Failing myocytes undergo electrophysiological remodeling, which is known to be the main cause of abnormal Ca2+ homeostasis. However, structural remodeling, specifically proliferating fibroblasts coupled to myocytes in the failing heart, could also contribute to Ca2+ cycling impairment. The goal of the present study was to systematically analyze the mechanisms by which myocyte–fibroblast coupling could affect Ca2+ dynamics in normal conditions and in HF. Simulations of healthy and failing human myocytes were performed using established mathematical models, and cells were either isolated or coupled to fibroblasts. Univariate and multivariate sensitivity analyses were performed to quantify effects of ion transport pathways on biomarkers computed from intracellular [Ca2+] waveforms. Variability in ion channels and pumps was imposed and populations of models were analyzed to determine effects on Ca2+ dynamics. Our results suggest that both univariate and multivariate sensitivity analyses are valuable methodologies to shed light into the ionic mechanisms underlying Ca2+ impairment in HF, although differences between the two methodologies are observed at high parameter variability. These can result from either the fact that multivariate analyses take into account ion channels or non-linear effects of ion transport pathways on Ca2+ dynamics. Coupling either healthy or failing myocytes to fibroblasts decreased Ca2+ transients due to an indirect sink effect on action potential (AP) and thus on Ca2+ related currents. Simulations that investigated restoration of normal physiology in failing myocytes showed that Ca2+ cycling can be normalized by increasing SERCA and L-type Ca2+ current activity while decreasing Na+–Ca2+ exchange and SR Ca2+ leak. Changes required to normalize APs in failing myocytes depended on whether myocytes were coupled to fibroblasts. In conclusion, univariate and multivariate sensitivity analyses are helpful tools to understand how Ca2+ cycling is impaired in HF and how this can be exacerbated by coupling of myocytes to fibroblasts. The design of pharmacological actions to restore normal activity should take into account the degree of fibrosis in the failing heart.Heart failure (HF) is characterized by altered Ca Heart failure (HF) is a major public health problem worldwide  and the 2+ handling in myocytes, since Ca2+ homeostasis is crucial for cell contraction and relaxation (2+ transient (CaT) with a slow rise time and a reduced rate of [Ca2+]i removal that prolong CaT duration and elevate the diastolic intracellular Ca2+ level , late Na+ current (INaL), transient outward K+ current (Ito), L-type Ca2+ current (ICaL), Na+ current through the L-type channel (ICaNa), K+ current through the L-type channel (ICaK), rapid delayed rectifier K+ current (IKr), slow delayed rectifier K+ current (IKs), inward rectifier K+ current (IK1), Na+/Ca2+ exchange current (INCX), Na+/ K+ ATPase current (INaK), background currents , and sarcolemmal Ca2+ pump current (IpCa). A detailed Ca2+ dynamics is also formulated in the model. Properties such as conductances determining ionic densities and membrane kinetics can be found in the original work , INaL conductance, the maximal flux of INCX and SR Ca2+ leak (Jleak), and the fraction of active binding sites of the Ca+2 calmodulin-dependent protein kinase II (CaMKa) were upregulated, while conductances of Ito and IK1, the maximal flux of INaK, and Ca2+ uptake via SERCA pump (JSERCA), and SR Ca2+-dependence of the steady-state activation of ryanodine receptor release  were downregulated. Further details about values and experimental references can be found in Supplementary Table S2.All simulations were performed at the cellular level. To study the electrophysiological behavior of cardiac myocytes, we used the most complete undiseased human ventricular AP model, developed by nal work . We intrnal work  and leadur group . Specifigap) flows from one cell to the other through gap junctions, driven by the potential gradient between the myocyte (VM) and the fibroblast (VF) membrane potential and regulated by a junctional conductance (Ggap).The increase in fibroblasts density due to aging and/or HF, and their interaction with myocytes were modeled using established cell–cell coupling equations. Fibroblasts were resistively coupled to one myocyte (Eq. 2), and a current (I+ current (IKr), an inward-rectifying K+ current (IK1), a Na+/K+ ATPase current (INaK), and a background Na+ current . The membrane potential is governed by the following ordinary differential equation:The electrical activity of fibroblasts was formulated using an active fibroblast model . The fibgap. Regarding fibroblast properties, the membrane capacitance (CF) was set to 6.3 pF and the resting membrane potential (EF) had a value of -49.6 mV as in The electrotonic interaction with the myocyte is included in the term IM = 153.4 pF vs CF = 6.3 pF). The value of Ggap was set to 3 nS for a single fibroblast, considered the nominal value in gap fivefold , calcium transient (CaT) duration from maximal upstroke to 80% of recovery (CaTD80), CaT rise time from 10 to 90% of upstroke (t10-90), and systolic and diastolic [Ca2+]i values. All biomarkers were calculated under steady state conditions after application of 1000 stimuli at 1 Hz.To evaluate the electrophysiological activity of myocytes, and particularly CaNa, INaL, Ito, ICaL, IKr, IKs, IK1, INaK, INCX, the SR Ca2+ uptake via SERCA pump (JSERCA), the SR Ca2+ release flux via RyR (Jrel), the SR Ca2+ leak (Jleak), and INab. These parameters were varied with a scaling factor obtained from a log-normal distribution of standard deviation (σ) equal to 0.3. This led to a 95% (±2σ) of parameters varying between 55 and 182% of its control value, representing inter-individual variability and drug-induced effects. A standard deviation of 0.1 was also considered in different sets of populations.The aforementioned models were used as the baseline to generate four populations of 300 different individuals. Inter-subject electrophysiological variability was represented by variating maximal ion current conductances with a scale factor, assuming that there are variations in the number of ion channels in the cell membrane between individuals . The var90 and systolic and diastolic [Ca2+]i values, in which electrical myocyte activity was considered normal. These limits were obtained from the baseline models as APD90normal ± /2 and after being compared to experimental studies  of parameters in the range of ±60% variation was used for this purpose. In this case, only the basic HF model (with and without coupled fibroblasts) was considered. We set limits for APD studies . Table 1Linear regression was employed to analyze biomarker sensitivities to electrophysiological variables. First, a univariate sensitivity analysis was conducted varying each parameter individually by a scaling factor of equal magnitude from the baseline models. The individual variation was set to ±60 or ±15% in different sensitivity analyses. This approach was based on our previous work .R2) indicated the predictive power of the simplified linear model, being R2 = 1 a good fit.Multivariate regression analyses were then performed using the generated populations of models. We related all the conductance scaling factors configurations (x) to each biomarker (y) with regression coefficients (b) that attempted to predict the indicators (Eq. 5). These coefficients were obtained by applying partial least squares (PLS) method to data as shown in Eq. 6 and after several transformations, such as log-transformations and z-score calculations . The obtBoth types of sensitivity analyses were compared with normalized sensitivities to study the similarities and differences in parameter identification as the most important contributors to each electrophysiological characteristic. Normalized sensitivities were calculated as the ratio between each sensitivity and the maximum absolute value obtained for a particular biomarker.The comparison between isolated and coupled myocytes was performed after adjusting the bar graphs to the standard deviation of the log-transformed biomarkers in N and HF conditions, to ensure equivalent percentages of change.Figure 1 illustrates two populations of models (n = 300) for endocardial human cells paced at 1 Hz, under normal (blue traces) and HF (red traces) conditions, with baseline models for the two respective populations indicated with black solid and dashed lines. The model population calibration process, meant to reproduce natural variability, generates a wide range of physiological AP waveforms and CaTs in both groups. This variability allows us to understand predicted drug effects by considering the HF cells with behavior most similar to normal cells, or vice versa. The electrophysiological remodeling applied to the basic ORdmm model to generate a baseline HF model reproduced the characteristic HF phenotype of prolonged APD and a slower Ca2+ dynamics with depressed systolic [Ca2+]i and elevated diastolic levels. In the generated normal and failing populations, significant differences between both conditions are observed in APs and CaTs, although some traces overlap. To understand the variability within the populations and predict what alterations might cause HF cells to behave more like normal cells, we performed a sensitivity analysis.Figure 2 represents the sensitivity of quantitative indicators of AP and CaT characteristics to changes in ion channels obtained in the univariate and multivariate sensitivity analyses. Sensitivities helped reveal the most important modulators of a determined electrophysiological property. High regression coefficients highlighted an important effect of a particular transport mechanism on Ca2+ indicators, taking into account the synergy between variables in the case of multivariate analysis. Our results showed that IKr was the parameter with the major impact on APD90 , while SERCA had the leading role in Ca2+ biomarkers . Sensitivities were normalized to allow comparison between the sensitivities obtained through a ± 60% univariate sensitivity analysis (light blue for N conditions and pink bars for HF conditions) with multivariate sensitivity analysis (dark blue for N conditions and red bars for HF conditions). In this comparison, results from both methodologies were consistent for APD sensitivities . Small differences were though observed in the case of Ca2+ biomarkers . For instance, Figure 2B shows that in HF, IKr seems to have a moderate impact on CaTD80 according to the univariate results (pink bars), while multivariate sensitivity indicates that the effect is lower (red bars). In other cases, with the univariate analysis, the relative importance of parameters can be altered between the N and HF conditions. This was the case for ICaL, INCX, and Jleak on t10-90 . Figure 2D shows how NCX has the main role in modulating systolic Ca2+ according to the univariate sensitivity analysis (light blue in N and pink bars in HF), while the multivariate sensitivity highlights SERCA and ICaL as the main modulators in N (dark blue bars) and HF (red bar) conditions, respectively, and the exchanger has a secondary role.Figure 2, light and dark blue vs pink and red), such as an increased impact of INaL on APD90 in HF with respect to normal conditions , a higher IKr influence on CaTD80 , and a decrease of SERCA modulation effect on systolic [Ca2+]i .Multivariate and univariate analyses also identified similar changes in parameter sensitivities from N to HF . For example, considering low variability in HF , both analyses (univariate and multivariate) highlighted ICaL as the most important parameter regulating systolic [Ca2+]i, while univariate sensitivities obtained with 60% variability (darker pink bar) highlighted INCX as the main regulator and underestimated the impact of ICaL.We further investigated the aforementioned discrepancies by performing additional sensitivity analyses with a lower conductance variability. Parameter variability was reduced to 15% in the univariate study and the standard deviation was decreased to 0.1 in the multivariate regression, resulting in sensitivities slightly different from those described above with higher variability . After reducing parameter variability, negligible differences were found when comparing both methods . This indicates that, with large parameter variability, and in the HF condition, significant non-linear relationships between parameter values and Ca2+ handling processes make the multivariable regression model less accurate.Coefficients of determination of the multivariable regression are shown in In summary, univariate and multivariate sensitivity analyses yield very similar results for low variability of parameters, which would correspond to natural inter-subject electrophysiological differences. Univariate analysis is less computational expensive and is thus a valid methodology under these conditions. However, when large variability is applied, which would respond to effects of drugs, significant discrepancies arise between both methodologies. Multivariate analysis should be more reliable, at least for biomarkers with coefficients of determination close to one. Indeed, multivariate analysis considers interaction between the different parameters of the ion channels altered.Figure 3 shows APD and systolic [Ca2+]i reduction in the normal baseline human endocardial model when fibroblasts were coupled (solid vs discontinuous blue trace), as well as in the failing baseline endocardial model (solid vs discontinuous red trace).When simulations were performed to electrically coupled fibroblasts to a myocyte, the myocyte’s AP and CaT were significantly changed. n = 300) generated for both N and HF conditions, the above-mentioned effects on APD and CaT were maintained, as shown in Figure 4. CaT traces in normal conditions  with coupled fibroblasts (discontinuous blue traces) overlapped with CaT traces in the absence of fibroblasts (solid blue traces).When fibroblasts were coupled to myocytes from two populations of models  decreased both in normal and failing conditions when fibroblasts were considered (discontinuous bars).To evaluate whether fibroblasts coupling would significantly change sensitivities of electrophysiological biomarkers to parameters variability, multivariate analyses were conducted on these new populations. When a high variability of parameters was considered (σ = 0.3), our results showed that in the presence of fibroblasts, biomarkers sensitivities to ionic variables slightly changed with respect to uncoupled myocytes. As displayed in Supplementary Figure S3), sensitivities hardly changed with respect to higher variability.To confirm these results, we decreased parameter variability to σ = 0.1 and obtained more accurate regression coefficients. In the new range of parameter variability . The population of models approach generates cells exhibiting a wide variety of physiological behaviors, including cells within the HF population that have APs and CaTs similar to those observed in healthy cells. Selecting this subpopulation and examining the distributions of parameters provides guidance for therapeutic targets. We identified parameter combinations restoring APs and CaTs to waveforms within a healthy range shown in Table 1 in Section “Materials and Methods.” In Figure 6A, red traces represent all models generated from the baseline ORdmm HF model (black dashed line) and blue lines are models satisfying healthy ranges of both AP and CaT biomarkers. Electrophysiological HF phenotype was restored in around 500 models when IKr, SERCA, and ICaL activities were enhanced, and NCX function decreased. Interestingly, NCX distribution presented several outliers at high scaling factors. When myocytes were coupled to fibroblasts , the role of SERCA, ICaL, and NCX was similar, whereas IKr modulation was not important.To analyze how failing APs and CaTs could be restored and brought to normal ranges, a larger population of failing models was generated  and without (dashed line) fibroblast coupling. To understand how these steady-state conditions were reached, we analyzed transient changes in myocyte [Ca2+] after fibroblast coupling, as shown in Figure 8. In these panels, isolated myocytes are initially at steady state, then fibroblasts are coupled beginning on the second beat. As shown in Figure 7C, Igap is an outward current flowing from the myocyte to fibroblasts, non-existing in the uncoupled myocyte. This outward current decreases membrane potential and has an indirect effect on voltage dependent currents such as ICaL. Indeed, we can observe that when fibroblasts are coupled (dashed traces) all currents and fluxes present a shorter duration, as well as AP, and a reduced peak , except ICaL. Despite the reduced duration of this current, the initial ICaL peak contributed to a larger Ca2+ influx through these channels (increase from 140 to 146 pC/μF). The integral of NCX was also computed and indicated an increase in inward NCX extruding more Ca2+ when fibroblasts were coupled (from 77 to 81 pC/μF). In the transient evolution shown in Figure 8F, the peak of ICaL exhibited a maintained increase compared with the uncoupled myocyte (shown in solid red). Due to the increased Ca2+ extrusion through NCX, however, [Ca2+]JSR progressively declined, leading to the decrease in [Ca2+]i peak observed in steady state.Fibroblast coupling decreased systolic Ca2+ impairment in HF, fibroblast to myocyte coupling does not alter the role of the main mechanisms regulating Ca2+ dynamics in myocytes, and (iii) drug action on ICaL and SERCA enhancement and NCX block would help CaT restoration in HF regardless of fibrosis presence.In this study, two methodologies were used to perform sensitivity analyses evaluating the effects of fibroblast–myocyte coupling under normal and HF conditions. Our main findings are that (i) univariate and multivariate analyses yield very similar results and low variability of parameters yields more reliable multivariate sensitivity analyses, (ii) despite exacerbating CaNaL, or the decrease of systolic [Ca2+]i sensitivity to SERCA in HF with respect to N conditions.Univariate and multivariate sensitivity analyses were performed using an initial large variability (±60%) and then smaller (±15%). At lower variability, changes in electrophysiological properties behaved linearly, making results from univariate and multivariate analysis more similar. Indeed, ionic currents and fluxes work synergistically to generate AP and CaT. Variables such as membrane potential and ionic concentrations link all ionic mechanisms in a way that a change in one parameter (conductivity in this case) has an effect on other parameters, and final differences in electrophysiological properties are the result of an interaction of all these variations. In addition, within the range of variation of a specific parameter, the effects on biomarkers can be different, and assuming linearity can become less accurate when a wide interval is considered. In fact, as electrophysiological remodeling in HF is simulated by changing parameters, the same scale factor applied in N and HF implies a different range of variation of such parameters, accounting for different sensitivities to biomarkers. This explains the increase in APD sensitivity to IMultivariate parameter sets, unlike varying one parameter at a time, can provide information about electrophysiological properties in a wide combination of parameters, which could be useful to evaluate the response of a drug in different individuals, instead of on a fixed baseline model. The high physiological or pathological variability of ionic parameters requires the analysis of the behavior in the whole range.2+ dynamics. We observed that univariate and multivariate sensitivities agreed in the most important parameters contributing to each biomarker. For instance, it was observed the strong impact of IKr on APD90, as CaL was highlighted with both methodologies, agreeing with experimental observations  changing to myofibroblast phenotype , and we observed similar effects, i.e., APD shortening and Ca2+-handling impairment. The improvement of myofibroblast models, incorporating Ca2+ dynamics, would certainly shed light into the understanding of Ca2+ dynamics alterations in the failing tissue.An important effect observed when simulating fibroblast–myocyte coupling is the marked APD shortening. Experimental studies showing the existence of gap junctions (connexin43) between fibroblasts and myocytes also revealed changes in AP waveform and conduction velocity due to a modulation of myocyte electrophysiology . Previou cycling . To date cycling . In the in vivo coupling, different degrees of fibrosis, and the difficult to quantify coupled fibroblasts in tissue has led to the exploration of different values of Ggap or a range of myocyte–fibroblast ratios , we observed that ICaL peak increased since the first beat, due to the immediate effects of Igap on AP. Initially, there was also an intracellular Ca2+ rise, as SR Ca2+ content was still elevated. The mechanisms to remove Ca2+ from the cytosol were then activated: SERCA and inward current through NCX (extruding Ca2+). NCX role, extruding Ca2+ out of the cell, becomes relevant in the transient state because if there is an imbalance with Ca2+ influx through ICaL channels, Ca2+ loss occurs. This mechanism might explain the reduced SR Ca2+ load in myocytes interacting with fibroblasts.Our findings showed that a higher Iller CaT . Therefo2+ impairment in HF by further reducing SR Ca2+ content in myocytes.Our findings suggest that fibroblasts increase Ca2+ dynamics in these cells. Ca2+ signaling has been observed in human cardiac fibroblasts although with different pathways to those in contractile myocytes (in vivo.Several limitations need to be considered when drawing conclusions from the present study. Although simulations of cellular electrophysiological behavior and systematic analyses of biomarkers complement and enrich experimental research, uncertainties in the development of the mathematical models employed might affect the outcome of the simulations. The limited availability of electrophysiology data from human fibroblasts has led to the use of a fibroblast model developed on the basis of adult rat ventricular tissue data, without taking into consideration potential changes in HF. Fibroblasts with the same characteristics were considered in normal and failing conditions, but electrophysiological remodeling in fibroblast currents could have an additional effect on myocytes . Anothermyocytes , and myomyocytes , but nei2+ can modulate the coupling current. If the modulation of the intercellular coupling by Ca2+ ions was taken into account, larger alterations in Ca2+ dynamics mechanisms could be observed. Any mechanism related to Ca2+ included into our model, such as realistic Ca2+ dynamics in fibroblasts, may affect the regulation of Ca2+ cycling in myocytes, and enhance the influence of fibroblasts.Gap junctional coupling between a myocyte and a fibroblast has been modeled by a simple conductance as in previous simulation studies of fibroblast-myocyte coupling. However, recent findings suggesting the interaction of connexin45 with CaM  highligh2+ release resulting from detubulation. However, a detailed model including local changes in membrane structure related to the spatial organization of ion channels in HF could provide more accurate results about Ca2+ impairment (Other structural modifications in HF, concerning myocytes, involve the loss of transverse tubules and reorganization of the cell membrane . We havepairment .2+ homeostasis is related to contraction and relaxation force, other mechanical factors, such as myofibroblast contractility when fibroblasts are differentiated into the activated form or extracellular stiffness due to the excessive accumulation of collagen in HF, can contribute to cardiac dysfunction. Our model only considers intracellular Ca2+ in myocytes at a cellular level to evaluate the contraction of the heart. The behavior in tissue or even in the whole organ could also be different. A defined architecture of the myocyte when considering spatial distribution would allow to take into consideration the distribution of gap junctions as well as the extracellular space which could modulate Ca2+ homeostasis as it does in electrical propagation (Although Capagation . We conspagation .Regarding sensitivity analyses, the non-linearities are the main issue in quantifying the impact of parameters on biomarkers. To restore the electrical activity and contraction of myocytes, the effect of some drugs can imply a high change in an ionic mechanism, and the predictive value of this methodology decreases with increased variability.2+ impairment observed in HF, which together with the arrhythmogenic activity of fibrotic tissue, can compromise the function of the myocardium. In the present study, the measures suggested to restore Ca2+ dynamics and contractility at cellular level are reliable because they are supported by experiments and according to our findings, tackling the electrophysiology remodeling in failing myocytes can also improve the effects of fibroblasts interactions.Nonetheless, despite these limitations, cellular simulations can shed light in the causes of CaMM, JF, and BT conceived and designed the study, and analyzed the data. MM performed simulations. JG and ES contributed to the methodology. MM and BT wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


During postgraduate education in pulmonology, supervisors are responsible for training residents in generic competencies such as communication, professionalism and collaboration, but their focus commonly lies more on medical-technical competencies. As an alternative approach to supporting residents to develop generic skills, we developed a personal mentoring program with a non-medical professional as mentor. In this study, the residents’ experiences with the mentoring program were evaluated.After an introductory session in which individual learning goals were established, pulmonology residents received at least six, 60–90-minute, individual, mentoring sessions largely consisting of feedback after being observed during daily clinical activities, over a period of 9 months. The residents’ experiences with mentoring were explored through in-depth interviews followed by a qualitative content analysis.From March to November 2016, ten residents in pulmonology completed the program. Despite initial scepticism, mentoring encouraged residents to reflect deeply on their professional interactions. This caused an increased awareness of the effects of their communication and behaviour on patients. Experimenting with communication and different behaviours in subsequent interactions felt rewarding and contributed to further development, resulting in increased self-confidence and job satisfaction.Mentoring residents by non-medical coaching was associated with improved residents’ proficiency in generic competencies. Workplace learning is the mainstay of postgraduate medical education applying to medical skills as well as to generic skills. In daily practice, residents develop generic competencies as they receive continuous informal feedback when communicating with patients, other residents, senior doctors, allied healthcare professionals and nurses .Clinical supervisors help residents to develop skills in these competencies. This can be practised by direct observation and feedback but this is not structurally implemented everywhere in postgraduate training , 3. SupeOther factors may limit the opportunities for residents to deliberately practise generic competencies, such as lack of supervision in busy medical practices and the supervisor’s dual and sometimes conflicting roles as teacher and mentor –8.The question arises whether supervisors are the most appropriate teachers for all residents and in all circumstances to support their development in generic competencies. To help residents to improve generic competencies, we developed a mentoring program with a non-medical coach observing residents in the workplace while performing daily clinical tasks and providing structured feedback. This study explored pulmonology residents’ experiences with this mentoring program.Residents working at the Department of Pulmonology of Isala, a large teaching hospital in Zwolle, the Netherlands, participated in the mentoring program. Their experiences with mentoring were evaluated by content analysis of semi-structured interviews carried out by an independent senior qualitative researcher after completion of the mentoring program and performed according to COREQ guidance principles.The mentoring program was designed and performed by a single coach trained in Facility Management, Leadership Development and Organisational Science, with 10 years’ experience in counselling, management coaching and interim management in both commercial and healthcare organisations.During mentoring, residents received applicable tips to improve their communication and collaboration skills. They were encouraged to reflect on their thoughts (for example their assumptions) and behaviour towards others, and to analyze this in order to develop more self-insight. They were challenged to vary and modulate their reactions to patients’ verbal and non-verbal communication. By try outs, they learned to anticipate and prepare subsequent interactions.The program started with a plenary meeting for all residents in which the aims and the content of the program were discussed. Subsequently, each resident participated in six individual 60–90-minute sessions with the mentor, over a timeframe of 9 months. During the first individual session, residents’ personal norms and values and motivations, background, learning goals and needs and expectations regarding the mentoring program were discussed.Subsequent individual sessions involved a brief preparation in which the resident discussed learning objectives with the mentor. The mentor observed the resident while performing daily clinical tasks , followed by a structured reflection and feedback session. Prior to each observation, the resident introduced the mentor to the patient, informed him/her about the mentoring program and asked the patient for consent regarding the presence of the mentor. During each encounter, the mentor did not intervene in any way. Individual sessions after each observed professional task also allowed time for mentor/mentee conversations on issues unrelated to the working context, the contents of which varied, depending on the resident’s personal needs and development phase.The study was approved by the Netherlands Association for Medical Education (NVMO) ethical review board (file number 606). Written informed consent was obtained from all participants.From March to November 2016, ten residents, in all phases of their training, completed the mentoring program. In Table All residents reported that the mentoring program had the most striking effects on their communication skills, not only with patients, but also with supervisors, colleagues and other healthcare professionals. They learnt to practise their communication techniques, try out new approaches, and apply structure to professional conversations. They also experienced an increased awareness of the effects of their communication and how it affected others.The residents perceived that their communication with patients had improved as a result of feedback and better preparation. In addition to discussing medical content, they felt more comfortable in expressing empathy with the patient’s context.The mentoring program increased residents’ consciousness regarding the importance of effective collaboration and their insight into their personal motivations, norms and values with respect to collaboration, whatever the stage of training. The mentoring sessions enabled them to discuss their emotions with supervisors, colleagues and other healthcare professionals and also respond to these emotions. This improved their ability to engage in constructive dialogue and to reach agreement on collaboration. Changes perceived in this domain were experienced as having a positive influence on the learning environment.The residents reported that mentoring improved their ability to identify and maintain boundaries in work-life balance. They developed a better understanding of their role in relation to their fellow residents, and how to assert their role within the group. The residents took more responsibility for optimizing work processes and educational activities. Mentoring helped, especially the younger residents, to increase self-confidence and assume their new role as doctors.The residents experienced mentoring as safe, credible and engaging. They characterized the mentoring as very personal, inevitable and with guaranteed feedback. They appreciated the strict focus on generic competences contrasting to the feedback they received from supervisors, which was largely based on medical technical aspects. Residents appreciated that—in contrast to their previous experiences with observations made by supervisors—they did not feel as if they were being assessed during the mentoring program.A mentoring program for generic competencies, added to pulmonology residency training, had a positive impact on the individual participants. Although the most profound results were reported in the domain of communication, residents also reported progress in professionalism and collaboration, independent of their phase of training.The perceived benefits of mentoring can most likely be explained by some key features of deliberate practice that were used to improve performance. The residents formulated learning goals in advance with the mentor. Repeated observations of learning activities with immediate feedback provided by the mentor, allowed the residents to try out newly learnt communication and collaboration skills . A body At the start of the mentoring program, some residents expressed scepticism on the usefulness of the program, which appeared to be based on their previous experiences with teaching communication and collaboration skills during preclinical training. This contrasted with the enthusiasm they expressed after completing the mentoring program and suggests initial unconscious incompetence in communication, collaboration and professionalism skills, and a limited trust in their ability to learn these skills.Isolated supervisor training, a competency framework and assessment instruments are apparently not enough for optimal clinical learning . The menMentoring of communication and behavioural skills performed by a non-medical professional worked well in our study. In comparison with clinical supervisors, the non-medical mentor provided more in-depth analysis and feedback about observed encounters with patients and colleagues. Unravelling the underlying causes of observed behaviour or communication appeared to be the trigger of residents’ behavioural changes and improvement of skills. In addition to a dialogue centred around deeper motivations, the mentor discussed follow-up measures and provided further help and guidance which are considered elements of ideal positive role modelling .The efficacy of mentoring is likely to rely on the quality of the relationship between the mentor and the resident, which should be focused on the resident and provide guaranteed emotional security . The perThe prolonged relationship with the mentor—lasting for up to 9 months—with repeated encounters created a feeling of relatedness, autonomy and competence in residents .This study was conducted in a relatively small group of residents in a single medical specialism and by one mentor. Currently, research is in progress to study the reproducibility of the success of this approach in larger groups and other disciplines.Despite initial scepticism, all residents in this study experienced increased self-efficacy in generic competencies through mentoring provided by a non-medical professional, whatever their phase of training.


Successful component alignment is a major metric of success in total knee arthroplasty. Component translational placement, however, is less well reported despite being shown to affect patient outcomes. CT scans and planar X-rays are routinely used to report alignment but do not report measurements as precisely or accurately as modern navigation systems can deliver, or with reference to the pre-operative anatomy.A method is presented here that utilises a CT scan obtained for pre-operative planning and a post-operative CT scan for analysis to recreate a computation model of the knee with patient-specific axes. This model is then used to determine the post-operative component position in 3D space.Two subjects were investigated for reproducibility producing 12 sets of results. The maximum error using this technique was 0.9° ± 0.6° in rotation and 0.5 mm ± 0.3 mm in translation. Eleven subjects were investigated for reliability producing 22 sets of results. The intra-class correlation coefficient for each of the three axes of rotation and three primary resection planes was > 0.93 indicating excellent reliability.Routine use of this analysis will allow surgeons and engineers to better understand the effect of component alignment as well as the placement on outcome. Dissatisfaction amongst total knee arthroplasty (TKA) is the result of a complex relationship between the patient anatomy, prosthesis design and position, and other patient-specific factors. Prosthesis malalignment has been linked to poor patient outcomes in which coronal and axial malalignment has been most closely studied , 2. To hComponent alignment refers to the angular difference between the prosthetic components and patient-derived antero-posterior (AP), medio-lateral (ML), and superior-inferior (SI) anatomic axes. This measurement has traditionally been the focus of post-operative analysis in TKA due to the ease of measurement –5. CompoThe pre-operative state of the patient is a critical source of missing data from most analyses which prevents accurate reporting of component position. Bony resections cannot be accurately determined from a post-op analysis alone, and as a result, there is very little data available on the outcome of TKA as a result of the modification of the anatomy , 7, highAlteration of the joint line and flexion/extension gaps is associated with a change in joint kinematics  and patiTraditional methods of assessing TKA component alignment, including short leg X-rays –16, longHere, we introduce a method of 3D reconstruction which utilises both a pre-operative and post-operative CT scan to determine the post-operative component position in TKA. The method may be extended to any joint replacement and is termed here the Australian Universal Resection, Orientation, and Rotation Analysis (AURORA) protocol. Landmarks and bone models unaffected by component flare obtained from the pre-operative scan are transformed into the post-operative frame of reference. Component position as defined by the landmarked patient-specific axes and bony resections are reported. The reproducibility and reliability of this method are presented and compared to other post-operative analysis techniques.A series of patients received long leg pre-operative CT scans for the routine pre-operative planning of TKA surgery  and to dAll patients investigated here had a TKA using OMNI APEX implants , from four different surgeons using four different techniques. Patients were randomly selected from a database of over 2000 TKA surgeries.The CT dose is calculated by multiplying the dose-length-product (mGy.cm) provided as supporting information with the CT scan, by the length of the CT scan in which the patient is imaged. The dose value is then converted to an effective dose based on anatomic conversion coefficients presented by Saltybaeva et al.  to allow3D reconstructed patient femur and tibia bones are generated within the pre-operative planning process through semi-automated segmentation, used to landmark and identify points of interest by biomedical engineers using the 3D imaging software, ScanIP . The patient bones are converted to stereolithography (STL) files and landmarked twice by different engineers. If any landmarks differ by a threshold value (in this case 4 mm), the landmark was reviewed by another trained engineer. Landmark references were used to define patient-specific bone axes and soft tissue attachment sites, see Fig. Using the post-operative full leg CT scan, the 3D post-operative femur and tibia bone sections unaffected by the component flare are segmented, see Fig. Euler transform matrices are obtained from the resulting registered pre-operative bones and used to transform the pre-operative bone landmarks into the post-operative CT reference frame. Using the transformed landmark references, component alignment and placement are determined within the local reference frames from the defined axes of landmarks identified pre-operatively.Two primary TKR patients were processed post-operatively twice by three engineers in a 2-week period. Patient CT scans were segmented and registered by an engineer and then reviewed by a second engineer. The same case was processed again by the initial engineer on another day at a different time of day and then reviewed by a third engineer. This process was repeated across the three engineers for the two cases with alternating reviewers, and a total of 12 registrations was then analysed see Fig. .Fig. 3FlComparison of component alignment angles in flexion/extension (FE), varus/valgus (VV), and internal/external (IE) rotation, and component placement values by measuring the femoral medial and lateral, distal and posterior condyles, and the medial and lateral tibial plateau was recorded. Reproducibility was assessed from these angular and resection measurements by determining the maximum difference and standard deviation from the mean calculated for each patient, with the 95% confidence interval defined across both cases.To describe the interobserver reliability, 11 TKR patients were processed post-operatively between two engineers. Each case was reviewed by a third and fourth engineer, with refinement of the bone and component registration made by the reviewing engineer if necessary. A set of 22 results was produced for the comparison of the three rotation axes across two components and six resection measurements. The intra-class correlation coefficient (ICC) was calculated for each of the measurements. An ICC value of 1 shows perfect reliability, values greater than 0.9 indicates an excellent result, 0.81 to 0.9 is very good, 0.76 to 0.80 is good, 0.5 to 0.75 is moderate, and < 0.50 is considered to show poor reliability , 27.The average effective radiation dose received per CT scan using this protocol is 1.24 ± 0.96 mSv. This dose is compared to other CT and radiography protocols in Fig. Using the AURORA protocol, patient movement in the CT scan may be detected at any point along the length of the bone. In previous methods, such as the Perth CT and Imperial protocols, movement in the mid-femur and mid-tibia will not be detected, leaving any measurements to propagate through the protocol as an error. In a database of CT scans obtained for the routine pre-operative planning of TKA, the rate of scans identified with movement over a 3-month period is 6.78% . Of this fraction, all movement in the scans were detected in the mid-femur and mid-tibia regions.The alignment reproducibility results generated from three engineers processing two cases at two different time points which were then QC checked are shown in Table The bony resection thicknesses are a proxy measure for the accuracy of measuring component placement and are shown in Table The rotational alignments and bony resections for the femur and tibial components reported for 11 cases performed twice (each time by a team of two different engineers) are shown in Figs. The maximum component alignment differences from the mean within this study are low compared to previous literature and provide a confidence interval up to tenfold narrower when compared to protocols in which individual CT slices were investigated , 22, 28,The resulting resection level measures of the femur and tibia also show high reproducibility, with the highest deviation seen for the medial tibial plateau resection at 0.5 mm and 0.3 mm between the two cases and confidence intervals of 0.6° and 0.4°, respectively. The magnitude of the error here, however, is only slightly above the other resections, indicating that there may not be a systematic reason for reduced accuracy when placing this component. Previous attempts have been made to investigate the effect on TKA outcome arising from resection levels. These studies have mainly focussed on the femur, particularly the posterior condylar offset , 34, 35.Across both femoral and tibial component alignments and bony resections, this 3D pre-operative registration process shows excelled reliability, in which all ICC values report greater than 0.93. The lowest reported ICC value of 0.93, resulting from the femoral axial IE rotation measure, is primarily due to the difficulty of post-operative registration of the femur component. The posterior condyles, which dominate the axial rotation positioning, of the APEX implant used in this study are thicker than the distal condyles (11 mm vs 9 mm) and tibial tray (~ 3 mm). As such, the CT flare is greater in these regions, reducing the accuracy of the registration. The ICC values reported here are consistently similar to or higher than other post-operative analysis techniques , 22, 36 The high reproducibility and reliability of calculating both component alignment and bony resections performed by surgeons can lead to a better understanding of the influences of component alignment and component placement. The current literature has thoroughly reviewed the influence of component malalignment and poor patient outcomes –39. MissThe proposed 3D registration process for post-operative analysis involves additional pre-operative CT imaging compared to other processes , 18. ThoManual translation and rotation of the pre-operative bones and component geometries into the post-operative CT scan is reasonably labour intensive, requiring on average 60 min to complete, before the registration is quality control checked by a second engineer with additional experience. Further refinement of the proposed post-operative analysis process could include the use of an automated registration method. A preliminary automated registration process using the iterative closest point (ICP) method  was perfJoint infection and component loosening are a cause of dissatisfaction and revision surgery. Joint infection can be identified by swelling of the joint and pathology reports; however, these are not always conclusive. Combining component position as determined using the AURORA protocol with SPECT imaging could identify bone metabolism associated with infection or component movement . AlthougThe proposed post-operative 3D registration method described here has some limitations. The current time taken for this analysis as mentioned is approximately 60 min; this represents a high engineering burden and must be reduced to improve use in routine analysis. Commercially, TKA component geometry varies between medical device manufacturers, forming a significant part of their IP portfolio, as such, the component geometries must be obtained from the implant companies, which may be difficult—limiting the generalisability of this technique to engineering firms with a close relationship with implant companies. The reproducibility analysis performed here utilises two cases processed at multiple time points by multiple engineers of equal training. To better understand the reproducibility, particularly when processing outlier or severely pathological anatomy, a greater number of cases should be analysed.Other methods to assess component position such as bi-planar X-rays followed by 2D to 3D registration offer a number of advantages over a CT, such as providing long leg assessments in a functional state . Such teComponent alignment has been of great interest in total knee arthroplasty; however, the focus has previously been on achieved component alignment and identification of malalignment without regard for the component placement or pre-operative anatomy. The method presented here uses a low-dose CT scan to analyse the position and rotation of all components in 3D space, with a comparison to the pre-operative anatomy, allowing surgical changes to the joint to be determined. The method shows excellent reliability and reproducibility by removing the sources of error that are typically associated with post-operative total knee arthroplasty analysis. Routine use of this analysis in TKA as well as other joint replacement procedures will allow surgeons and engineers to better understand the effect of component alignment as well as the placement on outcome.


Our results suggest developmental hypoxia improves cardiomyocyte anoxia-tolerance of juvenile turtles, which is supported by enhanced myofilament Ca2+-sensitivity and a superior ability to suppress ROS production. Maintenance of low ROS levels during anoxia might limit oxidative damage and a greater sensitivity to Ca2+ could provide a mechanism to maintain contractile force. Our study suggests developmental hypoxia has long-lasting effects on turtle cardiomyocyte function, which might prime their physiology for exploiting hypoxic environments.For some species of ectothermic vertebrates, early exposure to hypoxia during embryonic development improves hypoxia-tolerance later in life. However, the cellular mechanisms underlying this phenomenon are largely unknown. Given that hypoxic survival is critically dependent on the maintenance of cardiac function, we tested the hypothesis that developmental hypoxia alters cardiomyocyte physiology in a manner that protects the heart from hypoxic stress. To test this hypothesis, we studied the common snapping turtle, which routinely experiences chronic developmental hypoxia and exploits hypoxic environments in adulthood. We isolated cardiomyocytes from juvenile turtles that embryonically developed in either normoxia (21% O Often, 2 i, pHi and ROS, respectively, at room temperature. [Ca2+]i and pHi were measured simultaneously, by co-loading cardiomyocytes with Fura-2  and BCECF . ROS were measured separately by loading cardiomyocytes with DHE . Following the loading protocol, cells were resuspended in fresh isolation solution for 15–20 min to allow de-esterification. Cardiomyocytes were then placed in the recording chamber (at room temperature), perfused with normoxic saline and stimulated to contract at a frequency of 0.2 Hz, which is within the normal physiological range of heart-rates in common snapping turtles  ([Ca2+]e), from 1 to 5 mM , while simultaneously measuring relaxed cell length, according to a protocol by Wisløff et al. i), normalized Δ[Ca2+]i, diastolic [Ca2+]i, systolic [Ca2+]i, pHi, ROS production, cardiomyocyte shortening efficiency and times to rise and half-decay. For the GLM analyses, the between-group factor was developmental O2 (N21 versus H10), while time and treatment (normoxia and anoxia) were the within-group factors. A further GLM was used, followed by Sidak post hoc tests, to reveal the effect of the developmental O2 and experimental period  on cardiomyocyte shortening (the dependent variable), while controlling for Δ[Ca2+]i (the covariate). Finally, a mixed-effects, repeated-measures GLM, followed by Sidak post hoc tests, was used to reveal the effect of developmental O2 on myofilament Ca2+-sensitivity, with relaxed cardiomyocyte length as the dependent variable, while controlling for extracellular [Ca2+] (the covariate). Additionally, regressions were used to fit lines through the data. The Δ[Ca2+]i was calculated by subtracting the value of the diastolic [Ca2+]i from the systolic [Ca2+]i, and ROS values were normalized to the 5-min time-point and described as relative changes in production. All data are presented as means ±s.e.m. and considered significantly different, when p ≤ 0.05.Statistical analyses and graph plotting were carried out using S3.2 and treatment (anoxia and reoxygenation) on multiple aspects of juvenile turtle cardiomyocyte structure and function. The results are described below, and statistical support for the findings  are given in electronic supplementary material, tables S2–S7.The mixed-effects, repeated-measures GLMs revealed significant effects of developmental O(a)While the N21 and H10 turtles did not differ in body or heart mass , there w(b)2 on Δ[Ca2+]i and cell-shortening efficiency; a significant effect of treatment on cell shortening, Δ[Ca2+]i, pHi, ROS production, normalized Δ[Ca2+]i, systolic [Ca2+]i, time to rise and time to half-decay; a significant interaction between normoxia and developmental O2 on the Δ[Ca2+]i and cell-shortening efficiency; and a significant interaction between anoxia and developmental O2 on cell shortening, pHi, ROS production and cell-shortening efficiency. See electronic supplementary material, tables S3–S5, for full details of these statistical interactions.The mixed-effects, repeated-measures GLMs revealed a significant effect of developmental O(c)i of N21 and H10 cardiomyocytes were similar , but the average intracellular Ca2+ transient (Δ[Ca2+]i) was more than twice the size in N21 versus H10 cardiomyocytes (b); this meant the efficiency of shortening (% cell shortening as a function of Δ[Ca2+]i) was significantly greater in the H10 cohort (f). The large difference between Δ[Ca2+]i values can be explained by lower average N21 versus H10 diastolic [Ca2+]i levels, but similar average systolic [Ca2+]i; nevertheless, the differences in diastolic [Ca2+]i did not reach levels of statistical significance . Lastly, relative intracellular ROS production did not change during the control period (e).Under control conditions, cell shortening and pH similar a,d, but myocytes b; this m0 cohort f. The laificance c,d. Lastl period e.Figure(d)2 rapidly dropped inside the chamber to undetectable levels (within approx. 54 s), and N21 and H10 contraction declined in association with a reduction in Δ[Ca2+]i, pHi and ROS . However, while N21 cell shortening remained depressed throughout the anoxic period, H10 cell shortening recovered to control levels, after 15 min of anoxia (a), despite a significantly larger acidosis in the H10 cells (d) and a similar proportional decline in Δ[Ca2+]i to the N21 cohort (c). Therefore, the efficiency of cell shortening during anoxia tended to be higher in the H10 versus N21 cardiomyocytes; this effect was only statistically significant at the 25- and 30-min time-points (f). In both cell groups, the reduction in the Δ[Ca2+]i amplitude (b) was caused by a progressive increase in diastolic [Ca2+]i and a decrease in systolic [Ca2+]i, although these values did not reach levels of statistical significance . Anoxia also slowed the times to rise and half-decay of both cohorts .At the onset of anoxic perfusion, O and ROS a,b,d,e. f anoxia a, despit10 cells d and a s1 cohort c. Therefe-points f. In botmplitude b was cauificance c,d. Anox cohorts . It shou10 cells e.Table (e)2 was restored (approx. 63 s), N21 and H10 cardiomyocyte shortening recovered to pre-anoxic levels (a), in association with the restoration of pHi, ROS production , [Ca2+]i and the times to rise and half-decay 2+ transients, suggested that the H10 cohort has greater myofilament Ca2+-sensitivity. Therefore, we investigated the correlation between cardiomyocyte contraction and Δ[Ca2+]i . As expected, regression lines fitted to the data revealed that shortening is positively correlated with Δ[Ca2+]i, during all three experimental periods, with the exception of N21 cells in anoxia. However, when controlling for Δ[Ca2+]i, the extent of the shortening was greater in H10 cardiomyocytes, regardless of the experimental period . Furthermore, relaxed cardiomyocyte length decreased by a greater proportion in the H10 cohort, at the range of external Ca2+ concentrations tested (c). Collectively, our data suggest that the myofilaments of H10 cardiomyocytes have a higher sensitivity to Ca2+ than N21 cells. See electronic supplementary material, tables S6 and S7, for full details of these statistical interactions.The observation that H10 cardiomyocyte shortening was either equal to, or larger, than N21 cells, despite smaller CaΔ[Ca2+]i a,b. As el period a,b. Furts tested c. Collec4.f), higher myofilament Ca2+-sensitivity (figure 2e).Numerous studies across different vertebrate taxa show that developmental hypoxia alters cardiac morphology and function ,13,26–33figure 2sitivity  and a su(a)Previous work has shown developmental hypoxia is associated with cardiac enlargement in embryonic snapping turtles , but thi2+]i), compared with N21 cardiomyocytes (b). Differences in cardiomyocyte Δ[Ca2+]i can be explained by numerous factors, including Ca2+-influx pathways (e.g. L-type Ca2+ channel or the sarcoplasmic reticulum), efflux pathways (e.g. the Na+/Ca2+-exchanger) or buffering capacity . While we did not investigate these Ca2+-cycling mechanisms here, we found that H10 cardiomyocytes could still develop the same level of shortening as the N21 cells, despite the smaller Δ[Ca2+]i. Our findings suggest that these differences can partly be explained by improved myofilament Ca2+-sensitivity in the H10 cohort . For example, Ca2+-cycling is a major consumer of ATP, on account of the high energetic demand of Ca2+-removal pathways, such as the sarcoplasmic reticulum Ca2+-ATPase and the Na+/Ca2+ exchanger, which is indirectly dependent on the Na+/K+-ATPase i, which reduces myofilament crossbridge formation, and a profound intracellular acidosis, brought on by a respiratory and a metabolic acidosis, which reduces myofilament Ca2+-sensitivity [2+]i and a lower pHi than N21 cardiomyocytes. Evidently, prior exposure to developmental hypoxia improved cardiomyocyte anoxia-tolerance in juvenile turtles.N21 and H10 cardiomyocyte shortening was dramatically impaired within the first 5 min of the anoxic challenge. Consistent with this finding, c output ,41. Our sitivity . HoweverDanio rerio) exposed to hypoxia produce offspring that are more hypoxia-tolerant i remained relatively stable during anoxia, the mechanism underlying the rebound in cell shortening of H10 cardiomyocytes must be Ca2+-independent . Consistent with the hypothesis that developmental hypoxia enhances myofilament Ca2+-sensitivity, cell shortening in N21 and H10 cardiomyocytes was similar within the first 5 min of anoxia, despite the lower intracellular pH of the H10 cohort. However, the time course of the rebound in H10 cell shortening suggests separate mechanisms were activated to recover contractile function. One possibility is the suppression of ROS production. Low O2 tensions are associated with excessive ROS production, which is known to reduce cardiac force production, by directly modifying sarcomere proteins and reducing myofilament Ca2+-sensitivity i and ROS production all recovered to intermediate or pre-anoxic levels within the first 5 min of reoxygenation, despite the persistent increases in diastolic and systolic [Ca2+]i. These results suggest the turtle heart can withstand reoxygenation without apparent cellular injury, irrespective of developmental O2 tension.Although anoxia can produce extensive tissue damage in mammals, the greatest risk to cell survival occurs after reoxygenation, when the mitochondria produce a surge of ROS that can trigger apoptotic and necrotic cell death ,52. By c5.2 deprivation throughout their lifetime [2+-sensitivity and a superior ability to suppress ROS production. From an ecological perspective, these modifications might be beneficial to turtles and possibly other ectotherms, when they exploit hypoxic environments.North American freshwater turtles experience prolonged periods of Olifetime ,9. We ha


Although this is not the only mechanism involved in erythrocyte clearance, in this minireview, we focus on the following questions: Is the intracellular-free Ca2+ concentration and hence phosphatidylserine exposure dependent on the erythrocyte age, i.e. is the Ca2+ concentration, progressively raising during the erythrocyte aging in vivo? Can putative differences in intracellular Ca2+ and exposure of phosphatidylserine to the outer membrane leaflet be measured in age separated cell populations? Literature research revealed less than dozen of such publications with vastly contradicting results for the Ca2+ concentrations but consistency for a lack of change for the phosphatidylserine exposure. Additionally, we performed reanalysis of published data resulting in an ostensive illustration of the situation described above. Relating these results to erythrocyte physiology and biochemistry, we can conclude that the variation of the intracellular free Ca2+ concentration is limited with 10 μM as the upper level of the concentration. Furthermore, we propose the hypothesis that variations in measured Ca2+ concentrations may to a large extent depend on the experimental conditions applied but reflect a putatively changed Ca2+ susceptibility of erythrocytes in dependence of in vivo cell age.After about 120 days of circulation in the blood stream, erythrocytes are cleared by macrophages in the spleen and the liver. The “eat me” signal of this event is thought to be the translocation of phosphatidylserine from the inner to the outer membrane leaflet due to activation of the scramblase, while the flippase is inactivated. Both processes are triggered by an increased intracellular Ca The formation of erythrocytes named erythropoiesis takes about 7 days . It happIn a drop of human blood one can obviously find a composition of cells in all different ages. There is an age-dependent variation in their density. Young cells have a significantly lower density in comparison to old cells . Based oIt has been reported that physiological erythrocyte aging results in a decreased cell volume, size, and mean corpuscular volume . In addi2+, the associated phosphatidylserine exposure leads to erythrocyte clearance  realized by the Ca2+ pump. The passive Ca2+ influx is mediated through low capacity transport pathways with carrier properties  technique 2+concentration of 62 and 221 nM, respectively.Concerning the free intracellular Cantration . Combini2+ concentration compared with mature erythrocytes, whereas 2+ than mature cells but their intracellular Ca2+ concentration is not increased.In contrast, for the very young red blood cells, the reticulocytes 2+ concentration and in the phosphatidylserine exposure in dependence on erythrocyte age (based on density separated erythrocytes).2+ concentration as well as phosphatidylserine exposure present a linear relation relative to the density centrifugation fractions representing erythrocyte age. The regression values R2 for Ca2+ and phosphatidylserine of 0.94 and 0.92, respectively, speak for themselves (2+ was linear decreasing (R2 of 0.92), while the phosphatidylserine exposure was increasing (R2 of 0.80). The only differences between the two separate measurements (A and B) were the donors  and the composition of the solutions (see legend of 2) regression lines for different conditions, the data from 2+ concentration and phosphatidylserine exposure from erythrocyte age. In other words: Although the panels of Taken all these reports together, we have a very controversial situation. To further illustrate this conflicting situation, we present a reanalysis of previously published data . The peremselves . Surprisemselves , the int2+ concentration and also an increase in the phosphatidylserine translocation with cell age could be observed  incubation experiments showed that there was neither significant difference of the Ca2+ content (see above) nor a phosphatidylserine translocation depending on cell age when comparing age populations in pairs . However, since there is consistence in the reports concerning phosphatidylserine exposure (no change with cell age), we can conclude that the variation of the free intracellular Ca2+ concentration does not exceed 10 μM, which resembles the onset of the scramblase activation while the flippase is inactivated  are released from activated platelets and therefore resemble a physiological stimulation occurring in vivo . Under mechanical stress, prostaglandin E2 can even be released by the erythrocytes themselves , phosphatidylserine positive erythrocytes depict a rather quadratic dependence on cell age.There is a debate in the literature whether erythrocytes depict a cell age dependency in the response of lysophosphatidic acid stimulated . To this end, we present a reanalysis of data by 2+ signals  stimulation indicates such a dependence as a general property.Although we have no mechanistic hypothesis for this quadratic response, the consistency along the various types of physiological  and art2+ content of human erythrocytes is related to cellular aging at all. Nevertheless, intracellular Ca2+ is very likely be increased immediately prior erythrocyte clearance but since phosphatidylserine exposing erythrocytes are quickly cleared  solutions in an age-dependent manner  and not necessarily an increased Ca2+ concentration. The measurements in vitro may then reflect the experimental conditions addressing this susceptibility  increase in the intracellular Cat manner  and (2) e spleen . Therefolity cp. , which i2+ concentration we measure might thus be the product of an (in vivo) susceptibility for Ca2+ and the (in vitro) experimental conditions. Our measurements may therefore reflect the erythrocyte susceptibility for Ca2+ if we manage to keep the experimental conditions constant, which can be achieved in a laboratory but makes direct comparison of independent studies of different laboratories almost impossible. Furthermore, there might be conditions  that do not act on the susceptibility for Ca2+ (factor zero in the above-mentioned product).The Ca2+ in erythrocytes in dependence on cell age. Previous observations of highly variable Ca2+ concentrations after blood sample shipments (2+ measurements.This hypothesis has the potential to explain the greatly variable and partly contradicting experimental results concerning the dependence of intracellular Cahipments  support 2+ as we previously proposed for erythrocyte research in general (Finally, the conflicting reports reveal once more the need for intralaboratory and interlaboratory validation of protocols by quality controls in particular for the measurement of intracellular Ca general .All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The increase in [Ca2+]i regulates a number of ion channels and transporters that are required for establishing an osmotic gradient that drives water flow via aquaporin water channels in the apical membrane. The Store-Operated Ca2+ Entry (SOCE) mechanism, which is regulated in response to depletion of ER-Ca2+, determines the sustained [Ca2+]i increase required for prolonged fluid secretion. Core components of SOCE in salivary gland acinar cells are Orai1 and STIM1. In addition, TRPC1 is a major and non-redundant contributor to SOCE and fluid secretion in salivary gland acinar and ductal cells. Other TRP channels that contribute to salivary flow are TRPC3 and TRPV4, while presence of others, including TRPM8, TRPA1, TRPV1, and TRPV3, have been identified in the gland. Loss of salivary gland function leads to dry mouth conditions, or xerostomia, which is clinically seen in patients who have undergone radiation treatment for head-and-neck cancers, and those with the autoimmune exocrinopathy, Sjögren’s syndrome (pSS). TRPM2 is a unique TRP channel that acts as a sensor for intracellular ROS. We will discuss recent studies reported by us that demonstrate a key role for TRPM2 in radiation-induced salivary gland dysfunction. Further, there is increasing evidence that TRPM2 might be involved in inflammatory processes. These interesting findings point to the possible involvement of TRPM2 in Sjögren’s Syndrome, although further studies will be required to identify the exact role of TRPM2 in this disease.Salivary glands secrete saliva, a mixture of proteins and fluids, which plays an extremely important role in the maintenance of oral health. Loss of salivary secretion causes a dry mouth condition, xerostomia, which has numerous deleterious consequences including opportunistic infections within the oral cavity, difficulties in eating and swallowing food, and problems with speech. Secretion of fluid by salivary glands is stimulated by activation of specific receptors on acinar cell plasma membrane and is mediated by an increase in cytosolic [Ca In the absence of extracellular Ca2+, release from the ER causes a transient increase in [Ca2+]i that is not sufficient to maintain prolonged fluid secretion. The latter requires sustained increases in [Ca2+]i that is supported by Ca2+ influx into the cells. The primary function of the [Ca2+]i increase is to regulate the function of ion transporters and channels such as Na+/K+/2Cl− cotransporter 1 (NKCC1), Anoctamin 1 (ANO1), and Ca2+-dependent K+ (KCa), which cause vectorial transport of Cl− from the basolateral to the luminal side of the cell, and the generation of an osmotic gradient across the luminal membrane of the cell. The latter provides the driving force for water secretion through the apical membrane via the water channel, Aquaporin 5 (AQP5).Salivary glands secrete fluid composed of water and electrolytes in response to neurotransmitter stimulation of plasma membrane receptors that cause an elevation of cytosolic  and gate the channels. The main Ca2+ channels involved in SOCE in salivary gland cells are Orai1 and TRPC1. STIM1, an ER-Ca2+ binding protein, functions as the ER-[Ca2+] sensor and the gating component of both these channels i increases are essential for the regulation of salivary gland function, disruption of Ca2+ homeostasis, either in resting or stimulated cells, results in salivary gland dysfunction. Two major conditions result in loss of salivary gland function and tissue damage: primary Sjögren’s syndrome (pSS), a chronic autoimmune disease involving lymphocytic infiltration and loss of secretory function in salivary and lacrimal glands i. Muscarinic stimulation of saliva and tear secretion was downregulated in both TRPV4-deficient mice and in acinar cells treated with a TRPV4-specific antagonist (HC-067047). Furthermore, the temperature dependence of muscarinic salivation was shown to depend mainly on TRPV4. This study also showed a novel association between TRPV4, IP3Rs, and ANO1 that collectively contributes to the regulation of salivation and lacrimation.Regulation of cell volume in response to changes in osmolarity is critical in salivary gland fluid secretion. In response to carbachol (CCh) stimulation, cells undergo a decrease in cell volume, which then recovers via a regulatory volume increase (RVI). Conversely, hypotonic conditions lead to cell swelling and volume recovery via regulatory volume decrease (RVD). Both these processes depend on the water permeability of the cells, which in salivary gland cells is determined by the level of AQP5 in the membrane. A role for TRPV4 in RVD was previously reported by an earlier study reported by us . TRPV4 wlar Ca2+ . Anotherl glands . Activat2+]i in a cultured submandibular epithelial cell line. These results indicate that temperature-sensitive TRP channels are localized and distributed in acinar, ductal and myoepithelial cells of salivary glands, and that they might have a functional role in regulating and/or modulating saliva secretion. Further studies will be needed to characterize the exact role of temperature-dependent regulation of salivary gland function and the involvement of TRP channels in this mechanism.Additional TRP channels have also been identified in salivary glands. Immunohistochemistry has revealed the presence of TRPM8, TRPA1, TRPV1, TRPV3, and TRPV4 in myoepithelial, acinar, and ductal cells of the sublingual, submandibular, and parotid glands. Interestingly, perfusion of the entire submandibular gland with the TRPV1 agonist capsaicin (1 μM) via the submandibular artery significantly increased CCh-induced salivation, whereas perfusion with TRPM8 and TRPA1 agonists  decreased it. Application of agonists for each of the thermosensitive TRP channels increased i, which regulate Ca2+ signaling mechanisms that include regulation of ion channel activities, gene expression, secretion, apoptosis, and inflammasome assembly. TRPM2 also responds to warm temperatures that act synergistically with ADPR, NAD+, and cADPR at concentrations that otherwise cannot activate the channel i in response to radiation causes an increase in mitochondrial [Ca2+] and ROSmt but a decrease in mitochondrial membrane potential. This is accompanied by a relatively slower appearance of activated caspase-3 which persists for about a month after the treatment. In irradiated TRPM2−/− mice, the increases in [Ca2+]mt, ROSmt and activated caspase-3 are substantially attenuated. Importantly, TRPM2-dependent activation of caspase-3 is correlated with loss of STIM1. These interesting findings reveal that radiation-induced loss of salivary gland fluid secretion is mediated via a TRPM2-dependent pathway that impacts mitochondrial function and leads to irreversible loss of SOCE. Notably, cleavage of STIM1 by calpain and γ-secretase has been associated with stress and Alzheimer’s disease, respectively i as it is expressed in the plasma membrane of neutrophils, T and B lymphocytes, and dendritic cells. In T cells, cross-linking of cell surface receptors induces a rise of ADPR endogenously generated from NAD+ which can activate TRPM2 i and subsequent release of nitric oxide in microglia also depends on the TRPM2 channel function [2+ influx as the major ROS-induced Ca2+ signaling mechanism in macrophages [−/− mice [2+ influx is a critical step in coupling ROS generation to NLRP3 inflammasome activation and IL-1β maturation.Numerous proinflammatory cytokines are produced during the innate immune response to infection and inflammation, several of which have been linked with activation of TRPM2. Knockdown of TRPM2 attenuates LPS-induced production of IL-6, IL-8, IL-10, and TNF-α in THP1 monocytic cells. The corresponding decrease in LPS-induced Ca pathway . The proese mice . Evident−/− mice . Furthernfection . A recenfunction . Immune function . The prirophages , which r−/− mice . Thus, TTrpm2 knockout mice showed attenuation of inflammatory indicators such as production of CXCL2, neutrophil infiltration and ulceration [2+ influx via TRPM2 could represent a key inflammatory mediator in monocytes and in the epithelium of both salivary and lacrimal glands of SS patients. It will be very important to investigate the role for this channel in SS-induced salivary gland pathology. Establishing this will provide new strategies for treatment of the disease.Notably, Sjøgren’s Syndrome (SS) has been associated with overexpression of proinflammatory cytokines, including TNF-α, IL-7, IL-1β, IL-6, IL-10, IL-17, IL-18 and gamma-interferon (γ-IFN) ,140,141.ceration . Thus, R2+ signaling, ion transport, and water transport. Future studies should be focused on establishing the mechanisms underlying salivary gland dysfunction. Such studies should provide novel targets and strategies for treatment. On such target in salivary glands is TRPM2, which appears to be critically involved in radiation-induced irreversible loss of salivary gland fluid secretion. Based on data reported by us and others, potential therapeutic strategies could include manipulating channel activity, developing specific inhibitors of the channel or TRPM2-dependent signal transduction cascade. In the case of radiation-induced salivary gland dysfunction, we propose that inhibitors of TRPM2 or caspase-3, scavengers of ROS in the cytosol or mitochondria, as well as inhibitors of PARP1 could be used to protect against loss of function. Additionally, its ability to respond to ROS has made TRPM2 a potential therapeutic target for chronic inflammation and neurodegenerative diseases. TRPM2 ion channel and its gating molecule ADPR are previously unsuspected players necessary for robust cytokine production and innate cell activation during intracellular bacterial infection. These findings highlight the potential of the metabolic manipulation of ADPR levels or modulating TRPM2 activation modalities to exert immunomodulation. However, currently, direct evidence for TRPM2 involvement in Sjøgren’s Syndrome (SS) is lacking. Based on currently available data highlighting the role of TRPM2 in inflammatory process, it will be very important to assess whether TRPM2 contributes to Sjøgren’s Syndrome (SS). Identification of the role of other, including sensory, TRP channels in salivary gland function will also provide additional targets for modulating water secretion from the gland. Future studies should focus on these potentially novel and important roles of these TRP channels.Studies done over the past 30 years have provided a tremendous amount of information about the key molecular components that regulate salivary gland fluid secretion, including those involved in Ca