The micropapillary component in lung adenocarcinoma has a valid predictive role for patient prognosis. To investigate targeted clinical strategies, we collected 31 stage I lung adenocarcinoma samples and performed microdissection to separate micropapillary and non-micropapillary components, followed by whole exome sequencing. We focused on the genomic features, evolutionary advantages, and associated clinical implications of the micropapillary component, and we proposed and validated the possible presence of essential mutations, TP53 and ZNF469, in the formation of the micropapillary component. These features may be associated with the formation of high-grade invasive patterns in this component.Background: Micropapillary components are observed in a considerable proportion of ground-glass opacities (GGOs) and contribute to the poor prognosis of patients with invasive lung adenocarcinoma (LUAD). However, the underlying mutational processes related to the presence of micropapillary components remain obscure, limiting the development of clinical interventions. Methods: We collected 31 GGOs, which were separated into paired micropapillary and non-micropapillary components using microdissection. Whole-exome sequencing (WES) was performed on the GGO components, and bioinformatics analysis was conducted to reveal the genomic features of the micropapillary component in invasive LUAD. Results: The micropapillary component had more genomic variations, including tumor mutation burden, intratumoral heterogeneity, and copy number variation. We also observed the enrichment of AID/APOBEC mutation signatures and an increased activation of the RTK/Ras, Notch, and Wnt oncogenic pathways within the micropapillary component. A phylogenetic analysis further suggested that ERBB2/3/4, NCOR1/2, TP53, and ZNF469 contributed to the micropapillary component’s progression during the early invasion of LUAD, a finding that was validated in the TCGA cohort. Conclusions: Our results revealed specific mutational characteristics of the micropapillary component of invasive LUAD in an Asian population. These characteristics were associated with the formation of high-grade invasive patterns. These preliminary findings demonstrated the potential of targeting the micropapillary component in patients with early-stage LUAD. According to the latest statistics, lung cancer ranks first in terms of incidence and mortality among all malignant tumors in China, with lung adenocarcinoma (LUAD) accounting for approximately 60% of all lung cancer cases . With thThe MPP component is most common in ground-glass opacities (GGOs) and tends to be distributed at the margins of tumor invasion into normal tissue, with an unexpected inside-out growth pattern . The sidThis component is distributed in some tumors with characteristic mutations; for example, EGFR and BRAF have high mutation frequencies in MPP samples. This component is also enriched for mutations in certain functional pathways such as the PI3K pathway, which is an independent factor in the assessment of tumor recurrence ,10,11. EThis study aimed to better understand the overall information on mutations in this component and to gain insight into the role of mutational features in early lung adenocarcinoma in the process of cell differentiation and development. We collected a total of 31 GGO samples and performed whole-exome sequencing (WES) of different components, which was the basis to further elaborate the distribution ratios and mutation selection during the development of the MPP component as well as the differences in functional mutations, copy number variants (CNVs), and pathway mutations between tumor components.In this study, we used tumor tissue samples from 31 patients with lung malignancies who underwent radical lung cancer surgery at the Affiliated Cancer Hospital of Nanjing Medical University between February 2016 and February 2021 to study the mutational characteristics of tumor tissues. The patients′ postoperative tissue samples were pathologically confirmed to be lung adenocarcinoma-carrying MPP components. According to the eighth edition of the American Joint Committee on Cancer (AJCC)’s cancer staging, all samples collected were stage I. The lung cancer tissue samples were diagnosed by two pathologists, and microdissection was performed after microscopic pinpointing of the MPP component. The MPP component and non-MPP component were isolated from each sample.In addition, as control data for the genetic variants, we obtained peripheral blood mononuclear cell (PBMC) samples from 31 tumor tissue samples corresponding to patients. The study was approved by the ethics committee of Nanjing Medical University, and informed consent was obtained from the subjects for postoperative sample collection. The clinical information of the above patients is recorded in ® 3.0 Flurometer . A DNA small-fragment library was prepared according to the Agilent SureSelectXT Target Enrichment System method and procedure. After the library’s construction, an initial quantification was performed using Qubit 3.0, and the insert size of the library was measured using Agilent 2100. The insert size was determined to be as expected. After the insert size met the expectation, Q-PCR was performed using a Bio-RAD CFX 96 fluorescence quantitative PCR instrument and a Bio-RAD KIT iQ SYBR GRN to accurately quantify the effective concentration of the library (effective library concentration > 10 nM) to ensure the library’s quality. After the libraries passed QC, the raw data were generated in a FASTQ file format by running the double-end sequencing program (PE150) on the Illumina HiSeq sequencing platform according to the manufacturer′s instructions. The sequencing data quality for this project was assessed using the software FastQC v0.11.7, and the data were then compared with the reference genome using software such as BWA. We performed variant detection using GATK software and SNP and InDel annotation using ANNOVAR software [DNA was extracted from the tissue and blood samples using a FastPure FFPE DNA Isolation Kit and a FastPure Blood DNA Isolation Mini Kit V2. The DNA sample concentrations were measured using a Qubitsoftware ,13,14,15Based on the mutated MAF files, the known oncogenic signaling pathways in the TCGA cohorts  of the overall sample were enriched using OncogenicPathways in the maftools package . Each paTo assess the copy number status of the whole genome based on WES data, we modeled the copy number as an integer using the default parameters for SEQUENZA   while caThe dN/dScv package was used to calculate the dN/dS ratio for each component of each sample (including synonymous mutations) to infer the global mutational selection pressure during tumor development, and we selected samples that fit the dN/dScv calculation model for further analysis.The dispersion of the allele frequencies of somatic mutations in the whole exome sequencing data for each sample was measured using the mutant-allele tumor heterogeneity (MATH) method , and thip values were two-sided. The differences were considered significant when the p value was <0.05. Other figures were generated using the R package ggplot2.Statistical analyses were performed using R (v4.0.5). For comparisons of the continuous variables between groups, Wilcoxon rank-sum tests were used. For comparisons of the categorical variables paired between groups, McNemar chi-square tests were used. All reported To comprehensively study the mutational landscape of the MPP component and the transcriptomic characteristics of tumor cells in early-stage LUAD, we analyzed intraoperatively resected tumors and the corresponding peripheral blood samples from 31 patients with GGOs. The patients’ information is recorded in To capture the overall mutational landscape of the MPP components in GGOs, the somatic mutational profiles of different components within tumors are presented separately below and used as a basis for comparing differences between groups, followed by an assessment of the developmental characteristics of tumor cells based on mutational information.First, we observed that EGFR and TP53 were the main driver mutations in our study cohort, with most samples showing common driver mutations among different components (MPP and non-MPP) . The MPPWe calculated the CNV for each sample using Sequenza . HeatmapThe intratumoral heterogeneity (ITH) patterns of the paired components in each sample were quantified to estimate the component’s genetic correlations. As expected, the MPP component had higher ITH levels compared with those of the non-MPP component, consistent with the aggressive profile of the MPP component, suggesting an expansionary predominance of this component in early progression ,20 FiguI, as repThe MPP component has an apical secretory function and plays an important role in tumor infiltration and angiogenesis . To elucIn addition, the overall base variation layout at the whole-exon level in the MPP component as a whole was more consistent with the SBS13 profile in the Catalogue of Somatic Mutations in Cancer (COSMIC), suggesting an association with the activation of AID/APOBEC cytidine deaminase in cancer  Figure F,G, whilNext, we investigated the genetic changes specific to the MPP component in each patient in this cohort. As shown in Most of the histological subtypes from the same tumor sample source shared multiple genetic events, especially driver mutations, implying homology between the two components as well as pathological subtype differences in the tumor occurring after tumor driver mutations. In addition, the branch length of the MPP component was significantly longer than that of the non-MPP component, suggesting that more genomic alterations may be required for the germination process of the MPP component.p = 0.04), which is a known factor associated with poor prognosis and corresponds to genomic instability features, such as high CNV and TMB, in the MPP component. The gene-encoding zinc finger protein ZNF469 also showed a component-specific distribution (p = 0.07).Considering the role of specific mutations in the differentiation of tumor cells toward the MPP component, we counted the frequencies of the distributions of MPP branch mutations without trunk mutations in the samples based on the developmental tree . Within We selected single-nucleotide variants shared between different pathological subtypes of the same sample, which were aggregated with 636 mutated genes localized in all samples, and we further calculated the proportions of the distributions of each mutation using PyClone ,28. As edN/dS represents the ratio of non-synonymous to synonymous mutations and is a good means of quantifying the selection pressure during the evolution of the cancer genome. We used the standardized dn/ds algorithm (dN/dScv), filtered out the samples that fit the dN/dScv model, and calculated the ratio of the nucleotide substitution rate at non-synonymous to synonymous sites for each component in the sample to assess the significance of mutational selection pressure on each component during early progression ,30.The MPP component showed a significantly higher dN/dS ratio , suggestThe Pyclone results also showed higher abundances of TP53, ZNF469, TTN, and TENM4 mutations in the MPP components A–E. CombAlthough surgery is the primary route for the clinical treatment of patients with early-stage LUAD, the five-year recurrence rate after this treatment should not be ignored ,32,33. MWe reported the results of a comprehensive genomic analysis of the MPP component in GGOs. This component was initially considered to be homologous to the pathological components and therefore shared common driver mutations and copy number variants at common loci. However, this subtype carries a higher mutational load compared with that of the other components, as evidenced by TMB, CNVs, cancer cell fractions (CCFs), and variant allele frequency. Increasing evidence suggests that tumor cells accumulate mutations during proliferation and invasion and that CNVs at genetic loci play important roles in carcinogenesis and cancer progression .As reported previously, the MPP subtypes tended to show an aggregation of characteristic mutations  ,38; we aThe phylogenetic tree branches showed that tumor cells require more driver mutations during differentiation into MPP subtypes, which explains the increased mutational selection pressure in this pathological subtype. In addition to the oncogenic signaling pathway genes mentioned in this study, we also observed enrichment trends for TP53, ZNF469, TTN, and TENM4 in the MPP pathological subtypes. We verified that the less-studied ZNF469 was associated with poor tumor prognosis. These results support clinical observations and provide clinical ideas for precise treatment requirements.Our results demonstrate the concordance between mutational features and cellular functions. This concordance leads us to the next step of our study; that is, whether MPP subtype components with different driver mutations have significant and unique functional characteristics. A correlation between MPP subtypes and immunotherapy efficacy was reported , and immIn conclusion, we reported on the genomic analysis of MPP subtypes. The overall mutational profile of MPP subtypes showed high levels of TMB, CNVs, and CCFs and enrichment for prognosis-related genes such as TP53 and ZNF469. These results provide a solid genomic basis and potential target molecules for the accurate diagnosis and treatment of LUAD.


Mdmx and Mdm2 are two major suppressor factors for the tumor suppressor gene p53. In central nervous system, Mdmx suppresses the transcriptional activity of p53 and enhances the binding of Mdm2 to p53 for degradation. But Mdmx dynamics in cerebral infarction remained obscure. Here we investigated the role of Mdmx under ischemic conditions and evaluated the effects of our developed small-molecule Protein–Protein Interaction (PPI) inhibitors, K-181, on Mdmx–p53 interactions in vivo and in vitro. We found ischemic stroke decreased Mdmx expression with increased phosphorylation of Mdmx Serine 367, while Mdmx overexpression by AAV-Mdmx showed a neuroprotective effect on neurons. The PPI inhibitor, K-181 attenuated the neurological deficits by increasing Mdmx expression in post-stroke mice brain. Additionally, K-181 selectively inhibited HDAC6 activity and enhanced tubulin acetylation. Our findings clarified the dynamics of Mdmx in cerebral ischemia and provide a clue for the future pharmaceutic development of ischemic stroke. To date, although numerous clinical trials for stroke therapy have been accomplished, intravenous thrombolysis with recombinant tissue plasminogen activator (t-PA) remains the only clinical effective treatment2. However, the narrow therapeutic window and the rapid progression of brain injury following ischemia restrict its clinical application3.Stroke is the leading cause of human death and long-term disability all over the world5. In addition, p53-dependent neuronal apoptosis is associated with neuronal damage in both acute injury such as stroke and chronic neurodegenerative diseases8. p53 contributes not only to apoptosis but also to necrosis by controlling the interaction of cyclophilin D with permeability transition pores in mitochondria9. Accumulated research has shown that p53 plays a central role in neuronal cell death after cerebral infarction, and its suppression reduces ischemic cell damage15.The tumor suppressor gene p53 plays an important role in the development of the CNS16. Mdm2 is the predominant E3 ligase for p53 and degrades p53 by promoting ubiquitination of p53. On the other hand, Mdmx suppresses the transcriptional activity of p53 and enhances the binding of Mdm2 to p53 for degradation. The role of the Mdm2–p53 pathway after cerebral ischemia has already been reported18. Mdmx, a homologue of E3 ligase Mdm2, has been mainly studied as a p53-dependent cancer regulatory protein so far20, and most of the existing data are obtained from cancer studies21. Recent studies suggested an important role of Mdmx in normal neuron development. Mdmx modifications like Ring deletion22 and C462A knock in23 to alter its Mdm2 binding ability leaded to mice embryonic lethal. General Mdmx knock-out mice died at embryonic day 7.5–9.524, and conditional Mdmx deletion in CNS using a Nestin-Cre mouse model resulted in lethal deficits at embryonic day 17.525. Moreover, accumulative studies suggest an irreplaceable role of Mdmx not only in normal neuron development but also in various CNS diseases and therapies27. Neuronal stress signals induce Mdmx loss, whereas Mdmx knockdown favored neuronal apoptosis, suggesting Mdmx contributes to neurons survival in response to stresses28. In addition, Mdmx overexpression attenuates HIV-induced neurotoxicity29. In aggregate, these findings underscore a critical role of Mdmx in normal CNS development and maintenance.The two major suppressors of p53 are Mouse Double Minute 2 (Mdm2) and Mdmx 30, and most protein–protein interaction (PPI) inhibitors are peptides or antibody drugs. We previously reported new low-molecular-weight Mdmx–p53 disruptors and their anti-cancer activities31. Among them, S-2-benzamidophenyl 2-methylpropanethioate (K-181) and its free thiol derivative (K-181SH) preferentially inhibited Mdmx–p53 interaction over Mdm2–p53 interaction and suppressed the growth of cancer cells. We speculate that K-181, after being hydrolyzed to K-181SH in the body, interfered with the p53–Mdmx interaction through disulfide-bond formation between the free thiol groups of K-181SH and Cys77 in Mdmx in the p53-binding domain. Here, we investigated the effects of K-181 on ischemic stroke both in vitro and in vivo, and found that K-181 and its free thiol derivative K-181SH induced Mdmx up-regulation. More interestingly, K-181 and K-181SH showed an HDAC6 inhibitory effect. HDAC6 is a cytoplasmic Class II histone deacetylase and has a deacetylase activity on several cytoskeleton proteins including tubulin32. Dysregulated HDAC6 restores tubulin acetylation33, and correlates against neuronal microtubule instability34, cognition impairment35 and peripheral neuropathy development36, while HDAC6 inhibition attenuates neuronal damages under cell stresses37. Therefore, selectively targeting HDAC6 may alleviate neuropathic damages38.To the date, most of the pharmacological approaches aiming at reactivating p53 in cancer cells have focused on the interaction interface between p53 and Mdm2 or MdmxDespite countless efforts been made for development of drugs that inhibit the interaction of p53 with Mdm2 or Mdmx in cancers, the role of Mdmx after cerebral ischemia and its possibility as a therapeutic drug have not yet been reported. In this study, we clarified the dynamics of Mdmx after cerebral ischemia, and demonstrated the neuroprotective effects of our newly developed small-molecule PPI inhibitors by modifying the interactions between Mdmx and p53 and HDAC6 activity inhibition.Details for all methods are provided in the 8-week-old male C57BL/6J male mice were purchased from Charles River Japan, Inc. and raised under standard conditions of light (lights on: 8:00 a.m.–8:00 p.m.) and temperature . A totally of 130 mice were included in this study. All experimental procedures followed the guidelines and were fully approved by the Ethics Committee for Animal Experiments of Osaka University Graduate School of Medicine. All experiments were conducted in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.S-2-Isobutyramidophenyl 2-methylpropanethioate) and K-178SH, were synthesized according to the reported procedures31.Small-molecule PPI inhibitors, K-181, K-181SH, K-178  of the PPI inhibitors against the FLAG-p53 and GST–protein interaction measured by the modified ELISA were previously reported31.Inhibitory activities  using flanking BamHI and XhoI restriction sites. Briefly, AAV 293 were transfected with rAAV  and rAAV-ITR Mdmx or Mdm2 expression plasmid (single stranded genome) or pAAV-GFP Control Plasmid  and helper plasmid  by the calcium phosphate method. 120 h after transfection cells were harvested and vector purified using a standard iodixanol density gradient and ultracentrifugation protocol. Iodixanol was removed and vector concentrated in PBS by diafiltration using Amicon Ultra 100 kDa MWCO centrifugal devices . Vector was stored at − 80 °C until use. rAAV titers were determined by quantitative PCR and expressed as genome copies per ml (gc/ml). They AAV particles were applied to the primary neuronal culture at MOI of 5 × 1041. Construct for lentiviral vector (pLKO.1) expressing control shRNA (SHP002) and a shRNA against mouse p53 gene (Trp53) (Cat. TRCN0000173949) were purchased from Sigma-Aldrich. Briefly, 293FT cells were transfected with pCAG-HIVgp , pCMV-VSV-G-RSV-Rev  and a shRNA construct using the CalPhos™ Mammalian Transfection Kit (Takara). pCAG-HIVgp and pCMV-VSV-G-RSV-Rev were developed by the late Dr. Hiroyuki Miyoshi and provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan42. The lentivirus-containing medium was collected at 48 h and 72 h after transfection and ultracentrifuged at 70,000g for 140 min. The pellet was resuspended and applied for primary neuronal transduction at MOI of 10 at 5 days in vitro and incubated for 4 days.Lentivirus production was performed as previously described43. Mice were randomly distributed into K-181 and vehicle treated groups. General anesthesia was rapid conducted with 3% isoflurane and maintained at 1.5% via an open mask. The right middle cerebral artery was occluded for 60 min with a suture. The reperfusion of mice was verified by both laser-Doppler flowmetry (Advanced Laser Flowmetry) and a 2D laser blood flow imager . Laser-Doppler flowmetry was used to monitor cortical cerebral blood flow (CBF) by attachment to the exposure skull from pre until 15 min after operation, and mice with more than 30% of baseline during the first minute of occlusion were excluded. After the operation, mice were returned to their individual cages. During the whole surgery, the body temperature was monitored by a rectal probe and maintained at 37.0 ± 0.5 °C.Transient Middle Cerebral Artery Occlusion (tMCAO) was conducted as previously described44 was used. Spontaneous activity, symmetry of movements, symmetry of forelimbs, climbing wall of wire cage, reaction to touch on either side of trunk and response to vibrissae touch were scored as 0 to 3 points respectively. Lower score indicates worse neurological deficits. Neurological tests were evaluated by 2 researchers independently.After 48 h of recovery, foot fault test and neurological score were performed to evaluate mice neurological deficits. For the foot fault test, mice were placed on an elevated gridded platform above the surface and allowed to walk for 5 min. A foot fault was noted when the left forefoot misstepped and fell through the space between the grids. The percentage of left foot faults was measured for statistical analysis during the whole observation. For neurological score, a criteria described in a previous study45. Surface CBF was recorded by a laser speckle blood flow imaging system (Omegazone OZ-1). After general anesthesia, the skull was exposed by a midline scalp incision. The surface of the skull was wiped clean with saline-soaked gauze before recording. Color-coded CBF images were obtained in high-resolution mode.The measurement of CBF was conducted 24 h after tMCAO as previously describedAfter the total RNA extraction of brains and cultures using mirVana™ miRNA Isolation kit (Thermo Fisher Scientific), cDNA was prepared from 1 μg total RNA using the SuperScript VILO cDNA Synthesis Kit (Invitrogen). Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) was used for real-time PCR. 36B4 served as endogenous control. Relative expression was calculated using the ΔΔCt method with the QuantStudio 7 Real Time PCR System (Applied Biosystems). Primer sequences were described as below: p53: F: 5ʹ-GTATTTCACCCTCAAGATCC-3ʹ, R: 5ʹ-TGGGCATCCTTTAACTCTA-3ʹ. Mdmx: F: 5'-CCATCTGACGACATGTTTCC-3ʹ, R: 5'-TTACAAGCAGGACACGAAGC-3ʹ. 36B4: F: 5ʹ-TGTGTGTCTG CAGATCGGGT-3ʹ, R: 5ʹ-TGGATCAGCCAGGAAGGCCT-3ʹ.t test. Multiple groups comparisons were performed using one-way ANOVA with Bonferroni’s post hoc test. All data are presented as means ± standard deviation (SD).Comparisons between two separate groups were performed using unpaired non-parametric To investigate the role of Mdmx in stroke, we applied C57BL/6J wild-type mice to 60 min tMCAO, then investigated the Mdmx expression in mice brain Fig. A. Total 32 including Mdm2/Mdmx–p53 inhibitors, since Mdm2/Mdmx–p53 pathway could be linked to alteration of Mdmx expression. Firstly, we conducted inhibition assay of the small-molecule PPI inhibitors against Mdmx–p53 and Mdm2–p53 interaction , and a typical HDAC6 inhibitor Tubacin, were adopted as positive controls. As HDAC enzymes, HDAC1, HDAC4 and HDAC6 were used as representative HDAC class I (HDAC1/2/3/8), class IIa (HDAC4/5/7/9) and class IIb (HDAC6/10), respectively. HDAC enzyme activities were recorded and only compounds that inhibition (%) > 50 were defined to have an inhibitory effect. Compared to TSA, K-181 showed a selective inhibitory effect on HDAC6 activity but barely influenced HDAC1 and HDAC4 activity (Table Our group have so far synthesized multiple Histone Deacetylases (HDACs) inhibitorsTaken together, these results suggest K-181 and its derivatives have neuroprotective effects on ischemic stroke by disrupting Mdmx–p53 interaction and inhibiting HDAC6 activity Fig. . This maMany PPI inhibitors of interaction between p53 and its binding proteins have been developed in cancer. Despite the crucial role of p53 for cell death in neurological disorders, few reports have been made on the development of neuroprotective treatments with PPI inhibitors of Mdmx–p53 and Mdm2–p53. K-181, our newly developed small-molecule Mdmx–p53 inhibitor, reduced neuronal damage and attenuated the neurological deficits after stroke by increasing the expression of Mdmx protein.53. The function of p53 is impaired either by the mutations or multiple upstream negative regulators including in Mdm2 and Mdmx. Although many phase I, II and III clinical trials with small molecule compounds that reactivate p53 are currently underway, there remains certain limitations and concerns. Unlike its mutations difficult to target in cancers, p53 is often wild-type in mature neurons which are impaired by stroke and neurodegenerative diseases, and the discovery of therapeutic drugs targeting Mdm2/Mdmx–p53 interactions in clinics can be potentially useful in AML, multiple myeloma, and other hematological malignancies54.Somatic p53 mutations are found in approximately 50% of human cancers34. Nutlin-3a, a well-known Mdm2–p53 inhibitor with a molecular weight of 581, led to an increased vulnerability to ischemic neuronal injury, which could be reversed by p53 Knock-out. Furthermore, Patients harboring G allele in Mdm2 promoter (Single-Nucleotide Polymorphism 309 T>G) were reported to have a higher Mdm2 protein levels and better functional outcomes after stroke than those harboring the T/T genotype55.Multiple drugs have been synthesized to target Mdm2–p53 interaction up to nowResearches on Mdmx–p53 interaction inhibitor remain few. The first small-molecule Mdmx inhibitor is SJ-172550. However, we did not find a neuroprotective effect on ischemic neuronal damage by administration of SJ-172550 in this work. The novel small-molecular weight drug, K-181 preferentially disrupted Mdmx–p53 interaction over Mdm2–p53 interaction in mice brain and showed high binding affinity to Mdmx in vitro. Furthermore, K-181 oral administration increased Mdmx expression in vivo by a dose-dependent way, and exerted an inhibitory effect on p53, Mdm2 and E2F1 expression. In addition, K-181 showed a neuroprotective effect in acute ischemic stroke, which is supported by the downregulation of apoptotic regulators like cleaved Caspase-3 and proinflammatory factors like TNF-α.56. Previous studies have reported that Mdmx interacts with other apoptosis modulating proteins, such as E2F157, suggesting that Mdmx may play a critical role against neuronal death. Consistent with these reports, our study demonstrated that K-181 ameliorates mice neuronal death, accompanied with a decrease in E2F1 protein. Additionally, Mdmx has recently been reported to be p53-independently associated with CK1α activity58, DNA replication59, and ferroptosis60. Further investigation is needed on the involvement of these p53-independent mechanisms of Mdmx in cerebral ischemia.The neuroprotective effect of Mdmx may be both p53-dependent and p53-independent mechanism. Mdmx is phosphorylated at multiple sites in response to cell stresses, and these modifications play critical roles in different signaling pathways, especially in apoptosis and necrosis62, and Ricolinostat (ACY-1215) is the first oral selective HDAC6 inhibitor with reduced class I HDAC activity to be studied clinically for treatment of multiple myeloma63. Moreover, HDAC6 directly deacetylates Lys12064 and Lys381/38265 of p53 to coordinate p53-dependent signaling pathway, which further explains the neuroprotective effects of K-181 despite the p53-independent neuroprotection of K-181 induced Mdmx increase. The clarification of p53-dependent and p53-independent mechanisms of K-181 and Mdmx deserves further investigation.Interestingly, K-181 also had an inhibitory effect on HDAC6 activity by oral administration and restored tubulin acetylation. HDAC6 is involved in tau aggregation and phosphorylation in neurons and human AD brainAt present, most of PPI inhibitors are medium and large weight molecules, typified by peptides and antibodies. In this work, we discovered a small molecule PPI inhibitor K-181, and showed a neuroprotective effect of K-181 that disrupts Mdmx–p53 interaction and enhances Mdmx expression. Further studies are needed to clarify the underlying mechanism of its inhibitory regulation on Mdmx/Mdm2–p53 axis. This study may offer a novel insight into the future ischemic stroke therapy.Supplementary Figures.Supplementary Information.


Background: There is much scope to empower district hospital (DH) surgical teams in low- and middle-income countries to undertake a wider range and a larger number of surgical procedures so as to make surgery more accessible to rural populations and decrease the number of unnecessary referrals to central hospitals (CHs). For surgical team mentoring in the form of field visits to be undertaken as a routine activity, it needs to be embedded in the local context. This paper explores the complex dimensions of implementing surgical team mentoring in Malawi by identifying stakeholder-sourced scenarios that fit with, among others, national policy and regulations, incentives to perform surgery, career opportunities, competing priorities, alternatives for performing surgery locally and the proximity and role of referral hospitals.Methods: A mixed methods approach was used which combined stakeholder input – obtained through two group model building (GMB) workshops and further consultations with local stakeholders and SURG-Africa project staff – and dynamic modeling to explore policy options for sustaining and rolling out surgical team mentoring. Sensitivity analyses were also performed.Results: Each of the two GMB workshops resulted in a causal loop diagram (CLD) with an array of factors and feedback loops describing the complexity of surgical team mentoring. Six implementation scenarios were defined to perform such mentoring. For each the resource requirements were identified for the institutions involved – notably DHs, CHs and the party that would finance the required mentoring trips – along with the potential for scaling up surgery at DHs under severe financial constraints.Conclusion: To sustain surgical mentoring, it is important that an approach of continued communication, monitoring, and (re-)evaluation is taken. In addition, an output- or performance-based financing scheme for DHs is required to incentivize them to scale up surgery. Implications for policy makersMentoring of district hospital (DH)-level surgical teams is best done through a staggered, nation-wide roll-out of field trips, and a dedicated senior coordinator who is in regular contact with both central hospital (CH) and DH. It is critical to monitor surgical performance during periodic meetings with mentors and mentees, and to assess the acceptability and effectiveness of surgical team mentoring. Inclusion of in-service training and mentoring activities in the key performance indicators of both DHs and surgical specialists would improve their motivation to scale up safe surgery for rural populations. A mechanism of output- or performance-based financing of surgery is required to scale-up surgery at DHs and avoid unnecessary surgical referrals to higher level hospitals.  Implications for the publici) create a focal point with a dedicated national coordinator, (ii) routinely monitor DHs’ surgical performance, (iii) adequately reward both the mentors and the mentees, and (iv) introduce a financing mechanism through which DHs are financially compensated if they perform more surgery. Scaling up surgery at district hospitals (DHs) in order to make safe surgery more accessible for rural populations requires mentoring of DH surgical teams through periodic field visits by teams of senior surgical staff who are usually based at central hospitals (CHs). For such surgical team mentoring to be effective and carried out efficiently, there is need to , as part of DH-level surgical teams that further comprise anesthesia providers, theatre nurses and support staff.2 Non-specialists face a multitude of barriers to providing surgery, such as shortages of supplies, poor infrastructure, regulatory ambiguities, and lack of training and supervision,3 which poses challenges to their retention for work in rural areas.4 In Malawi, a country with 18 million inhabitants of whom 84% live in rural areas, there are only 42 surgical specialists, mostly working in the central hospitals (CHs) of Blantyre and Lilongwe. Most  care at Malawian DHs is provided by a cadre of non-physician clinicians, called clinical officers in Malawi.5 The European Union-funded SURG-Africa project was designed to demonstrate an effective way to improve district surgical capacity using locally available resources. It has implemented and evaluated surgical mentoring and supervision between 2018 and 2020.6 This intervention consisted of two complementary parts: periodic mentoring trips of DH surgical teams by specialists from CHs; and a managed remote surgical consultation network based on WhatsApp.7 The rationale behind the intervention was that surgical teams at DHs would be empowered in terms of professional knowledge, skills and confidence to undertake a wider range and a larger number of surgical procedures. Through this two-pronged intervention it was expected that surgery would become more accessible to rural populations and that the number of unnecessary referrals to CHs would decrease. Worldwide, millions of people lack access to safe surgery.8 Firstly, the intervention  needs to fit with the needs and features of the local setting . In SURG-Africa, ensuring a good fit was addressed through a participatory action research stage, prior to the start and during implementation of the mentoring intervention.9 It entailed the design and shaping of the mentoring model, along with periodic reviews and adaptations of the intervention, where needed, based on workshops with mentors and mentees. Secondly there needs to be a fit between the intervention and the broader ecological context . Such a fit is important for a sustainable uptake and scaling up of the intervention in routine practice. The aim of this paper is to explore stakeholder-sourced scenarios that would optimize the second fit. The central research question was: what would be the most suitable scenario for a continuation and possible further expansion of surgical team mentoring in Malawi? Based on the findings from two group model building (GMB) workshops and further consultations with local stakeholders and project staff we defined six implementation scenarios. For each scenario we explore future resource requirements for the institutions involved: DHs, CHs, and the party or stakeholder that would finance the mentoring trips. We also explore likely ‘soft’ consequences of scenarios, based primarily on GMB findings. From these we formulate policy recommendations. Although the managed clinical network continues, the mentoring trips funded through SURG-Africa ended in early 2020, which coincided with the onset of the coronavirus disease 2019 (COVID-19) pandemic. The sustainability of this intervention’s effects hinges on the willingness and ability of the Malawian healthcare system to continue surgical mentoring once the Ministry of Health (MoH) approves the resumption of field visits. Such a continuation will depend on two conditions. Two GMB workshops were held in September 2019: one in Nsanje district, the southern-most district of Malawi, selected on purpose because of its remoteness  and particular interest in surgical mentoring; the other in Salima, the venue for a national-level workshop. The Nsanje workshop explored the requirements for performing district-level surgery and the possible consequences of scaling up surgical services locally, ie, Nsanje DH. A total of 24 participants drawn from Nsanje district council, the DH, and several nearby health centers participated in the workshop. Both clinicians and administrators participated. In Salima, the focus was on the dynamics of surgical mentoring, its sustainability in Southern region, where the SURG-Africa supported mentoring intervention was being implemented, and its replicability in the country’s two other regions. The total of 23 workshop participants included representatives from the Directorate of Clinical Services of the MoH, surgical mentors from two CHs and clinical staff from several DHs, most of which had received surgical mentoring visits. Among the participants several cadres  were represented.10 This method required participants to take some time to think by themselves and then write down on paper cards the factors affecting (in our case) surgical scale-up and sustainability of the mentoring. Going around the room, participants were asked to mention the factors they had identified. These were added to a causal loop diagram (CLD) that was projected on a screen in real-time so participants could immediately see their contributions and correct the facilitating team in case their input was misunderstood. After all factors were added to the screen, causal relationships between factors were discussed and added by the group. The model building was completed when no further factors or relationships were contributed, after which the group reviewed the whole diagram. Upon completion of the diagram in Salima, two sub-groups of participants discussed options for sustaining the intervention, guided by the following three questions: what are the conditions for sustaining the intervention, what could be done to ensure that these conditions are fulfilled, and which party/stakeholder is in the best position to do this? Although the topic matter differed between the two workshops, the same general process was followed. Both workshops were facilitated by a moderator (author LB), a ‘model builder’ , and a note taker (MI). Each workshop started with the facilitating team describing the purpose of the workshop and soliciting active participation and having an open mind for the experiences of fellow participants. A visual model was then developed in several rounds of variable elicitation, each starting with the nominal group technique.t0 = 0 being the first month of the SURG-Africa project ; t1 being the end of SURG-Africa supported mentoring activities in March 2020 as per plan, which coincided with the suspension of all field trips in Malawi due to the COVID-19 pandemic; and t2 as the end of the 5 years horizon, in early 2023. We considered 3 main stakeholders: DHs, CHs, and the party that would take over financing of the mentoring trips from SURG-Africa. We included 23 government-owned DHs, denoted with i . We estimated the resource requirements of sustaining mentoring over time with a horizon of 5 years and counted in months with i to its referral hospital in month t was expressed as ri(t). From previous studies we know that the intervention increases local team skills and confidence, which in turn decreases patient referrals to the CH.7 The effectiveness of the intervention  is denoted with x(t) and is measured from 1  to 0 . The optimal effectiveness is unlikely to be 0 as appropriate referrals are essential in a functioning surgical system. We assume that a change in the number of referrals has two resource-related effects for DHs. Firstly, there will be savings as any avoided referral will reduce the DH’s expenditure on fuel, vehicle servicing, and travel allowances, with cri denoting the cost per referral from DH i. Secondly, with each avoided referral the DH may incur more costs for doing local surgery, with csi the cost per major surgery at DH i. The costs per month Ci(t) is thus: The referrals that are sent from DH γ is the percentage of avoided referrals that is operated on at the DH . Parameter α is the percentage of avoided referrals that is treated conservatively and thus does not receive surgery. Instead they are treated at the ward where the incurred costs are less than a full operation, ie, βcsi with β a number between 0 and 1. Mentoring is cost-saving from the DH perspective if Ci(t) < 0. Here, j (denoted as Rj) we summed up all referrals based on the existing referral patterns in Malawi: The resource implications of surgical mentoring trips for CHs are as follows. First of all, there is the reduction of referrals received from DHs and the associated reduction in bed occupancy in wards. To calculate referrals to each CH J is the set of DHs that refer to CH j and pij is the percentage of referrals from DH i that go to CH j. The latter term was included to accommodate the fact that some DHs refer cases to multiple CHs. The total impact on bed occupancy, oj, was calculated by multiplying incoming referrals with the average length of stay of a surgical patients in a hospital l, which means that oj(t) = Rj(t)l. The second effect on CHs is the time spent on mentoring trips by specialists. To calculate this, we defined f as the number of mentoring trips undertaken per DH per year. We assume each mentoring trip takes two working days, as in the SURG-Africa model. Then the average number of days a CH would need to perform per month is: Where j. We assume all DHs in Southern region would receive mentoring visits from QECH, with no role in surgical mentoring for Zomba CH, as there is only one available mentor there. This means that Kamuzu CH and Mzuzu CH would mentor all DHs in their respective regions linear models on best available data and filled in missing data with these. An overview of the sources for several key inputs can be found in 11 For the number of referrals in the central region we used a study by Maine and colleagues.12 Based on interviews with DH managers/clinicians we assumed 40% of all referrals in DHs are surgical.13 To estimate the number of referrals in the Northern region we fitted a loglinear regression model using distance to referral center and district population as independent variables. Data on district population was derived from the 2018 Malawian census and distances were calculated using Google maps. When DHs were known to refer to multiple CHs (with some percentage) we used the weighted average as distance to referral center in the regression. From SURG-Africa data were available on the actual number of referrals from hospitals in the Southern region. The effectiveness of the intervention includes enhanced team work and increased capacity and confidence of DH-level surgical teams. We assumed that it takes time to build capacity and that without mentoring this capacity dissipates. In our models we therefore assume that the monthly number of referrals from a mentored DH follow an exponential decay function and that referrals return to normal with the same dynamics if there is no mentoring:N0 is the effectiveness at the start of the intervention , and λ the exponential decay constant. Parameter λ was estimated through a loglinear model based on referral data collected at QECH between 2018 and 2020.11 Note the inclusion of a 11 We therefore assumed that no more than two-thirds of surgical referrals could be reduced. Based on the study by Mwapasa et al cited earlier the percentage of avoided referrals that underwent surgery locally was set at 25% and the percentage of avoided referrals that were managed conservatively was set at 10%.7 Trainer capacity at CHs was derived from an unpublished capacity assessment conducted in 2019 .We assumed the average length-of-stay of a referred surgical patient at a CH was 6 days.11 Here, 13 Where no empirical data were available, the cost per referral for DHs and the cost of a mentoring trip were imputed using a loglinear regression model that used distance to referral center and mentoring center. The marginal costs for each additional procedure, csi, was calculated from an earlier costing study.14 We adjusted the unit cost in that study by subtracting the capital costs and adjusting for case-mix to obtain the marginal recurrent costs. For hospitals where this study did not provide data, we imputed using the mean marginal recurrent cost. The costs of the intervention cmi for hospitals in the Southern region was estimated using SURG-Africa budget and expenditure data. Data from periodic field trip reports and vehicle logs were used to differentiate between expected costs and actual expenditures. Expected fuel usage was set at 9 kilometers per 1 liter of fuel, with an extra 3-5 liters per trip depending on the distance to be covered. A fuel price of 850k MWK  per liter was assumed. For the expected cost per mentoring trip for DHs in central and northern regions we fitted a linear model using distance to mentoring facility as the sole predictor. As the project used government rates for the travel-subsistence allowance, we assumed these costs would not change if mentoring was taken up by the healthcare system. We assumed most costs associated with mentoring visits are variable, ie, that costs would be negligible when no visits occurred . The cost per referral for Nsanje, Mwanza and Mulanje  was obtained from an earlier study conducted in the SURG-Africa project. As some parameters are stochastic we ran each scenario 1000 times and used the mean model outcome.β), % costs incurred at ward (α), % avoided referrals operated locally (γ), % referrals that are surgical (ρ), and speed constant for mentoring effectiveness per trip (λ). The first four parameters are percentages, so we varied them from 0% to 100%. The fifth and last one (λ) is an exponential decay constant derived from a loglinear model based on referral data. We varied it from 0 (signifying that the intervention had no effect) to the lower bound of its 95% confidence interval . The impact of these parameters was evaluated for the following outcomes: total referrals, total DH costs for surgery, total DH costs for surgical referrals, total mentoring costs, and total (extra) case load for CHs. To determine if our assumptions regarding certain key parameters might have influenced model outcomes, we ran univariate deterministic sensitivity analyses, involving 5 parameters: % avoided referrals managed conservatively  of district level surgery’ . Although there are many interlinking factors, several themes emerge. First of all, the basic physical requirements for surgery are: supplies, equipment, infrastructure, and electricity. Secondly, GMB participants mentioned the importance of staff and their motivation (variables in green). A third theme is the motivation of patients (blue variables). All these themes affect and are affected by the standards of district level surgery. Finally, we see that several (long term) outcomes of improved standards of surgery are mentioned: reduced referrals to QECH, reduced morbidity and mortality, and an alleviation of surgical need. Some broad issues that affect many variables in the CLD were also mentioned: competing priorities for DHs, sources of finance for surgery, and budget ceilings. The CLD shows many self-reinforcing feedback loops, reflecting changes in surgical standards that can have long-term growth effects. Examples of this are a more surgically self-sufficient DH staff after they have been trained and mentored; increased interest of donors to contribute if standards go up. However, there are also self-correcting feedback loops that make it hard to achieve and retain gains, such as overloaded staff, inadequate infrastructure/supplies and reliance on external funding for supplies and training.effects of the mentoring visits and the remote surgical consultation network are improved relations and communication between surgically active personnel in central and DHs, which in time could lead to improved care at DHs, and a subsequent reduction in surgical referrals from DHs to CHs. This would in the long run have financial implications for the DH. If the number of surgical referrals decreases, DHs will incur less costs for referrals but more costs to perform surgery themselves. The identified requirements for a successful intervention are: motivation of mentors and mentees, coordination, and funding of mentoring trips. These, in turn, result in several policy requirements for the mentoring intervention to be sustained. First of all, much of the (continued) effectiveness depends on good communication between CH-level mentors and DH-level mentees, and their respective hospital management teams. The core of the CLD from the Salima workshop  consists From the GMB sessions and further discussions among participants and project researchers, 6 possible scenarios were defined.  The projections of referrals in the different scenarios are presented in  The costs of mentoring are highest in the intense national roll-out scenario and lowest in the scenario where mentoring is only sustained in Southern region. The mentoring ‘investment’ per avoided referral goes up when the intervention is expanded to Central and Northern regions. From the DHs perspective, mentoring is cost-saving in 3 scenarios and it breaks even in other 2. There is a difference in the expected savings between hospitals: DHs that are farther away from their referral center are more likely to save money on avoided referrals . When th Participants of the Salima workshop considered the MoH the most appropriate party to fund and coordinate surgical mentoring. They recommended that the MoH establishes a focal person/desk that coordinates mentoring and other activities in support of district-level surgery. Inclusion of  mentoring as an activity in the annual District Implementation Plans, with a dedicated budget line, could be instrumental to sustain the mentoring program. Important areas for investment are in the training of adequate numbers of mentors, incentives for better staffing at the district level , and adequate supplies for both mentoring and regular surgical duties. It was noted that a risk during SURG-Africa was that some DHs accumulated cases they are ideally expected to perform routinely on their own (or even cases that should otherwise be referred to the higher centers) with the expectation that the mentors would come and do them during the mentoring visit. In other cases there were not enough patients booked for the training. For these reasons, terms of reference should be developed for mentors and mentees that focus on good planning and make it clear that the visits aim to enable local teams to do more surgery through teaching and mentoring.6 Although there are  no financial costs to it, moderation is needed to ensure the forum remains functional. After SURG-Africa ends, coordination needs to be done by specially assigned staff. If coordination is lacking it may happen that trips are cancelled because DHs are ready or because visits are planned on days that do not align with mentor duties. A recommendation from GMB participants was that CH should employ a check list or a manual they can use before travelling to the DH to make sure that the DH is actually capable of receiving them in terms of available mentees and supplies. It seems more cost-effective to have four visits per DH per year than 6. This is because in both cases referrals will decrease to the same level , only at a slightly different speed. This comes at more costs and at a larger probability of cancellation. To reach the optimal reduction in referrals it is more important that mentoring is sustained over a long time than that it is rolled out quickly and intensely. It is also important that the managed clinical network remains functional, because mentors and mentees can use it to discuss cases. Whether or not cancellations affect the costs of the intervention depends mainly on how funds for mentoring are disbursed. If they are conditional on successful completion of field trips, cancellation of such trips may be harmful to the extent that they may induce inappropriate surgical referrals. If, on the other hand, funds for mentoring are disbursed regardless of successful completion of trips then there is a risk of dwindling motivation and wastage. In any case, trip cancellations would undermine learning effects and erode motivation of mentees, especially if DH staff have prepared themselves to receive and work with the mentors. Insufficient support on the side of CHs may be a risk. GMB participants mentioned that the absence of a specialist surgeon is immediately noticeable, while the benefits of fewer referrals are observed only after the mentees will have gained additional knowledge, skills and confidence. This has an opportunity cost and it may increase the workload on fellow specialist surgeons at the CH. On the other hand, in the long-term surgical mentoring might be expected to reduce the number of unnecessary surgical referrals and decongest the CH. The estimated resource requirements suggest that the trainer capacity required for mentoring would be substantial, especially in a scenario of nationwide roll-out with 6 trips per DH per year. This finding is in line with the Salima participants’ recommendations for CHs: focus on training and availability of mentors, and involve more specialties in the mentoring teams.7 As specialized surgery tends to be more expensive, costs are expected to go up when the number of unneeded referrals from DHs decreases. In the resource requirement forecast models, the return on investments for CHs are substantial, but we calculated these numbers across the time horizon. From  For DHs, the main recommendation from the stakeholders in Salima was that DHs should try to ensure the availability of mentees and make a firm commitment to work with the mentors. The feasibility of these two recommendations is contingent on mentee motivation while it may be threatened by staff transfers. In the Salima GMB, mentee motivation was dependent on there being a mutual understanding between mentors and mentees that there was a fair share of benefits. It was mentioned that some mentees had complained that mentors received an allowance while they themselves did not. Mentors, on the other hand, argued that their allowance, based on government rates, was only to cover incurred expenses; and more importantly, that mentees would need to see the opportunity to increase their knowledge and skills as the main motivation to take part in the mentoring intervention. Staff transfers are a common occurrence in Malawi, both within and between hospitals. With changing local teams, mentors found it hard to establish relationships with mentees and mentor them. It was also mentioned that some DHs tended to save up complex surgical cases for the mentors to operate on during mentoring visits. Although patients benefited from this, it would undermine the mentoring’s main aim of strengthening local surgical capacity. From earlier work we know that the financial situation at many Malawian DHs is dire, with many being dependent on donor funds and in debt with local suppliers. A major risk to the sustainability of the mentoring intervention is therefore the DHs’ financial capacity to undertake surgery on patients that they would no longer refer after having increased their technical capacity as a result of mentoring. Our resource estimates suggest that for many DHs , the financial savings of fewer referrals may be about equal or less than the additional costs they would incur from performing more surgery locally . For abo Whether or not the intervention is cost saving from a DH perspective hinges on the uncertainty in our estimate of the cost of a major surgery at the DH level.  Supplementary files 2 to 6 present graphs produced in the sensitivity analyses that show the effects of different parameter values on five outcomes. Overall the model outcomes are most sensitive to the estimates of mentoring effectiveness and the percentage of referrals labeled as surgical. It appears there is little room for improvement on just the intervention side . This figure shows that a lower level of effectiveness implies more surgical referrals. Although higher effectiveness (relative to our estimate) would further lower surgical referrals, the effect is small. All parameters that influence referrals automatically have an impact on the costs of referrals, the costs of surgery, and the work load of CHs; and this is proportional to the change in referrals . Regarding impact of the percentage of surgical referrals on total referrals, Figure A4 shows that with a lower percentage of patients requiring surgical care, total referrals go up . Another important observation is that the costs of mentoring remain the same, regardless of its effectiveness ; it means that the intervention would be less cost-effective, as fewer referrals are avoided for the same investment.the articulations, workarounds and muddling through that keep the show on the road are not footnotes in the story, but its central plot.”15 For sustainable policy, it is often not wise to rely on completely articulated ‘sustainment plans’ that spell out actions for the coming years because the potential outcomes of complex systems are hard to predict. The healthcare system is composed of many actors, each with their own perspective and sphere of influence. Chandler argues that many rules guide the behavior of a healthcare system change over time and that these are hard to identify.16 This makes intervening hard and likely to have unintended consequences. She argues that instead of creating new rules in the system through intervention (plans), implementers instead should focus on finding ways to incentivize actors so that new  behavior emerges. From our study we conclude that there is a strong case for the MoH in Malawi to work with its CHs towards institutionalization of district-level surgical team mentoring. Although the system-wide benefits of such mentoring are acknowledged by all stakeholders, the incentives may not be directly apparent or even be insufficient, which would preclude a nation-wide roll-out. When surgical specialists engage in mentoring activities, CHs may immediately feel the loss of specialist time, with the benefit of reduced referrals coming only later. DHs may not be incentivized to do more surgery (even if their teams become more skilled/confident) if the costs of doing surgery locally exceed the savings gained from referring fewer patients, and especially if the government does not compensate DHs for these additional costs. In this study we investigated issues surrounding policy adoption of a surgical mentoring intervention in Malawi. We used both qualitative stakeholder input and quantitative modeling to inform possible scenarios for sustaining and rolling out surgical mentoring. Policy adoption and the articulation of new mandates in a complex adaptive system such as the healthcare system in a resource-constrained environment is a topic of much recent debate. In a commentary on the role of complexity in health services research, Greenhalgh notes that “ We further argue that for the institutionalization of surgical mentoring a staggered nationwide roll-out is most suitable. Although the forecast model suggests that a staggered roll-out would be slower to yield benefits than an immediate national roll-out, it provides more time to develop, test and learn, and to implement coordination and monitoring mechanisms for sustainable surgical team mentoring. For the development of such mechanisms several recommendations can be made:Coordination is best done by a dedicated focal point in the MoH with specially assigned senior staff who are in regular contact with both CHs and DHs. As cancellations of mentoring trips may have negative effects , it is important to ensure DHs are ready to receive the mentors  and that cancellation due to conflicting mentor or mentee duties is minimized. Disbursement of travel-subsistence allowances is best made conditional on trip completion and delivery of trip reports. Such reports can serve as a tool for monitoring compliance to agreed mentoring procedures. If reports are incomplete, submitted late or filled out by junior staff, this may be a sign of eroding compliance. It may be beneficial to include in-service training and mentoring in the key performance indicators for both DHs and surgical specialists for motivation purposes. 9 it was found critical for mentors and mentees to jointly finetune the intervention through periodic meetings, to improve both its acceptability and effectiveness. In a continuation of participatory action research work done under SURG-Africa, If surgical mentoring will actually result in more DH-level surgery, there may be a need to coordinate (more) closely with district health administrators  so as to secure adequate resources. A system-wide issue that may undermine the sustainability of mentoring, even if it is well-coordinated, is the financial situation of DHs. DHs in Malawi provide most services  free of charge despite the costs they incur for supplies, repairs, utilities, and kitchen services. In our models about 50% of DHs would incur losses for each avoided referral, especially those close to referral centers. This may discourage them from scaling up surgery unless they are compensated somehow for the surgical services they provide. Surgery not being a national programme in Malawi and not having a dedicated budget line, there is no such compensation. We would therefore argue for a kind of output-based or performance-based resource allocation: the more surgery a hospital provides, the more (government) budget they would be entitled to. The major strength of this study is that it combined stakeholder input with evidence and dynamic models to explore policy options. The ideal circumstances of evidence-based policy are, like in many real-world instances, not present in this study. This means that we had to make do with best available evidence and/or make assumptions based on expert input. The question is, of course: was there any alternative? Nation-wide randomized controlled trials of surgical mentoring policies are not feasible. We would argue that results from a mixed-method approach such as the one employed here are valuable for policy exploration as long as they are not taken as absolute evidence for future planning and budgeting, but rather as an entry point for further improvements and roll-out. The main limitation of this study is that for the post-2020 forecasts we assumed other major system factors to remain constant, such as the need for surgery and the number of available mentors. A pointed and unanticipated example of this limitation is the COVID-19 pandemic, which is having a major effect on Malawi’s healthcare system but was not considered at the GMB workshops. A second limitation is that the models used data and perspectives from the Southern region mostly. It is likely that our forecasts for the Central and Northern regions would have been different, had we had access to empirical data from those regions. Another limitation is that we did not take surgical referrals from health centers (rather than from DH) or inter-CH referrals into account in the quantitative models. Finally, we did not take into account any residual effects from the remote surgical consultation network which is likely to continue under all scenarios. As such, we expect it would affect all scenarios equally and not significantly impact our findings. This paper explored the complex dimensions of implementing surgical team mentoring. Although there are tangible system-wide benefits, including benefits for patients, the complexity of the healthcare system in Malawi makes sustaining and rolling out surgical mentoring a non-straightforward affair. To institutionalize surgical mentoring, it is important to take an approach of staggered roll-out, coordination, provision of appropriate incentives, and periodic monitoring. In particular, an output- or performance-based financing scheme for DHs would be required to incentivize them to scale up surgery. We thank the GMB workshop participants for their valuable contributions. We are thankful for the assistance of Dr. Monic Lansu of Radboud University in preparing the GMB workshops. Ethical approval was granted by the College of Medicine Research and Ethics Committee (COMREC) on February 14, 2018 (P.01/18/2336). Authors declare that they have no competing interests. Conception and design: HB, LB. Acquisition of data: HB, MI, GM, CP, PN, JG, and LB. Analysis and interpretation of data: HB, MI, and LB. Drafting of the manuscript: HB. Critical revision of the manuscript for important intellectual content: all authors. Statistical analysis: HB. Obtaining funding: NM, EB, RB, JG, and LB. Supervision: LB. This work was performed under the SURG-Africa project, funded by the European Union’s Horizon 2020 Programme for Research and Innovation, under grant agreement no. 733391.Supplementary file 1. List of 23 Government-Owned District Hospitals.Click here for additional data file.Supplementary file 2. Sensitivity Analysis of Parameter Effects on Total Referrals .Click here for additional data file.Supplementary file 3. Sensitivity Analysis of Parameter Effects on Costs of Referrals for District Hospitals .Click here for additional data file.Supplementary file 4. Sensitivity Analysis of Parameter Effects on Cost of Surgery for District Hospitals .Click here for additional data file.Supplementary file 5. Sensitivity Analysis of Parameter Effects on Total Mentoring Costs .Click here for additional data file.Supplementary file 6. Sensitivity Analysis of Parameter Effects on Total Case Load at Central Hospitals .Click here for additional data file.


Recently, irreversible electroporation has shown promising results for the treatment of cardiac arrhythmias. However, reversible and irreversible effects of pulsed electric field on cardiac myocytes remain poorly understood. Here, we evaluated the influence of a monophasic single electric pulse (EP) on the contractility, Ca2+ were simultaneously monitored for up to 20 minutes after EP application in Fura-2 loaded left ventricular myocytes. Lethal voltage thresholds were determined using 100 µs and 10 ms pulses and by discriminating cell orientation with respect to the electric field.Isolated rat left ventricular myocytes were electroporated using single monophasic EP of different durations and voltages. Sarcomere length and intracellular Ca2+ which was dependent upon the voltage delivered to the cell. Intermediate-voltage EP  increased sarcomere shortening, Ca2+ transient amplitude, and diastolic Ca2+ level measured 1 minute post-EP. Although sarcomere shortening returned to pre-EP level within 5 minutes, Ca2+ transient amplitude decreased further below pre-EP level and diastolic Ca2+ level remained elevated within 20 minutes post-EP. Spontaneous contractions were observed after sublethal EP application but their frequency decreased progressively within 20 minutes. Lethal EP voltage threshold was lower in myocytes oriented perpendicular than parallel to the electric field using 100 µs pulses while an opposite effect was found using 10 ms pulses.Electroporation led to an immediate increase in intracellular Ca2+ homeostasis as a function of the EP voltage. Moreover, EP-induced lethality was preceded by a large increase in intracellular Ca2+ and was dependent upon the EP duration, amplitude and left ventricular myocytes orientation with respect to the electric field. These findings provide new insights into the effect of pulsed electric field on cardiac myocytes.Sublethal EP affected rat left ventricular myocytes contractility and disrupted Ca Electroporation is a promising novel energy modality for safe, efficient cardiac arrhythmia ablation.Pulsed-electrical fields may induce both reversible and irreversible electroporation when applied to the myocardium but these effects remain poorly characterized.2+ and depends on the pulse amplitude and duration.Irreversible electroporation is preceded by a large increase in intracellular CaCell orientation with respect to the electrical field is a determinant of myocyte’s lethality induced by electroporation.2+ and contractility in isolated myocytes.Sublethal electroporation is associated with perturbations in intracellular Ca1 Technologies leveraging pulsed electric field (PEF) to achieve reversible and IRE have been developed in a broad range of medical specialties including oncology,6 neurology,10 dermatology12 and most recently, cardiology.16 Specifically, the use of PEF to ablate aberrant cardiac tissues via IRE is rapidly emerging as a promising energy modality in the treatment of cardiac arrhythmias. Traditional cardiac ablation techniques involve the use of thermal energy modalities such as radiofrequency ablation and cryoablation.18 One of the most common procedures within cardiac catheter ablation is pulmonary vein isolation. Pulmonary vein isolation aims to isolate atrial fibrillation triggers located in the pulmonary veins via selective ablation of this area. Although pulmonary vein isolation using thermal ablation technologies are approved and generally effective and safe clinical procedures for the treatment of atrial fibrillation, they are still plagued by both safety and efficacy issues. For example, thermal-based energy modalities used for pulmonary vein isolation procedures may cause collateral and detrimental tissue damage leading to pulmonary vein stenosis, phrenic nerve paralysis, esophageal fistulas, or bronchial damage. Incomplete lesion formation can result in pulmonary vein reconnection and arrhythmia recurrence.19Electroporation is a phenomenon which is associated with transient increase in membrane permeability and is achieved by cell exposure to high-voltage electric pulses (EPs). When cells are exposed to electric field strengths and duration that exceed a threshold, the increase in membrane permeability and disruption of cell homeostasis will lead to cell death. This is described as irreversible electroporation (IRE). Cells following exposure to electric fields below the IRE threshold may however regain homeostasis and survive.23 For example, in a recent preclinical study on PFA, a significantly reduced risk of pulmonary vein stenosis was reported in comparison to radiofrequency in a canine model.24 Radiofrequency ablations resulted in vagus nerve, esophageal, and lung injury, whereas this was not observed with PFA.,24 Although mechanisms of cell death after EP are complex,25 cell death mechanisms (and dynamics) of cardiac cells in response to PEF are not well understood. That said, it is generally known in the field of IRE that cell death is a function of the electric field.26 Because the ultimate goal of PFA is IRE, the application of high voltages to membranes may also induce reversible effects.1 Reversible electroporation is inevitable in vivo given the electric field distribution around electrodes.,28 Considering that electric field strength drops as the distance from the electrodes increases, it can be expected that a volume exists adjacent to the electrodes above the IRE threshold where all cells will die. Surrounding that ablated volume, there may be a region of tissue where transient or reversible effects will be induced. In this context, our understanding of the reversible and IRE effects on cardiac cellular function related to PEF application is desperately needed. In nerves, electrode proximity and voltage (field strength) can dose-dependently increase neural stunning times of paced phrenic nerves after PFA application.29 Previous defibrillation research has demonstrated transient effects on cardiac cell permeability in the early 2000s, such  stunning phenomenon has not been described in the context of PEF aimed to ablate cardiac tissue.31 It is possible that reversibly effected, that is “ stunned” zones could be observed clinically and would be interpreted as acute procedural success. But if such stunned zones recover, the ablation therapy could fail due to incomplete lesion formation/electrical isolation with subsequent pulmonary vein reconnection. Moreover, the orientation of elongated cells in electric field affects EP,39 which can play a significant role in ablating heart tissue where structure (position of cells) and function are strongly connected.Pulsed field ablation (PFA) is a term used to describe the use of IRE to ablate cardiac tissues for the treatment of cardiac arrhythmias. PFA utilizes hundreds to thousands of volts applied to electrodes on a catheter either in a monopolar or bipolar fashion/mode to induce cell death via membrane hyperpermeabilization mechanisms. PFA holds great promise and potential advantages over traditional energy modalities and may be able to alleviate/mitigate limitations of the thermal-based ablation modalities including collateral damage. It has been hypothesized that cardiac cells may be more sensitive to IRE than other cell types, such as nerve cells or smooth muscle cells, and thus less collateral injury is expected with PFA. This hypothesis is also supported by absence of side effects in preclinical as well as clinical studies.2+ in excitation-contraction coupling, we hypothesized that varying doses of PEF will modulate both Ca2+ levels and mechanical function of isolated cardiac myocytes. Gaining knowledge of EP effect on contractility and Ca2+ is becoming more relevant as PFA is now in clinical development and is speculated to become the preferred energy modality for cardiac ablation.,43Due to the central role of Ca2+ transient (CaTr) and recovery or cell death over a clinically relevant time frame .44 Studied variables included pulse parameters , pacing, as well as cell orientation in the applied electric fields.Thus, the goal of this study was to characterize reversible and irreversible effects of high voltage PEF on isolated left ventricular myocyte’s (LVM) contractile function, CaThe data that support the findings of this study are available from the corresponding author upon reasonable request. All experiments were performed in accordance with the European Union council directive 2010/63/EU for the protection of animals used for scientific purposes following local ethical committee approval.2, 0.4 NaH2PO4, 5 HEPES, 10 Glucose, 10 Creatine, 20 Taurine (pH 7.4 with NaOH), and 0.75 mM CaCl2. The hearts were then perfused with the isolation solution supplemented with 0.1 mM Ethyleneglycol- bis(β-aminoethyl)-N,N,N′,N′-tetraacetic Acid (EGTA) and finally with the same solution with the addition of collagenases  and proteases  fwor 8 to 10 minutes. The left ventricles were then dissected, cut into small pieces and gently shaken at 37 °C in the enzymatic solution. Myocytes were collected by filtration through a nylon gauze and were used within 8 hours.45Male Wistar rat (250–350 g) were anesthetized  and euthanized. Hearts were quickly excised, mounted on a Langendorff system, and perfused at 8 mL/min (37 °C) through the aorta with an oxygenated isolation solution containing (in mM): 130 NaCl, 5.4 KCl, 1.4 MgCl2, 1 MgCl2, 10 glucose and 10 Hepes (pH 7.4 with NaOH) at 34 °C to 37 °C . Cardiomyocytes were paced via field-stimulation to contract at 1 Hz  and sarcomere length continuously monitored through a video-detection system (IonOptix Corporation). In some experiments, intracellular Ca2+ ([Ca2+]i) level was simultaneously monitored in myocytes loaded with 4 µM Fura-2 Fura-2 acetoxymethyl ester (Invitrogen) (Invitrogen). Briefly, following excitation at 340 and 380 nm, the emission light was collected at 510±40 nm through a photomultiplier tube connected to a spectrophotometer system . The ratio of the emitted light in response to 340 and 380 nm excitation was used as an index of [Ca2+]i. Sarcomere shortening expressed as a percentage of resting sarcomere length, diastolic intracellular Ca2+ level and CaTr amplitudes were analyzed using IonWizard (IonOptix Corporation).46 For pacing-induced activity, all measurements were performed at steady state and the analysis was done on averaged trace of 10 consecutive beats.LVM were placed in a perfusion chamber mounted on the stage of an inverted microscope  and continuously perfused with a Tyrode solution (mM): 140 NaCl, 5 KCl, 1.0 CaCl47 Electric field intensity (E) estimated as a voltage-to distance ratio and given in V/cm is provided for easier comparison. PEF were applied as a single monophasic EP of 100 µs duration at low (80 V [E=200 V/cm]), medium (140 V [E=350 V/cm]) voltage. At these field strengths, both contractility and [Ca2+]i were monitored independently of cell orientation relative to the PEF. High-sublethal voltage EP (100 µs duration) were applied either between 300 and 440 V (E=750–1100 V/cm) to cells oriented parallel to the electric field or between 140 and 200 V (E=350–500 V/cm) to cells oriented perpendicular while monitoring contractility and [Ca2+]i. Pacing was stopped just before the EP application, and myocytes were left unpaced for 1 minutes after EP delivery. Sarcomere length and [Ca2+]i were monitored during this unpaced period. After 1 minute, myocyte pacing was resumed to assess contractile and [Ca2+]i recovery. Then, pacing was stopped and resumed every 5 minutes over a 20-minute period to monitor the presence of spontaneous contractions . Cells that recovered were then exposed to voltages of increasing intensity to identify the lethal voltage values. Another set of experiments was conducted in which EP was delivered under continuous pacing at 1 Hz. During this investigation, both sarcomere shortening and [Ca2+]i parameters were evaluated every 5 minutes over a 20-minute period . We established the lethality profile by applying a single 10 ms EP at different voltage amplitudes (50–140 V). A complementary metal oxide semiconductor camera connected to the side port of the microscope  was used to image isolated cardiac myocytes within the field of view prior and ≈30 s after EP application. The percentage of rod-shaped striated cells which died upon EP application  was calculated separately for parallel- and perpendicular-oriented myocytes with respect to the electric field.Only rod-shaped myocytes showing clear striations were used. When the role of cell orientation with respect to the electric field was studied, only cells showing axis angle with respect to electric field of 0±10°  and 90±10° (perpendicular) were used for the experiments. Myocytes were exposed to EP at various voltage amplitudes using 2 platinum electrodes (4 mm spacing) connected to both a field-stimulator and a PEF generator  producing an electric field used for either pacing and electroporation, respectively.t test, paired t tests, or repeated-measure 1-way ANOVA or their respective nonparametric tests were used to compare our conditions. Differences were considered significant for P<0.05.Experimental data are expressed as means±SEM; n represents the number of experiments; N represents the number of animals used for the experiments. Samples were tested for normal distribution with d’Agostino-Pearson. Statistical analysis was performed using GraphPad Prism version 6.0 . Unpaired 2+]i traces are presented in Figures 2+]i reaching higher level than systolic [Ca2+]i observed when pacing at 1 Hz resulting in a large sarcomere shortening. At low-voltage amplitudes, the first peak CaTr recorded after EP was similar in amplitude to the first transient obtained upon resuming field-stimulation after an unpaced period and was reminiscent of postrest potentiation.48 At intermediate-voltage amplitudes, the first CaTr after EP was slightly greater than postrest first CaTr . This PEF-induced immediate increase in [Ca2+]i was followed by a fast decrease in [Ca2+]i accompanied by sarcomere relaxation similar to that observed upon pacing LVM. However, this relaxation was incomplete and [Ca2+]i remained elevated for several seconds before returning closer to initial diastolic levels within 1 minute and beyond . Representative traces showing sarcomere length and [Ca2+]i traces at different times after exposure to an intermediate voltage amplitudes are shown on Figure 2+ levels increased at 1 minute following exposure to 140 V and did not return to baseline level within 20 minutes  with the exception of sarcomere shortening being significantly reduced 10, 15, and 20 minutes after EP.Typical sarcomere length and Ca+i traces2+ levels found in cells for both orientations at lethal voltages were significantly different , that is, avoiding potential cumulative effect of multiple pulses being applied to the same cells. We also assessed the effect of longer-lasting electroporating pulses on lethality in both perpendicular and parallel-oriented myocytes with respect to electric field. Quiescent myocytes were exposed to single 10 ms electroporating pulses of different voltage amplitudes and lethality was assessed by image analysis . Interestingly, for this pulse duration, parallel myocytes were more sensitive to PEF-induced cell death than perpendicular cells . This led us to assess the effect of high voltage, sublethal PEF on contractility and [Ca2+]i at distinct voltage amplitudes for these 2 cell subpopulations, that is, 300 to 340 V for cells oriented parallel and 140 to 200 V for cells oriented perpendicular to the electric field. At 1 minute post-EP with high voltage, sublethal pulses, 60% of perpendicular, and 55% of parallel cells presented elevated diastolic [Ca2+]i level and remained unresponsive to pacing. In cells that responded to pacing at 1 minute post-EP, a significant increase in sarcomere shortening was observed in perpendicular but not in parallel myocytes following 1 minute after EP . At this time, high voltage pulses did not significantly alter CaTr amplitudes in either cell groups but there was a trend for an increase in diastolic Ca2+ level in both parallel and perpendicular myocytes . At 5 minutes after applying a high voltage amplitude pulse, a significant increase in sarcomere shortening was observed in LVM for both orientations  and contractions which progressively disappeared with time whatever the voltage . This constitutes an important step to better understand mechanisms involved in cardiac PFA. As clinical ablation therapies based on the principles of IRE are being developed, it is important to understand the underlying cellular effects of irreversible but also of reversible electroporation in cardiac myocytes.This study provides new insights into the effects of PEF on LVM function. We determined for the first time the influence of PEF on myocyte contractile function and i to ensure fine-tuned diastolic (low [Ca2+]i) and systolic (high [Ca2+]i) cycles in normal heart function. In our study, we found that EPs perturbed intracellular Ca2+ homeostasis. First, we found an elevated [Ca2+]i plateau which followed EP application before returning to lower diastolic levels in LVM exposed to low-, intermediate-, and high-sublethal voltage EPs. This intracellular accumulation of Ca2+ depended directly upon EP voltage amplitudes and was associated with cell death when subsequent PEF of increasing amplitude were applied to myocytes. Such a PEF voltage-dependent effect was likely related to an increase in sarcolemma pore density which is known to increase with increasing electric field strengths.38 The degree of membrane (electro)permeabilization is a key factor determining the fate of the cell after exposure to high voltage amplitudes; either cell death (IRE) or recovery (reversible electroporation). Indeed, this physiological process (cell recovery) is crucial to maintain cellular function and to preserve the integrity of the plasma membrane as well as the Ca2+ handling machinery55 to limit undesirable exchange of ions and other components between intracellular and extracellular media. Here, we could not determine the Sarco/endoplasmic calcium ATPase (SERCA) or sodium-calcium exchanger (NCX) function in myocytes immediately after EP application as these would not respond to pacing at this point. However, at 1 minute after EP—or 5 minutes for high voltage EPs—the CaTr decay time constant, an index of function of Ca2+ removal pathways, was similar to that measured prior to EP for all sublethal voltages. It has been previously shown that the recovery times of cells/tissues exposed to electroporating pulses depend mainly on the pulse settings , the cell orientation and the experimental buffer.,56 Note that myocytes partially recovered their capacity to normally respond to the pacing as early as 1 minute after EP for low- and intermediate-voltage amplitudes. However, after this time, we also identified spontaneous contractions which were associated with spontaneous intracellular Ca2+ releases and elevated diastolic Ca2+ levels. Those spontaneous contractions were more frequent when myocytes were exposed to high voltage EP, indicative of a possible membrane defect (cell membrane and/or sarcoplasmic reticulum). Abnormal intracellular Ca2+ responses have been described upon exposing mouse ventricular myocytes to repetitive nanoseconds pulse-width PEFs.37 In our study, we observed these events already after the application of a single monophasic EP, but these may be expected also after a train of pulses being delivered. Further work will be required to confirm the effect of PEFs on Ca2+ homeostasis at the tissue level in coupled myocytes and evaluate their arrhythmogenic potential. To our knowledge, no arrhythmic event has been reported from clinical and preclinical studies using PFA. We can speculate that the large increase in diastolic Ca2+ level induced by PEFs may induce a closure of the connexins and reduce cell-to-cell coupling until intracellular Ca2+ levels are regulated. This would prevent the propagation of the spontaneous arrhythmogenic Ca2+ events to the surrounding myocardium. It must be noted that EP appeared to be reversible (20 minutes after EP) and may thus indicate the cells’ ability to regulate their Ca2+ homeostasis to maintain their function. Interestingly 5 to 20 minutes is also time reported for cell membrane resealing in electroporation in vitro.,57 Previous studies demonstrated similar time courses of recovery after the exposure to high-voltage amplitudes.61 Here, despite an initial increase in sarcomere shortening and CaTr upon resuming pacing, there was a trend for a decrease in these parameters below their control level and a rise in diastolic [Ca2+]i within a 20-minute time frame after EP. These results suggest myocytes may not completely recover from PEF application within the observed 20 minutes although further experimental investigations are required to evaluate myocyte recovery in the longer term.It is well established that the cardiac contractility is modulated by a specific equilibrium of intracellular Ca2+ influx, mitochondrial damage leading to ATP depletion, reactive oxygen species production, and an increase of the cell content leakage inexorably leading to cell death.66Irreversible electroporation, resulting in cell death, caused by electroporating pulses is mainly related to increased permeabilization of the plasma membrane which occurs when the membrane potential reaches supraphysiologic values. Indeed, when the membrane potential exceeds a critical value, a disruption of the plasma membrane structure can trigger a detrimental biological process which is manifested by the formation and expansion of large hydrophilic pores, massive Ca67 The specific membrane structure of LVM with transverse tubules may also contribute although this will require further investigations. However, when applying 10 ms pulses the opposite effect was observed, that is, cells oriented parallel were more sensitive to PEF-induced cell death then cells oriented perpendicular to the electric field. Parallel cells being more sensitive than perpendicular is in line with previous study in which authors also identified a lower lethal threshold in ventricular myocytes oriented parallel to the electric field using 10 ms pulses.,39 For millisecond pulses, it has been suggested this could be related to the fact that the maximum change in membrane potential occurs at the cell ends which are close to the electrodes and to the threshold for electroporation which is proportional to the cell length in the direction of the field.38Interestingly, when applying 100 µs pulses, we observed that cells oriented perpendicular were more sensitive to the PEF-induced cell death than cells oriented parallel to the electric field. The elongated geometry of LVM may contribute to this effect as suggested in silico by others.38 This discrepancy may be related to the different type of cells and their respective geometries and membrane properties. The mechanisms underlying this different sensitivity to EP in parallel and perpendicular cells according to pulse duration are not well explained but may involve differences in the way pores are formed37 or membrane curvature.67 Identifying the pulse parameters for which parallel and perpendicular cells share a similar sensitivity to IRE, may facilitate the creation of more homogeneous lesions within the thickness of tissues with heterogeneously-oriented cells such as in the atrial wall.A similar change in sensitivity of cells to PEF according to their orientation has recently been unveiled in cardiac-like cell lines, where short and long pulses had opposite effects, a similar observation as in our study—although the crossover pulse duration appeared to happen at shorter pulse durations.,69 In order to carefully identify the effects of PEFs on cardiac myocytes we used monophasic single pulses of varying duration and amplitude. However, clinical IRE protocols typically involve biphasic train of pulses. These protocols may require higher field strength as suggested by former studies in noncardiac cell lines.,71 The mechanisms by which PEFs affect Ca2+ homeostasis and contractility described in the present study are likely to be similar. Furthermore, it is also well established that there are physiological or pathological ionic current remodeling and Ca2+ handling changes according to the species and age that may influence PEF-induced Ca2+ changes and interpretations.73The study was conducted on isolated rat LVM. Different cardiac cell types  or species  could be associated with different effects. For example, atrial myocytes and Purkinje cells are known to have different structures with less transverse tubules compared with ventricular myocytes.2+ changes in IRE myocyte. Myocyte capacity to recover from a monophasic PEF was time-dependent and was related to pulse voltage and duration. Irreversible effects, that is, cell death, involved a large increase in [Ca2+]i and strongly depended on myocyte orientation within the electric field. Further work is however necessary to expand the knowledge from monophasic single pulses of 100 µs and 10 ms pulses used in our study to multiple pulse frequencies and biphasic waveforms on cardiac myocytes.We identified reversible effects of PEF on isolated LVM excitation-contraction coupling during a clinically relevant time scale and defined the impact of CaWe thank Celine Ayez for taking care of the animals.This study was funded by Medtronic.Drs Sigg and Stewart are employees of Medtronic. Dr Miklavčič is a Medtronic consultant. Drs Batista Napotnik and Miklavčič received a Medtronic grant. The other authors report no conflicts.Table S1Figures S1–S4