient in small animals. In contrast, lipopolysaccharide administration in mice also slowed the decrease in the Ca transient, but this was associated with decreased SERCA activity due to sulphonylation.152 This was accompanied by a small decrease in diastolic [Ca2+]i over the range of 1 to 6 Hz, the explanation for which is not clear.An important clinical situation that produces heart failure and depressed myocardial contractility is sepsis. Cecal ligation and puncture in the rat slowed the decay of the Ca transient; this was attributed to increased frequency of Ca sparks and accompanied by decreased systolic and increased diastolic [Ca2+]i and force. More work is needed at physiological rates to characterize this.Although it is not easy to draw conclusions from the above work on patients and animal models with HFrEF, it does appear that in the majority of studies where physiological rates have been studied, there is a frequency-dependent increase in diastolic i and the amplitude of the Ca transient. These effects were attributed, at least in part, to increased Ca2+ leak through the RyR (seen as increased Ca spark frequency) and decreased NCX activity.155 In the other, although the animals and isolated ventricular trabeculae had impaired diastolic function, isolated myocytes, taken from the same hearts, showed lower diastolic [Ca2+]i and shortening, suggesting that the main cause of mechanical dysfunction involved passive mechanisms rather than Ca2+ handling.156 Modeling studies have pointed out that the maintained ejection fraction in HFpEF could be achieved despite a decrease in systolic [Ca2+]i due to the compensatory effect of concentric ventricular hypertrophy.157 Another, recently developed, model of HFpEF is that of an inbred rat with a hypertrophic heart. This has increased diastolic and systolic [Ca2+]i accompanied by an increase in the L-type Ca current.127 It is, therefore, possible that the increase in both diastolic and systolic [Ca2+]i augments Ca2+ efflux to compensate for the increased influx. In the absence of measurements, however, it is impossible to exclude a contribution from effects of [Na+]i mediated via NCX. Interestingly, the rate of decay of the Ca transient was accelerated arguing against decreased SERCA activity.A major issue with studying HFpEF in animals has been the difficulty of producing an appropriate model.2+]i, attributed to altered NCX possibly due to increased [Na+]i.158 A subsequent study159 showed a slowing of the rate of decay of both cell shortening and the systolic Ca transient but no effect on the level of either systolic or diastolic [Ca2+]i. The experiments were, however, performed at a slow rate. This study also found that acute administration of the NCX inhibitor SEA0400 accelerated the decay of the systolic Ca transient, but the mechanism was unclear. Another study from this group found that in vivo administration of another NCX inhibitor (ORM-11035) also accelerated relaxation160—a result consistent with studies on the Dahl salt-sensitive rat where the improved relaxation produced by NCX inhibition was attributed to an effect on fibroblasts, decreasing fibrosis.161Kidney disease is a risk factor for HFpEF, and this has been modeled experimentally by removing 80% of renal tissue resulting in prolonged ventricular relaxation and elevated end-diastolic pressure. Early work found elevated diastolic i during stimulation at 1 Hz.163 It is unclear why diastolic [Ca2+]i should decrease. Some studies have found decreased L-type Ca current,164 which may decrease average [Ca2+]i. In addition, the slowing of decay of the Ca transient will increase average [Ca2+]i thereby allowing a lower diastolic [Ca2+]i as long as there is sufficient time for [Ca2+]i to fall in diastole. A similar study found a decrease in resting [Ca2+]i in unstimulated cells.165 This argues for alterations of background Ca2+ influx or NCX/PMCA. It should, however, be noted that there is evidence that the depression of contractility in the streptozotocin rat may be independent of changes of [Ca2+]i.166 Metabolic dysregulation is also associated with development of HFpEF. A recent study found that the ZSF-1 obese rat had elevated diastolic but similar systolic [Ca2+]i compared with controls.167 Mitochondrial [Ca2+] was also elevated and suggested to partly compensate by stimulating metabolism but also to result in adverse consequences of mitochondrial overload. This article also reported a decrease in diastolic [Ca2+]i with increasing stimulation rate—a result that differs from other studies reviewed here. Atria from the ZSF-1 obese rat also show impaired function but no effect on either the rate constant of decay of the Ca transient or diastolic [Ca2+]i was observed.168 Finally, a recent publication has introduced a mouse model of HFpEF using a combination of high-fat diet and nitrosative stress, which seems to mimic many of the features of the human condition.169 It would be useful to use this model to study [Ca2+]i.As mentioned above, diastolic dysfunction is clinically observed in diabetes mellitus. Work on a streptozotocin rat model, with normal systolic and impaired diastolic function, found a decrease in the rate constant of decay of the systolic Ca transient due to decreased SERCA activity.170Work on ventricular strips from patients with cardiac hypertrophy but normal ejection fraction has found interesting results. When the stimulation rate was increased, preparations from hearts showing LV hypertrophy developed increased diastolic force, and this was abolished by the contractile uncoupler butanedione monoxime suggesting that it resulted from myofilament activation. When SR function was inhibited by the SERCA inhibitor cyclopiazonic acid plus ryanodine, there was an elevation of diastolic force, which was much greater in those preparations that had previously developed significant diastolic force with raised frequency.171). Many of these mutations increase the sensitivity of the myofilaments for [Ca2+]i. This, alone, would increase diastolic force, but, in addition, the increase in Ca2+ buffering slows the decay of [Ca2+]i, elevating end-diastolic [Ca2+]i,29 and, therefore, diastolic force/pressure.Finally, hypertrophic cardiomyopathy can also result in a heart failure syndrome. While this is a separate disease entity to HFpEF, it also leads to diastolic dysfunction. Hypertrophic cardiomyopathy is often an inherited condition that can result from mutations in the sarcomeric proteins, which make up the thick and thin filaments, (reviewed in2+]i increases in heart failure. There are at least 2 (nonexclusive) explanations for this. (1) As discussed in an earlier section, any decrease in the systolic Ca transient will decrease Ca2+ efflux during systole, requiring a compensatory increase in diastolic [Ca2+]i. (2) Another explanation is provided by the increase in [Na+]i commonly observed in heart failure,172 which will decrease Ca2+ efflux on NCX, thereby requiring an increase in average [Ca2+]i, which may, in part, be provided by increased diastolic [Ca2+]i. Consistent with this, elevation of [Na+]i by inhibition of the sodium pump increased diastolic force at elevated stimulation rates.143 Further evidence linking NCX to diastolic function came from work on ventricular strips from failing human hearts showing that the greater the expression of NCX, the better the diastolic function.173 The increase in diastolic force and [Ca2+]i can also be attenuated by the drug ranolazine—a blocker of the late sodium current that decreases [Na+]i.174 Similarly, work on rats found that ranolazine reversed the diastolic impairment produced by the anticancer drug doxyrubicin.175 In canine myocytes, experimental ischemic heart failure increased diastolic [Ca2+]i at elevated rates, and this was normalized by ranolazine or tetrodotoxin.176 Work on mice found that overexpressing CaMKIIδC decreased systolic and increased diastolic force.35 The decrease of systolic [Ca2+]i has been attributed to excessive phosphorylation of RyRs leading to diastolic Ca2+ leak177 as evidenced by increased Ca2+ spark frequency. Again, these effects were reversed by ranolazine thereby linking them to changes of [Na+]i.178 In contrast, a recent study found that ranolazine had no effect on diastolic force in ventricular muscle taken from patients with HFpEF, arguing against a role for changes of [Na+]i.179Although, as discussed above, there is considerable variation between studies, the consensus appear to be that diastolic [Ca2+]i has been suggested in the cardiomyopathy observed in the mdx mouse—a model of Duchenne muscular dystrophy. Here, the elevated diastolic [Ca2+]i is normalized by Gd3+ suggesting that it originates from Ca2+ entry through TRP channels.77As mentioned in an earlier section, a different explanation of elevated diastolic i. These changes could, therefore, account for HFrEF . D.C. Hutchings is an academic clinical lecturer supported by the National Institute of Health Research.None.