Lowering extracellular Ca2+ induced hemichannel opening, which was associated with cAMP signaling pathway activation and increased renin production. Blockade of hemichannels with inhibitors or downregulation of Cxs with siRNAs abrogated the activation of cAMP pathway and the elevation of renin. Further analysis revealed that cAMP pathway activation was blocked by adenylyl cyclase inhibitor SQ 22536, suggesting an implication of adenyl cyclase. Furthermore, the participation of hemichannels in the activation of the cAMP signaling pathway was also observed in a renal tubular epithelial cell line NRK. Collectively, our results characterized the hemichannel opening as a presently unrecognized molecular event involved in low Ca2+-elicited activation of cAMP pathway and renin production. Our findings thus provide novel mechanistic insights into the low Ca2+-initiated cell responses. Given the importance of cAMP signaling pathway in the control of multiple cellular functions, our findings also highlight the importance of Cx-forming channels in various pathophysiological situations.Connexin hemichannels play an important role in the control of cellular signaling and behaviors. Given that lowering extracellular Ca In contrast to other secretory cells, in which an increase in Ca2+ usually promotes secretion, renin secretion is inversely related to the Ca2+ concentration in JG cells [The renin-angiotensin system is a major regulatory system controlling extracellular fluid volume and blood pressure. The rate-limiting enzyme in this hormonal cascade is renin, which is secreted into the circulation by renal juxtaglomerular (JG) cells of the afferent arteriole. Renin secretion is controlled by several factors, such as blood pressure, sodium chloride load, sympathetic nerves, hormones, cytokines, and vasoactive materials. Although the signal pathways underlying the actions of these factors differ, it is generally accepted that a change in intracellular CaJG cells ,4,5,6,7.2+ regulates renin have been the focus of many previous investigations [2+ regulates renin through modulation of adenylyl cyclase activities. Low Ca2+ enhances the activities of the Ca2+-inhibitable isoforms of adenylyl cyclases in JG cells, thus increasing intracellular cyclic adenosine monophosphate (cAMP) and stimulating renin synthesis [2+ suppresses adenylyl cyclase and inhibits renin [The molecular mechanisms by which Caigations ,2,4,5,8.ynthesis ,2,8. Oppts renin ,10,11. cts renin ,13.Gap junctions (GJs) are intercellular channels that permit the direct exchange of ions, secondary messengers, and small signaling molecules among neighboring cells. Each GJ channel is composed of two hemichannels that reside in the plasma membrane of two closely apposed cells. Each hemichannel is composed of six protein subunits termed connexin (Cx). Up to now, more than 20 different Cx molecules have been identified. Gap junction-mediated intercellular communication (GJIC) plays an important role in the regulation of cell signaling and functions ,15,16. I+), glutamate or prostaglandin E2 (PGE2) [2+, a situation that stimulates renin, also activates hemichannels [2+-induced activation of cAMP pathway and renin production.Besides intercellular GJ channels, the non-junctional Cx hemichannels are also activated under various pathophysiological situations and participate in the regulation of cellular behaviors through the release of mediators such as cAMP, adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD2 (PGE2) ,23,24,252 (PGE2) . Second,channels ,29,30,31channels ,33,34. T2+ in mouse renin-secreting cells. Given the importance of the cAMP signaling pathway in multiple pathophysiological situations, the activation of the pathway by hemichannels provides a novel mechanistic insight into the actions of Cx hemichannels and suggests that Cx channels are critically involved in the control of many cellular processes.Here, we present our results that hemichannel opening is requisite for the effective activation of the cAMP signaling pathway-induced low Ca2+, we have used a renin-expressing cell line As4.1. This cell line was isolated from a mouse renal tumor and has been well characterized for the synthesis, storage and secretion of renin. We first confirmed that As4.1 cells expressed functional Cx molecules. Based on the previous reports and availability of antibodies [To determine hemichannel activity after removing the extracellular Catibodies , we immu2+ activated hemichannels [2+-free medium initiated a rapid exchange of small molecules between the inside and outside of the plasma membrane, as indicated by the influx of LY and efflux of ATP. In the presence of the hemichannel inhibitor heptanol, this exchange was almost completely blocked. These results indicate that Ca2+ deprivation activates hemichannels in As4.1 cells.We and others have previously reported that the removal of extracellular Cachannels ,29,30,312+ deprivation-induced renin production, we first examined the influence of hemichannels on renin-regulating cAMP pathway [2+ elevated vasodilator-stimulated phosphoprotein (VASP) and cAMP response element binding protein (CREB) phosphorylation, two validated substrates of cAMP-dependent protein kinase A (PKA) [To determine the role of hemichannels in Ca pathway ,11. Figu A (PKA) ,36. This2+-initiated CREB phosphorylation . Anti-phospho-cAMP response element-binding protein  and anti-CREB (#21052-2) were purchased from Signalway Antibody . Anti-phospho-vasodilator-stimulated phosphoprotein  and anti-Cx40 antibody (#AB1726) were obtained from Calbiochem . Anti-renin antibody was bought from AnaSpec  and also gifted by Prof. T lnagami . FITC-conjugated swine anti-rabbit immunoglobulin (#F0205) was purchased from DAKO . Anti-Cx43 (#C6219), anti-β-actin (#A3854), heptanol, lucifer yellow (LY), trypsin and all other chemicals were obtained from Sigma-Aldrich Japan .2+ and Ca2+-free situations, cells were exposed to Ca2+-free DMEM  with or without supplementation of 1.8 mM Ca2+.The renin-expressing As4.1 cell line was obtained from the American Type Culture Collection (ATCC No. CRL2193). For maintenance and passage, cells were cultured at 37 °C in RPMI-1640 with L-glutamine, phenol red and HEPES medium  supplemented with 5% fetal bovine serum (FBS). For comparison of cell responses between normal CaImmunocytochemical staining for Cx40, 43 and 45 was performed as previously reported ,29. In bGJIC was assessed by transfer of the membrane-impermeable fluorescent dye, LY after single cell microinjection using an automated microinjection system , as described previously .2+-free medium in the presence or absence of 0.05% LY for 20 min. The cells were then rinsed and fixed with 3% paraformaldehyde. Immunofluorescent images were captured using a CCD camera attached to an Olympus IX71 microscope .Hemichannel permeability was evaluated by the cellular uptake of LY as described previously ,29,30,31ATP was measured using a luciferin/luciferase bioluminescence assay kit (Molecular Probes). The intensity of chemiluminescent signal was determined by a luminometer  as described previously ,29,30,31Western blot was performed using an enhanced chemiluminescence system ,29,30,312+-free medium for the indicated time. Cellular protein was extracted and subjected to western blot analysis of the targeted proteins.Cells were transiently transfected with siRNA specifically targeting against Cx37 (Mm_Gja4_1 FlexiTube siRNA), Cx40 (Mm_Gja5_1 FlexiTube siRNA), Cx43 (Mm_Gja 1_2 HP siRNA) and Cx45  or a negative control siRNA (AllStars Negative Control siRNA) at a final concentration of 20 nM using Hyperfect transfection reagent for 24 h . After tt-test. The analysis was performed using SigmaStat statistical software . p < 0.05 was considered to be a statistically significant difference.Values are expressed as mean ± SE. Comparison of two populations was performed using a Student’s


A secondary malignancy is the most serious complication in lung cancer (LC) survivors. This study aimed to evaluate the clinicopathological features, predictable risk factors and survival of patients with LC who developed therapy-related acute myeloid leukaemia (t-AML).Patients from the Surveillance, Epidemiology, and End Results (SEER) database diagnosed with t-AML after LC between 1975 and 2015 were included. Standardized incidence ratios (SIRs) were used to perform multiple primary analyses. The risk of t-AML development among LC patients was assessed using a logistic regression model. Kaplan–Meier analysis was used to construct overall survival (OS) curves. Cox regression was used to assess the influence of various prognostic factors.A total of 104 patients with t-AML after LC-targeting chemotherapy were included. The median latency period to the development of t-AML was 35.5 months. The calculated SIR of t-AML was 4.00. Chemoradiotherapy, small cell lung cancer (SCLC), or localized/regional-stage LC was a risk factor for the development of t-AML. The median OS was only 1 month, and those younger than 65 years were predicted to have a better OS time.t-AML is a rare but serious late complication in LC patients and is associated with a poor prognosis. It is necessary to carry out long-term follow-up and screen for t-AML in LC patients, especially among those undergoing both radiotherapy and chemotherapy, with SCLC or with localized/regional-stage LC. Among cancers, lung cancer (LC) has the highest incidence and mortality rates worldwide. The estimated number of new cases of LC in the United States in 2019 was 228,150, and the estimated number of deaths was 142,670 [The development of therapy-related acute myeloid leukaemia (t-AML) has always been considered a rare but highly serious complication of cytotoxic chemotherapy and radiotherapy . It is eThe purpose of this study was to identify the clinicopathological features of LC-related t-AML and predictable risk factors and survival of patients with LC who developed t-AML by analysing the Surveillance, Epidemiology, and End Results (SEER) database. The findings will offer treatment guidance for physicians.In this retrospective population-based study, information for patients diagnosed with t-AML after treatment for primary LC was obtained from the SEER-9 database. The SEER database is a system of cancer registries that incorporates high-quality data. In the present study, we included 9 cancer registries  as released in November 2018. This cohort covers approximately 10% of the total US population. According to the limited-use protocol of SEER research data, these data can be used publicly, and approved by the institutional review committee was not required for our research.Subjects diagnosed with t-AML after a primary LC diagnosis were identified from the multiple primary-standardized incidence ratio (SIR) method in SEER*Stat (version 8.3.6). From 1975 to 2015, we first screened patients whose malignant tumours were located in the lung by using the selected function of multiple primary SIRs. All of these patients with LC had received chemotherapy during LC treatment. From this group, we further selected patients who developed t-AML after LC diagnosis during this period. The following criteria were used to exclude patients from this study: (1) age less than 20 years; (2) history of malignancy in addition to LC; (3) diagnosis of AML within 2 months after LC diagnosis; and (4) incomplete survival information.Demographic parameters were extracted, including sex, age, ethnicity, and the period of diagnosis, as were histopathological LC data, including stage, pathologic grade and histologic type. The SEER historical stage was used for LC staging , 13. It SIRs, excess risk (ER) and person-years at risk were chosen for primary analyses, as generated from the multiple primary-SIR analysis. SIRs were used to compare the incidence of AML in primary LC patients who had received chemotherapy after LC diagnosis to the incidence of AML in the general population. We also computed the ER to estimate the number of excess t-AML patients per 10,000 persons per year.Categorical data were compared using the chi-square test, and the results are expressed as proportions. Odds ratios (ORs) of t-AML development risk in patients with LC were obtained using a logistic regression model. OS curves were established by Kaplan–Meier analysis and compared using the log-rank test. Finally, Cox regression was employed to evaluate the effect of various prognostic factors on OS.P value < 0.20 in univariate analysis were included in the multivariate logistic or Cox regression model. Multicollinearity was tested using the variance inflation factor (VIF) method and, a VIF ≥5 was not permitted. The statistical analysis was performed using “R” version 3.6.2 (http://www.r-project.org) and SPSS 24.0 . All P values were two-sided and considered statistically significant at < 0.05.Variables that had a P = 0.332). Over time, the number of LC patients with or without t-AML increased to varying degrees, especially those with t-AML, with a sevenfold increase. The proportion of LC patients who developed t-AML was higher in those who underwent surgery (P = 0.006) or radiotherapy (P = 0.001) during LC treatment and in those with localized/regional-stage LC (P < 0.001) than in those who did not.One hundred and four patients with t-AML after LC diagnosis and 158,541 patients with primary LC who did not develop t-AML between 1975 and 2015 in the SEER-9 database were included. Table The calculated SIR of t-AML was 4.00 (95% CI: 3.28–4.82), with an ER of 3.66 cases per 10,000 persons. Regarding patients with t-AML, SIRs were increased to various degrees for different LPs, calendar periods and ages at diagnosis  . Nonetheless, the 2-year and 5-year OS rates were very low in both groups, with no statistically significant difference . The median OS of patients with t-AML based on stratification by the period of diagnosis  was not increased  (P = 0.625) . In total, 97.1% of the patients died before the end for this study. The median OS was only 1 month (95% CI: 0.4–1.6) (Table P = 0.012). However, patients with t-AML caused by localized/regional LC or who were recently diagnosed with LC during the 2005–2015 period did not have a longer OS. Other factors, such as sex, LP, pathologic grade, surgery, radiation and pathological type of LC, showed no predictive value for OS.Multivariable analysis of the 104 patients with t-AML showed that only age < 65 years had predictive value for a longer OS (hazard ratio (HR): 0.59; 95% CI: 0.40–0.89; The risk of developing t-AML in survivors of LC and their survival conditions are increasingly raising concern because the use of active modern therapies and the number of survivors is drastically growing. Our research used the latest data provided by the SEER database to evaluate clinicopathological features, risk and survival among patients with t-AML after LC. To the best of our knowledge, this information has never been previously reported. According to our results, among the different age, LP, period of diagnosis and sex groups, patients with t-AML after LC had higher SIRs. Multivariable analysis showed that within the LC patient group, pathological classification of LC, stage and radiotherapy were indicators of t-AML development. Finally, patients with t-AML had a very poor prognosis, with a median OS of only 1 month, and only age < 65 years was a factor for a longer OS. No difference in median OS was found in other subgroups, including LC stage and period of diagnosis subgroups.Eighty percent of all patients with t-AML were diagnosed within the first 5 years after their LC diagnosis; the median LP from LC diagnosis to t-AML diagnosis was 35.5 months. The other 20% of patients with t-AML were diagnosed more than 5 years after their LC diagnosis, emphasizing the necessity of long-term follow-up for patients with LC. The most common cause of t-AML is exposure to alkylating agents or radiotherapy, usually after an LP of approximately 5–7 years , and patExcept within the first year after LC diagnosis, patients with LC had higher SIRs of developing t-AML than the general population, regardless of their sex, age, LP and diagnosis period. This emphasizes the universality of high risk of t-AML development in LC patients. In the primary LC cohort, multivariate analysis showed that those who received both chemotherapy and radiotherapy had an increased risk of developing t-AML compared with those who received chemotherapy alone. This finding was consistent with previous reports , 18. NevAdditionally, the pathological type and stage of LC were found to be important factors affecting the occurrence of t-AML. The cumulative risk of t-MDS/t-AML after intensive treatment targeting SCLC has been reported to be 14% at 4 years after diagnosis  and 25% Our observations did not reveal difference in the risk of developing t-AML after LC during different calendar periods, in contrast to what has been previously reported , 19. In We found that patients with t-AML had a very poor prognosis, with a median OS of only 1 month. With the advancement of supportive therapy over the past 40 years, the application of new chemotherapy drugs, such as CPX-351 , and theSeveral factors can potentially explain the poor outcome of t-AML. First, the survival rate of primary LC is already relatively low. Therefore, such leukaemias will further reduce the possibility of long-term survival in LC. Second, patients with t-AML are usually older, leading to weaker immunity and more age-related complications. Third, previous chemotherapy for LC may result in the depletion of haematopoiesis reserves, causing a longer bone marrow (BM) suppression time and more serious treatment-related complications after t-AML treatment. Finally, molecular and cytogenetic abnormalities associated with conventional chemotherapy resistance, such as TP53 mutations, are common in patients with t-AML, leading to a poor prognosis , 26. AltAn unanticipated finding was that only age younger than 65 years predicted improved OS in patients with t-AML. Several studies have shown that older age indicates a worse OS among patients with t-AML , 28. ThiThere are several limitations in our study. First, four patients developed AML 10 years after the diagnosis of LC. Due to the lack of data, including cytogenetics and molecular genetics, in the SEER database, there is no way to accurately determine whether the occurrence of AML is due to chemotherapy/radiotherapy or is de novo AML. Moreover, these genetic characteristics may affect the prognosis of patients with t-AML. Second, in 1999, the World Health Organization lowered the threshold of BM blast percentage required for AML diagnosis from 30 to 20%. However, it is still debated whether the natural history and responsiveness to therapy of patients with 20–30% BM blast is comparable to that of patients with > 30% BM blast AML, and this may affect the analysis of t-AML in different periods. Third, the SEER database does not include all initial chemotherapy and radiotherapy information because some treatments may have been performed outside the hospital or medical centre that reported the case to the registration centre. Thus, the conclusion of this study may not represent all patients with LC receiving chemotherapy and radiotherapy. Fourth, no information about the type, dose and course of chemotherapy drugs in the SEER database is available, and information regarding whether patients with t-AML received palliative treatment or active treatment and whether haematopoietic stem cell transplantation was used is not provided. These factors may affect the analysis of high-risk factors and prognostic factors of t-AML.In conclusion, t-AML is a rare but serious late complication of patients with LC and has a poor prognosis. The conclusions of our study are valuable when explaining the risk of developing t-AML among patients with LC. The benefit of chemoradiotherapy and risk of developing t-AML should be weighed by physicians when selecting chemoradiotherapy regimens. It is necessary to carry out long-term follow-up and screening of t-AML among patients with LC, especially those receiving both radiotherapy and chemotherapy, with localized/regional-stage disease or SCLC. Furthermore, a prospective, multicentre study is urgently needed to improve the prognosis of patients with t-AML after LC. Additionally, uploading and sharing more patient data from various medical centres around the world would contribute to international cooperation to increase our understanding of such patients and find better treatment methods.


This process requires a considerable amount of energy and the active participation of mitochondria, which encompass ∼30% of the cell volume and represent the major source of ATP in the heart. Importantly, in adult cardiomyocytes, mitochondria are distributed in a highly orderly fashion and strategically juxtaposed with SR. By taking advantage of the vicinity to Ca2+ releasing sites, they take up Ca2+ and modulate ATP synthesis according to the specific cardiac workload. Interestingly, with respect to SR, a biased, polarized positioning of mitochondrial Ca2+ uptake/efflux machineries has been reported, hinting the importance of a strictly regulated mitochondrial Ca2+ handling for heart activity. This notion, however, has been questioned by the observation that, in some mouse models, the deficiency of specific molecules, modulating mitochondrial Ca2+ dynamics, triggers non-obvious cardiac phenotypes. This review will briefly summarize the physiological significance of SR-mitochondria apposition in cardiomyocytes, as well as the pathological consequences of an altered organelle communication, focusing on Ca2+ signaling. We will discuss ongoing debates and propose future research directions.In cardiomyocytes, to carry out cell contraction, the distribution, morphology, and dynamic interaction of different cellular organelles are tightly regulated. For instance, the repetitive close apposition between junctional sarcoplasmic reticulum (jSR) and specialized sarcolemma invaginations, called transverse-tubules (TTs), is essential for an efficient excitation-contraction coupling (ECC). Upon an action potential, Ca Though ER and SR membranes/lumen are continuous, protein distribution is polarized, with completely different kinetics of Ca2+ uptake and release between the two compartments. As an example, ryanodine receptors (RyRs) and calsequestrin are enriched in the SR, whereas inositol-trisphosphate receptors (IP3Rs) and calreticulin in the ER  is a specialized, differentiated domain of the endoplasmic reticulum (ER), generated from a reorganization of the ER membranes during myogenesis . SR consn the ER . Importan the ER .As to mitochondria, in adult cardiomyocytes, they are densely packed between jSR and parallel tubules of longitudinal SR, tethered to both SR domains. Specifically, seminal ultrastructural studies revealed, by electron microscopy (EM), a highly orderly organelle juxtaposition between SR and mitochondria, maintained by electron-dense filamentous tethers, whose length is approximately 10 nm . The molIn the early postnatal days, SR-mitochondria contacts are scarcely observed, possibly suggesting a non-essential role for cell viability; yet, their developmentally regulated appearance hints a structural and/or functional advantage. However, while the ultrastructural features of this coupling have been well defined in the heart, on the other hand (and in contrast with other tissues) its functional significance is still debated. Below, we briefly review these aspects.2+ releasing channels (RyR2 in cardiomyocytes) and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) argues against a significant contribution of mitochondrial localization to the process of mitochondrial Ca2+ uptake (see below). Conversely, some experimental evidence suggests this latter could take advantage of the strategic organelle positioning. Indeed, RyRs do not homogenously distribute in the SR membrane, but are specifically recruited on the jSR subdomain facing the TTs (2+ channels (VOCCs), originating the “Ca2+ releasing unit” (CRU). In these interface regions (dyadic clefts), TTs and jSR juxtapose with a gap of ∼10–15 nm, where electron-dense structures, representing RyR “feet,” are observed . In turn, RyR opening triggers a massive Ca2+ release from the SR into the dyadic cleft, promptly spreading in the whole cell and ultimately responsible for cell contraction. Of note, mitochondria are excluded from the dyadic clefts, being tethered to jSR on the opposite side , and then the OMM . This latter is endowed with a Kd for Ca2+ of ∼15 μM and is composed by the pore-forming subunits (MCU and/or the dominant negative isoform MCUb), by the essential component EMRE  and by additional regulatory subunits residing in the mitochondrial intermembrane space  several RyR feet fuel CICR and amplify Ca2+ microdomain, up to 100 μM (2+ buffers) enhance Ca2+ spreading; (d) longitudinal SR elements, frequently connecting the two jSR cisternae located at the opposite sides of a TT  the of a TT , may limjunction . Thus, tpulation , with th+/Ca2+ exchanger , while minimizing the energetic cost  inevitably associated with mitochondrial Ca2+ uptake  has been demonstrated to reduce the extension of SR-mitochondria contacts, while increasing organelle distance, reducing caffeine-induced SR to mitochondria Can damage . The oppn damage . Similarionality . However2+ transfer in the heart is concerned, in the microdomain that bathes the ends of the mitochondria closer to the CRUs, mathematical models calculated a [Ca2+] of ∼10-20 μM  and types of stimulations  used in the different studies. This topic has been extensively reviewed and the interested readers are referred to a comprehensive contribution , mitochondria promptly take up Ca2+ (rise time ∼49 ms), whereas their Ca2+ efflux is relatively slow   in organisms endowed with lower heart rates, such as humans .2+ transients in the heart, they have been consistently suggested to stimulate ATP synthesis, thus matching the increased energy demand upon cell contraction  and, ultimately, ATP synthase , the organelle coupling with longitudinal SR may provide a structural advantage to fuel SERCA (which is abundant in this SR domain) with both mitochondria-derived ATP  increases cytosolic Ca2+ peaks and contraction during spontaneous Ca2+ pacing, whereas MCU overexpression triggers the opposite effects , but also indirectly, by modulating ATP availability.In addition to the role played in the modulation of cardiomyocyte bioenergetics, mitochondrial Ca effects . Similar animals . Finallym of MCU . Howevermyocytes . As disclability . Therefo2+ signaling in cardiomyocytes. However, recent experimental evidence cast doubts on its overall significance. In particular, three different mouse models, in which whole organism or cardiac mitochondrial Ca2+ accumulation was severely compromised by manipulating MCUC composition . Moreover, it should be noted that, although the process of rapid mitochondrial Ca2+ uptake is severely (if not completely) compromised in these models, yet the resting mitochondrial Ca2+ levels are not deeply affected , mediating Ca2+ uptake, has been reported to be expressed in the IMM of heart organelles and activated at low cytosolic [Ca2+]  and Ca2+ signal and cell metabolism has tremendously increased our understanding of cardiomyocyte physiopathology, yet prompting additional and unpredicted questions. The intimate relationship between SR and mitochondria appears critical to regulate mitochondrial Ca2+ uptake. Nevertheless, whether mitochondria take advantage of their strategic positioning to efficiently detect Ca2+ microdomains on a beat-to-beat basis is still matter of debate. Species-specific differences, partly due to diverse heart rates, may dramatically impact on the complex balance between Ca2+ uptake/efflux dynamics, resulting in substantially different outcomes. Moreover, the lack of severe phenotypes in mice lacking key MCUC components suggests that additional routes, guaranteeing a sufficient, albeit minimal, Ca2+ signal in the mitochondrial matrix might exist. Alternatively, unknown compensatory mechanisms may occur (see for example, the dramatically different effect of NCLX deletion in adult vs newborn mouse heart; In the last decade, the rapid growth in our knowledge of the mechanisms regulating the complex balance between mitochondrial Ca2+ transfer, lipid metabolism is known to be modulated by ER-mitochondria tethering; yet, as far as SR-mitochondria juxtaposition is concerned, this aspect has been largely neglected in literature. Similarly, the impact of SR-mitochondria contacts on autophagy, as well as on the regulation of mitochondrial dynamics and morphology, has not been sufficiently explored in the heart. Clearly, an in-depth investigation of the molecules regulating SR-mitochondria coupling appears urgent to study these issues. The development of novel techniques, enabling to simultaneously measure with sufficient spatial and temporal resolution different parameters, will offer the opportunity to further raise the bar, allowing to precisely evaluate the impact of these pathways in different cardiac pathologies.In addition to CaMR and RF revised the literature, wrote and discussed the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli and may be involved in the survival of susceptible strains of E. coli in the presence of antibiotics, through the hydrolysis of the β-lactam antibiotic. In this study, OMVs isolated from β-lactam-resistant E. coli and from mutants, lacking porin or β-lactamase, were evaluated to establish if the porins or β-lactamase in OMVs were involved in the degradation of β-lactam antibiotics. OMVs isolated from E. coli deficient in β-lactamase did not show any degradation ability against β-lactam antibiotics, while OMVs lacking OmpC or OmpF showed significantly lower levels of hydrolyzing activity than OMVs from parent E. coli. These data reveal an important role of OMVs in bacterial defense mechanisms demonstrating that the OmpC and OmpF proteins allow permeation of β-lactam antibiotics into the lumen of OMVs, and antibiotics that enter the OMVs can be degraded by β-lactamase.Gram-negative bacteria have an outer membrane inhibiting the entry of antibiotics. Porins, found within the outer membrane, are involved in regulating the permeability of β-lactam antibiotics. β-lactamases are enzymes that are able to inactivate the antibacterial properties of β-lactam antibiotics. Interestingly, porins and β-lactamase are found in outer membrane vesicles (OMVs) of β-lactam-resistant  Since the discovery of penicillin, antibiotics have been responsible for prolonging human life and advancing human medicine. However, antibiotic-resistant bacteria, also known as superbugs or multi-drug resistant (MDR) bacteria, have emerged due to the indiscriminate misuse of antibiotics ,2. O’NeiE. coli can help β-lactam-susceptible E. coli avoid the effects of β-lactam antibiotics through hydrolysis. In addition, porins (OmpC and OmpF) and β-lactamase (Blc1) were seen to be upregulated in OMVs of β-lactam-resistant E. coli compared to OMVs of β-lactam-susceptible E. coli [E. coli (RC85+) , referred to as outer membrane vesicles (OMVs), into the external environment during both in vitro growth and in vivo infection ,8,9,10. 0 nm, ref E. coli . Therefo (RC85+) . In the + by knocking out each of these genes. The successful deletion of blc1, ompC, and ompF in mutant RC85+ strains was confirmed by PCR amplification shown in + . An antimicrobial sensitivity test was conducted with the mutant strains to determine whether changes in their antibiotic resistance occurred compared to RC85+  of all β-lactam antibiotics was reduced. In the case of ΔompC, there was no difference in MIC levels relative to RC85+, apart from the MIC for cefazolin, which was enhanced, whereas inactivation of the ompF gene conferred more resistance to cefoperazone, cefazolin, and cefalexin in the mutant compared with RC85+.To establish if Blc1, OmpC, or OmpF are involved in the OMVs’ ability to degrade β-lactam antibiotics, mutants were produced from RC85own in + . The delto RC85+ . In the + were isolated after incubation under the same culture conditions. Electron micrograph analysis exhibited the similarity of OMVs isolated from the mutants and RC85+ in size with the spherical structure  of OMV protein (+ OMVs (64.4 mU/mg). OMVs from Δblc1 cells displayed the lowest β-lactamase activity of 2.7 mU/mg.Differences in β-lactamase activity between OMVs from RC85ver time a. Since  protein b. The β-+ and mutants  on the growth of β-lactam susceptible E. coli (RC85) cells in the presence of a growth-inhibitory dose of six β-lactam antibiotics . RC85 treated with OMVs from the ΔompC mutant grew in all antibiotics tested, but their growth was slower than the samples containing RC85+ OMVs. Furthermore, RC85 incubated with ΔompF OMVs grew after 24 h in cefoperazone and after 18 h in cefazolin, which was slower than that obtained with the ΔompC OMVs, while no growth was detected in the presence of the other four antibiotics  over the 36 h culture period. On the other hand, RC85 incubated with Δblc1 OMVs did not show any growth when each of the six antibiotics was present. After the growth curve experiment (E. coli (RC85) received antibiotic-resistant substances through OMVs during the experiment in E. coli at the species level using the MALDI-Biotyper .To determine if the loss of porin or β-lactamase proteins from the OMVs influences the degradation of β-lactam antibiotics, we investigated the effect of OMVs from RC85ibiotics . When RCperiment , all sam+, Δblc1, ΔompC, and ΔompF in their ability to degrade the different β-lactam antibiotics tested. With all six β-lactam antibiotics examined, RC85+ OMVs showed the highest hydrolytic activity, followed by ΔompC then ΔompF OMVs, while no change in antibiotic concentration was noted with Δblc1 OMVs. These results imply that β-lactamase is the most important factor in the degradation of β-lactam antibiotics by RC85+ OMVs, alongside porin, specifically OmpF, which showed higher permeability to all six β-lactam antibiotics tested when compared to OmpC.Concentrations of β-lactam antibiotics were measured at specific time points in a cell-free system to determine whether β-lactam antibiotics could be hydrolyzed by the OMVs . CompareE. coli than from β-lactam-susceptible E.coli, and only OMVs from β-lactam-resistant E. coli were found to degrade β-lactam antibiotics [E. coli to degrade β-lactam antibiotics. Our results suggest that it is not the loss of β-lactamase but the loss of porin from the outer membrane of the OMV that influences the yield of OMVs obtained. The loss of porin does not affect the β-lactamase activity of OMVs but the loss of β-lactamase dramatically eliminated β-lactamase activity by the OMVs. Thus, the presence of β-lactamase and porin in the OMVs plays a significant role in the direct hydrolysis of β-lactam antibiotics.We previously showed that porin proteins and β-lactamase enzyme are more abundant in OMVs isolated from β-lactam-resistant ibiotics . TherefoE. coli plays an important role in the growth of susceptible bacteria by degrading β-lactam antibiotics before they can affect the bacteria [+ directly degrade β-lactam antibiotics to protect sensitive strains from antibiotic environments  showed instability, with increased OMVs production due to a structural deficiency of the outer membrane [Several studies have demonstrated that the loss of porins from the bacterial outer membrane can impact the production of OMVs. For example, Mcbroom et al. (2006) indicated that relative OMV production from an e E.coli . A deleti mutant . Valeru ild-type . In linemembrane . TherefoE. coli has developed four major mechanisms to resist the inhibitory effect of β-lactam antibiotics: inactivation of the antibiotics by enzymes, alteration of the active site of PBPs (penicillin-binding proteins), decreased permeation of the antibiotics and increased efflux of the antibiotics [β-lactam antibiotics are widely used antibiotics that are highly effective in combating bacterial infections . These iibiotics ,40. β-laibiotics . Porin pibiotics ,43,44.E. coli mutants showed increased resistance to several β-lactam antibiotics, such as ampicillin, cefalotin, cefoxitin, ceftazidime, aztreonam, and imipenem [An observed decreased in the resistance of the β-lactamase mutants to β-lactam antibiotics compared with the wild-type was due to reduced β-lactamase activity ,46. Previmipenem . The absimipenem . Our finE. coli OMVs [OmpC and OmpF are considered as the leading transport porins that assist penetration of most β-lactam antibiotics ,51,52,53oli OMVs . Diffusioli OMVs ,55. Chemoli OMVs . Among tE. coli because of the greater number of charged residues in the OmpC channel than in that of OmpF [E. coli in the presence of antibiotics, the group treated with ΔOmpC OMVs grew faster than the group treated with ΔOmpF OMVs  broth or LB agar. Broth cultures were grown at 37 °C with orbital shaking. Growth was monitored by measuring absorbance at 600 nm (OD600) using an xMark microplate spectrophotometer .Antimicrobial-sensitive oli RC85 , antimicli RC85+ , and mutE. coli Gene deletion Kit”  and the chloramphenicol resistance gene (CmR) was amplified from pKINGeo/ccdB, which was designed in our laboratory [R gene as a selection marker, because β-lactam-resistant E. coli RC85+ is resistant to ampicillin. Fragments 1 and 3 were amplified from pRed/ET (amp), while fragment 2 was amplified from pKINGeo/ccdB (R). The “Quick & Easy E. coli Gene Deletion Kit” was used to construct the gene deletion mutant strains according to the manufacturer’s protocol, with some modifications [R) expression plasmid was transformed into the E. coli strain RC85+ by electroporation (Bio-Rad MicroPulser) at 1800 V with a 4 ms pulse rate. Transformants (RC85+ + pRedET) were selected on LB agar containing 35 μg/mL chloramphenicol  and grown overnight at 30 °C. A bacterial colony was selected from the plate and cultured in LB medium containing 35 μg/mL chloramphenicol overnight at 30 °C. Transformant cultures were re-incubated in super optimal broth (SOB) conditioned with L-arabinose  at a final concentration of 0.3% (w/v) at 37 °C until an OD600 of 0.2 was obtained to induce pRedET. Induced cells were harvested by centrifugation for 30 sec at 16,000 × g in a cooled microfuge benchtop centrifuge and re-suspended in chilled 10% (v/v) glycerol. This process was repeated five times before electroporation. Competent RC85+ cells were mixed with generated hygromycin cassettes flanked by homology arms to replace the DNA fragment (E. coli (RC85+). Colonies from gene deletion mutants were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry  [E. coli.Plasmid pRed/ET (amp) was obtained from the “Quick & Easy boratory . An FRT-Geo/ccdB . Anotherications . The pRefragment . Electrofragment . PCR proGermany)  to confi+. Eight β-lactam antibiotics, namely amoxicillin, ampicillin, cefalexin, cefazolin, cefoperazone, cefotaxime, cloxacillin, and methicillin (Sigma-Aldrich) and five other classes of antibiotics, including amikacin, colistin, kanamycin, nalidixic acid, and streptomycin (Sigma-Aldrich) were selected for this. The MIC of each antimicrobial agent was determined using the broth-dilution method in 96-well plates [Minimum inhibitory concentration (MIC) values were used to compare relative resistance levels of mutant strains to those of RC85l plates  according for 20 min, and the supernatant was filtered through 0.45-μm pore-sized vacuum filters. The filtered supernatant was concentrated by ultrafiltration using a QuixStand Benchtop system . This was then centrifuged at 150,000× g at 4 °C for 3 h, and the OMVs purified on a continuous sucrose density gradient at 120,000 × g at 4 °C for 18 h. The OMV band was removed and centrifuged for 3 h at 150,000× g at 4 °C. The final OMV pellet was washed and resuspended in 10 mM Tris-HCl (pH 8.0) and filtered through a 0.2-μm filter. All purification steps were performed at 4 °C. The protein yields of OMV samples were measured using a Pierce BCA protein assay kit . Transmission electron microscopy (TEM) of OMVs was performed as previously described [Purification of OMVs was performed as previously described . Brieflyescribed  using a escribed  using a +, Δblc1 RC85+, ΔompC RC85+, and ΔompF RC85+ cells on the cytotoxicity of β-lactam antibiotics was monitored by assessing the growth of OMV-treated RC85 cells. The β-lactam antibiotics used were: penicillin family (ampicillin and amoxicillin), first-generation cephalosporin , and the third-generation cephalosporin (cefotaxime and cefoperazone). The following six antibiotics were used at concentrations known to inhibit RC85 growth: ampicillin, 30 μg/mL; cefotaxime, 1.25 μg/mL; cefoperazone, 4 μg/mL; amoxicillin, 12 μg/mL; cefazolin, 8 μg/mL; and cefalexin, 16 μg/mL. The MIC of β-lactam antibiotics against the RC85 were listed in 5 CFU/mL) were inoculated into medium containing one of these antibiotics and 5 μg/mL of the respective OMV sample. RC85 in the antibiotic-free medium was used as a positive control, while the negative control consisted of bacteria and growth-inhibitory concentrations of the respective antibiotics. All tubes were incubated at 37 °C with shaking at 150 rpm. All experiments were performed in the dark to exclude the effect of light on the stability of the antibiotics used. The bacterial growth curves at OD600 were recorded at 3-h intervals up to 36 h using an xMark microplate spectrophotometer. Experiments were performed using three independent sets of bacterial cultures. The bacterial cultures were inoculated onto TSA with or without the respective same concentrations of antibiotics to confirm whether the susceptible bacteria could survive by antibiotic-resistant gene transfer via OMVs. Colonies from each cultured sample  on TSA without antibiotics were randomly selected and identified by MALDI Biotyper [The effect of OMVs on the growth of bacteria in the presence of β-lactam antibiotics was performed as previously described with slight modifications . The effBiotyper  to check+ and mutant strains, a colorimetric β-lactamase activity assay kit  was used according to the manufacturer’s instructions. The assay is based on the hydrolysis of nitrocefin, a chromogenic cephalosporin producing a colored product that can be measured spectrophotometrically (OD490). A buffer of 10 mM Tris-HCl (pH 8.0) was used as a negative control and lyophilized positive control included in the kit was used. The quantity of enzyme capable of hydrolyzing 1.0 μM of nitrocefin/min at 25 °C corresponds to 1 U of β-lactamase. To liberate β-lactamase from the lumen of OMVs, each obtained OMVs were sonicated from 5 min (the effective sonication time on release β-lactamase from E. coli) [g at 4 °C for 20 min. Equal concentrations of each OMV sample (2.5 μg) were dispensed into the wells of a clear flat-bottomed 96-well, and nitrocefin and buffer (provided in the kit) were added to make a final volume of 100 μL. The absorbance at OD490 was immediately measured in kinetic mode for 60 min at 25 °C. For all measurements, three independent experiments were performed. A standard curve was generated using 0, 4, 8, 12, 16, and 20 nmol of nitrocefin, and the specific β-lactamase activity of each sample was expressed in milliunits/milligram of protein.To test the differences in β-lactamase activity between OMVs from RC85E. coli) , cooled E. coli) , and cen+ and mutants on the degradation of the six antibiotics listed above in a cell-free system were analyzed by liquid chromatography/electrospray ionization mass spectrometry . A 5 μg/mL sample of respective OMV in PBS was mixed with ampicillin (30 μg/mL), cefotaxime (1.25 μg/mL), cefoperazone (4 μg/mL), amoxicillin (12 μg/mL), cefazolin (8 μg/mL), or cefalexin (16 μg/mL). Filtered PBS containing the respective antibiotics without OMVs was used as a positive control. All samples were incubated at 37 °C with shaking at 150 rpm and diluted 20-fold prior to analysis. The concentrations of antibiotics were recorded at specific time points  in triplicate. For LC-MS/MS, LC-MS grade water  containing 5 mM ammonium formate (Sigma-Aldrich) and 0.1% formic acid  (v/v) (solution A) and LC grade methanol (Burdick & Jackson) containing 5 mM ammonium formate with 0.1% formic acid (v/v) (solution B) were used as the mobile phase, at an initial A:B ratio of 30:70 or 50:50, depending on the antibiotic of interest. The compounds were separated using a Poroshell 120 EC-C18 column . Isocratic elution with phases A and B was followed by 3 min of total chromatography. The flow rate was 0.2 mL/min, the column temperature was 30 °C, and 99.99% pure nitrogen gas was used for desolvation. For the quantification of antibiotics, at least two or more transitions were selected for each analyte and the positive electric spray ionization (ESI+) was used with the multiple reaction monitoring (MRM) mode. The MassHunter software  was used to process the LC-MS/MS data and quantification of the analytes.Measurement of β-lactam antibiotic concentrations was carried out as previously described , with slp < 0.05.Statistical analysis was performed using Graphpad Prism, version 8.1.1. . Significant differences were determined by One-way Analysis of Variance (ANOVA). Data are presented as mean ± standard deviation (SD). The difference was considered statistically significant at All data generated or analyzed during this study are included in this published article and its


Here, we report a unique acute myeloid leukemia (AML) bone marrow-derived mesenchymal stem cell (MSC) with both mesenchymal and endothelial potential, which we have named Mesenchymal Cancer Stem Cells (MCSCs). These MCSCs are CD90-CD13-CD44+ and differ from MSCs in isolation, expansion, differentiation, immunophenotype, and cytokine release profile. Furthermore, blocking CD44 inhibited the proliferation and cluster formation of early MCSCs with lower ICAM-1 protein levels. Similar CD90-CD44+ cancer stem cells have been reported in both gastric and breast cancers, which grew in floating spheres in vitro and exhibited mesenchymal features and high metastatic/tumorigenic capabilities in vivo. Our novel discovery provides the first evidence that certain AMLs may be comprised of both hematopoietic and stromal malignant cells. Targeting MCSCs and their cytokine release has potential as a novel therapeutic approach in AML. To the EditorAcute myeloid leukemia (AML) most commonly occurs in older adults  and has A new concept of niche-induced oncogenesis has been proposed that is based on the observation that artificial genetic perturbation of mesenchymal stem cells (MSCs), and their progenies leads to leukemogenesis of myelodysplasia and AML in transgenic mice , 8. In tThe AML bone marrow donor is a 32-year-old female with a past medical history of diabetes and hypothyroidism. She was admitted to the emergency department with intermittent chest pain, shortness of breath, and gum bleeding, and diagnosed with AML with myelomonocytic differentiation. Peripheral blood smears showed leukocytosis with ~ 52% blasts/equivalents and marked thrombocytopenia. FISH showed normal cytogenetics. An AML panel was positive for mutations of DNMT3A, NPM1, and NRAS without FLT3-ITD.During ex vivo isolation of MSCs from this patient BM, we observed two different cell populations. The early plastic-attached cells were CD90+CD13+CD44+ MSCs Fig. , and relFinally, we performed a proteome assay of supernatants from MSC and MCSC cultures. The release of key angiogenic cytokines like VEGF and growth factors such as FGF basic and PDGF were significantly increased in the P7 MCSC culture Fig. b. To evaIn summary, we provide novel evidence of MCSCs as the cellular origin of angiogenesis and cytokine release in an AML patient’s BM. More research with additional patients is needed to discover if this phenomenon is unique to this one AML patient or has broader ramifications for AML as a whole. Our data suggest that future therapeutic strategies should also be developed to target MCSCs and their cytokine release to achieve stringent complete remission and prevent AML progression and relapse. The existence of MCSCs might be applicable to other cancers as well.Additional file 1: Supplementary Figures. Supplementary Figure 1.  There is no significant difference in differentiation capabilities of P4 MSCs and P4 MCSCs during ex vivo cultures. A) MSCs Panel: FACS plot of CD34-CD13+MSCs, which differentiated into bone , fat (phase bright), and cartilage (Alcian blue staining). B) MCSCs Panel: FACS plot of CD34-CD13+MCSCs, which differentiated into bone , fat (phase bright), and cartilage (Alcian blue staining). Supplementary Figure 2. Comparison of Cell Proliferation of MSCs and MCSCs. A) The P10 MCSCs were found to proliferate much faster than P10 MSCs. ** P<0.01; B) These rapidly proliferating MCSCs do not express cleaved Caspase3  and continue to express strong CD44+. Supplementary Figure 3. Anti-CD44 monoclonal antibodies inhibited the cluster formation and proliferation of P5 MCSCs. A) Phase bright images of floating clusters from P5 MCSCs without treatment. B) Phase bright images of floating cells from P5 MCSCs with treatment of anti-CD44. C) Aggregate cluster count data from P5 MCSCs treated with anti-CD44 or without treatment. D) Aggregate cluster count data from P7 MCSCs treated with anti-CD44 or without treatment. Where applicable, data are means ± SEM from each group and were analyzed by Student t-test. *P<0.05; N=3. E) Proteome comparison (mean pixel density) of ICAM-1 between supernatants from P7 MCSC and P5 MCSC cultures. *P<0.05Additional file 2: Materials and Methods. Discovery of proangiogenic CD44+mesenchymal cancer stem cells in an acute myeloid leukemia patient’s bone marrow


The effect of entropy at low noises is investigated in five-strategy logit-rule-driven spatial evolutionary potential games exhibiting two-fold or three-fold degenerate ground states. The non-zero elements of the payoff matrix define two subsystems which are equivalent to an Ising or a three-state Potts model depending on whether the players are constrained to use only the first two or the last three strategies. Due to the equivalence of these models to spin systems, we can use the concepts and methods of statistical physics when studying the phase transitions. We argue that the greater entropy content of the Ising phase plays an important role in its stabilization when the magnitude of the Potts component is equal to or slightly greater than the strength of the Ising component. If the noise is increased in these systems, then the presence of the higher entropy state can cause a kind of social dilemma in which the players’ average income is reduced in the stable Ising phase following a first-order phase transition. The large number of payoff parameters causes difficulties in systematic investigations and in the classification of possible behaviors as well. These difficulties can be reduced by recognizing that two games are equivalent if their payoff matrices can be transformed into each other by exchanging some strategy labels.Multi-player evolutionary games are simple models that can be employed to study a wide range of systems across several diverse disciplines ,6,7,8,9.f matrix  if the pn strategies, generalized coordination games are composed of two-strategy coordination subgames between all possible strategy pairs [The linear decomposition of payoff matrices ,12,13,14volution ,25,26,27gy pairs . When thgy pairs ,29,30, sgy pairs  or the Agy pairs ) that exgy pairs ,34,35. ISo far, we have tacitly considered cases where the maximal payoff is unique or degenerate in such a way that equivalent phases possess similar features and symmetries at low noises. What happens when the latter conditions are not satisfied? Which of the possible equilibria will be preferred? For equal average payoffs in the ordered strategy arrangements, it will be shown that these systems select one of those ordered phases that have higher entropy. Furthermore, entropy can have a similar role even when the payoffs of the ordered phases are tuned away from being equal.In this paper we investigate the stabilization effects of entropy in a five-strategy coordination game where the subgames of the first two and the last three strategies are identical to a ferromagnetic Ising and a three-state Potts model, respectively. It is also assumed that the players receive zero income if they choose strategies belonging to different subgames. Using Monte Carlo simulations and cluster variation methods, we show that one of the “Ising phases” is stabilized by its higher entropy at low noises if the payoffs are identical in all five ordered strategy arrangements. Furthermore, this system undergoes an Ising-type phase transition when the noise is increased. Evidently, any small extra payoff in the “Potts phase” can stabilize it in the low noise limit. In the rest of the paper we describe the model in detail and quantitatively study its phase transitions for several payoff values.n available pure strategies to play in all four games. If we represent these strategies with the n Cartesian unit vectors of an n-dimensional Euclidean space, then the income of the player at site x can be calculated asx, the summation runs over this player’s nearest neighbor sites  and last three strategies  are an Ising and a three-state Potts model, respectively. Opposing players whose strategies belong to different subgames both receive zero payoff. We note again that we use pure coordination-type games for which the sum of payoffs is zero in each row and column of the payoff matrix. Consequently, the elements of our Potts payoff matrix are shifted by a uniform irrelevant constant in comparison with the standard diagonal Potts payoff matrix ,37. If th of α/2 . Our anaThe present game is a potential game because its payoff matrix is symmetric  affecting the decision-making abilities of players. This rule exponentially favors higher payoffs, provided that the opponents do not modify their strategies [If the evolution of strategy profile rategies ,29,30,35The main advantage of the application of the logit rule is that the repetition of the above elementary strategy changes will drive potential games into the Boltzmann distribution ,34 when ith strategy (K) the system evolves into a disordered state in which strategies are chosen by almost equal shares of the population, or more precisely, all five strategy frequencies approach K goes to infinity. On the other hand, strategy choices are homogenized in the low-noise limit, and we find the system in one of the competing ordered phases. When The macroscopic behavior of this system can be quantified by the average proportions i at any site. Obviously, these probabilities are normalized, that is, i and strategy j occupying neighboring sites. Both methods evaluate independent equilibrium configuration probabilities as extremal points of a thermodynamic potential Mean-field and pair approximations belong to the family of cluster variation methods ,38,39,40On a square lattice, We used different methods to find the extremal points of the approximated thermodynamic potentials. In the mean-field case a direct numerical approach was employed: At extremal points the partial derivatives of the free energy with respect to one-site probabilities should vanish, that is,We have to keep in mind the nonnegativity and normalization properties of probabilities as well. These equations can be solved numerically. Our results are presented in We also employed Monte Carlo simulations  to numerThe competing Ising and Potts phases were studied using ordered initial states (homogeneously prepared Ising or Potts states) due to the apparent metastability of the ordered strategy arrangements. This way we could reduce the transient time necessary for the slow domain growing process that forms ordered phases if the system is started from a random initial state. However, the application of the latter, slower method was unavoidable when we wished to determine the critical temperature of the first-order transition between the Potts and Ising phases, which turned out to be To illustrate the domain growing process, we have performed some time-consuming simulations starting from random initial strategy distributions near the transition point. ture see a the Potture see b, one ofFirst, we sketch the general features that are already captured by mean-field approximation, and then move on to highlight finer details revealed by pair approximation and Monte Carlo simulations. From the Monte Carlo results, we extract the critical exponents of the system’s order-disorder phase transitions. Finally, we argue that the first-order transitions between the ordered phases are driven by entropy.Equation , stable K is increased. The phase transition between the Potts and the Ising phases is of the first order and it is driven by the higher entropy of the Ising phases.Evidently, the increase of 1.05 see b,c, and In agreement with expectations, Mean-field approaches neglect the correlations between (fixed) neighbors, but their predictions may be applied to well-mixed populations. The common feature of the present games is that the average potential is zero in the disordered phase at the level of the mean-field approximation. Moreover, in the Ising (Potts) phase the Potts (Ising) strategies do not contribute to the average potential. Pair approximation, however, takes into consideration the relevant nearest neighbor correlations and gives more adequate predictions, as shown by comparisons with Monte Carlo data in The predictions of mean-field theory are justified by both pair approximation and Monte Carlo simulations at low temperatures that typically cause point defects in the ordered phases. The latter methods, however, predict a differently structured disordered phase where Ising strategies are chosen slightly more often than Potts strategies due to the correlations favoring the choice of identical neighboring strategies. It is worth mentioning that in the analytic continuation of the disordered solutions the equal frequencies of Potts strategies tend to zero as In general, cluster variation methods overestimate the critical temperatures of continuous order-disorder transitions but give a more accurate approximation of first-order transition points between ordered phases. This expectation is confirmed by comparing the plots of In order to characterize the continuous transitions, we have investigated the critical algebraic behaviors hidden in the quantities  and 1.1 ,44. All  Onsager  for the  Onsager ,16 for aWhy is the Ising state still preferred near the order-disorder transition point when n-strategy elementary coordination game is an extension of the original coordination game [n is greater than the threshold The stabilization effect of entropy can also be observed in elementary coordination games ,45 that ion game ,46,47,48d nth=27 , then thd nth=27 ,50,51, an is increased, critical temperatures should become lower because of the faster increase in the entropy content of the disordered phase.If decoupling of independent game components holds at the mean-field level, then the above-described results should give us an idea about what happens in games made up of an Ising and an n−-strategyIn this paper, we have studied a five-strategy evolutionary potential game that pits an Ising-type and a three-state Potts-type coordination game against each other. This interaction combines the two games as independent subgames of the first two and last three strategies, respectively. The application of the logit strategy update rule has allowed us to utilize various well-known concepts and methods of statistical physics. The systematic investigation of the spatial system is restricted to a model where the players are located at the sites of a square lattice and repeatedly play the five-strategy game against their nearest neighbors. Due to this choice, the observed properties of the stationary states can be compared with universal features characteristic of the original Ising and Potts models.The game is defined by a single parameter Independently of the preferred ordered phases at low noises, the system undergoes a continuous  order-disorder phase transition. The quantitative analysis of the order parameters indicates that in most of the cases these continuous transitions preserve the universal behavior of the corresponding (Ising or Potts) model. Additionally, we have observed non-universal behavior in a narrow range of parameters where the interplay between these two different transitions seems to be relevant. It is worth mentioning that similar non-universal critical phase transitions were also reported in several versions of the Ashkin-Teller model ,52,53,54We have found that the first-order transition from the Potts state to the Ising state is accompanied by a loss of average income for


Exposing hippocampal cultures to low-magnesium media is a well-accepted in vitro model of status epilepticus. Using this model, it was shown that endoplasmic reticulum Ca2+ uptake was significantly inhibited in homogenates from cultures demonstrating electrophysiological seizure phenotypes. Calcium uptake was mainly neuronal. However, glial Ca2+ uptake was also significantly inhibited. Viability of neurons exposed to low magnesium was similar to neurons exposed to control solutions. Finally, it was demonstrated that Ca2+ uptake inhibition and intracellular free Ca2+ levels increased in parallel with increasing incubation in low magnesium. The results suggest that inhibition of Mg2+/Ca2+ ATPase-mediated endoplasmic reticulum Ca2+ sequestration contributes to loss of intracellular Ca2+ homeostasis associated with status epilepticus. This study describes for the first time inhibition of endoplasmic reticulum Mg2+/Ca2+ ATPase in a mixed primary hippocampal model of status epilepticus. In combination with animal models of status epilepticus, the cell culture model provides a powerful tool to further elucidate mechanisms that result in inhibition of Mg2+/Ca2+ ATPase and downstream consequences of decreased enzyme activity.Loss of intracellular calcium homeostasis is an established mechanism associated with neuronal dysfunction and status epilepticus. Sequestration of free cytosolic calcium into endoplasmic reticulum by Mg This has been demonstrated in both animal i physiology and cellular dysfunction in hippocampal neurons examined acutely after exposure to 3 h of low Mg2+ solution. It was demonstrated that functioning of key Ca2+-mediated enzymes, such as Ca2+/calmodulin-dependent protein kinase II i levels were significantly elevated throughout and acutely after 3 h of exposure of hippocampal cultures to low Mg2+ media [2+]i would have profound consequences for Ca2+-dependent enzymes and protein trafficking.Exposure of primary hippocampal cultures to low Mgigations ,13,14,15igations ,17, throinase II , and Ca22+]i homeostasis in neurons is sequestration of Ca2+ from the cytosol to the lumen of the endoplasmic reticulum (ER) mediated by the ER membrane-bound sarco/endoplasmic reticulum Mg2+/Ca2+ ATPase (SERCA). SERCA is a member of the p-type ATPase family which requires adenosine triphosphate (ATP) to transport Ca2+ from the cytoplasm to the ER lumen against a concentration gradient. Briefly, Mg2+-ATP binds to SERCA forming a high-energy phosphorylated intermediate by reaction of the gamma phosphate of ATP to an aspartate residue of the catalytic domain [2+ molecules into the ER from the cytoplasm for every ATP molecule hydrolyzed into adenosine diphosphate + phosphate. Our lab showed in several models of neurological disorders associated with loss of [Ca2+]i homeostasis that SERCA-mediated Ca2+ uptake into microsomes (ER vesicles) isolated from brain homogenates was significantly inhibited i dysfunction due to prolonged depolarization/repolarization events.SE was induced in primary hippocampal cell cultures as described previously . Brieflyightness ,25,26. N2+ solutions as described above. Cultures were then transferred to the stage of an Olympus IX-70 inverted microscope equipped with phase contrast optics and were perfused continuously (gravity perfusion) at 1 mL/min with low Mg2+ or control solutions throughout each experiment. Temperature was maintained at 34–35 °C using a heating stage . Patch electrodes with a resistance of 2–5 MΩ were pulled from borosilicate glass capillaries  using a Brown-Flaming PC-80 puller  and then fire polished. Electrodes were filled with a solution containing 140 mM potassium gluconate, 1 mM MgCl2, 10 mM sodium HEPES, pH 7.2, and osmolarity adjusted to 310 ± 5 mOsm with sucrose. Recordings were conducted in the whole-cell current-clamp mode i were measured using the ratiometric, high-affinity fluorescent calcium indicator, fura-2 acetoxymethyl ester , employing modified procedures previously established i became sustained and elevated in hippocampal cells as a result of prolonged seizures and that inhibition of ER SERCA Ca2+ uptake was involved [2+ homeostasis leads to the unfolded protein response [2+ solution [There are a multitude of conceivable repercussions of inhibition of ER SERCA-mediated Ca neurons . It was  neurons ,46. It winvolved ,30. Thusinvolved  and unchresponse . It was response  and inhiresponse  neurotrasolution . Therefo2+ sequestration into ER vesicles was significantly inhibited in association with a 3 h exposure to low Mg2+ conditions. Viability of neurons following incubation in both control and low Mg2+ solutions for 3 h was not different. Both the initial rate and steady-state activity of SERCA were inhibited. The glial bed that the neurons were grown on also showed inhibition of Ca2+ uptake, but the inhibition observed in the mixed neuronal/glial cultures was mostly neuronal in origin. The degree of SERCA inhibition was concomitant with duration of low Mg2+ exposure as was the level of neuronal cytosolic Ca2+, as Mg2+ exposure increased from 1 h to 3 h, inhibition of SERCA and levels of cytosolic Ca2+ likewise increased.Utilizing a well established cell culture model of status epilepticus in hippocampal neurons, it was shown that SERCA mediated Ca


Consistent with reduced contractility and calcium (Ca2+) currents, the cytosolic Ca2+ ([Ca2+]i) transient from ARDKO animals was smaller and decayed slower. Importantly, no changes were observed in membrane resting potential, AP amplitude, or the inward K+ current. Finally, we modified our existing cardiac ECC computational model to account for changes in the ARDKO heart. Simulations suggest that cellular changes in the ARDKO heart resulted in variable and dyssynchronous Ca2+-induced Ca2+ release therefore altering [Ca2+]i transient dynamics and reducing force generation. In conclusion, chronic sympathetic hyperactivity impairs ECC by changing the density of several ionic currents (and thus AP repolarization) causing altered Ca2+ dynamics and contractile activity. This demonstrates the important role of ECC remodeling in the cardiac dysfunction secondary to chronic sympathetic activity.The sympathetic nervous system is essential for maintenance of cardiac function via activation of post-junctional adrenergic receptors. Prolonged adrenergic receptor activation, however, has deleterious long-term effects leading to hypertrophy and the development of heart failure. Here we investigate the effect of chronic adrenergic receptors activation on excitation-contraction coupling (ECC) in ventricular cardiomyocytes from a previously characterized mouse model of chronic sympathetic hyperactivity, which are genetically deficient in the adrenoceptor α2A and α2C genes (ARDKO). When compared to wild-type (WT) cardiomyocytes, ARDKO displayed reduced fractional shortening (~33%) and slower relaxation (~20%). Furthermore, ARDKO cells exhibited several electrophysiological changes such as action potential (AP) prolongation (~50%), reduced L-type calcium channel (LCC) current (~33%), reduced outward potassium (K Coordination of sympathetic activity through cardiovascular reflexes enables accurate, short and long term control of blood flow and pressure2. Furthermore, a physiological increase in sympathetic activity is a determinant for cardiovascular responsiveness to exercise or other stress stimuli . In the heart, SNS effects are mainly through stimulation of β-adrenergic receptors (β-AR)3.Activation of the sympathetic nervous system (SNS) exerts several effects on the cardiovascular system. These include accelerating heart rate, increasing cardiac contractility, reducing venous capacity, and promoting vasoconstriction of resistance arteries4 triggers the activation of compensatory mechanisms such as renin-angiotensin-aldosterone system (RAAS), the cytokine system, and the SNS5. Regarding SNS, continuous or excessive stimulation of β-AR in the myocardium might trigger several deleterious effects. For example, hyper-activation of β1AR receptors can drive cardiomyocyte hypertrophy, mitogenesis of cardiac fibroblasts, and even apoptosis5. Such effects arising from increased sympathetic tone are described as critical factors for the progressive development of various cardiovascular diseases including heart failure (HF)6.The decline in the cardiac contractile capacity after an acute myocardial infarction or chronic pressure overload6.There is strong evidence supporting the role of increased sympathetic tone in the progression of several cardiac dysfunctions. Less understood, however, is the role of increased sympathetic tone in the modification, conduction, and propagation of the electrical stimulus throughout the cardiac muscle. Additionally, details regarding increased sympathetic tone on cardiac ion channel function, calcium handling, and cell contraction are poorly elucidated and existing experimental models are mostly unsuitable for the study of the role of SNS in the observed deleterious cardiovascular diseaseset al., 19997 showed that the presynaptic α2A and α2C receptors modulate release of neurotransmitters in the SNS terminals. The removal of these receptors leads to sympathetic hyperactivity and increased plasmatic norepinephrine, causing mice to be prone to developing cardiac hypertrophy, exhibiting reduced physical capacity, impaired cardiac contractility, changes in cardiomyocyte ultrastructure, and higher mortality with age progression after 5 months7. Due to these characteristics, the adrenergic receptor double knock-out (ARDKO) animal model is well suited for the study of the role of sympathetic tonus increase in development and progression of cardiac disease.The present study explored the role of chronic sympathetic hyperactivity (CSH) in the remodeling of the excitation-contraction coupling (ECC). We utilized a genetic model of sympathetic hyperactivity created by knocking out α2A and α2C adrenergic receptors. While α2 adrenergic receptors are absent in the cardiac myocytes, they provide inhibitory feedback in adrenergic synapses and are an essential factor for the control of sympathetic tone. Hein 2+ handling.Using this ARDKO animal model we investigated the influence of CSH on cardiac ECC and found that CSH induces an electrochemical remodeling in ventricular cardiomyocytes (VCMs) which included action potential (AP) prolongation, altered ion currents, and impaired cellular contractility and Ca8. The animals were obtained from the School of Physical Education and Sport, University of São Paulo, São Paulo, SP, Brazil.All experimental procedures were conducted according to guidelines from both the Brazilian Society of Laboratory Animal Science (SBCAL) and the United States’ National Institutes of Health (NIH) Animal Care Guide. The project was approved to the Animal Use Ethics Committee of the Federal University of Minas Gerais (CEUA - UFMG) without restriction to the proposed procedures (Protocol No. 340/2013). Two animal groups were used, the ARDKO  in C57BL6/J genetic background, and wild-type (WT) controls . At the age of seven months, ARDKO mice had cardiac dysfunction associated with exercise intolerance and a higher mortality rateFor indirect measurement of heart rate (HR) and mean arterial pressure (MAP) in WT and ARDKO animals (7 months old), we used a rodent plethysmograph . The mice were contained using a heated acrylic chamber and the body temperature was maintained at 35 ± 2 °C with constant monitoring by an infrared thermometer. The animals’ tail was then fitted to a rubber cuff that was adapted to the proximal region and connected to the sphygmomanometer to inflate and deflate automatically at fixed intervals of 50 seconds. Near the cuff was attached a pulse transducer that captured the signals and recorded them on a computer. The measurements only started after 5 minutes of acclimatization. MAP and HR were calculated automatically by the software from the data obtained from 3 consecutive measurements.9. In this process, hearts were perfused with Ca2+-free, cell-isolation buffer (CIB), containing (in mM): 130 NaCl, 5.4 KCl, 0.5 MgCl2, 0.33 NaH2PO4, 22 glucose, 25 HEPES, and 0.4 EGTA (pH 7.4), maintained at 37 °C for 3 min to clean the heart. After this process, the Ca2+-free CIB solution is replaced by CIB (without EGTA) containing 0.3 mM CaCl2, 1 mg ml−1 collagenase type II, 0.06 mg ml−1 protease type XXIII, and 0.06 mg ml−1 trypsin, which was perfused for 3–5 minutes. The heart was then removed from the cannula and the ventricles were separated, cut into small pieces, and then subjected to an additional 8 min of digestion at 37 °C, with CIB supplemented with collagenase, trypsin, and protease. After these two steps of enzymatic digestion, the cells were isolated from the tissue by light agitation using a glass pipette. This solution containing the isolated cells was then filtered, centrifuged at 100 g for 30 sec, resuspended in CIB solution containing 1.2 mM CaCl2 and 2 mg/ml bovine serum albumin (BSA), and maintained in this solution for 10 min at 37 °C. Finally, the cells were centrifuged again (100 g for 30 s) and maintained in a Tyrode’s solution with the following composition . VCMs were used for experiments within 6 hours of isolation. VCMs with rounded edges and other visible signals of membrane damage or VCMs not responsive to electrical stimulation were not used for experiments.To obtain VCMs, the animals were euthanized by decapitation and hearts were carefully removed via thoracotomy, cannulated through the aortic trunk, and then mounted in a Langendorff constant pressure system, where they were perfused in a retrograde flow systemThe cellular contractility was measured by overall cell shortening using an edge detection system with high-speed camera  at room temperature . Cells were placed in a perfusion chamber filled with Tyrode’s solution (see above) mounted on an inverted microscope  and connected to two parallel metal electrodes immersed in the solution to provide the electric field stimulation . The stimulation frequency ranged from 1 to 3 Hz depending on the experiment to be performed. A separated subset of cells was exposed to isoproterenol  prior to the contractility experiments.The records of the APs and ionic currents was performed using the patch clamp amplifier EPC-10  and recorded on a computer via the PACTHMASTER acquisition software  in the “Current clamp” and “Voltage clamp” modes. All experiments were performed at room temperature. To record the ion currents, the internal and external solution were specific for the current to be studied  and using solutions designed to prevent contamination with Cl–currents10. The NCX current was obtained after the subtraction of those currents before and after the Ni2+ application, thus yielding only the Ni2+-sensitive membrane current component. For LCC currents, the cells were stimulated with square voltage steps (300 ms in duration) from −80 to +60 mV in 10 mV increments, every 10 seconds. For outward potassium currents, the protocol consisted of square voltage steps (3 seconds in duration) from −60 to +70 mV in 10 mV increments, every 10 seconds. The inward potassium currents were elicited with a square voltage steps (3 seconds in duration) from −120 to −50 mV in 10 mV increments, every 10 seconds. In a subset of LCC current recordings, cells were pre-treated with isoproterenol  before the measurements.To trigger AP initiation (in current-clamp mode), the myocytes were stimulated with a brief depolarizing current (1 nA for 2 ms) and the changes in the membrane voltage was observed for 950 ms (at 1 Hz stimulation) and 320 ms (at 3 Hz). For the recording of NCX current, whole-cell membrane currents was measured using a voltage ramp ranging from −90 mV to +60 mV before and after exposing the cell with Ni2+]i transient recording, VCMs were loaded with Fluo-4 acetoxymethyl (AM) for 30 minutes (20–25 °C) at a concentration of 5 μM in Tyrode’s solution. For NO and ROS measurements, the VCMs were loaded for 30 minutes using the DAF-FM  or DCFH-DA  at concentrations of 10 and 3 μM, respectively. After the loading period, VCMs were then centrifuged and resuspended in Tyrode’s solution, where they remained for another 10 minutes allowing for de-esterification of the probes. The confocal images were acquired using an LSM 510 META confocal microscope  from the Center for Acquisition and Image Processing of UFMG (CAPI - ICB - UFMG). VCMs were placed in a perfusion chamber filled with Tyrode’s solution and the probe was excited by an argon laser (488 nm). Emitted light was filtered through a long pass filter (505 nm) and collected by a photomultiplier tube. Similar to the cellular contractility experiments, an electrical stimulus with a frequency of 1 Hz was used to induce the [Ca2+]i transients. The acquisition of the [Ca2+]i signals was performed using the confocal in line-scan mode (1.84 ms/line) on middle portion of the myocyte avoiding the nuclei. Since the basal fluorescence (FO) was not different between groups, the changes in fluorescence (F) were normalized by the initial fluorescence (FO) and displayed as F/FO. For NO and ROS measurements, cellular fluorescence was measured using a Nikon Eclipse Ti microscope . DAF and DCF  were excited by a Xeon light source  filtered at 480/30 nm, and emitted light was filtered through a 535/40 nm band-pass filter before being collected by an EMCCD camera . The acquisition parameters  were kept the same throughout all the experiments. After subtraction of background fluorescence, the mean fluorescence intensity of each cell was measured. The DCFH oxidation to DCF is highly non-specific and responds to reactive species beyond just H2O211. Due this fact, changes in DCF florescence are interpreted as changes in the cellular redox environment  rather than direct changes exclusively in H2O2 production.For confocal microscopy and . Finally, Hodgkin-Huxley style gating variables are used to generate the various sarcolemmal membrane currents associated with the mouse, cardiac AP. Force and sarcomere length were calculated based on a previously published Markov chain formulation16. ECC gain is calculated by dividing the integrated RyR2 flux by the integrated LCC flux. See our previous publication for more details17. For ARDKO simulations, parameters were modified according to Table The computational model of cardiac excitation-contraction coupling (ECC) used here is based on our previously published work19. Similar to these previous findings, we observed that ARDKO animals displayed increased heart rate (HR) and mean arterial pressure (MAP) secondary to the chronic sympathetic hyperactivity  in both stimulation frequencies 1 and 3 Hz) tested  we used confocal microscopy to record [Ca2+]i dynamics in quiescent and electrically stimulated cells. [Ca2+]i waves were observed in only 8.7% and 11.4% of WT and ARDKO cells, respectively. Within this group, ARDKO appeared to have a higher [Ca2+]i wave frequency (0.28 ± 0.10 waves 10 s−1) over their WT counterparts (0.12 ± 0.05 waves 10 s−1), however this was not statistically different between groups. When electrically stimulated (1 Hz), ARDKO VCMs had reduced [Ca2+]i transient amplitudes when compared to their WT counterparts  of WT and ARDKO VCMs using a whole-cell patch-clamp technique. A marked prolongation of the AP was observed in ARDKO Fig. . SpecifiCSH is associated with reduced outward K+currents and increased NCX current.+ currents as well as the NCX current. Similar to the LCC current, outward K+ current density is reduced in the ARDKO cells when compared to WT   and sustained (Iss) currents have a larger contribution to the sum of the outward K+ currents in ARDKO. The inward rectifier currents are key elements during later phases of AP repolarization and also help maintain adequate resting membrane potential. Consistent with the unchanged membrane resting potential of ARDKO, no significant change in those currents could be detected  Fig. . Althougted Fig. .2+ extrusion from the cytosol, NCX is an electrogenic transporter and can therefore influence the transmembrane potential. This is most commonly observed during the repolarization phase of the cardiac AP. ARDKO VCMs had an increase (55%) in the current sensitive to 5 mM of Ni2+ (a blocker of NCX activity) over WT  animals. While 3-month-old ARDKO animals displayed reduced body weight compared to the WT, there was no alteration in their heart/body weight ratio to indicate heart pathology. Consistent with this, we observed that 3 month old ARDKO VCMs presented similar cellular contractility (compared to WT) in both stimulation paradigms and upon activation of the βAR Table . Additio+ and Ca2+ currents along with increased NCX current) to create a representation of WT and ARDKO conditions. Importantly, we observed that altering only these key parameters account for the AP prolongation and reduced force generation in ARDKO  to the ARDKO phenotype, we performed simulations where each current alteration was implemented individual. These simulations demonstrate that: (1) the reduced LCC current primarily drives changes in Ca2+ transient   diminished IK+ currents drive elevated [Ca2+]i transient amplitude and AP prolongation (as expected) but also increase force production  within a CRU yielding the activation of the CRU RyR2 cluster to generate a Ca2+ spark . Furthermore, the diminished Ca2+ spark fidelity observed in the ARDKO compared to WT  but slightly altered systolic levels for WT and ARDKO .To better understand the individual contribution of the experimentally observed, membrane current changes  elain Fig. 20; and fion Fig. . Interessee Fig.  suggestsIn this study we demonstrated that the genetic model of CSH provided by the ARDKO mouse induces contractile dysfunction in VCMs by remodeling of various ion channels and transporters essential for the ECC. These changes were observed before a severe hypertrophy and without compromised adrenergic signaling.7. The reduced contractility displayed by ARDKO hearts is a common point on various cardiac disorders including HF, especially at higher heart rates or in the presence of catecholamines21. In this study, the model of endogenous sympathetic overactivity generated by deletion of adrenergic α2A and α2C receptors reduced fractional shortening and contraction and relaxation velocity (see above) which correlates well with past observations6.We used a genetic model of sympathetic hyperactivity caused by the knock-out of α2A and α2C adrenergic receptors. This double knock-out leads to sympathetic hyperactivity and increased plasmatic norepinephrine, and in the long term these animals are prone to developing cardiac hypertrophy, reduced physical capacity, impaired cardiac contractility, altered VCM ultrastructure, and higher mortality with age progression8, which is consistent with the proposed role of these receptors in cardiac maladaptation in ARDKO model. Furthermore, the cardiac ECC remodeling in the ARDKO, is also consistent with other experimental models in which prolonged β-AR stimulation by isoproterenol resulted in reduced cardiac contractility25. While cardiac ultrastructure, capacity, and contractility have already been extensively characterized, here we provided specific evidence for ECC alterations and identified the underlying mechanisms responsible for the macroscopic changes previously reported in this experimental model6.The cardiac dysfunction in this animal model can be attenuated by β-AR antagonists treatment 27. In the heart, this desensitization is regulated by G-protein-coupled receptor kinase 2 (GRK2), which is elevated in HF28. While, the upregulation of GRK2 in the adrenal medulla also play a critical role in the elevated circulating levels of catecholamines associated to HF30. However, at the time points included in our study (3 and 7 months), CSH had not impaired β-AR signaling. When exposed to isoproterenol, cardiomyocytes from WT and ARDKO had similar contractile characteristics and LCC current density, in contrast to the reduction observed under normal conditions. Furthermore, prior in vivo observations, showed no difference in the maximal chronotropic response between WT and ARDKO mice when challenged with isoproterenol6. Since GRK2 and β-AR expression levels are unknown in ARDKO mice, the molecular background for β-AR signaling is not clear in this CSH model. In the future, the availability of such data combined with the results shown here will better characterize the role of CSH in driving cardiac remodeling. Taken together this suggests that CSH can compromise cardiac ECC prior to β-AR desensitization. However, we do not exclude the possibility that CSH can impair β-AR signaling if sustained for longer periods than those explored in this study.One characteristic of the HF pathology is the abnormal β-AR signaling via desensitization of those receptors31. AP prolongation, however, fails to maintain force generation at later stages of HF and becomes a driving factor for the onset of arrhythmic events33. Here, we show that CSH leads to a similar AP prolongation and that reduced outward K+ currents and elevated NCX activity underlie this profile. In parallel, sarcolemmal LCC current density is reduced in the ARDKO model with significant implications for CICR dynamics and cell contraction.Another important change found in animals with CSH was an increase in AP duration. This is consistent with early stage HF studies where AP prolongation is commonly observed and is believed to be an important cellular mechanism for maintaining force generation as a compensatory mechanism after the contractile dysfunction associated with HF+ currents associated with CSH are also observed in other models of HF34. Following a detailed analysis of the contribution of the various K+ current subtypes, we found no changes in delayed rectifier and inward rectifier currents. In larger mammals, these subtypes are important regulators of AP duration, and in late stage HF, decreased inward rectifier currents play an important role in the generation of arrhythmias35. However, delayed rectifier K+ currents have low expression in small rodents and play no significant role in the AP prolongation observed in the CSH-induced model. In fact, the major contribution to down-regulation of outward K+ currents in our experiments was a reduction in Itof. The reduction in Itof is another common maladaptation related to K+ channels in HF. Note, however, that while this current significantly regulates AP duration in small rodents it plays a minor role in larger mammals despite being responsible for the initial repolarization phase (phase 1) of the cardiac AP37. Phase 1 governs the initial Ca2+ influx through LCC and therefore CICR and contractility. Hence, a more prominent phase 1 repolarization due to a higher density of Itof would likely increase Ca2+ influx (due to elevated driving force for Ca2+ entry) thereby triggering more Ca2+ release from the SR and consequently improving myocyte contraction38. Conversely, down-regulation of Itof would likely contribute to the reduced contractility observed during HF and in the ARDKO model.The reduced K2+ levels have been previously described in the ARDKO experimental model18. In this study, we provide new evidence associating increased NCX current with the prolongation of the AP in the ARDKO. This increase in NCX levels might represent a compensatory mechanism for the decreased SERCA function highlighted by slowed decay of Ca2+ transients in ARDKO. Such a mechanism would act as a means for the cell to attempt to maintain normal relaxation rates39. Note that in ARDKO, reduced SERCA levels have also been shown to be partly compensated for by increased phospholamban phosphorylation (in Thr17 but not Ser16)40. The increase in NCX levels may also play an important role in HF when in reverse mode, which can aid in Ca2+ entry during the systolic phase to augment the pathologically diminished SR Ca2+ release41. Finally, NCX transport is electrogenic and therefore NCX activity can alter the cardiac AP. This is especially true in HF and in larger animals, where NCX current can drastically alter the shape of the AP and drive arrhythmias43.Increased NCX expression and its role in controlling intracellular Ca44. Furthermore, an increase in ROS availability, as identified here, has been linked to an reduced Itof current density46 and increased activity of NCX41 which would promote the AP prolongation observed in the ARDKO myocytes. Furthermore, an unbalanced cellular redox state reduces SERCA function and increases the SR Ca2+ leakage via RyR2, which can lead to Ca2+ mishandling and arrhythmias48.Consistent with a prominent role of NCX is evidence in healthy mice that an inducible overexpression of NCX1 causes a pronounced prolongation of the AP without significantly changing the resting potential2+ currents49, however, others observe a maintenance of the macroscopic current22. This mismatch may be due to a non-uniformity of isoproterenol administration, where some results vary according to the animal model, dose, and time of treatment. The reduced Ca2+ currents induces a directly contractile dysfunction process50. However, in HF, reduced cell contractility could not be well correlated with decreased LCC current density39 which appears more frequently in end-stages of HF32. In addition, decreased expression of LCC was not observed in all cases51. However, there is a considerable redistribution, with a decrease in Ca2+ current in the t-tubule membrane, likely due to t-tubule remodeling, with a concomitant increase in sarcoplasmic membrane surface area22. This t-tubule remodeling is an important factor in the transition between a hypertrophic state and HF, as it has close relationship with contractile function52.In addition to the reduced fractional shortening and contraction and relaxation velocity displayed by the endogenous sympathetic hyperactivity model used here, a reduction in LCC current density was also observed. Some isoproterenol-based sympathetic hyperactivity models show decreased Ca49. 2) increased expression or activity of phosphatases  in the ARDKO heart that are known to reduce LCC phosphorylation levels and LCC currents19. Additionally, the expression of some kinases important for the modulation of LCC  are not altered in the animal model used in this study19. Given the critical nature of LCC in initiating CICR during systole to generate the [Ca2+]i transient, we decided to investigate how this reduced LCC current would alter CICR dynamics in the ARDKO heart.Here, in the ARDKO heart we observed an approximately two-fold reduction in peak LCC current density (see above). The two likely mechanisms that contribute to this reduced LCC current are: 1) cellular hypertrophy, and the resulting increase in cell capacitance, found in the ARDKO model and other models involving the exogenous β-adrenergic stimulation 6, this deficit was highlighted here via a significant decrease in single cell fractional shortening from freshly-isolated, field-stimulated, VCMs. While the marked decrease in LCC currents described above is a likely contributor to this reduced shortening we investigated further using confocal recording of [Ca2+]i transients.Consistent with past studies we observed a significant decrease in contractile function in the ARDKO hearts. While commonly observed via reduced ejection fraction2+]i transient amplitude and asynchrony of initial Ca2+ release. Using a computational model of cardiac ECC, we observed increased variability in timing of Ca2+ sparks leading to a significant reduction in Ca2+ release synchrony and higher incidence of delayed Ca2+ sparks during the [Ca2+]i transient in ARDKO heart, which contributed to slowed [Ca2+]i transient decay. Also, by isolating individual currents, the simulations revealed some complex interactions between the various components of ARDKO-based remodeling. This was especially notable when relating to the opposing influences of the reduced IK+ and LCC currents on the systolic Ca2+ transient and force generation. Since this Ca2+ mishandling and impaired force generation underlies contractile dysfunction39, our experimental and computational data combine to better characterize the role of CSH in cardiac dysfunction and disease progression.Reduced LCC current in ARDKO hearts impairs CICR therefore causing reduced [Ca2+ signaling. These changes are accompanied by a prolongation of the cardiac action potential caused by reduced outward K+ currents and increased NCX current. These changes along with diminished LCC current drive dysfunctional CICR behavior at the molecular level which underlies the macroscopic contractile deficits. These results have provided novel insights into the role of CSH in the progression of cardiovascular disorders.Chronic sympathetic hyperactivity (CSH) induced by deletion of the α2A and α2C adrenergic receptors reduces contractility in VCMs by an impairment of CaSupplementary Information.