Ciona robusta , a model organism with a simple neural circuit, a single pair of MNs (MN2L/MN2R) was determining the rhythm of its spontaneous early motor behavior . MN2s are known to be one of the main components of Ciona CPG, though the neural activities of MN2s in the later larval period (St.25-) were not yet investigated. In this study, we investigated the neural activities of MN2s during their later stages and how they are related to Ciona’s swimming CPG. Long-term simultaneous Ca2+ imaging of both MN2s with GCaMP6s/f (St.22-34) revealed that MN2s continued to determine the rhythm of motor behavior even in their later larval stages. Their activities were classified into seven phases (I-VII) depending on the interval and the synchronicity of MN2L and MN2R Ca2+ transients. Initially, each MN2 oscillates sporadically (I). As they develop into swimming larvae, they gradually oscillate at a constant interval (II-III), then start to synchronize (IV) and fully synchronize (V). Intervals become longer (VI) and sporadic again during the tail aggression period (VII). Interestingly, 76% of the embryos started to oscillate from MN2R. In addition, independent photostimulations on left and right MN2s were conducted. This is the first report of the live imaging of neural activities in Ciona’s developing swimming CPG. These findings will help to understand the development of motor neuron circuits in chordate animals.Motor neurons (MNs) are one of the most important components of Central Pattern Generators (CPG) in vertebrates , 1911, 84(572), 308–319). However, it is unclear how the neural activities of these components develop during their embryogenesis. Our previous study revealed that in  Central Pattern Generator (CPG) is a locomotor network of neurons distributed along the vertebrate spinal cord, which generates rhythmically patterned outputs in the absence of rhythmic inputs . CPG is The mechanisms for generating rhythmic neuronal bursts during swimming behaviors are already well-studied with CPG of lampreys. In the case of lampreys, it was revealed that the swimming-related burst generators are distributed along their spinal cords, inhibitory neurons are involved in pattern generation, and their contralaterally extending neurons are GABAergic . In zebrCiona larvae are already revealed  family, one of the crucial components of the presynaptic active zone and Ca2+ homeostasis in flies and humans, were also expressed in Ciona , which are considered to be homologous with Mauthner cells, project axons contralaterally to MN2 .Recent research  revealedCiona. MN2 neurons show Ca2+ oscillation with an -80 s interval in the early tailbud stage (-St.24). This activity was cell-autonomous, because MN2 could oscillate even at a dissociated single-cell level. This Ca2+ oscillation coincides with unilateral tail muscle contracting behaviors observed in tailbud stages . In addition, the bursts of the action potential in MN2 gradually become tuned to the rhythmic ipsilateral muscle contractions occurring in each ETF  and independent photostimulations on each MN2 were conducted for the first time. This enabled us to compare the differences between MN2L and MN2R activities and their synchronicity. This is the first report of the live imaging of neural activities in Ciona’s developing motor circuit, which is assumed to be its swimming CPG.The purpose of this study is to uncover the neural activities of MN2s in later developmental stages. When compared to Brown’s Half Center Model, MN2s could be estimated to be one of the main components of Ciona robusta  adults (wild type) (ld type)  were regEggs collected in seawater were fertilized and kept at room temperature for 30 min. At 30 min post-fertilization, eggs were centrifuged, and the seawater was completely replaced with 0.77 M mannitol in 10% seawater . Plasmid-construct (around 600–1000 ng/μl) was combined with the egg containing 0.77 M mannitol mixture. Electroporation was performed according to the previous study . In the Ciona embryos by their dorsal side was invented. The methods were as follows. Embryos were collected by a glass pipette and fixed on a glass-based Petri dish . The direction of the embryos was modified by making a weak water stream with glass pipettes or by gently touching the embryos with a pulled glass needle. When the larvae started to swim, micro cover glass  was placed on the embryos and fixed on the glass-based dish with grease . An Olympus fv1000 microscope was used, and the images were processed by an Olympus fv1000 viewer. Excitation was performed at 488-nm to visualize GCaMP and photostimulate ChR2, and 559-nm to visualize mCherry. Olympus 20× or 40× oil immersion lenses were used. Room temperature was maintained at 20°C. Laser power density was measured by the optical power meter 3664  and optical sensor 9742 (HIOKI).2).Single-cell photostimulation of MN2 after St.27 (swimming larva) was performed with Channelrhodopsin-2  attached to a stereoscopic microscope . Room temperature was kept at 20°C. Images were captured at 200–400 frames per second (fps). Tail curvature was quantified from these images.The swimming behavior of Ciona swimming behavior, images obtained by high-speed camera were processed with the open-source programs Fiji  and MATLAB (MathWorks). Image denoising, binarization, and skeletonization were processed by Fiji and exported as a .BMP image sequence. Skeleton pruning was processed in MATLAB. The midline of the larva was extracted, and the curvature of the midline was calculated with the position of three points on the midline: start-(A), mid-(B), and end-(C) (To quantify the tail movement of  end-(C) . The equFluorescent intensity was detected by the AQUACOSMOS software (Hamamatsu Photonics). Data were analyzed with MATLAB  and Excel . The fluorescent intensity data of each ROI  was impo2+ burst timings of MN2L and MN2R, Rayleigh tests (https://github.com/circstat/circstat-matlab). The time point of the MN2R Ca2+ peak was assigned as the phase value of 0°, while that of the next MN2R Ca2+ peak was assigned as the phase value of 360°. Vector . TwentyCiona embryos were fixed on a Petri dish on their dorsal side. However, in previous studies most of the embryos were captured from the lateral side, or studies used embedded larvae for live imaging . This method enabled live-imaging of both MN2L and MN2R without restricting its natural embryonic development or tail muscle movement.To capture images of both left and right MN2s using an inverted microscope,  imaging . Therefo2+ oscillations at both MN2L and MN2R were measured even after St.24  were calculated  could be divided into seven phases  depending on the intervals and the synchronicity of MN2L and MN2R Ca2+ transients. Each phase is defined as follows  change, Calculated . Ca2+ os follows :In Phase I, MN2L and MN2R oscillate sporadically . Phase IIn Phase II, intervals of MN2L and MN2R bursts begin to converge to a constant value (-40 s) . Phase IIn Phase III, MN2L and MN2R oscillate at constant intervals . Phase IIn Phase IV, MN2L and MN2R oscillate at constant intervals . Burst tIn Phase V, burst timing of MN2L and MN2R synchronizes  and osci2+ bursts (>30 s) were found to occur sporadically  were found to occur sporadically . In addition, a significant change was found in the R-values between Phase IV (0.391) and the later phases , implying that the MN2L and MN2R Ca2+ bursts completely synchronize after Phase V.A Rayleigh test of uniformity was conducted and circular plots for each phase were created N = 5, . It is n2+ transients of MN2L and MN2R, tail movements were quantified by a high-speed camera from Phase III to Phase VI (N = 3). A spontaneous unilateral muscle contraction seen before the swimming larva stage is referred to as Early Tail Flicks  , tail muscle contractions occurred only unilaterally , suggesting that Phase VI larvae can continuously contract their tails and swim for a longer period than in Phase V. These tendencies correspond with the intervals and durations of MN2 activity captured by Caoff value of GCaMP6s , the reff = 3.9 ) was use2+ transients were tracked with DeepLabCut  are seen during Phase V, at the timing of MN2L and MN2R’s synchronized Ca2+ elevation.Tail movements during the CaepLabCut . As a re notched , especia notched , indicat2+ oscillation coincided with Ca2+ elevation in the ipsilateral tail muscle; it was also shown that MN2 stimulation was necessary to evoke tail flicks in St.24 , the tail flicked to the right side, meaning that the right-sided muscle contracted ) . On the 2+ oscillation continues to the tail absorption period. From the synchronization of MN2L and MN2R Ca2+ bursts, MN2s’ Ca2+ transition was divided into seven phases , MN2R and MN2L work independently as a pattern generator for each of the ipsilateral muscles without any transmissions between these left and right neurons. However, as Ca2+ transitions change from Phase I to Phase III, intervals of MN2L and MN2R bursts converge to a constant value (-40 s). There was an individual difference in which one MN2 started to oscillate earlier than the other MN2, though MN2R had a higher tendency to be earlier than that MN2L . Entrainment starts from Phase III.  mature, the contralateral axons connect MN2R with MN2L, reinforcing the entrainment of their Ca2+ oscillations, which eventually synchronize to the other MN2.1) As other contralateral neurons in 2+ oscillations to a 40-s interval independently, without help from other neurons.2) MN2s themselves contract their CaIn Phase I, Ca2+ oscillation.Future work is required to understand the mechanisms for this frequency convergence of MN2R and MN2L Ca2+ oscillation may be involved in its axon growth rate. Axon outgrowth requires axon guidance molecules, though such cues used for both MN2 and ACIN in Ciona remain unknown.Meanwhile, the axon growing speed observed during Phases I to IV was asymmetric; axon outgrowth of the earlier oscillating motor neurons was faster than that of others . This imEphrin, and Semaphorin 3A, have been primarily found in the notochord of Ciona tailbud embryos   . These m2+ bursts start to synchronize. Though this synchronization is intermittent, the change implies that information exchange has started between MN2L and MN2R to make either of them coincide with the other MN2’s burst timing.The significant difference between Phases III and IV is that in Phase IV, MN2L and MN2R Ca2+ bursts start to synchronize permanently. The high-speed recording of the tail muscle contraction also reveals that Phase V belongs to the swimming larva stage, in which the somatic left and right tail muscles contract alternately and generate a continuous tail-beating ≒ swimming behavior , followed by negative phototaxis  , and 177 neurons. For instance, Groups I and II photoreceptors are responsible for phototaxis and gravitaxis, respectively, and activate swimming via pr-AMG RNs, which project to MGINs (It is known that wnwards) . These pto MGINs . Phases In Phase VII, MN2 oscillates sporadically although it is under metamorphosis and its tail is absorbing. The tail becomes immotile in this developmental stage , suggestCiona motor circuit, it could be hypothesized that the Ca2+ activity of MN2R and MN2L is reciprocally driven by the receipt of contralateral inhibitory inputs from other neurons, such as ACIN. However, it was shown that the Ca2+ burst timings of MN2L and MN2R were synchronous in the swimming larvae, though their tail beating was left-right alternate. The possible reasons for this are as follows:2+ transitions were obtained with a frame rate of 0.2- sec/frame. Asynchronous Ca2+ bursts of MN2L and MN2R could be recorded with a higher frame rate camera or by transducing Ca2+ sensors with a higher sensitivity.1) In this study, fluorescent images of Ca2) In the later swimming larvae, muscle contraction may still be finely tuned by MN2 action potentials (as described in When Brown’s Half Center Model is adapted to the 2+ transitions were obtained with a minimum frame rate of 0.2 s/frame. Realtime membrane potential imaging of both MN2s in future research is adequate to understand the coordination between them. Though Ca2+ bursts of MN2 were synchronized, each Ca2+ spike occurring inside one Ca2+ burst may not be synchronized when recorded with a higher frame rate or by transducing other sensors with a higher sensitivity.However, in this study, fluorescent images of Ca


The mean AP-cycle length (APCL) and APCL variability inform on the effectiveness of clock coupling. Endogenous ATP metabolite adenosine binds to adenosine receptors  that couple to Gi protein-coupled receptors, reducing spontaneous AP firing rate via Gβγ signaling that activates IKAch,Ado. Adenosine also inhibits adenylyl cyclase activity via Gαi signaling, impacting cAMP-mediated protein kinase-A-dependent protein phosphorylation. We hypothesize that in addition to IKAch,Ado activation, adenosine impacts also Ca2+via Gαi signaling and that both effects reduce AP firing rate by reducing the effectiveness of the Ca2+ and membrane clock coupling. To this end, we measured Ca2+ and membrane potential characteristics in enzymatically isolated single rabbit SANC. 10 µM adenosine substantially increased both the mean APCL  and AP beat-to-beat variability from 5.1 ± 1.7% to 7.2 ± 2.0% (n = 10) measured via membrane potential and 5.0 ± 2.2% to 10.6 ± 5.9% (n = 40) measured via Ca2+ (assessed as the coefficient of variability = SD/mean). These effects were mediated by hyperpolarization of the maximum diastolic membrane potential (membrane clock effect) and suppression of diastolic local Ca2+releases (LCRs) (Ca2+-clock effect): as LCR size distributions shifted to smaller values, the time of LCR occurrence during diastolic depolarization (LCR period) became prolonged, and the ensemble LCR signal became reduced. The tight linear relationship of coupling between LCR period to the APCL in the presence of adenosine “drifted” upward and leftward, i.e. for a given LCR period, APCL was prolonged, becoming non-linear indicating clock uncoupling. An extreme case of uncoupling occurred at higher adenosine concentrations (>100 µM): small stochastic LCRs failed to self-organize and synchronize to the membrane clock, thus creating a failed attempt to generate an AP resulting in arrhythmia and cessation of AP firing. Thus, the effects of adenosine to activate Gβγ and IKACh,Ado and to activate Gαi, suppressing adenylyl cyclase activity, both contribute to the adenosine-induced increase in the mean APCL and APCL variability by reducing the fidelity of clock coupling and AP firing rate.The spontaneous action potential (AP) firing rate of sinoatrial nodal cells (SANC) is regulated by a system of intracellular Ca Adenosine (ado) is an endogenous cardiac metabolite, generated cysteine . Ado concysteine .via ado-activated A1 and A3 receptors signaling that like muscarinic receptor M2 stimulation leads to activation of inhibitory GTP-binding protein to dissociate into its Gαi and Gβγ subunits. These effects, respectively, result in both the inhibition of adenyl cyclase (AC) and activation of GIRK4 K+ channels that conduct IKACh and IKAdo currents  that hyperpolarize the SANC membrane potential (CaL and If caused by β-adrenergic receptor stimulation (f in the absence of β-adrenergic stimulation and slows pacemaking in SANC similar to acetylcholine (ACh)  .KAch,Ado and/or If are blocked (tertiapin-Q and/or Cs), the AP firing rate reduction in rabbit SANC still remains substantial within the broad range of physiological concentrations of ACh . Thus, 2+-calmodulin activated AC-protein kinase-A (PKA) and CaMKII signaling is widely accepted , that activate inward Na+/Ca2+ exchanger current (INCX) (f and ICaT to initiate the diastolic depolarization (2+ channels (Cav1.3) (2+-induced Ca2+ release (CICR), and continued spontaneous LCRs from the SR form an ensemble Ca2+ signal that accelerates diastolic membrane depolarization via INCX activation (m) depolarizes sufficient to open Cav1.2 L-type Ca2+ channels, an AP is ignited, which (via CICR) induces a relatively synchronous activation of Ca2+ release channels (ryanodine receptors), resulting in a global cytosolic Ca2+ transient  , which prization . Feed-fo(Cav1.3) , Ca2+-intivation . When thransient . AP firi2+ clock , affectscoupling . The avecoupling .2+ cycling, reducing Ca-calmodulin-activated AC-PKA-CAMKII signaling , they both exert their downstream effects via a signaling cascade that involves the same Gi protein coupling . The SAN region was cut into small strips (∼1.0 mm wide) perpendicular to the crista terminalis and excised as reported previously (2+-free solution containing (in mM): 140 NaCl, 5.4 KCl, 0.5 MgCl2, 0.33 NaH2PO4, 5 HEPES, 5.5 glucose, (pH = 6.9) and incubated on shaker at 35°C for 30 min in the same solution with the addition of elastase type IV  collagenase type 2 , Protease XIV  and 0.1% bovine serum albumin . The SA node preparation was next placed in modified Kraftbruhe (KB) solution, containing (in mM): 70 potassium glutamate, 30 KCl, 10 KH2PO4, 1 MgCl2, 20 taurine, 10 glucose, 0.3 EGTA, and 10 HEPES (titrated to pH 7.4 with KOH), and kept at 4°C for 1 h in KB solution containing 50 mg/ml polyvinylpyrrolidone . Finally, cells were dispersed from the SA node preparation by gentle pipetting in the KB solution and stored at 4°C.SANC were isolated from male rabbits in accordance with NIH guidelines for the care and use of animals, protocol # 34-LCS-2019 (as previously described) . New Zeaeviously . The fin2+ dynamics within isolated single rabbit SANC were measured by 2D imaging of the fluorescent Ca2+ indicator, Fluo-4. Cells were loaded with 5 μM Fluo-4AM  for 15 min at room temperature. Fluo-4AM was subsequently washed out of the chamber with bathing solution contained the following (in mM): NaCl 140, HEPES 5, NaH2PO4 0.33, KCl 5.4, MgCl2 1.0, glucose 5.5, CaCl2 1.8; titrated to pH 7.35 with NaOH. Ca2+ signals were measured within the ensuing 30 min at 35°C ± 0.1°C. Temperature was controlled by an Analog TC2BIP 2/3Ch bipolar temperature controller from CellMicroControls (United States), which heated both the glass bottom of the perfusion chamber and the solution entering the chamber (via a pre-heater). It is important to note that Ca2+ imaging was not performed immediately after cells were taken from 4°C KB solution, but following an adaptation time of 20–30 min, during which cells settled in the perfusion chamber, attach to the glass, and were loaded with the Ca2+ indicator. Ca2+ signals were recorded only from rhythmically firing SANC at baseline (CV<10%). Thus, the measured cells were truly pacemaker cells by their classical morphological shapes and by the presence of rhythmic spontaneous beating, and therefore other cell types  were excluded by these criteria.CaFluo-4 fluorescence was detected using a high-speed PCO. edge 4.2 CMOS camera  mounted on a Zeiss inverted microscope  with a x63 oil immersion lens and a fluorescence excitation light source . Fluo-4 fluorescence excitation  and emission light collection  were performed using the Zeiss filter set 38 HE. To avoid phototoxicity, Fluo-4 was excited only for short periods of time (<20 s) . Data acm was measured in the current clamp configuration using an Axopatch 200B amplifier (Molecular Devices). Patch pipette resistances ranged between 3–5 MΩ, and pipettes were filled with a solution containing (in mM): K+ gluconate 120, NaCl 5, MgATP 5, HEPES 5, KCl 20; titrated to pH 7.2 with KOH. Amphotericin B  was added into the pipette solution as the pore-forming agent. The liquid junction potential was calculated by pClamp software (Molecular Devices) to be 13 mV and included in data analysis.V2+ transient peak and the onset of the LCR occurrence. The “LCR size” is given as the LCR full propagation path in µm2. The computer program also provides the LCR ensemble signal  to objectively, automatically, and rapidly analyze the individual and ensemble behavior of the LCRs . The pro2+ signal only. Vm and Ca2+ were also recorded simultaneously in a subset of SANC. Both Vm and Ca2+ recordings were electronically synchronized. The Vm measurements and 2D Ca2+ signals were obtained before, during, and following washout of 10 µM ado . We measured SANC APCL using two parameters: AP intervals or AP-induced Ca2+ transient intervals that were closely correlated   , increas (10 µM) . In resp washout . The CV  washout . To conf minutes .m was measured, the ado-induced increase in APCL was washable after 10 min (max), and decreased the mean diastolic depolarization slope . With ado, there was an increase in the number and percentage of the smallest LCRs. Correspondingly, there was a decrease in larger LCRs. Ado also prolonged the mean LCR period, consistent with the idea that the period of the coupled-clock system increased.The results of statistical analysis of LCR characteristics are presented in 2+ transient cycle length and mean LCR period in response to ivabradine, a funny current inhibitor  of the LCR period and APCL (It has been previously shown that a concurrent increase in beat-to-beat variability (CV) accompanied an increase in the mean AP-induced Canhibitor . The incand APCL .2+ signal    because d as TTI . Note a d as TTI . Therefod as TTI  and thatmversus Ca2+ permit closer inspection of simultaneous time-dependent changes occurred in Vm and Ca2+. Vm and Ca2+ throughout the AP cycle inform on the electrochemical gradient oscillation that underlies each AP cycle  than in the same cells in the presence of ado (300 msec) . The time from ignition onset to TOP  is the duration of the ignition phase. Ado prolonged the time-to-ignition (MDP to ignition phase onset) from 160 msec at baseline to 300 msec with ado. The ignition phase duration decreased with ado from 400 msec at baseline to 310 msec with ado Pane A). Peak Vm and Ca2+ transient amplitudes (labeled 4 in Panels A and B) occur at later times with ado (640 ms) than baseline (610 ms) (Panel A). AP repolarization and Ca2+ transient decay initiates sooner at baseline than during ado (Panel A). Peak Vm amplitude, labeled 4 in Panels A and B, occurred at a lower Ca2+ level but later in time in ado (640 msec) than baseline (610 msec) (Point 1 in 00 msec) . Progres10 msec) . The tim2+ and Vm signals during an AP cycle. This hysteresis is greater with ado than at baseline. Whereas the difference of the duration of the AP ignition period is 560–610 msec (1.1 times greater) and time to 90% decay is 890 msec and 960 msec (1.08 times greater), the main difference to the time domain and percent differences between ado and baseline is the time-to-ignition onset. The major factor increasing hysteresis between ado and control in the phase-plane diagram is due to a 2-fold increase in the time-to-ignition onset in ado (300 ms) and control (160 ms). The time-to-ignition onset is regulated by the pumping of Ca2+ into the SR. This rate of Ca2+ cycling into the SR to achieve the threshold required for spontaneous LCRs to occur and self-organize, is the degree to which it influences Vm time-to-ignition onset.Note that the degree of hysteresis indicates the overall uncoupling of CaCaL, If, IKACh,Ado (membrane clock components) and SR Ca2+ pumping (Ca2+ clock), and demonstrated the their synergistic effects to reduce the AP firring rate  and junctional SR (CajSR) substantially drop . Ado indirectly effects the Ca2+ clock via its effect on the membrane clock to slow SANC firing rate, commensurate a decrease in net SR Ca2+ influx and intracellular Ca2+, i.e. oscillatory substrate for Ca2+clock. Thus, changes in SANC firing rate were due to direct effects on both clocks and indirect effects of both clocks on each other. Numerous feedbacks and feedforwards of this process occurred until a new equilibrium was reached.The present study demonstrates that ado increases APCL and APCL variability of SANC by directly and indirectly effecting both membrane and Ca via Gαi  to reducvia ACh. ACh and ado have different membrane receptors, but likely target the same IKAch,Ado channels via Gβγ, hyperpolarizing the cell membrane and extending the time of diastolic depolarization  is a specific Ca+-ATPase . CPA has pumping . The slo pumping . The CPAon slope . Thus, w1 and A3) coupled to Gi proteins , reflecting clock uncoupling. One important interplay happens with respect to SR Ca2+ loading. On the one hand, the longer SR Ca2+pumping via SERCA during the longer diastolic depolarization in the presence of ado stimulation favors a larger SR Ca2+ loading. On the one hand, ado via Gαi signaling attenuates AC activity that leads to lower levels of cAMP and cAMP-dependent PKA activity. This results in phospholamban de-phosphorylation that ultimately slows SERCA Ca2+ pumping  deplete cell of Ca2+. Thus, the factor of longer SERCA pumping competes with slower SERCA pumping and cell Ca2+ depletion. Our numerical modeling takes into account all these positive and negative contributions to SR Ca2+ loading and shows that SR Ca2+ content is expected to decrease, both the junctional SR and network SR .Our numerical modeling results  provide proteins . Importaergistic . The eff pumping . Thus, wKACh,Ado by tertiapin had no effect; its effect begins to occur at higher concentration of ACh, i.e., at about the half maximum dose of ACh  activation of muscarinic cholinergic receptor (or adenosine receptors), at which IKACh,Ado is not activated. It is important to note, however, that interpretation of our experimental and numerical modeling results obtained in single SANC to SAN tissue is complicated. For example, it was shown  have a high surface-to-body mass, causing them to lose a large amount of heat. In order to maintain 37°C mice and rats employ nonshivering thermogenesis, which activates sympathetic stimulation to burn brown fat to produce heat. A biproduct of the excessive sympathetic dischargers is the elevation of heart rate in small rodents (mice and rats). For example, maintaining mice in thermo-neutral metabolic environment 30°C vs room temperature 20–24°C markedly reduces the heart rate as much as 40% .2+ transient cycle length was, on average, 13% longer in cells loaded with Ca2+ indicator Fluo-4 than cells not loaded with Fluo-4. This may reflect Ca2+ indicator buffering intracellular Ca2+. Nevertheless, ado effects on mean AP-induced Ca2+ transient and APCL variability were similar to the effects of ado on APCL and APCL variability recorded via perforated patch clamp in the absence of Ca2+ indicator.The AP-induced CaK,Ado, ICaL, If and other specific mechanisms and molecules have not been experimentally studied here and merit future investigations. As mentioned above, cAMP-mediated effects of ado have been strongly associated with a level of sympathetic stimulation. Therefore, Ca2+ signaling (Ca2+ clock) could be also important in the setting of sympathetic stimulation and represents an important topic for future studies.While the aim of the study was to address the coupled-clock system, specifically LCRs and the membrane potential in ado effects, the biochemical pathways and interplay of LCRs with I2+ signaling for tissue function, and especially for the shift of pacemaker location, has been demonstrated in recent studies of S100B effects in intact SAN (One important SAN phenomenon is the shift of pacemaker location within the SAN tissue in response to different perturbations, including ado . While otact SAN .Finally, it is important to note that while we traditionally discuss average data and illustrate the mechanisms in a numerical model of a SANC, in reality, SANC are a naturally heterogenous population of cells  that hav


Soft, wearable, powered exoskeletons are novel devices that may assist rehabilitation, allowing users to walk further or carry out activities of daily living. However, soft robotic exoskeletons, and the more commonly used rigid exoskeletons, are not widely adopted clinically. The available evidence highlights a disconnect between the needs of exoskeleton users and the engineers designing devices. This review aimed to explore the literature on physiotherapist and patient perspectives of the longer-standing, and therefore greater evidenced, rigid exoskeleton limitations. It then offered potential solutions to these limitations, including soft robotics, from an engineering standpoint.A state-of-the-art review was carried out which included both qualitative and quantitative research papers regarding patient and/or physiotherapist perspectives of rigid exoskeletons. Papers were themed and themes formed the review’s framework.Six main themes regarding the limitations of soft exoskeletons were important to physiotherapists and patients: safety; a one-size-fits approach; ease of device use; weight and placement of device; cost of device; and, specific to patients only, appearance of the device. Potential soft-robotics solutions to address these limitations were offered, including compliant actuators, sensors, suit attachments fitting to user’s body, and the use of control algorithms.It is evident that current exoskeletons are not meeting the needs of their users. Solutions to the limitations offered may inform device development. However, the solutions are not infallible and thus further research and development is required.The online version contains supplementary material available at 10.1186/s12984-022-01122-3. In the UK, 6.8 million people live with mobility-related disabilities; the leading causes of which are musculoskeletal conditions and stroke , 2. PersImpaired mobility can have widespread effects on an individual’s quality of life as participation challenges impact their work, social life and activities of daily living (ADLs) . MobilitDevelopment in wearable powered exoskeletons offers a potential solution to traditional rehabilitation challenges , 19. An Many of these existing rigid exoskeletons were initially developed to provide maximal assistance to those with complete paralysis resulting from spinal cord injury. Interest has increased in the exoskeletons that can provide sensory-guided motorised lower limb assistance for person’s with stroke , 28. TheDespite these proposed advantages, rigid exoskeletons have not been widely adopted clinically . AlthougSoft-robotics is an emerging field with capabilities to address the limitations of bulky, rigid and heavy exoskeletons through designing wearable, soft devices that are lightweight, compliant and flexible, resulting in safe human interaction, suitable for body assistance , 38. SofIf the advantages of soft robotics are to be realised and implemented effectively and transitioned rapidly into clinical and community settings, the engineering and clinical gap must be reduced. Consequently, this state-of-the-art review aims to identify the limitations of existing exoskeletons as perceived by clinicians and patients, and discuss the potential soft-robotic solutions. The review informs FREEHAB, a project which aims to design wearable, assistive soft-robotic devices for people with impaired mobility (project website - The Right Trousers ). This cThe "Initially, the researchers individually scoped out the literature, to highlight areas of potential interest for review and used this initial exploration to define the aim, and subsequent scope, of the review. It was evident that there was limited literature on patient and physiotherapist perspectives on hard and soft exoskeletons See the "" sectionet al. (2014) [In order to gain a greater insight into users’ perspectives, literature with qualitative and quantitative studies were included in the review. As exoskeletons have progressed significantly within recent years, it was preferable to have more recent papers. However, the Wollf . (2014)  paper wa. (2014)  regardinThe review aimed to focus on finding solutions for exoskeleton issues that were user-led. Therefore, the review was led by a health researcher (LM) without an engineering background and without (at this time) active involvement in the technologies of soft exoskeletons. It was important that the engineers did not outline the perspectives of the users, as they would bring their own bias due to their knowledge of the technologies that they could develop.LM independently searched and identified relevant literature using the databases MEDLINE, AHMED and CINHAL. Snowballing of papers was also carried out. LM collated the literature into a summary table and the selected literature was also discussed with MC and AT researchers with a physiotherapy and occupational therapy background, respectively. Themes were identified, which were recurring patterns regarding patient and physiotherapist perspective of exoskeletons across the papers. which included the themes which would become the framework for presentation of findings  and viewed videos of people walking with assistance from the device . The stu’s study  .A secondary safety concern, with expression limited to one physiotherapy student, but with particular relevance in light of the COVID-19 pandemic, was the ease of cleaning the device . PredomiCompliance as a physical property is one of the most important required features in developed exoskeletons, facilitating adaptability, comfort and safe interaction with human body . Series The decrease in rigid components can be observed throughout the history of developments in exoskeletons using a variety of soft robotics technology to pursue totally soft exoskeletons, which is safer, more comfortable and friendly to users  . This waIn terms of the design considerations for engineers, it is essential that the device is easy to put on and take off. A soft trouser, which integrates soft robotic actuators,  is easier to wear and reduces unproductive time during therapy. The challenge remains for the soft structure to effectively interface with the body and to provide sufficient torque/force and tension/compression to deliver mechanical assistance as required . A lightStudies commonly referenced weight, size and placement of exoskeletons. In the Vaughan-Graham et al. study , both stReducing the weight of exoskeletons has been a key focus for engineers designing rehabilitative and assistive devices. Existing rigid portable exoskeletons aim to restore mobility of disabled patients, examples include Exo-H2, Indego, ReWalk and EksoNR, have masses between 11 and 25 kg . They caPlacement of body attachments is another critical part of exoskeletons. Key anchors, such as the shoulder, the iliac crest of the hip and the plantar surface of the feet, are defined as effective body locations onto which to attach assistive devices . These sCost of exoskeletons was a consideration shared by both physiotherapists and patients , 25, 34.To the authors’ knowledge, there is no literature on cost evaluation of exoskeletons or use of cost reducing materials. However, the soft exoskeletons can generally separate into two major parts: a suit with integrated soft actuators, and a power-supplying unit. Presently, there is an increasing trend to use more fabrics to create soft exoskeletons . These sAppearance of exoskeletons was a patient consideration; however, it was ranked as the least important device criteria for users of wheelchairs (3.23 mean importance out of 5) , 34. TheExisting soft exoskeletons have been designed with aesthetics and discretion as important considerations, while maintaining high body-assisting performance . TextileThis state-of-the-art review has highlighted a range of patient and physiotherapist perceived concerns and design considerations regarding rigid and soft exoskeletons. Patient and physiotherapists had reservations regarding rigid exoskeletons’ safety, such as falls, despite not having an experience that would warrant this or having seen wider evidence that demonstrates that falls were only present in testing of early exoskeleton prototypes . The sigFrom an engineering perspective, the first challenge is developing new soft actuators that are lightweight, compliant and sufficiently powerful enough to deliver smooth and safe assistance for natural mobility , 83. TheBoth physiotherapists and patients were concerned that if there were only one exoskeleton device that was made to fit all patients, it may result in unnatural movements , 25, whiWe perceive that the future exoskeletons will become totally soft and naturally integrate with normal clothing. Their actuators and body attachments will be combined to create multifunctional assistive clothing with capabilities of stiffness variability and morphology deformation on every areas of the suit. For example, at no assistance, the entire suit turns soft, improving ease of donning and doffing. Moreover, it is perceived to be able to harvest energy gained from passive shape deformation and heat loss released from the body surface during daily activity. Also, when activated, certain areas of the suit can be controlled to become stiffer to support loads on lower limbs or to prevent undesired movements and joint misalignment. In contrast, other areas can actively vary their stiffness to deform, e.g. contract, elongate, bend and twist, and enable actuators to effectively transmit forces to assist required body movements and also prevent joint misalignment. In addition, active morphology deformation can enable suit adaptability to specific users and self-fitting for comfortable usage by controlling shrinking and loosening of the suit to fit the user’s body. A variety of developed textile actuators was classified depending on their purpose and reviewed in .Physiotherapists and patients all highlighted the importance of having a device that was lightweight, to prevent the user’s gait pattern being altered  and to aThe cost of the device was important to both patients and physiotherapists, in both terms of cost for the patient to buy for personal use, and also the purchasing by clinics , 25, 31.Although the appearance of rigid exoskeletons was commonly discussed by patients, their views varied, with some feeling appearance was irrelevant if the device allowed them to live their life, and others not wanting to feel stigmatised by a visible device . This deTo build the next generation rehabilitative device, we propose that developments are required in i) building efficient soft robotic actuators, ii) fabricating comfortable body attachments, iii) improving sensor technology and iv) developing robust adaptive control strategies.Similar to the key metric performance of human skeletal muscles, soft fluidic and electrically-driven actuators have high potential to become future smart artificial muscles due to their high stress, strain and bandwidth, addressing the requirements to create the future soft exoskeletons .Soft fluidic actuators have backdrivability (behaving like a spring), low cost and high specific force and power, despite low efficiency around 30% because of losses in the fluidic-to-mechanical energy conversion process , 93. TheSoft electrically-driven actuators are another potential candidate for future soft exoskeletons due to their high actuation performance, fast and quiet operation, and high efficiency , 93. ForAuxetic or transformable structures has been explored to develop ankle and knee braces that are lightweight and comfortable, similar to a textile or garment . ExploitThe conditions of body attachment, such as size, shape and attachment point, play a crucial role in patient comfort that form an essential focus of evaluation for the performance of soft wearable exoskeletons. As previously discussed, the safe placement for attachments of the soft exoskeletons on the user’s body is a shoulder, hip and feet . In addiAlthough not considered by the patients and physiotherapists, we perceive that breathability is an important feature of body attachment and can significantly enhance comfort while wearing the soft exoskelton. This can be achieved by temperature and humidity exchanges on the skin surface. For example, the suit can possess self-deformation, which is sensitive to skin surface temperature and humidity and autonomously deforms its structure to inhale/exhale surrounding air for cooling/warming of the suit .Typically, exoskeletons require a sensing system to be highly accurate, fast, and robust to disturbances. The sensing system is accountable for three fundamental features: (1) measuring force generation, (2) monitoring body motions, and (3) evaluating assistance efficiency. First, the soft exoskeletons require sensors to measure their actuators’ outputs, which are then fed back to their controller to deliver accurate assisting force and strain. Sensors are also used to prevent undesired damage, for example, stopping actuation when high forces and out-of-range motions are detected.Second, IMU  sensors are commonly used to track and simulate body movements by their attachment across body joints. With the development in a real-time data-analysing system, IMUs were able to precisely predict walking and sit-to-stand activities , 117. AlWe envision that all of these functional sensors need to be soft and aesthetically embedded within soft exoskeletons to comprehensively monitor the user’s body. The future of assistive soft robotic clothing may include multiple layers that contain actuating, sensing and control units, operating synchronously to facilitate a person’s improved mobility.As a result of the body motion information acquired from suit sensors, a virtual mobility simulation can be simultaneously created. Additionally, an open source platform that can capture the motion from the suit with plug-and-play capabilities may facilitate rapid analysis of patient mobility patterns. The integration of the above technologies with an online data communication and service has the potential to track mobility improvements and enable remote patient consultations with physiotherapists.Exoskeletons must always generate the appropriate amount of assisting forces at the right time for effective assistance without negatively impacting a user’s mobility. As previously shown, human-in-the-loop optimisation was developed as an advanced control algorithm which can automatically adapt the assisting strategy based on the user’s mobility, consistently delivering effective, harmless and optimal assistance , 70. Altwhat about devices make them ‘effective’ and for who, and what outcome measures class this ‘effectiveness’.Implementation will only be successful if devices are themselves effective and that this can be proven in clinical and usability trials. A recent Cochrane review  of 62 trHowever, measuring effectiveness is a problem that is widespread across the robotics field. A systematic review  exploredhow therapists use devices and why therapists use devices in the way they do; this is essential to not only design robotic assistive devices that meet therapists’ needs, but also to create guidelines that can facilitate successful implementation of such devices. Notably, the need to understand the therapist’s clinical reasoning and the devices outcomes are interconnected. As Vaughn et al. [Fundamentally, there must be an understanding of n et al.  study exThis review identified that physiotherapists felt that training professionals in exoskeleton use had to be multi-faceted, and that the training was often negatively perceived to be draining. Stephenson and Stephens  exploredWe present state-of-the-art technologies and examples of current rigid and soft exoskeletons which have been designed to address the needs highlighted by physiotherapists and patient users. This review has demonstrated a range of issues with current rigid exoskeletons, with limitations related to device safety, one-size-fits-all feature, ease of use, weight, placement, cost, and appearance. We provide a concept of future exoskeletons and soft-robotics solutions classified into four key factors: soft robotic actuators, body attachments, sensing system and control algorithm. The solutions proposed are extensive and the considerations when designing future soft exoskeleton devices are complex.Soft pneumatic-driven and electrically-driven actuators are perceived as having the highest potential for future soft technology exoskeletons thanks to their high flexibility, speed of response and portability, and stress and strain performance that matches or exceeds human muscles. Applying compliance to all components of exoskeletons can increase users’ comfort and help prevent harm if assistance is misaligned or unsafe movements occur. Variable-stiffness capability can stiffen certain areas of the soft exoskeletons to maintain and deliver sufficient force transmission through the device to effectively assist lower limbs. This property also provides the devices with adaptability to deform themselves to fit to various specific users’ requirements, increasing ease in donning/doffing. The assistive device will use advanced sensors to detect user intention and an adaptive control algorithm will learn users’ patterns of movement over time to better respond when needed. Future devices must have these features in order to be fit for purpose in time-restricted therapy sessions, which was a key challenge of current exoskeletons. Textiles are recommended as the basis for developing future soft robotic exoskeletons, enabling them to resemble normal clothing, and enhancing aesthetics and user acceptance.We hope that the findings of this work may facilitate engineers in designing softer, more comfortable and more effective exoskeletons that meet the multi-faceted needs of their patient and physiotherapy users. In light of the findings of this review, it is strongly recommended that any assistive device development is supported by continuous user involvement and consultations.Additional file 1. Additional figures and tables.


Space-time adaptive processing (STAP) is an effective technology in clutter suppression and moving target detection for airborne radar. Because airborne radar moves at a constant acceleration, and there is a lack of independent and identically distributed (IID) training samples caused by the heterogeneous environment, using the conventional STAP methods directly cannot ensure a good performance. To eliminate these effects and improve the performance of clutter suppression, a STAP method based on a sparse Bayesian learning (SBL) framework for uniform acceleration radar is proposed here. This paper introduces the signal model of the uniform acceleration radar. To promote the sparsity, a generalized double Pareto (GDP) prior is introduced into our method, and the estimation of hyper parameters via expectation maximization (EM) is given. The effectiveness of the proposed method is demonstrated by simulations. Space-time adaptive processing (STAP) ,2 is a tUnfortunately, it is known from the Reed–Mallet–Brennan (RMB)  rule thaTo solve this problem, many algorithms have been proposed to ameliorate the performance of clutter suppression.From the start of the STAP technique application, reduced-dimension and reduced-rank methods ,5,6,7,8,Over the past twenty years, the sparse recovery STAP (SR-STAP) methods ,16,17,18The sparse Bayesian learning (SBL) algorithms proposed by Tipping  have beeIn this paper, for the case of accelerated motion of the radar platform, the direct application of the traditional STAP method cannot achieve the equal performance of clutter suppression. Thus, we will improve the performance of the STAP method with the sparse Bayesian framework. Unlike most M-SBL-STAP methods that use Gaussian distribution priors and Laplace distribution priors, we used the generalized double Pareto (GDP)  prior inBased on the sparse Bayesian learning using the generalized double Pareto prior, a hierarchical Bayesian model built for the STAP is proposed here.Improving the performance of clutter suppression for the radar platform that moves at a constant acceleration.Compared with other STAP methods, the simulation results and performance analysis are given to demonstrate the effectiveness of the proposed method.The main contributions of this paper are listed as follows:The work is organized as follows: Notations: Boldface uppercase and boldface lowercase letters are used to represent matrices and vectors, respectively; This paper considers the airborne radar system equipped with a uniform linear array (ULA) of Neglecting the effect of range ambiguity, a general model for the received data of the P caused by the Then, the delay of receiving the echo from the scattering point Hence, assuming that the clutter component and the noise component are mutually uncorrelated, the clutter-plus-noise covariance matrix (CNCM) can be written as:In order to obtain the best performance for target detection under the criterion of the maximum SINR, the optimal STAP weight vector of the Combining the sparse recovery techniques with the STAP to make full use of the sparse characteristics of the clutter data, the requirements of the training samples can be significantly reduced. In the application of sparse recovery, first we discretize the angle-angular two-dimensional plane uniformly into According to the sparse recovery algorithms, it can be inferred that the problem of representing In this section, a STAP method based on the sparse Bayesian learning (SBL) framework using GDP prior for the uniform acceleration radar is presented here.To begin, the likelihood of measurement Each column in In , the schDue to the introduction of the parameter, we need to model the new parameter It has been demonstrated in  that theThe prior of Based on the Bayesian theorem, the posterior density of The posterior In the E-step, according to the Equation (17), covariance and mean are given as:In the M-step, we update the hyper-parameters by maximizing the expectation of The expansion of Equation (20) is marked as Equation (21). In Formula (21), The derivative of Setting Equation (22) to zero to obtain the update formula of Similarly, the derivative of Setting Equation (24) to zero to obtain the update formula of In addition to the update of the hyper-parameters, we also need to update the noise edure in :(26)(σ2)The M-GDP-STAP algorithm is shown in In this section, a simulation of the results is provided to demonstrate the effectiveness of the proposed method. Ward’s clutter model is adopted here, and the clutter-to noise ratio is set to be 40 dB. The 400th range gate is chosen to be the CUT. We set the numbers of spatial and temporal grid points to In order to evaluate the clutter suppression performance of the proposed method, we used the metric of improvement factor (IF), which is a widely used indicator to assess the performance of our proposed method and other reference methods, defined as follows:The proposed method is compared with the multiple orthogonal matching pursuit (M-OMP-STAP), the multiple focal underdetermined system solver (M-FOCUSS-STAP), and the iterative adaptive approach (IAA-STAP). In addition, we also added the traditional STAP algorithm based on the angle Doppler compensation (ADC-STAP) for comparison. As shown in To better illustrate the performance, From From We compared the average running time of different algorithms and the results are listed as follows: A total of 100 independent Monte Carlo trials were taken to obtain the average running time. Although the M-SBL and the M-GDP have shown a great performance in IF curves and clutter spectrums, the running time of the M-GDP is much less than that of the M-SBL. Although the running time of some algorithms is far less than that of the M-GDP, their performance cannot be accepted, such as the M-OMP, the M-FOCUSS and the M-IAA. The average running time is shown in In this paper, in order to improve the clutter suppression for the radar platform that moves at a constant acceleration, we study the model of sparse Bayesian learning and introduce the generalized double Pareto distribution prior into the SBL-based STAP framework. Compared with the classical sparse-based STAP method, the GDP prior can promote the sparsity of the sparse coefficient matrix, which will enhance the sparse recovery. At the end of this paper, the simulation results and the performance analysis are given to demonstrate the effectiveness of the proposed method.We outline a few future research directions. For airborne radar with a higher DOF, the computational complexities of the SBL-based algorithms will be greatly improved; thus, the corresponding fast algorithm will be one of the directions the research will take. In addition, the derivation of algorithms suitable for two-dimensional array radars is also a direction worthy of research.


The ultimate goal of prolonged infusion of beta-lactam antibiotics is to improve the outcome of infectious diseases. However, merely increasing target attainment (or the %fT>MIC) is unlikely to lead to improved clinical outcome for several reasons. First, the PK/PD index and target are dynamic entities. Changing the PK (as is the case if prolonged instead of intermittent infusion is used) will result in different PK/PD targets and even PK/PD indices necessary to obtain the same level of bacterial cell kill. Second, the minimal inhibitory concentration is not a good denominator to describe either the emergence of resistance or toxicity. Therefore, we believe a different approach to antibiotic dosing is necessary. In this perspective, we introduce the concept of the maximum tolerable dose (MTD). This MTD is the highest dose of an antimicrobial drug deemed safe for the patient. The goal of the MTD is to maximize bacterial cell kill and minimize the risk of antimicrobial resistance and toxicity. Unfortunately, data about what beta-lactam antibiotic levels are associated with toxicity and how beta-lactam antibiotic toxicity should be measured are limited. This perspective is, therefore, a plea to invest in research aimed at deciphering the dose–response relationship between beta-lactam antibiotic drug concentrations and toxicity. In this regard, we provide a theoretical approach of how increasing uremic toxin concentrations could be used as a quantifiable marker of beta-lactam antibiotic toxicity.The surge in antimicrobial resistance and the limited availability of new antimicrobial drugs has fueled the interest in optimizing antibiotic dosing. An ideal dosing regimen leads to maximal bacterial cell kill, whilst minimizing the risk of toxicity or antimicrobial resistance. For beta-lactam antibiotics specifically, PK/PD-based considerations have led to the widespread adoption of prolonged infusion. The rationale behind prolonged infusion is increasing the percentage of time the beta-lactam antibiotic concentration remains above the minimal inhibitory concentration (% Increasing drug resistance rates and the scarcity of new antibacterial drugs pose a serious threat for the clinical utility of antimicrobial drugs . In respThe scientific advances in the field of antimicrobial dose optimization have mainly been determined by pharmacokinetic (PK) and pharmacodynamic (PD) principles. PK/PD is the science relating the effect of drug exposure (PK) to an outcome measurement (PD) . For antIn recent years, a wealth of evidence emerged, demonstrating that the PK of beta-lactam antibiotics in critically ill patients is significantly different from the beta-lactam PK observed in healthy volunteers or non-critically ill patients . The patf) of the antibiotic drug remains above the minimal inhibitory concentration (MIC) is the PK/PD index of choice (fT>MIC) [10 reduction of colony-forming units (CFU/mL)) is called the PK/PD target [Beta-lactam antibiotics are considered time-dependent antibiotics and the time (T) that the unbound fraction ((fT>MIC) ,12. By cD target . ImportaD target .fT>MIC and target attainment rates  infusion of beta-lactam antibiotics is extending the duration of infusion in order to increase the %nt rates . The ultfT>MIC has been clearly demonstrated [The ability of prolonged infusion to increase the %nstrated ,15. Unfonstrated ,17. Othenstrated ,18, lowenstrated ,20, a fanstrated , increasnstrated  or impronstrated  with pronstrated ,25,26. Cnstrated .Pseudomonas aeruginosa hollow-fiber infection model for piperacillin. A dosing of 3, 9 and 17 g of piperacillin, either via intermittent (0.5 h infusion duration) or extended infusion (4 h infusion duration) was simulated. The targets (in Cmin/MIC ratios) reported for stasis, 1-, 2- and 3-log10 kill and the suppression of resistance for extended infusion were consistently higher compared with the targets documented for intermittent infusion (Klebsiella pneumoniae. For the Kp69 strain (with an MIC of 1 mg/L), a Cmin/MIC ratio of 1.09 with intermittent infusion was sufficient to avoid resistance development, while for the extended infusion a Cmin/MIC ratio of 3.18 was necessary. These examples illustrate that different PK/PD targets may apply for the same reduction of CFU when different modes of infusion are used.Prolonging the duration of infusion to increase the target attainment depends on the assumption that the PK/PD index and target by itself are static and are independent of the mode of infusion used. However, this theory has been challenged, and attaining the same PK/PD target with a different mode of infusion does not necessarily imply an equal level of bacterial cell kill . For exainfusion . In addiThe concept of dynamic PK/PD indices and targets in terms of changing beta-lactam antibiotic concentrations have previously been described ,32,33,34fT>MIC as the PK/PD index of beta-lactam antibiotics is related to the short half-life (and therefore the PK) of most of these drugs. In situations where the half-life is prolonged, for example in patients with kidney failure, fAUC/MIC was found as the best predictor of the antibacterial effect of beta-lactam antibiotics [fT>MIC was the PK/PD index best related to the antibacterial efficacy [fT>MIC is the PK/PD index of choice for amikacin in mice with a normal kidney function , as opposed to fAUC/MIC in mice with an impaired kidney function .Intriguingly, the optimal PK/PD index is also dependent on PK, as described by Nielsen et al.  and Krisibiotics . Even moefficacy . The facefficacy , fT>MIC fT>MIC with a prolonged infusion of beta-lactam antibiotics will be of no use when resistance development is concerned. Indeed, determinants other than the mode of infusion, such as the pathogen involved, the duration of therapy and the initial inoculum size, may be much more important for regrowth [An optimal dosing regimen would allow maximal antibacterial effect, whilst minimizing drug toxicity and the risk of resistance development. Nevertheless, most of our beta-lactam antibiotic dosing regimens were based upon PK/PD targets and indices for bacterial cell kill alone. However, the recent literature has illustrated that we may need different antibiotic exposures (illustrated by different PK/PD targets and indices) for the suppression of resistance, as opposed to bacterial cell kill ,41. Moress 10 times the MIC) to avoid toxicity is not relevant. As Lau et al. [Finally, a PK/PD index or target linked to bacterial cell kill will tell us nothing about the risk of toxicity, as toxicity for a patient is not associated with susceptibility. Hence, using a PK/PD target . In addition, higher dosing will lead to higher tissue concentrations, which is important in critically ill patients, given the high variability of tissue penetration to different foci of infection ,52. FinaClostridioides difficile infection [Translation into practice would require knowledge of the concentrations associated with beta-lactam toxicity and, preferably, toxicity would be easily measurable . To datenfection . Of thesnfection . Approxinfection ,54. The Crystal nephropathy, which is a result of antimicrobial precipitation and crystallization in the renal tubuli has been documented with high amoxicillin concentrations, but is assumed to be very rare and a specific drug level linked to crystallization has not been defined ,56.Hypersensitivity is a common side-effect of beta-lactam antibiotics, but is likely not linked to the dosing regimen or drug concentration. Acute interstitial nephritis and drug-induced liver injury (DILI) are immune-mediated idiosyncratic reactions, and it is therefore assumed that these reactions are also not linked to the drug concentration. Whether or not myelotoxicity is dose-dependent is a matter of debate .Not unsurprisingly, beta-lactam through concentrations are related to a decline in kidney function ,50,51. IWith respect to the theory of the ‘maximum tolerable dose’, modeling beta-lactam concentrations (PK) as well as modeling uremic toxin concentrations (PD) as two dynamic parameters (pharmacokinetic/toxicodynamic modeling), analogous to the PK/PD models incorporating dynamic bacterial growth in response to changing antibiotic concentrations, may circumvent the issues we currently experience with static PK/PD indices and targets .fT>MIC will not, by definition, lead to increased bacterial cell kill given that the PK/PD index and target are not static entities. Moreover, merely prolonging the duration of infusion in an attempt to increase the %fT>MIC is likely irrelevant when it comes to suppression of regrowth and avoidance of toxicity. In the future, administering a maximum tolerable dose instead of a (minimum) dose that has been developed to achieve a predefined PK/PD target for efficacy only, may help preserve our antimicrobial armamentarium. Currently, the specific levels of beta-lactam drug toxicity are ill-defined and therefore research focusing on the pharmacodynamics of beta-lactam antibiotic toxicity is urgently needed. A first step in this process should be measuring the uremic toxin concentrations in patients receiving beta-lactam antibiotics. These data can then be used to develop a pharmacokinetic/toxicodynamic model, which in turn could inform clinicians on the maximum tolerable dose. The patients with advanced kidney disease are at risk of both high uremic toxin concentrations, as well as high beta-lactam antibiotic concentrations and therefore represent a study population of interest for the purpose of developing such a pharmacokinetic/toxicodynamic model.An ideal antibiotic dosing regimen maximizes bacterial cell kill, whilst minimizing drug toxicity and the risk for resistance development. In critically ill patients, the finding of low beta-lactam antibiotic concentrations due to PK variability has led to the adoption of prolonged infusion to increase target attainment. From a purely PK/PD point of view, increasing the duration of the infusion to increase the %


This mechanism allows cilia to sense GABA and initiate cilia-specific Ca2+ signaling through cilia-localized GABAB1 receptors.Sanchez, Incedal et al. identify a mechanism that enables excitable cells to utilize Ca B1 receptors are strongly enriched at the base of the cilium, but are mobilized to more distal locations upon agonist binding. Using cilia-targeted Ca2+ indicators, we find that activation of GABAB1 receptors induces selective Ca2+ influx into primary cilia through a mechanism that requires voltage-dependent Ca2+ channel activation. Islet β-cells utilize cytosolic Ca2+ increases as the main trigger for insulin secretion, yet we find that increases in cytosolic Ca2+ fail to propagate into the cilium, and that this isolation is largely due to enhanced Ca2+ extrusion in the cilium. Our work reveals local GABA action on primary cilia that involves Ca2+ influx and depends on restricted Ca2+ diffusion between the cilium and cytosol.The primary cilium is an organelle present in most adult mammalian cells that is considered as an antenna for sensing the local microenvironment. Here, we use intact mouse pancreatic islets of Langerhans to investigate signaling properties of the primary cilium in insulin-secreting β-cells. We find that GABA In the 2+ dynamics within these functional micro-organs. Contrary to the vast majority of studies on the primary cilium in immortalized cell lines, the current work enabled the study of cilia signaling in a preparation that preserves both tissue architecture and function. Using this model, we unveiled cilia-specific Ca2+ activity driven, at least in part, by activation of metabotropic GABA receptors with a dependence on Ca2+ extrusion, which isolates the cilium from contaminating cytosolic Ca2+. We describe here for the first time a cilia-localized class C GPCR, the GABAB1 receptor, and show that it exhibits a distinct distribution with enrichment toward the cilia base. We also show that agonist stimulation mobilizes receptors to more distal parts of the cilium and triggers ciliary Ca2+ transients in a putative non-canonical fashion.In the present work, we used intact endocrine pancreatic islets of Langerhans to measure ciliary CaA) and metabotropic (GABAB1/2) GABA receptors, but the localization of these receptors within β-cells of intact pancreatic islets of Langerhans has not been determined. Since GABA can reach the islets both through the circulation and through local release from β-cells, receptor segregation might be a mechanism by which the islet cells can control the response depending on the source of GABA. Immunostainings of mouse islets showed the expression of all three GABA receptors. Whereas GABAA and GABAB2 receptors displayed diffuse localization with enrichment in cytoplasmic vesicles, GABAB1 receptors instead localized to a single, small, elongated compartment with an average length of 2 µm and a width of <400 nm as determined by stimulated emission depletion (STED) imaging , also presented with cilia enriched for GABAB1 receptors, but in contrast to islets the receptors were not restricted to the cilium base but occupied the entire cilium membrane . To evaluate the properties of this Ca2+-indicator, we permeabilized clonal MIN6 β-cells expressing Smo-GCaMP5G-mCh and superfused the cells with Ca2+-buffers while recording GCaMP5G and mCherry fluorescence using total internal reflection fluorescence (TIRF) microscopy. This technique enabled simple access to primary cilia protruding from the cell surface and also extended recordings of cilia Ca2+ due to low photobleaching. We observed stepwise increases in ciliary Ca2+ concentration, with half-maximal and maximal effect at 0.6 and 3 μM, respectively  initiated ciliary Ca2+ signaling in the form of distinct Ca2+ spikes without accompanying changes in the cytosolic Ca2+ concentration in MIN6 β-cells expressing Smo-GCaMP5G-mCh  and calculated their density over defined periods of time  sensors based on Epac1  strongly inhibited depolarization-induced Ca2+ influx in mouse islet β-cells  was without effect  glucose-containing buffer, we observed the occurrence of spontaneous ciliary Ca2+ flashes  and GABAB (CGP35348) receptor antagonists were without effect on the spontaneous cilia Ca2+ signaling, but washout of the antagonists resulted in a pronounced increase in Ca2+ activity , while the responses in the cell bodies were similar, showing that Ca2+ buffering within the cilium may help to shape the Ca2+ signals. Taken together, these results show that the cilium is isolated from cytosolic Ca2+, and that this at least in part is due to an enhanced Ca2+ extrusion, which is Na+ dependent and sensitive to amiloride.If the primary cilia of the islet cells utilize Carization . This ine cilium . The size cilium . The ratartments . Similar+ sensor . Next, we cilium . Interese cilium . This ef removed . The eff removed . To detee cilium . The extdilation . Togethe2+ activity in a more physiological context, we exposed Smo-GCaMP5G-mCh–expressing islets to a buffer containing 11 mM glucose, a concentration at which insulin secretion is strongly stimulated. This resulted in an immediate and very pronounced increase in GCaMP5G fluorescence in the islet cell cilia (2+ influx and was not seen with soluble GCaMP (2+ concentration change. One possibility is that ATP generated by glucose metabolism affects the fluorescent properties of GCaMP5G (2+ indicator (Lyn11-R-GECO) did however not reveal any direct impact of ATP on either GCaMP5G or R-GECO fluorescence , whereas more distal regions were phase-shifted . Ca2+ changes in dendritic spines do not propagate into the dendrite, largely due to efficient extrusion, and this mechanism is required for normal synaptic function . This implies that GABAB1 receptors have important functions independent of the G-protein-coupled GABAB2 receptor, and that these functions may depend on its ciliary localization. In some neurons, GABAB receptors are functionally coupled to voltage-dependent Ca2+ channels, and activation of the receptors have both stimulatory and inhibitory effects depending on the type of cell and channel   pre-bleach intensity), F∞c (asymptote of fluorescence recovery of the whole cell), Fbg (mean background intensity), F∞ (asymptote of the bleach ROI), and F0 (bleach ROI post-bleach intensity). The same approach was used for photorelease of caged Ca2+ in islet cells that had been preincubated for 30 min in imaging buffer supplemented with 5 µM of the AM-ester-form of o-nitrophenyl-EGTA (Thermo Fisher Scientific).FSub-diffraction microscopy was performed with a Stedycon STED super-resolution instrument (Abberior Instruments) with an Olympus BX63F upright motorized microscope body. The objective was a 100× / 1.56 NA (Olympus) and the STED depletion laser had a wavelength of 775 nm. Emission filters were 575–625 and 650–700 nm corresponding to Star 580 and Star Red fluorophores, respectively.2+ indicator 5HT6-GGECO1, we were unable to record pre-stimulatory activity in islets expressing bPAC. This was instead assessed in parallel control experiments performed on islets lacking bPAC expression. Activation of cGMP production was achieved by 561-nm light exposure delivered through evanescent wave excitation on the stage of a TIRF microscope (see below for details). Activation light was delivered in pulses (100 ms every 2 s) and the impact on Ca2+ signaling was determined using the co-expressed cilia-targeted Ca2+ indicator 5HT6-GGECO1.Islets were transduced with adenoviruses carrying the blue-light-activated adenylate cyclase (bPAC) or the yellow-light-activated guanyl cyclase (RhGC) under the control of cytomegalovirus (CMV) promoters. Activation of cAMP production was achieved by 491-nm light exposure delivered through evanescent wave excitation on the stage of a TIRF microscope (see below for details). Activation light was delivered in pulses (100 ms every 2 s). Because of spectral overlap between activation of bPAC and excitation of the cilia-targeted Ca2+ and were indicated and depolarized with 30 mM KCl.Islet compression experiments were performed with a custom-built manipulator referred to as the cell press . The dev2 2.6, MgCl2 1.2, KCl 5.6, Hepes 10, and glucose 3; pH 7.4, 300 mOsm). The pipette solution was puffed toward the cilia by gently applying positive pressure with the mouth. A second borosilicated pipette with the same resistance was used to induce plasma membrane depolarization by puffing a solution containing (in mM): NaCl 63, CaCl2 2.6, MgCl2 1.2, KCl 8.6, Hepes 10, and glucose 3; pH 7.4, 300 mOsm. The mechanical stimulation pipette was located >5 µm from the cilia head and the high potassium pipette 10–15 µM from the cell body at the opposite side of the mechanical stimulation pipette. The mechanical stimulation was intermittently applied every 1 s during 225 s. During the recording, the cilia were allowed to rest from the stimulation 1–3 times to see possible changes at the cilia fluorescence baseline. Then, cells were let to rest 25 s without stimulation before inducing membrane depolarization for 200 s. The high potassium solution was puffed by applying constant positive pressure (3.7 psi) controlled by a Pico Spritzer . The experiments were carried out at 32°C. The samples were observed with a 40× Zeiss objective with a 1.2 NA. A Prime 95B CMOS camera (Teledyne Photometrics) was used to acquire the images, and GCaMP and mCherry fluorophores were excited by 473- and 561-nm lasers (Cobolt). Red and green channel images were acquired simultaneously at a 2 Hz frequency, and the emission light was separated onto the two halves of the camera chip using an image splitter  with a cutoff at 565 nm  and emission filters . Cilia and cell body intensities over time were analyzed by homemade Macros in Fiji (ImageJ). Results are reported as the Δ(GCaMP/mCherry) intensity ratio.For mechanical stimulation of MIN6 cell cilia, we used borosilicate pipettes of 0.5–1µM diameter to puff extracellular solution locally . Next, a wash with 2% BSA in PBS for 5 min was followed by 1 h incubation with secondary antibodies (same way as primary). Subsequently, samples were washed with 2% BSA in PBS for 5 min and mounted using ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). To preserve islets structural integrity, they were embedded into a 2% agar in PBS gel after being put into mounting medium.2+ imaging data (TIRF) was performed by drawing ROIs containing the cilium from where mean values of pixel’s intensities were extracted from each time frame. Kymographs were created using the corresponding function in Fiji; for ratiometric ones, the GCaMP kymograph was divided by the corresponding mCherry kymograph. For the analysis presented in 2+ activity in the cilium was analyzed using the Neural Activity3 . Samples were tested for normal distribution using Kolmogorov-Smirnov test to fulfill the criteria for performing Student’s t test , and logarithmic transformations were performed in cases of non-normal distributions. Where suitable, non-parametric Wilcoxon signed-rank tests were performed instead. For small data sets, non-parametric Mann–Whitney U-tests were performed instead. For multiple comparisons where data distribution was normal, one-way ANOVA was used followed by Tukey’s or Dunnett’s post hoc tests. When data distribution was non-normal, Friedman test or Kruskal–Wallis test with Dunn’s multiple comparisons was used instead. Fisher’s exact test was used for categorical data. Graphs show mean values ± SEM unless otherwise stated. We did not perform a power analysis to predetermine sample sizes. The experiments  and control experiments showing that overexpression of this sensor does not interfere with Hedgehog-dependent exit of Patched from the cilium or with Hedgehog-mediated cilia lengthening. 2+ clearance mechanisms in the cilium and confirmatory experiments using an alternative cilia-localized Ca2+ indicator. 2+-independent effects of glucose on Ca2+-indicator behavior in the primary cilium.Review HistoryClick here for additional data file.


The intracellular calcium handling system of cardiomyocytes is responsible for controlling excitation-contraction coupling (ECC) and has been linked to pro-arrhythmogenic cellular phenomena in conditions such as heart failure (HF). SERCA2a, responsible for intracellular uptake, is a primary regulator of calcium homeostasis, and remodelling of its function has been proposed as a causal factor underlying cellular and tissue dysfunction in disease. Whereas adaptations to the global (i.e. whole-cell) expression of SERCA2a have been previously investigated in the context of multiple diseases, the role of its spatial profile in the sub-cellular volume has yet to be elucidated. We present an approach to characterize the sub-cellular heterogeneity of SERCA2a and apply this approach to quantify adaptations to the length-scale of heterogeneity (the distance over which expression is correlated) associated with right-ventricular (RV)-HF. These characterizations informed simulations to predict the functional implications of this heterogeneity, and its remodelling in disease, on ECC, the dynamics of calcium-transient alternans and the emergence of spontaneous triggered activity. Image analysis reveals that RV-HF is associated with an increase in length-scale and its inter-cellular variability; simulations predict that this increase in length-scale can reduce ECC and critically modulate the vulnerability to both alternans and triggered activity.This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’. T. T2.1γ(rat r = 1 b and σ2⋅ (b)2+ dynamics model [This study used a simplified version of the O'Hara-Rudy dynamic human ventricular model ,29,30, ics model . SERCA2af(x) over a multi-dimensional space in which each point x ∈ ℝn takes some random value from a domain of real numbers [σ2, and correlation length-scale, λ, to reflect physiological boundaries on these parameters [c), which can be produced at a discretization resolution corresponding to that of the three-dimensional computational model: each voxel  of the spatial map represents one CRU, and the local Jupmax scale factor is given by the associated value in the expression map (d).Given the distribution of SERCA2a expression observed . Any number of unique GRFs may be produced using the same parameter set, enabling an assessment of the impact of structural arrangement on Ca2+ handling dynamics; except where otherwise stated, three independent maps were generated and implemented at each length-scale.It was observed  that whereas some small inter-cell variation in relative whole-cell SERCA2a expression was present, the significant difference was in correlation length-scales. In order to isolate the impact of length-scale only, isotropic maps  were generated with the same whole-cell mean SERCA2a expression and whole-cell input variance, σ) of the distribution  or, equivalently, by the range defined by ±3σ. In the present study, a σ of 0.3 was implemented for all simulations at all length-scales, corresponding to the ±3σ range of 0.1–1.9. Note that this rescaling was applied after the normalized (σ2 = 1) GRF was produced.It is important to note that the length-scale and the total degree of heterogeneity/variation are independent of each other; the length-scale determines the spatial correlation of the values within the range defined by the total variation of the distribution. The degree of heterogeneity can be controlled by the standard deviation ( (c)2+ transient (CaT) under normal pacing and pro-arrhythmogenic conditions, corresponding to CaT alternans and spontaneous Ca2+-release events (SCRE).In this study, we quantified the length-scales describing the sub-cellular distribution of SERCA2a in rat healthy and RV-failure myocytes. Simulations were then performed to assess the impact of different heterogeneity parameter profiles on the intracellular Ca (i)λ = 1, 3, 5, 10 µm), three maps were used for a total of 12 heterogeneous maps and the homogeneous control.Expression maps were loaded into the MSCSF  and pace (ii)2+ transient alternans were studied by applying rapid pacing in combination with multiple parameter combinations which are known to promote alternans [NLTCC, or a reduction to the channel open transition rate, LTCCPO) and SERCA2a , rapid pacing (basic cycle length = 400 ms) was applied in combination with a functional model of isoprenaline (ISO) which comprises enhanced LTCC activity (×2) and SERCA2a activity (×1.75) as well as enhanced K+-currents to maintain AP duration [2+ loading and the uncertainty in the degree of enhanced activity owing to ISO, we also introduced a condition in which In order to induce SR-Caduration . Owing t. 3 (a)N myocytes = 5 LV and 11 RV) and MCT (N = 6 LV and 7 RV) treatment.In total, 29 datasets were analysed, including stacks and single images from both left ventricular (LV) and RV rat cardiomyocytes taken from animals which underwent the control  to range between 1 and 4 µm in control cells (mean = 2.277 ± 0.143 µm) and between 2 and 11 µm in MCT cells (mean = 4.930 ± 0.588 µm). A significant difference in λ of these cells was found , thus the observed remodelling in HF [figure 2b): RV cells were observed to have a higher correlation length-scale in both control and HF remodelling  than LV cells . Throughout this paper, an increase in length-scale is considered as an increase in heterogeneity, as it corresponds to larger patches of high/low channel expression, although we recognize that ‘increased heterogeneity’ can be ambiguous in these cases. Thus, from herein, ‘increased heterogeneity’ is synonymous with an increase in λ.Correlation length-scales, observed a to rangng in HF –13 has t (b)a), congruent with the expectations of correlation length-scales. In control pacing, whole-cell CaTs  generally decreased in magnitude as length-scale increased (b) despite the maintained global expression of SERCA2a. The reduction in the CaT was ultimately attributable to the diastolic SR-Ca2+ load, which was significantly reduced compared to the homogeneous condition as length-scale increases (b). An increase in length-scale was also associated with an increase in inter-cellular variability of both the CaT magnitude and diastolic SR-Ca2+ load.The length-scale parameter input had a clear impact on the spatial properties of the three-dimensional GRFs used to perform simulations a, congruncreased b despitencreases b. An incc). This spatial variation, and its inter-cellular variability, was increased with length-scale.The reduction in CaT magnitude associated with longer length-scales is accompanied by an increase in the spatial variation of the CaT throughout the cell, as a direct consequence of local SERCA2a function: regions of high/low SERCA2a spatially correlated with more rapid/slower decay of the CaT c. This s2+ did not exhibit such a simple relation to local SERCA2a expression and was dependent on the time within the cycle : during early refilling stages, the regions with high SERCA2a expression exhibited the most rapid refilling and thus larger local SR-Ca2+; however, by late-stage refilling towards the end of the cycle, regions of low-SERCA2a expression exhibited the highest SR-Ca2+ load owing to a combination of diffusion within the SR (from high to low-SERCA2a regions) and continued uptake in these low-SERCA2a regions owing to local cytosolic Ca2+ remaining high (figure 4a). The increased SR-Ca2+ load in these regions should reduce the activity of Jup relative to this cytosolic Ca2+, providing a potential mechanism by which SR diffusion can reduce the overall activity of Jup in heterogeneous map conditions.The spatial profile of SR-Ca2+ (b) was evaluated by varying the time-constant of diffusion within the SR in a single heterogeneous SERCA2a map . Larger time-constants (i.e. slower diffusion) led to a smaller reduction in the SR-Ca2+ relative to the homogeneous model than smaller time-constants, supporting the feasibility of the above proposed mechanism. However, even at time-constants that effectively block SR diffusion within the time-scale of the cardiac cycle, the SR-Ca2+ load was still reduced compared to the homogeneous control, indicating that this mechanism alone does not fully explain the observations. Nonlinearity in the dependence of Jup on both cytosolic- and SR-Ca2+  could also contribute. This was evaluated by assessing the diastolic SR-Ca2+ load in homogeneous cell models with the global expression of SERCA2a scaled (c): increasing SERCA2a expression led to a relatively smaller increase in diastolic SR-Ca2+ than the reduction that was observed when SERCA2a was reduced (c). Thus, averaging a normally distributed variation in SERCA2a would decrease the SR-Ca2+ load compared to the homogeneous, control condition; indeed, this is the case in the simulations where SR diffusion was effectively blocked. These two factors therefore combine to produce the overall observed reduction in SR-Ca2+ and consequently the CaT magnitude.The impact of intra-SR diffusion on cell-averaged SR-Ca2+ b was evaa scaled c: increa reduced c. Thus,  (c)2+ handling system were more substantially affected by pacing rate, with the differences between homogeneous and heterogeneous conditions generally enhanced at rapid pacing rates compared to slower pacing rates . The nature of the rate dependence can also be affected: in homogeneous conditions, there was observed an increase in the CaT peak at more rapid pacing rates, whereas in the heterogeneous maps, pacing rates above 130 bpm demonstrated a reduction in the CaT peak.SERCA2a heterogeneity had a negligible impact on the rate dependence of the AP . Properties of the Ca (d)a), although the fewest/smallest alternans were observed at a length-scale of 10 µm across all conditions. There was also a substantial degree of inter-map variation at each length-scale , i.e. the magnitude of alternans, and indeed whether or not they appeared, was dependent not only on the length-scale but also the specific features of the map, leading to increased inter-cellular variability. Despite the difference between some of the parameter combinations being very small , these disparities could lead to opposing behaviour. This indicates the high sensitivity of the emergence of alternans to model conditions and provides an explanation for the impact of SERCA2a heterogeneity: it can either push the cell into or out of the phase-space necessary for alternans, thus either inducing them where they were not present, or inhibiting them where they were present.Introducing heterogeneous underlying SERCA2a expression maps either inhibited alternans present in the homogeneous model, or induced alternans under conditions where they were not present in the homogeneous model. This shift  occurs generally across the range of parameter combinations considered a, althoub): those regions which were or were not active during the small beat were largely consistent across subsequent small beats. Analysis of the local CaT in individual CRUs reveals that this regularity is primarily determined by local diastolic Ca2+ (b) and local SR-Ca2+ : in regions of low SERCA2a, intracellular uptake is slow and thus decay of the CaT is slow; local diastolic Ca2+ therefore remains higher at the time of the next excitation, and, as with normal pacing, this is associated with higher local SR-Ca2+ loads; the RyRs are therefore more robust to reactivation. In the homogeneous model, however, there is no significant regular variation in diastolic Ca2+ or SR-Ca2+ throughout the cell and thus the alternans mechanism in this condition is not directly determined by local Ca2+.Whereas alternans in homogeneous cells demonstrated essentially random spatial properties , the introduction of SERCA2a heterogeneity reduced the random nature of the alternans and introduced a broadly regular structure b: those lic Ca2+ b and loc (e)2+ loading protocols described above, SCRE activity was detected by measuring characteristics of any wave exceeding a suitable threshold (greater than 0.135 µM) in cytosolic Ca2+ over the quiescent period. Delayed after depolarizations (DADs) and triggered action potentials (TA) were detected by measuring characteristics of any depolarization in transmembrane potential which exceeded suitable thresholds for each type of behaviour .Following the application of the rapid pacing SR-Caa); however, no definitive pattern emerged which correlated with the length-scale itself. Rather, any introduction of heterogeneity at any length-scale increased the TA count relative to the homogeneous condition: mean TA count for the homogeneous model was 0.20 ± 0.06, compared to 0.44 ± 0.07 for λ = 1 µm (p < 0.05), 0.36 ± 0.07 for λ = 3 µm (p < 0.05), 0.58 ± 0.08 for λ = 5 µm (p < 0.01) and 0.5 ± 0.08 for λ = 10 µm (p < 0.01). When SCRE did occur, the mean magnitude of the spontaneous CaT (SCaT) did not differ significantly between different length-scales. However, the magnitude did vary significantly between individual maps, both within and between length-scales (a).In condition 1 , the introduction of SERCA2a heterogeneity increased the count and probability of TA occurring a; howeveh-scales a.Figureb) heterogeneous SERCA2a expression yielded a significant decrease in mean peak SCaT magnitude as length-scale increases. No significant differences were observed in the TA count for short length-scales , but at length-scales corresponding to HF cells , a significant reduction in the TA count was observed, from 0.55 ± 0.05 in.the homogeneous condition to 0.32 ± 0.1 for λ = 5 µm (p < 0.05) and 0.05 ± 0.04 for λ = 10 µm (p < 0.01).In contrast with condition 1, in condition 2  the direct impact of local SERCA2a efflux, affecting the magnitude of Ca2+ which propagates to neighbouring CRUs; and (ii) the secondary effect of local SERCA2a activity determining local SR-Ca2+ load, which itself influences the probability of spontaneous or triggered Ca2+ sparks. These two mechanisms were isolated by performing simulations in which, following pacing to steady state, either homogeneous SR-Ca2+ load was imposed across the cell while maintaining the heterogeneous SERCA2a map, or the homogeneous SERCA2a map was imposed across the cell while maintaining the heterogeneous distribution of SR-Ca2+ load. These data (c) reveal that it is primarily the heterogeneous localized SR-Ca2+ load which determines the changes to SCRE activity, rather than the direct impact of SERCA2a on Ca2+ wave propagation. This explains the requirement of increased global SERCA2a for an increase in TA in heterogeneous maps, as it is these conditions which sufficiently load local regions of the SR-Ca2+ to both induce and maintain spontaneous Ca2+ waves; in the ISO-only condition, the loss of SERCA2a function and reduced whole-cell SR-Ca2+ load associated with increased λ  but may also originate from opposing locations (i.e. high SERCA2a), as a consequence of more rapid refilling in these regions during the Ca2+ wave .There is a causal but complex correlation between local SERCA2a expression and the nucleation sites for spontaneous Ca. 4 (a)2+ release events and incidences of spontaneous transmembrane depolarizations in the heterogeneous models 2+ uptake in failing human hearts [2+, such as increased SR-Ca2+ leak through the RyRs [3Rs) [2+ load and reduced magnitude of the CaT, as well as an increase in the spatial heterogeneity of the CaT itself. Moreover, heterogeneous SERCA2a expression resulted in an increase in inter-cellular variability, another feature commonly associated with HF [2+ is a consequence of reduced activity of intracellular uptake in heterogeneous conditions owing to a combination of the inherent nonlinearities in the dependence of Jup on intracellular- and SR-Ca2+, and the impact of intra-SR diffusion.HF is associated with a loss of contractile performance, underlain by reduced efficacy of CICR. The function of SERCA2a is strongly correlated with a decrease in SR-Can hearts  and propn hearts –38, somen hearts , whereasn hearts . Other mRs [3Rs) . This pr with HF ,42. Our  (c)Previous studies have shown the importance of CRU coupling and the inherently random dynamics of sub-cellular CaT alternans ,43, whicet al. [2+ dependence. This leads to largely the same spatial pattern of the CaT on subsequent small beats, contrary to what is observed in homogeneous cells. This difference in the fundamental underlying mechanisms of CaT alternans may have critical implications for the most effective and safe method to manage these phenomena.The present study adds to this discussion by also demonstrating that heterogeneous magnitude of SERCA2a in different regions of the cell can also constrain the random spatial nature of CaT alternans, suggesting a shift of mechanism from the 3Rs described by Qu et al.  , protective  or both . It will be important to establish whether remodelling of SERCA2a heterogeneity precedes, follows, or is concomitant with remodelling of whole-cell channel expression. (d)IK1 is observed in HF and promotes the emergence of TA from underlying SCRE [2+ loading: above a threshold, increased length-scale (as observed in HF) was associated with an increase in SCRE and TA count; below this threshold, an increase in length-scale inhibited the emergence of TA. In either case, inter-cellular variability in the emergence of TA was substantially increased. HF conditions, such as fibrosis and reduced IK1, may significantly reduce the minimal substrate required for cellular TA to manifest in tissue and thus the increased presence of individual cells which are pro-TA could underlie increased arrhythmia triggers in HF. Further investigation is required at the systems level to determine whether an increase in SERCA2a heterogeneity in HF contributes to increased arrhythmia triggers.HF is generally associated with increased cellular triggers, which may manifest as focal excitations in whole-heart-inducing arrhythmia . A reducing SCRE ,46. Howe2+ waves (through increased SR-Ca2+ load) but also inhibit them (through impairing inter-CRU Ca2+ propagation and increasing the SR threshold).The observed complex and biphasic impact of SERCA2a on SCRE is consistent with previous modelling and experimental studies –49, whic (e)a). To ensure no differences owing to imaging modality, only confocal microscopy images produced by the authors were used in this study.There are a number of limitations associated with the present study, pertaining to the experimental data analysis and the simulation results. There is inherent spatial variation present within the imaging datasets owing to the quality of staining and differences in imaging conditions which may contribute towards estimation of correlation length-scale. This is mitigated by the down-sampling procedure which averages out this data over a resolution 10–50 times larger than the original image a. To ensb). The final values of correlation length-scale are a statistical mean and standard error for each cell. Approximately 40% of the cells in this study were single images, with 60% having six or more images, and 30% having 20 or more. All cells analysed for this study were done so to the maximum possible extent; z-axis integration ensured both single images and stacks were comparable while ensuring three-dimensional features were captured. All images within a stack underwent the same processing step determined suitable for all images within that stack. This method may also measure anisotropy within sub-cellular expression; however, owing to the sizes of processed datasets, there was lower confidence in the estimations for anisotropy for the cells analysed in this study. For this reason, only isotropic analysis was considered in this study. It is likely that longitudinal-transverse anisotropy is a feature in sub-cellular heterogeneous expression at the micron scale, and this may contribute to the large error sizes in the cells with a higher correlation length-scale.The variogram fitting procedure works better with larger datasets; owing to the processing required by the image analysis step, some of this data is lost. This included condensing the data into two-dimensional, motivated by the variability in image quality and cell morphology, as well as the limited availability of data with a sufficient number of slices. To ensure reliable values were obtained from this analysis, each cell was analysed three times, each time requiring 50 successful variogram fits using a range of binning parameters suitable for each dataset b. The fiJup, is ultimately regulated by both SERCA2a and PLB expression, and therefore the assumption that local SERCA2a expression directly correlates with Jup magnitude is an over-simplification. It would be more correct to state that the heterogeneity maps implemented in the simulations represent Jup rather than SERCA2a. It would therefore be valuable in future studies to generate these maps based on combined analysis of SERCA2a and PLB, as it is unknown whether their heterogeneity will spatially correlate.One major component which was not accounted for in the present study is the SERCA2a inhibitor phospholamban (PLB). The intracellular uptake flux, λ = 1, 3, 5 and 10 µm) were chosen for the computational study as they represented the range of heterogeneity observed in the image analysis study. Observing the full range of integer length-scales may have provided a smoother gradient of behaviour in length-scale; however, owing to the scope of this project, computational tractability and the range covered by this choice of length-scales, it was determined that they were sufficient to reveal the full range of emergent behaviour. Similarly, the total extent of heterogeneity was not varied within the present study, and expression was assumed to follow a normal distribution; it would be important to see if (and in what way) the impacts of length-scale are affected by both the total heterogeneity and the skew of the distribution.Four correlation length-scales  and other pro-arrhythmogenic conditions, such as atrial fibrillation and ageing. It is noteworthy that the observed differences between control and HF are very similar in both RV and LV, despite this being an RV-HF model, indicating that this could possibly be a general and common feature of HF and perhaps other diseases.The present study implemented models and data from multiple species, i.e. using experimental data from rat and a human-based computational model. This was motivated by the fundamentally mechanistic aims of the study in combination with the models of human ventricular electrophysiology being more robust and better developed than those of the rat. We note that in simulations, cell-specific heterogeneity maps were not used. Rather, maps at different length-scales were implemented, covering the range observed in the data, enabling the general mechanistic relationship between length-scale and dynamics to be elucidated. Future studies which aim to provide cell-specific insight, for example, in explaining specific functional data, would be better performed using data and models from the same species.2+ handling transporters (such as NCX and RyRs), as this will undoubtedly influence local flux balance and SR-Ca2+ loading, as well as in combination with the global remodelling of Ca2+ handling and ion-current channel expression. Furthermore, translating the impact on inter-cellular variability into tissue models would provide more substantial insight into the impact of SERCA2a heterogeneity and increased inter-cellular variability on the emergence of arrhythmia.Whereas the present study focussed on heterogeneity at the macroscopic (micron) scale, super-resolution (nanometre) properties of heterogeneity and variability, such as clustering and co-localization distances with other channels, will also probably be highly important for governing local function . Moreove. 52+-transient alternans and spontaneous Ca2+-release. We have therefore established that the spatial profile of SERCA2a in the sub-cellular volume, and potentially that of other Ca2+ handling transporters, is a property which may be remodelled in cardiovascular disease and can contribute to observed pathophysiology of function.The present study has quantified the remodelling of SERCA2a sub-cellular heterogeneity in RV-HF. It demonstrates a general increase in the correlation length-scale, and its inter-cellular variability, with HF. These changes were predicted to contribute to reduce CICR under normal pacing conditions, as well as modulating, sometimes critically, the emergence of Ca