Oryctolagus cuniculus); however, initial findings have found they can increase their serum 25-hydroxyvitamin D3 (25-OHD3) following 12 h of artificial UVB exposure. Current husbandry recommendations for rabbits do not include specific UVB lighting requirements. Rabbits are a common pet and research model and are frequently housed indoors without access to natural UVB lighting. Rabbits that are chronically vitamin D deficient may develop mineral deficiencies that can lead to poor calcification of the teeth and skull, predisposing these animals to dental abnormalities, bone infections, and other debilitating diseases. While initial results suggest artificial UVB is positive for rabbits, UVB can also be detrimental to the health of vertebrates. The aim of this study was to determine if shorter-duration UVB exposure could also increase 25-OHD3 concentrations. Rabbits were provided 6 h of artificial UVB daily for 14 days, and there was a significant increase in 25-OHD3 concentrations over time. These findings affirm that rabbits can use short-duration artificial UVB to increase 25-OHD3 concentrations.Vitamin D is an important hormone that can be acquired through diet or exposure to ultraviolet B (UVB) radiation. Few studies have evaluated the effects of UVB radiation on vitamin D concentrations in the domestic rabbit (Oryctolagus cuniculus), both methods appear viable, but there is limited research evaluating the effects of UVB on this species. Previous studies found that 12 h of artificial UVB radiation significantly increased 25-hydroxyvitamin D3 (25-OHD3) concentrations over time. While these findings suggest UVB can be beneficial in rabbits, this form of radiation can also be detrimental to vertebrates. The purpose of this study was to determine if shorter-duration UVB could elicit a similar physiological response in rabbits while minimizing potential negative effects. Six rabbits were used for this pilot study. The baseline serum 25-OHD3 was measured for each rabbit and following 14 days of 6 h/day exposure to artificial UVB, a second 25-OHD3 sample was collected. There was a significant increase (p = 0.001) in serum 25-OHD3 over time . This study affirmed that 6 h of UVB produced 25-OHD3 concentrations similar to those found in rabbits exposed to 12 h of UVB. Future studies should continue to determine how the duration of UVB exposure affects 25-OHD3 concentrations.Vitamin D is an important hormone that can be acquired through diet, exposure to ultraviolet B (UVB) radiation, or a combination of these methods. In domestic rabbits ( Oryctolagus cuniculus) do not include specific lighting requirements beyond a 12–14 h photoperiod [3 (25-OHD3) concentrations were significantly higher in the UVB-exposed rabbits following the 14-day exposure [3, suggesting that UVB exposure should be considered for captive rabbits.In the wild, rabbits are exposed to all three beneficial components of sunlight, including ultraviolet, visible, and infrared light. However, current husbandry recommendations for captive domestic rabbits  had a significantly higher risk of developing cutaneous SCC than nonwhite cats, with the greatest effect on the ears and nose, or areas with higher exposure and little to no fur [Mus musculus) that were chronically exposed to UVB radiation at 280–320 nm were found to develop systemic immunosuppression, which led to the development of primary skin cancer [While UVB light has been found to play an important role in the endogenous synthesis of vitamin D in vertebrates, it is not without risk, as direct UVB exposure can cause photodermatitis, erythema, structural damage to the eye, and cancer ,6,7,8,9.n cancer . These fChinchilla lanigera) exposed to 12 h/day of artificial UVB irradiation for 16 days with no reported adverse effects [The irradiance and duration of exposure to UVB can increase the risk for adverse effects ,10. Emer effects . A longe effects . While t3 concentrations over time compared with their baseline concentrations and that the post-exposure 25-OHD3 concentrations following 6 h of exposure would not significantly differ from a previous study following 12 h/day exposure under similar conditions for 14 days [The purpose of this study was to evaluate if shorter duration daily exposure to UVB at similar low irradiances to those in previous studies would elicit a similar physiological response in the rabbit while minimizing potential negative effects. The specific hypotheses being tested in this study were that rabbits exposed to 6 h of artificial UVB radiation for 14 days would significantly increase their 25-OHD3 concentrations of 50 nmol/L, and a standard deviation (SD) of 15 nmol/L between the baseline and 14-day sampling periods. Each rabbit served as its own control in this experimental study and received the treatment of UVB light exposure. No inter-individual controls (non-UVB-exposed rabbits) were used because all of the available animals (only 6 were available) were required to meet the sample size determination requirements noted in our calculations. As there were no inter-individual controls, we used historic baseline and control data for comparisons (see single-sample t-test) to our results.This experimental, non-randomized study was performed under the regulations and policies established by the Institutional Animal Care and Use Committee (IACUC 14-265) at the University of Illinois . Six dwarf mixed-breed juvenile rabbits  with pigmented haircoats  from a private source  were used for this study. The sample size used for this study was based on the following a priori information: an alpha = 0.05, a power = 0.8, an expected difference in 25-OHDAll the animals were weighed and examined to determine whether they were healthy. The rabbits were housed in same-sex pairs in 71 cm × 44 cm × 41.5 cm plastic-bottomed wire cages  on pine bedding . Fresh water was provided ad libitum in a sipper bottle. The rabbits were offered unlimited timothy grass hay  and ¼ cup of timothy-based antibiotic-free pellets daily . The cage substrate, water, and food were replaced daily. General room lighting was provided by a non-UVB-producing fluorescent light for a 12 h photoperiod. The temperature of the room was maintained at 23–27 °C (73–80.6 °F). The rabbits did not have exposure to artificial UVB lighting or natural sunlight prior to the study.After the initial acclimation period, each rabbit was anesthetized with 5% isoflurane , and 1 L/min oxygen was delivered using a facemask. The rabbits were maintained on 1–2% isoflurane with 1L/min oxygen during blood collection. A total of 1 mL of blood  was collected from the cranial vena cava using a 22- to 25-gauge needle fastened to a 3 mL syringe. All blood collection was performed between 1700 h and 1900 h. Once the sample was collected, each animal was recovered on oxygen and all animals recovered uneventfully. Animal restraint, anesthesia, and venipuncture followed the recommended guidelines outlined in Mitchell and Tully [g for 10 min within 90 min of collection. During the 14-day study, UVB radiation was provided by two full spectrum commercial compact bulbs  externally fixed to the top of the cages. UVB radiation was measured by a radiometer  within the cage at the level of the animals at 9 points in a grid pattern along the perimeter and directly under the lights on days 1, 8, and 14 from 1700 h to 1800 h. At the distance the bulbs were secured, the rabbits received 15–50 microwatts/cm2 of UVB radiation in the range 290–310 nm. UVB exposure was divided equally over two time points (approximately 0600 h–0900 h and 1500 h–1800 h) to mimic exposure at dawn and dusk. Rabbits were observed daily for abnormal behavior and signs of photophobia. On day 14, a second blood sample from each rabbit was collected using the same methods described for baseline sampling. All serum samples were transported on wet ice to the authors’ laboratory and stored at −80° C. Once the second set of samples was collected and processed, all samples (baseline and day 14) were submitted together (within 7 days of second samples being collected) on wet ice to Michigan State University  to measure 25-OHD3 concentrations using a radioimmunoassay [Blood samples were placed into non-anticoagulant microtainers  and centrifuged at 4000× 3 concentrations were significantly different over time or by sex; the interaction term time x sex was also included. A single-sample t-test was also used to determine if the baseline and 14-day 25-OHD3 concentrations were significantly different from a previous study using similar husbandry conditions and artificial UVB lights for 12 h/day for 14 days [p < 0.05 was used to determine statistical significance.The distributions of the data were evaluated using the Shapiro–Wilk test, skewness, kurtosis, and q-q plots. Because the data were normally distributed, the mean, SD, and minimum-maximum (min-max) values are reported. A repeated measures ANOVA was used to determine if the 25-OHDp = 0.001) in the 25-OHD3 concentrations over time for all of the rabbits after being exposed to 6 h of UVB radiation (p = 0.93) or the interaction term . There was no significant difference in the baseline 25-OHD3 concentrations in the rabbits from this study (27.7 nmol/L) compared with those exposed to the same type of artificial light at baseline  for those reported in Emerson et al. [All six rabbits served as their own control. The rabbits had mean bodyweights of 411 g (range 350 to 455 g) and 619 g (range 495 to 710 g) at the baseline and 14-day sampling periods. Weight gain was expected due to the age of the animals. There was a significant difference . These results are important because if the duration/exposure of UVB is shortened and similar 25-OHD3 concentrations are achieved, it may be possible to reduce some of the potential adverse effects associated with UVB exposure. Ultimately, longitudinal studies will be needed to confirm the potential risk.The results of this study confirm the authors’ first hypothesis that juvenile rabbits exposed to 6 h of artificial UVB light for 14 days would significantly increase their serum 25-OHDMetabolic bone disease is a common nutritional disorder seen in captive species. Nutritional osteodystrophy results from prolonged deficiencies of calcium or vitamin D or from an inappropriate calcium-to-phosphorus ratio in the diet ,13,14. C3 concentrations in these patients. These vitamin-D-deficient rabbits developed hypocalcemia, hypophosphatemia, and hyperparathyroidism [3 concentrations <20 ng/mL (50 nmol/L) [3 concentrations <12 ng/mL (30 nmol/L) [3 in rabbits to better ascertain the health of these animals and assist with interpreting the results of studies such as the one reported here.Previous studies that have diagnosed rabbits with hypovitaminosis D reported undetectable serum 25-OHDyroidism ,15. In h nmol/L) ,18. In a nmol/L) . Based o nmol/L)  studies 3 concentrations to a 12 h UVB exposure. Ultimately, it is important to establish a reference range for 25-OHD3 concentrations in domestic rabbits to determine the amount of UVB exposure required to achieve a healthy state. Unfortunately, a larger study population is required to establish a reference range, with a minimum of 20–40 subjects but preferentially >120 study subjects, based on the recommendations of the American College of Veterinary Clinical Pathologists [3 concentrations were not different between the cases and controls for Emerson et al. [3 were noted in the rabbits exposed to UVB, the authors believe the sample size limitation did not impact the results. Additionally, the 25-OHD3 concentrations reported in Watson et al. [p = 0.62), while the values for the controls were different . A direct comparison between baseline 25-OHD3 concentrations was not made between Watson et al. [Current lighting recommendations for captive rabbits are limited to maintaining a 12 h photoperiod ; however2) of UVB being provided. In the present study, rabbits were exposed to 15–50 µwatts/cm2 for 6 h/day. These values are similar to those recorded for Emerson et al. [2) and Watson et al. [2). Due to the similar amounts of UVB exposure, the results of the present study reinforce that a shorter length (6 h/day) of UVB exposure should generate similar 25-OHD3 concentrations as 12 h of exposure. To reduce the likelihood of adverse effects, reducing the total quantity of UVB is considered prudent. Future studies should evaluate the effects of lower quantities of UVB, in addition to the length of exposure, to further refine the amount of UVB required to achieve the desired effect in rabbits.In addition to considering the length of time an animal is exposed to UVB, it is important to consider the amount  [3 concentrations in the present study and the control rabbits in Emerson et al. [The concept of exposing rabbits to artificial UVB to generate vitamin D is fairly recent ,4,12. Whaptivity ,15. Histaptivity ,22,23. Taptivity . The res0 IU/kg) ,23,25. T3 concentrations in these juvenile rabbits were not significantly different from another population of juvenile rabbits housed under the same conditions [3 concentration, as did their kits [There were no significant differences between the sexes in this study. This was not surprising because the rabbits were juveniles 10–12 weeks) at the time of the study, although these animals were expected to become sexually mature shortly after this age [ weeks atnditions ; thus, ieir kits . As note3 concentrations in 140 Finnish pet rabbits [n = 46) had a mean serum 25-OHD3 of 27.9 ng/mL (69.75 nmol/L), which is similar to the findings for rabbits with access to artificial UVB in the present study, Emerson et al. [t-test , there is no significant difference between the two populations. The mean serum 25-OHD3 concentrations for all the Finnish pet rabbits (n = 140) was 26.0 ng/mL (65 nmol/L), with a range of 4.5–67.5 ng/mL (11.25–168.75 nmol/L). Diet was significantly (p = 0.001) associated with serum 25-OHD3 concentrations in this population, while access to the outdoors with potential exposure to UVB radiation did not reach statistical significance. There were several limitations to this study that could have influenced the results, including differences in breeds, sexes, ages of rabbits, reliance on owner questionnaires to determine groups, length of outdoor access, types of enclosures, and the ratio of sunny versus shaded areas. Additionally, 15 different brands of commercial feed were provided, with vitamin D concentrations ranging from 700–2000 IU/kg. This cross-sectional study illustrates the complexity of factors that can affect 25-OHD3 concentrations in pet rabbits and the importance of further investigating the role of UVB on the health of captive rabbits.In addition to the artificial UVB studies noted previously, there have been recent attempts to characterize the value of natural UVB for captive rabbits. A study in Finland evaluated serum 25-OHD rabbits . RabbitsAnother study in France utilized an online survey distributed to pet rabbit owners to determine the influence of outdoor exposure on dental disease . The aut3 concentrations in captive rabbits after only 6 h/day of artificial UVB for 14 days and that there is no difference in the 25-OHD3 concentrations between 6 h/day exposure and 12 h/day exposure (over 14 days and 6 months of exposure), there remains much still to answer regarding what are normal concentrations of 25-OHD3 for captive rabbits and how we can reduce the likelihood of adverse effects associated with UVB exposure in these animals. At this time, based on these results and the results of Watson et al. [As expected, the results of this study have generated more questions than answers related to the importance of artificial UVB for captive rabbits. While the authors answered their two hypotheses for this study, that artificial UVB can increase 25-OHD


We used Langendorff’s model to monitor the susceptibility of aging  hearts to IR, with a specific focus on Ca2+-handling proteins. IR, but not aging itself, triggered left ventricular changes when the maximum rate of pressure development decreased in 24-month-olds, and the maximum rate of relaxation was most affected in 6-month-old hearts. Aging caused a deprivation of Ca2+-ATPase (SERCA2a), Na+/Ca2+ exchanger, mitochondrial Ca2+ uniporter, and ryanodine receptor contents. IR-induced damage to ryanodine receptor stimulates Ca2+ leakage in 6-month-old hearts and elevated phospholamban (PLN)-to-SERCA2a ratio can slow down Ca2+ reuptake seen at 2–5 μM Ca2+. Total and monomeric PLN mirrored the response of overexpressed SERCA2a after IR in 24-month-old hearts, resulting in stable Ca2+-ATPase activity. Upregulated PLN accelerated inhibition of Ca2+-ATPase activity at low free Ca2+ in 15-month-old after IR, and reduced SERCA2a content subsequently impairs the Ca2+-sequestering capacity. In conclusion, our study suggests that aging is associated with a significant decrease in the abundance and function of Ca2+-handling proteins. However, the IR-induced damage was not increased during aging.During aging, heart structure and function gradually deteriorate, which subsequently increases susceptibility to ischemia–reperfusion (IR). Maintenance of Ca Cardiovascular diseases are the leading cause of death worldwide, taking more than 17.9 million lives each year , with more than four out of five deaths due to heart attack or stroke . The rol2+ ), where V is the velocity, Vmax is the maximum Ca2+-ATPase activity, and KCa is free calcium concentration [Ca2+] at Vmax/2.Cardiac sarco-endoplasmic reticulum Caprotocol  on the PDot blot analysis was used to visualize 314 kDa inositol-1,4,5-triphosphate receptor and 560 kDa ryanodine receptor. Homogenates were diluted to 4.0 µg/µL and 2.5 µL drops (10.0 µg of protein) were spotted onto a 0.45 µm nitrocellulose membrane . After drying, primary and secondary antibodies were applied to membranes according to the immunodetection procedure described below.® SD Semi-Dry Transfer Cell apparatus  for 80 min. To quantify the total protein, the resulting blots were incubated in red-colored Ponceau S for 5 min. Non-specific binding was blocked overnight with 5% non-fat dry milk in Tris base saline with 0.05% Tween 20 (TBS-T).Proteins (40 µg per sample) from tissue homogenates were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis with 8%, 10%, or 15% gels (based on Mw of target protein) under denaturing conditions (3 min at 95 °C). Separated proteins underwent a semi-dry transfer at 60 mA per gel onto nitrocellulose membranes using Trans-BlotThe next day, blots were washed three times in TBS-T (the washing solution was used between each step) and incubated with the following HRP-conjugated primary antibodies: anti-VDAC1  for 1 h; anti-GRP75  and anti-NCX1  for 2 h; anti-PLN  for 3 h; anti-MCU , anti-SERCA2a , anti-RyR , and anti-IP3R  overnight.Then, membranes were incubated with the appropriate secondary antibodies for 1 h: rat anti-mouse IgG-HRP  for VDAC1, GRP75, and PLN; goat anti-mouse IgG-HRP  for SERCA2a; goat anti-rabbit IgG-HRP  for NCX1, MCU, RyR, and IP3R. Finally, the immunoreactive proteins were visualized after 5 min exposition to Clarity™ Western ECL chemiluminescent substrate in the dark using a ChemiDoc™ XRS Imaging System and quantified in the Image Lab™ program . Results were normalized to total protein and expressed as relative protein levels in arbitrary units (AU).https://www.R-project.org/, accessed on 15 December 2021) ver. 4.0.5., with the aid of several libraries, most notably, lme4 and DHARMa. To make the research reproducible, the analyses were written in an R notebook. The data were summarized by the mean, median, standard deviation, and inter-quartile range. A spaghetti plot was used to visualize the longitudinal data. A quantile–quantile plot with a 95% confidence interval constructed by the bootstrap method was used to assess the normality of the continuous variables. Repeated-measures two-way ANOVA was fitted to the data using the linear mixed model, which in the Wilkinson Rogers notation can be expressed as response~treatment*age + (1|id). The deviance residuals were used for the diagnostic analysis of the model . The marginal means were used to derive the estimates of the mean and its 95% confidence interval and pairwise post hoc comparisons with the Tukey adjustment of p-values. Marginal and conditional R2 were used to measure the effect size. An interaction plot was used to visualize the marginal means. Results are presented as mean ± standard deviation (SD) and findings with a p-value below 0.05 were considered statistically significant.Data were explored and analyzed in R (The effect of IR on parameters of contractile function in aging hearts is summarized in p < 0.001) from 1506.4 mmHg/s to 843.7 mmHg/s after IR. The initial value of the maximum rate of relaxation (LV −dP/dt) was maintained during aging. However, the response to IR was different among the individual age groups, with the most sensitive 6-month-old hearts reaching only 62.6% of pre-ischemic value (** p < 0.01) in comparison to the 15- and 24-month-old groups  activity in sarcoplasmic reticulum. We recorded very stable Ca2+-dependent activity profile changes in 24-month-old hearts in comparison to 6- and 15-month-old hearts exposed to IR. At higher free [Ca2+] concentrations (1.0–5.0 μM), 6-month-old hearts were able to reach 83.2% of the maximum velocity measured in control-perfused hearts. On the other side, the Ca2+-binding ability of 15-month-old hearts was highly affected in response to IR reaching only 63.9% of control. This points toward impaired Ca2+-sequestering capacity of old cardiomyocytes  in senescent individuals compared to adults , which exists in a dynamic equilibrium of oligomeric states ranging from pentamer to monomer. In our study, the formation of PLN oligomers and monomers was age-dependent in response to ISCH or IR. Total PLN elevation accelerates cytotoxic effects, causing inhibition of Caed to IR B. Contra2+-regulating proteins in aging cardiomyocytes subjected to IR, with a specific focus on the communication between ER/SR and mitochondria. Ca2+ release channels, IP3R and RyR, had the same trend of expression changes with a decrease in RyR level by 43.6% after IR in the 6-month-old group, but with age, there was significant suppression of both proteins , such as a 3.6-fold decrease in the Na+/Ca2+ exchanger NCX1  might be associated with prolonged SR–Ca2+ transients [2+-transporting capacity and overall ATPase activity. Since Ca2+ influx into the SR is an ATP-dependent process (~15% of cardiac energy usage), overexpression of SERCA2a could increase energy requirements in 24-month-old rats, but lower ATP reserve may contribute to contractile dysfunction.Age had a significant impact on basal Caansients ,32. On tansients , which hansients  and exis2+-ATPase activity at low free Ca2+ concentrations up to 0.1 μM in 15-month-old hearts, with the ratio of PLN to SERCA2a contents higher in comparison to 6- or 24-month-old hearts. Since a comparative study  reported that only 40% of SERCA2a is normally regulated by PLN [2+ concentrations in 15-month-old. This points towards impaired Ca2+-sequestering capacity of aged cardiomyocytes subjected to IR. Ca2+-ATPase activity in 6-month-old post-ischemic hearts was affected at higher (2–5 μM) free Ca2+ with unchanged SERCA2a protein levels. The overall PLN, as well as the ratio of PLN to SERCA2a, massively increases after IR in the 6-month-old group, which can slow down Ca2+ reuptake by SERCA2a. These changes are mostly seen in senescent 24-month-old rats [Previous studies have shown that the inhibitory activity of PLN is attributed to its monomeric form, as seen in PLN mutagenesis experiments  or recipd by PLN , the eleold rats ,33. Moreold rats , leaving2+ is essential for ECC, and RyR2 regulates Ca2+ release from SR to the cytosol [2+ overload [2+ leakage from SR and subsequent Ca2+ overload [2+-releasing protein, IP3R, during IR suggested that its cardioprotective effect could be relevant for therapeutic strategies aimed at modulating the interaction of mitochondria and SR [2+-regulatory proteins allows Ca2+ transfer to mitochondria [2+ [2+ from the sarcoplasmic reticulum and contribute to mitochondrial Ca2+ overload. Recently, the VDAC1–citrate synthase co-immunostaining study confirmed an increase in VDAC1 per unchanged mitochondrial density [Sarcoplasmic reticulum-derived Ca cytosol . It was overload . On the overload . We havea and SR . The prochondria , where Vdria [2+ . In the  density . The pre density  with the density .2+ uptake by mitochondria [2+ uniporter multi-protein complex mediates Ca2+ uptake into the matrix and subsequently activates the synthesis of ATP [GRP75 as a modulator of the IP3R–VDAC1 complex increases the efficiency of Cachondria . The inns of ATP . Throughs of ATP  and funcs of ATP . AlthougThe limitation of this study is that experiments were performed only on male rats. Therefore, we cannot exclude that, due to hormonal effects, the tolerance to ischemic injury in female rats will differ from that observed in the present study. The presented experimental model is suitable to monitor acute ischemic injury that leads to complete recovery in time. For this reason, the phenomenon of stunning may lead to a delay in post-ischemic recovery of cardiac function, and prolonged periods of reperfusion will also be appropriate for testing.2+-handling proteins. However, the IR-induced damage was not increased during aging. These findings do not support the frequent view on the role of aging in the susceptibility to heart ischemia–reperfusion injury. Nevertheless, they suggest that SERCA2a, RyR, and other Ca2+-handling proteins as well as proteins involved in SR–mitochondria cross-talk may serve as suitable target molecules for therapy of age-associated alterations in heart function.In conclusion, our study suggests that aging is associated with a significant decrease in the abundance and function of Ca


In the absence of extracellular Ca2+, the SERCA inhibition typically leads to a decrease in ER Ca2+ with the corresponding transient increase of cytosolic Ca2+  as well as inherited diseases, such as immunodeficiency, neutropenia and tubulointerstitial kidney disease  is one of the two main reservoirs for releasable Ca cytosol   in Drosophila , which is futher activated by Ca2+ efflux from the ER. Normalization of the ER ATP/ADP ratio allows BiP to limit the Ca2+ leakage via binding to Sec61 channels and thus inactivates the regulatory circuit. However, the details of this signal transduction pathway are still somewhat controversial  and Ca2+ oscillations. Next, Pick et al. present their quantitative data and kinetics of thapsigargin-induced Ca2+ efflux from the ER and SOCE as its consequence on the Ca2+ dynamics in HEK293 cells. Parys and Van Coppenolle focus our attention on the Sec61 channel as the most abundant and ubiquitous ER Ca2+ leak channel and its various roles in health and disease. This brilliant review also introduces the various interaction partners of the Sec61 channel that are involved in tight control of the leak (including Sec62). Dagnino-Acosta and Guerrero-Hernandez add another control mechanism of the Sec61 channel, i.e., phosphorylation, which at least in smooth muscle cells is mediated by protein kinase C (PKC). Coming back to the role of the Sec61 channel and its modulators in disease, the contribution by Zimmermann et al. on tumor diseases is highly recommended to bridge the gap from bench to bedside. This paper is also an excellent introduction for two original articles by Radosa et al. as well as Körner et al. respectively, which deal with Ca2+ efflux from the ER as a novel target of anti-metastatic and anti-proliferative therapy in head and neck cancer and the oncogene SEC62 as a prognostic marker in patients with ovarian malignancies, respectively. Staying with human medicine, the Research Topic is finished off by Liu et al. who discuss the current views on the biophysical and physiological properties of the ER membrane protein PKD2, which is also termed polycystin-2 or TRPP2, and on how PKD2 contributes to ER Ca2+ homeostasis in cell physiology and to autosomal polycystic kidney disease in pathophysiology. Notably, PKD2 represents one of the additional channels for Ca2+ efflux from the ER, which be highlighted to exist in addition to the Sec61 channel in the Introduction to this editorial.In this Research Topic, renowned international experts in the area of cell biology and human medicine report on their mechanistic and medical insights into various aspects of the Ca


Aging impairs the function of the central circadian clock in mammals, the suprachiasmatic nucleus (SCN), leading to a reduction in the output signal. The weaker timing signal from the SCN results in a decline in rhythm strength in many physiological functions, including sleep–wake patterns. Accumulating evidence suggests that the reduced amplitude of the SCN signal is caused by a decreased synchrony among the SCN neurons. The present study was aimed to investigate the hypothesis that the excitation/inhibition (E/I) balance plays a role in synchronization within the network.2+) imaging, the polarity of Ca2+ transients in response to GABA stimulation in SCN slices of old mice (20–24 months) and young controls was studied.Using calcium (Ca2+ concentration, with higher Ca2+ levels in SCN neurons of old mice, indicating an alteration in Ca2+ homeostasis in the aged SCN. We conclude that the change in GABAergic function, and possibly the Ca2+ homeostasis, in SCN neurons may contribute to the altered synchrony within the aged SCN network.We found that the amount of GABAergic excitation was increased, and that concordantly the E/I balance was higher in SCN slices of old mice when compared to young controls. Moreover, we showed an effect of aging on the baseline intracellular Ca Based on molecular feedback loops, individual SCN neurons generate  rhythms . This ci rhythms . Through rhythms . Misalig rhythms . Aging p rhythms . Vice ve rhythms . UnderstIn both humans and animal models, age-related changes often lead to a reduction in behavioral activity levels and fragmented sleep–wake rhythms , a longein vivo and ex vivo studies showed a significant reduction in the amplitude of the ensemble electrical activity rhythm of the aged SCN , which is expressed in almost all SCN neurons . Althoug2+ transients in response to GABA stimulation in SCN slices of old mice (20–24 months) and young controls during the day, and tested our hypothesis that aging leads to an increment in excitatory responses to GABA. We confirmed that the E/I balance shifted towards more excitation in SCN slices of old mice compared to young controls. Furthermore, we found an effect of aging on the baseline intracellular Ca2+ concentration ([Ca2+]i) and intracellular Ca2+. The cells measured from the SCN slices of old mice showed a higher [Ca2+]i during the day compared to the cells from the young controls. Analysis of Ca2+ transients’ kinetics in response to depolarizations showed slower increase reaching higher peak amplitudes in old SCN neurons, suggesting higher buffer capacity, but also larger Ca2+ influx compared to young. This may contribute to high basal [Ca2+]i and dysfunctional Ca2+ signaling in the old SCN neurons amplifying the effect of increased E/I ratios.Here, we investigated the aging effect on the GABAergic E/I balance by measuring intracellular calcium levels. Specifically, we determined the polarity of Ca2.2.1.N = 11) and old  male C57BL/6 mice  were housed in a climate controlled environment  with full-spectrum diffused lighting with an intensity between 50 and 100 lux (Osram truelight TL) and ad libitum access to food and water throughout the experiment. The mice were kept in groups of 2–4 mice on an equinoctial photoperiod of 12:12 h light–dark (LD 12:12) cycle. Mice older than 20 months received, in addition to the regular food, hydration and nutritional gels as supportive care. The animals were kept under these conditions for at least 4 weeks prior to the ex vivo experiments. Ex vivo experiments were performed within a 4-h interval centered around the middle of the day, zeitgeber time (ZT) 6.Young  and saturated with 95% O2–5% CO2. Coronal hypothalamic slices containing the SCN (250 μm) were cut using a vibratome  and sequentially maintained in a holding chamber containing regular, oxygenated ACSF (CaCl2 increased to 2 mM and without MgCl2). The slices were incubated in a water bath (37°C) for 30 min and were then maintained at room temperature until the start of the recordings.After decapitation, brains were quickly removed and placed into modified ice-cold artificial cerebrospinal fluid (ACSF), containing (in mM): NaCl 116.4, KCl 5.4, NaH2.3.2+ indicator dye fura-2-acetoxymethyl ester . First, the slices were transferred from the holding chamber to a 35 mm petri dish and reviewed under the microscope. The side of the slide that contained (the larger part of) the SCN was placed up and the ACSF was removed from the petri dish. One drop of highly concentrated Fura-2-AM (998 μM) was placed on the SCN of each slice for 1 min after which 1 mL of a mix of ACSF containing 7 μM Fura-2-AM was added to the slices. The slices stayed in this mixture for 1 h on room temperature while maintained saturated with 95% O2–5% CO2. The slices were then rinsed four times with fresh ACSF before being transferred back into the holding chamber where they stayed another hour on room temperature. After this loading protocol, the slices were moved (one by one) to a recording chamber  mounted on the fixed stage of an upright fluorescence microscope  and constantly perfused with oxygenated ACSF (2.5 mL/min) at room temperature. The indicator dye was excited alternatively at wavelengths of 340 and 380 nm by means of a monochromator . Emitted light (505 nm) was collected by a 40x objective and detected by a cooled CCD camera , and images were acquired at 2 s intervals. It can be expected that Fura-2 will label neurons as well as astrocytes, however the morphology of Fura-2-filled SCN astrocytes is distinct from SCN neurons  was applied to identify healthy neurons, but also to determine the Ca2+ buffer capacity of the cell. Cells with at least 10% increase in [Ca2+]i in response to K+ were considered to be healthy. The experiments as well as the analysis were accomplished using imaging software .Neurons in brain slices were bulk-loaded with the ratiometric, membrane permeable Ca neurons , and thetrocytes . We are 2.4.2+ concentration was calculated. Neuronal Ca2+ responses were further analyzed using IGOR Pro . Cells with an amplitude less than 10% of baseline values in response to elevated levels of K+, or cells with instable  baselines or baselines >600 nM were excluded from analyses. These criteria will eliminate cells that are severely dysfunction or in apoptosis, but we still expect to be able to record aging-related changes in the SCN slices. Previous work has shown that aging induces rather specific loss of neuronal function  but not overall diminishing of neurons or even GABAergic signaling  and averaged per group. In total we measured 924 cells in 27 slices from 9 old mice and 1,204 cells in 26 slices from 11 young mice.Single-wavelength images were background subtracted and ratio images (340/380) were generated. Regions of interest were generated to define cells and mean ratio values were determined, from which the intracellular Caignaling . The tra2+ transients in response to high-K+ application were analyzed using a custom-made Python script (3.8.10). Basal intracellular Ca2+ concentration ([Ca2+]i) before the GABA application was represented by the mean of 10 measurements before treatment. The amplitude of [Ca2+]i transients was defined as the difference between peak and baseline value. Values extending more than three times the standard variation in the data set were excluded. The time constant of the rising phase (tau) was calculated by fitting an exponential curve into the interval between the start of the high-K+ rise  and the maximum value of [Ca2+]i for the high-K+ transient. Fitted curves with an r-squared value lower than 0.95 were excluded.Ca2+]i baselines and high K+ responses were tested using generalized estimating equations (GEE) models, with brain slice as a grouping variable. The resulting p-values underwent Bonferroni correction to account for multiple testing. When single variable comparisons were drawn between the two age groups, two-sided, unpaired t-tests with Welch’s correction were performed. Differences with p ≤ 0.05 were considered significant.Statistical analyses were performed using GraphPad Prism  and IBM SPSS statistics version 25 . The effect of age on the GABAergic response types, [Ca3.3.1.2+ transients in SCN slices from old (20–24 months) and young (2–4 months) mice (2+]i in response to GABA application (non-responders). In another subset of SCN neurons we recorded a biphasic response to GABA. The amplitude of the GABAergic responses did not differ between the old and the young SCN neurons . The percentages of the different GABAergic response types differed between old and young animals. Old SCN slices exhibited significantly more excitatory responses to GABA as compared to SCN slices of young controls . This increase in GABAergic excitation leads to an increase in the E/I balance in old SCN slices, as compared to young SCN slices . These results show that aging affects the polarity of responses to GABA.We investigated the effect of aging on the GABAergic activity in SCN neurons by recording GABA-induced single cell Cahs) mice . In each3.2.Several studies have shown that the amount of GABAergic excitation varies between the different SCN areas in rats . TherefoN = 9, young: 19.98 ± 4.14%, N = 5, p = 0.001, inhibition; old: 36.81 ± 5.53%, N = 9, young: 60.62 ± 4.89%, N = 5, p = 0.04).The calculated E/I balance was also significantly different between young and old in the posterior SCN slices . No differences in GABAergic responses or E/I balance were observed in the central part of the SCN slices of young and old mice . There were also no significant differences in GABAergic responses between the dorsal and ventral SCN, both for the old and young mice  in SCN neurons from old and young mice during the day. We found higher baseline [Ca2+]i levels in cells of the old SCN, when compared to the young SCN neurons . We also wondered if GABAergic response types were correlated to different baseline levels of [Ca2+]i. Interestingly, the age-related increase in basal [Ca2+]i was restricted to cells responding to GABA with either inhibition or excitation, but not found in non-responding cells or cells with a biphasic response to GABA. Baseline [Ca2+]i was higher in old SCN neurons that exhibited GABAergic inhibitory as well as excitatory responses, when compared to young SCN neurons .To examine whether the baseline [Ca2+ homeostasis in the SCN neurons is affected by age and the GABAergic response type is correlated to the baseline [Ca2+]i in young and old SCN. To further investigate this, we analyzed the kinetics and amplitude of the high-K+ response, which can be influenced by alteration in the intracellular Ca2+ buffering system. We found that the rise time of the Ca2+ influx increased in aging SCN neurons (p = 0.045), while there were no differences in amplitude of the peak response (p = 0.205). However, we found regional differences in amplitude of Ca2+ transients between young and old SCN with larger impact in the central and dorsal regions on the amplitude of the response . We could not find a significant change in rise time in the subregions . This implies an increased buffering capacity of the SCN causing the slower rise, but also an increased net Ca2+ influx in the central subregion leading to a larger response during excitation of the neurons.These results suggest that Ca4.2+ levels in the mammalian pacemaker. Our results demonstrate that aging affects the response polarity, and thus the function, of GABA, which is the most abundant neurotransmitter in the SCN. We measured significantly more GABAergic excitatory responses in SCN slices from old mice when compared to slices from young controls. Particularly, in the posterior part of the old SCN we found significantly more excitation and significantly less inhibition compared to the young SCN. Accordingly, we recorded increased E/I ratios in SCN slices from old mice. We also demonstrate that the baseline [Ca2+]i is higher in SCN cells from old mice, compared to young mice. This is interesting considering that Ca2+ is an important intracellular signaling molecule and critical for molecular rhythm generation  and the membrane potential (Vm)  . Proteinial (Vm) . There iial (Vm) , howeverial (Vm) . Anothernor role . Our res4.1.in vivo and ex vivo studies showed a significant reduction in the amplitude of the ensemble electrical activity rhythms of the aged SCN that is likely the consequence of decreased synchronization within the SCN network  approved the protocol .AHOE, SM, and JHM designed the study. AHOE, PdTG, and AC performed the experiments. PdTG developed analysis tools for calcium transients. AHOE, PdTG, AC, and AD performed the analysis of the data. AHOE, SM, JHM, and PdTG wrote the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by funding from Velux Stiftung (project grant 1029 to SM) and by funding from ERC (adv grant 834513 to JHM). This study is part of the doctoral thesis by AHOE .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In response to the changing availability of nutrients and oxygen in the bone marrow microenvironment, acute myeloid leukemia (AML) cells continuously adjust their metabolic state. To meet the biochemical demands of their increased proliferation, AML cells strongly depend on mitochondrial oxidative phosphorylation (OXPHOS). Recent data indicate that a subset of AML cells remains quiescent and survives through metabolic activation of fatty acid oxidation (FAO), which causes uncoupling of mitochondrial OXPHOS and facilitates chemoresistance. For targeting these metabolic vulnerabilities of AML cells, inhibitors of OXPHOS and FAO have been developed and investigated for their therapeutic potential. Recent experimental and clinical evidence has revealed that drug-resistant AML cells and leukemic stem cells rewire metabolic pathways through interaction with BM stromal cells, enabling them to acquire resistance against OXPHOS and FAO inhibitors. These acquired resistance mechanisms compensate for the metabolic targeting by inhibitors. Several chemotherapy/targeted therapy regimens in combination with OXPHOS and FAO inhibitors are under development to target these compensatory pathways. Thus, novel therapeutic strategies that are more effective and carry a lower risk of organ damage than current treatments are urgently needed.Acute myeloid leukemia (AML) comprises a highly aggressive, biologically heterogeneous group of hematopoietic disorders involving one or more cytogenetically or molecularly abnormal cell clones. It is primarily a disease of older adults. The standard of care for relapsed and refractory AML has progressed minimally in the past 30 years, with survival rates of less than 12% for patients over 65 years old-4. Highly proliferative AML cells, drug-resistant AML cells, and leukemia stem cells (LSCs) that remain quiescent have been shown to depend on oxidative phosphorylation (OXPHOS). LSCs differ from bulk leukemia cells in that they possess stem cell characteristics including abnormal self-renewal capacity and drug resistance. Persistence of LSCs in the BM microenvironment after chemotherapy is considered an important factor in AML relapse. Both quiescent AML cells and LSCs survive through metabolic activation of fatty acid oxidation (FAO) along with OXPHOS in their mitochondria. Hence, the reprogramming of energy metabolism processes in AML cells is recognized as a potential therapeutic target. Inhibition of OXPHOS and FAO can disrupt metabolic homeostasis, increase reactive oxygen species (ROS) production, and cause apoptosis in AML cells,6,7. However, inhibition of this altered energy metabolism triggers various adaptive mechanisms in AML cells through their interaction with BM stromal cells. Thus, the BM microenvironment provides a setting in which secondary resistance to OXPHOS inhibition can develop, thereby contributing to the survival of AML cells. Therefore, strategies combining chemotherapy and specific molecular targeted therapy may have promise for eliminating BM-resident AML cells and LSCs.AML cells always face two major metabolic challenges: their high rate of proliferation imposes increased bioenergetic demands, and fluctuations in the availability of external nutrients and oxygen in the bone marrow (BM) microenvironment threaten cellular survival. In the BM, AML cells constantly modulate their metabolic state to adapt to this fluctuating microenvironment, shifting between quiescent, proliferative, and differentiated statesIn this review, we summarize the current state of knowledge about mitochondrial OXPHOS and fatty acid metabolism in residual AML cells in the BM microenvironment. We further describe the molecular mechanism by which AML cells acquire resistance to OXPHOS and FAO inhibitors. Finally, we evaluate potential therapeutic regimens combining OXPHOS and FAO inhibitors to target the metabolic vulnerabilities of BM-resident chemoresistant leukemia cells and LSCs.-9.Whereas circulating AML cells are effectively eliminated by drug treatment, AML cells residing in the BM acquire resistance to chemotherapy. The BM microenvironment provides growth factors for leukemic cells, promotes immunosuppression, and supports leukemic cell survival. In response, leukemia cells adapt their metabolic state to this constantly changing environmentEnergy metabolism encompasses the molecular pathways whose products are involved in cellular energy production in the form of ATP. In leukemic cells, energy metabolism relies on OXPHOS and associated catabolic pathways, including glycolysis and fatty acid metabolism. The energy required for ATP production is produced by the mitochondrial potential, which causes protons to reenter the mitochondria through complex V. Fatty acid metabolism also supplies acetyl-CoA to the tricarboxylic acid (TCA) cycle through FAO .,10. Actively proliferating AML cells respond to their increased energy and substrate demands via upregulation of OXPHOS, glycolysis, and related biochemical pathways. In turn, their bioenergetic efficacy strongly depends on extrinsic signals from the microenvironment.Recently, the molecular mechanisms by which AML cells undergo metabolic reprogramming and those underlying the antileukemic efficacy of OXPHOS inhibitors have been demonstrated-14. Indeed, AML cells so strongly depend on OXPHOS for metabolism that they might cause hypoxia in the BM environment. These hypoxic niches are expanded, in part, through activation of the transcription factor hypoxia-inducible factor 1α (HIF-1α),15. Transcriptional complexes often include metabolic enzymes, which locally supply substrates and cofactors to these complexes. For this reason, OXPHOS itself and the transcription factors that regulate it are attractive targets for novel therapeutic interventions.The leukemic BM microenvironment is generally hypoxic; during disease progression, hypoxic areas in the BM expand,18 and subsequently validated in solid tumors. Across cancers, cancer stem cells share two important features: they can self-renew and produce differentiated progeny. The OXPHOS-dependent survival mechanism of LSCs is common to several solid tumor stem cells. For example, pancreatic cancer stem cells use OXPHOS for survival by accumulating the transcription coactivator peroxisome proliferator-activated receptor coactivator-1α (PGC-1α), which enhances mitochondrial biogenesis and the oxygen consumption rate and is sensitive to inhibitors of mitochondrial respiration. Reliance on OXPHOS has also been observed in other solid-tumor stem cells, including those in brain and breast cancers.The persistence of LSCs and treatment-resistant AML cells in the BM remains the major cause of failure to eradicate AML. Cancer stem cells were initially identified in AML. The transcriptional and epigenetic signatures of leukemia-initiating LSCs are largely mutation-independent-25. Instead, rewired cellular metabolism has been increasingly recognized to play a significant role in LSC maintenance and treatment resistance in AML. In LSCs, several components of the electron transport chain (ETC) complexes I and V have been shown to be more abundant than in normal hematopoietic stem cells (HSCs). Notably, AML LSCs overexpress antiapoptotic BCL-2, which has been shown to regulate ATP/ADP exchange across the mitochondrial membrane by facilitating regulation of voltage-dependent anion channels and adenine nucleotides,,29 preventing the loss of coupled mitochondrial respiration during apoptosis.Several recent studies have revealed how AML LSCs exploit OXPHOS. However, several clinical trials have shown that the efficacy of these OXPHOS inhibitors is limited,32. In trials of several types of solid tumors, one putative complex I inhibitor, carboxyamidotriazole, had no clinical benefit. Mouse studies of BAY87-2243, a novel complex I inhibitor, demonstrated antitumor activity and no toxic effects, but the phase I trial was terminated because of unexpected toxic effects. These findings indicate that OXPHOS inhibitors have a narrow therapeutic window and emphasize the need to better understand how the BM microenvironment enables AML cells to become resistant to the metabolic stress caused by OXPHOS inhibition.Understanding the crosstalk between AML cells and their microenvironment is critical to targeting the pathways involved in the metabolic reprogramming of chemoresistant AML cells and LSCs. In preclinical studies, most AML models responded to inhibition of OXPHOS via targeting of ETC complex Iin vivo models have shown that this transfer reestablishes respiration and promotes tumorigenesis. In OXPHOS-dependent AML cells, OXPHOS inhibition induced formation of tunneling nanotubes that enabled this mitochondrial DNA trafficking from BM stroma cells to AML cells. In the formation of tunneling nanotubes, a filopodium-like protrusion is extended from one cell to another. This process is positively regulated by activation of motor proteins such as Rho GTPases through actin polymerization,37 and filopodia formation through focal adhesion[38]. In addition, a recent study showed that a transmembrane complex gap junction channel opens under ROS-induced oxidative stress via PI3K-Akt activation to enable the transfer of mitochondrial DNA from stromal cells in the BM to HSCs [.One such mechanism occurring in the tumor microenvironment is the horizontal transfer of mitochondrial DNA from host to tumor cells. Studies using  to HSCs [39].,40. As the process by which damaged mitochondria are segregated for elimination by autophagy, mitochondrial fission is central to mitophagy-44. Cellular metabolism and cell survival require efficient mitophagy,45. Because of the centrality of these processes in the maintenance of mitochondrial function, both mitochondrial fission and crosstalk with BM stroma cells via tunneling nanotubes may be another mechanism by which AML cells develop resistance to OXPHOS inhibition.OXPHOS inhibition-induced horizontal transfer of mitochondria from BM stromal cells to AML cells is accompanied by endogenous mitochondrial fission and elimination of damaged mitochondria by mitophagy, both of which contribute to AML cell survival,48. However, LSCs are able to maintain their stemness by mitochondrial fission and mitophagy, which balance mitochondrial functions such as energy production, ROS generation, and apoptosis regulation. Given these competing pressures, LSCs have a fragile mitochondrial network. Thus, blocking both OXPHOS and other metabolic pathways is a promising strategy for overcoming OXPHOS resistance associated with the BM microenvironment. Two possible targets include the enzyme ASS1 and the lipid metabolism protein LRP1, both of which are overexpressed in OXPHOS inhibitor-treated AML cells in vivo. ASS1 is essential for the biosynthesis of arginine, and LRP1 contributes to hemin-induced autophagy in leukemia cells,51. Further investigations will improve our understanding of how these enzymes shape the responses of LSCs to the metabolic and energetic effects of OXPHOS inhibition.LSCs have low rates of energy metabolism and cannot upregulate glycolysis after OXPHOS inhibition. Thus, they are particularly sensitive to OXPHOS blockade. However, metformin carries a risk of severe lactic acidosis,54, which is a safety concern for cancer patients. In addition, because high OXPHOS levels play a key role in cytarabine resistance, treatments combining cytarabine with OXPHOS inhibitors might be more effective than monotherapy with either type of agent. Recently, a novel lipoate analog, devimistat, an inhibitor of two key TCA cycle enzymes, the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes,56, showed a satisfactory safety profile in AML patients. In a phase I study, patients with relapsed or refractory AML who received a combination of devimistat with cytarabine and mitoxantrone had a complete remission rate of 50%.Several repurposed drugs have been shown to inhibit OXPHOS. Biguanides, including metformin, are routinely used for diabetes treatment and have been proposed for use in cancer because they inhibit complex I of the ETC in cancer cells. These are encouraging findings that indicate that this approach would be particularly effective for older patients with the highest unmet medical needs. In sum, the judicious use of novel OXPHOS inhibitors in combination treatments may add to armamentarium of currently available therapeutics.In a phase II study of this combination, responses were observed in older patients but not in younger patients. In addition, RNA sequencing analysis showed a decrease in expression of mitochondria-related genes with aging, suggesting that age-related reduction in mitochondrial quality may be related to devimistat response. While it was reported that BM adipocytes occupy approximately 60% of the BM in 65-year-old individuals, the development of BM adipocytes varies across different skeletal regions, and single-point iliac biopsy may not represent the BM environment of the skeletal system containing the red marrow and yellow marrow. In a previous study, leukemic cells have been shown to colonize in both red and yellow marrow regions, adhere to the cortical bone in the spine, and have enhanced activity in the proximal/distal femur. In addition, radiation therapy accelerates the differentiation of mesenchymal stem cells into adipocytes in BM. Such temporal and spatial changes in the BM microenvironment may play a key role in leukemia’s dynamic adaptation of FAO and in leukemia cells’ interactions with BM stromal cells.Adipocytes in the BM microenvironment support the survival of several types of tumor cells by stimulating FAO through fatty acid transfer. FAO is metabolically activated to promote leukemic cell survival by remodeling and lipolysis of BM adipocytes. FAO is an essential source of mitochondrial NADH and FADH2 for the ETC and provides acetyl-CoA to the TCA cycle to produce ATP. BM adipocytes supply long-chain fatty acids, which are then taken up into the cytoplasm via the scavenger receptor CD36,66. Fatty acids activation is a two-step reaction. In the first step, the fatty acids form acyl-CoA in the cytoplasm. Then, FAO breaks down acyl-CoA to form acetyl-CoA in the mitochondria. Carnitine O-palmitoyltransferase 1 (CPT1) catalyzes a rate-limiting step of FAO; this enzyme conjugates fatty acids to carnitine, which is required for fatty acids to translocate from the cytoplasm to the mitochondria. The internalized fatty acids are further transferred to the AML cell nucleus by the lipid chaperone fatty acid-binding protein 4 (FABP4). In the nucleus, the fatty acids ligate to peroxisome proliferator-activated receptor γ (PPAR). Activated PPAR induces downstream target genes, including CD36, FABP4, and the antiapoptotic BCL2 [.AML cells generally obtain fatty acids for FAO from the extracellular microenvironment through lipolysis of stored triglyceridestic BCL2 [69].,71. Therefore, CD36 and CPT1 are potential targets for AML. A CD36 neutralizing antibody inhibited metastasis of human melanoma and breast cancer cells, and inhibition of CPT1 caused mitochondrial damage leading to cell death in primary AML cells.As with AML cells, the specific BM microenvironment created by the interaction between LSCs and stromal adipocytes supports the metabolic demands of LSCs. LSCs induce adipocyte lipolysis, which drives FAO in LSCs and facilitates their survival,74. Ovarian cancer cells also survive and proliferate in an adipocyte-rich microenvironment. When primary human omental adipocytes were co-cultured with ovarian cancer cells, the adipocytes underwent lipolysis, and FAO was induced in the cancer cells. These processes are mediated by adipokines including interleukin-8 and by upregulation of FABP4 both in adipocytes and ovarian tumor cells. In studies of leukemia, AML cells co-cultured with BM adipocytes exhibited higher levels of FABP4, and knockdown of FABP4 prolonged survival in a mouse model of leukemia. Thus, FABP4’s critical role in cancer cell survival involves its interactions with adipocytes.FABP4 is important in FAO and cancer cell survival in both solid and hematologic cancers. Adipocytes are known to serve as fatty acid reservoirs in breast cancer and melanoma,77. Ovarian cancer cells have been found to upregulate HSL phosphorylation, thereby stimulating the release of free fatty acids from adipocytes. AML blasts also induce HSL phosphorylation and, thus, activation of lipolysis in BM adipocytes.Activation of β-adrenergic receptors, along with a G protein-coupled cascade that stimulates the lipolytic enzyme hormone-sensitive lipase (HSL), induces lipolysis of adipocytes. AMPK, an important modulator of energy metabolism, is activated upon ATP depletion. Its functions include upregulation of fatty acid uptake, FAO, and regulation of autophagy,79. Levels of adiponectin, much of which is supplied by BM adipocytes, have been shown to increase during cancer therapy, and to promote chemotherapy resistance in myeloma cells via inducing adipokine secretion of adipokines and activating AMPK-dependent autophagy,82. AMPK also positively regulates responses of an antiapoptotic chaperone heat shock protein that binds to denatured and unfolded proteins and promotes protein refolding or degradation to support AML cell survival. In sum, leukemic cells often rely on fatty acids when they undergo metabolic stress, and the NADH and FADH2 generated by FAO support ATP production, redox homeostasis, biosynthesis, and cell survival.BM adipocytes also increase AML cells’ expression of adiponectin and its downstream target, AMP-activated protein kinase (AMPK), a stress response kinase. LSCs rely on fatty acid uptake and consumption to shape their adaptation to the conditions of the BM microenvironment, their response to drugs, and their development of drug resistance. Several such mechanisms have been identified. Mitochondrial uncoupling in AML cells negatively regulates Bak-dependent mitochondrial permeability transition. In a study using samples from patients with relapsed AML, LSCs acquired the ability to counteract the loss of amino acid metabolism by upregulating FAO. Specifically, this mechanism may underlie the development of resistance to treatment with azacitidine/venetoclax, a common induction regimen used mainly in older patients with AML,86. In addition, a preclinical study demonstrated that cytarabine-resistant AML cells had enhanced FAO and OXPHOS. Thus, targeting the metabolic vulnerabilities of chemoresistant LSCs, such as their dependence on FAO, may be a useful strategy for eradicating these cells.FAO is involved in the interactions between LSCs and BM stromal cells. Numerous studies have reported the anti-AML effect of inhibition of CPT1, the major rate-limiting enzyme in FAO,84,87,88. CPT1 positively controls FAO by conjugating fatty acids with carnitine to transfer fatty acids into the mitochondrial matrix. Etomoxir is a pharmacological inhibitor of CPT1A, one of the isoforms of CPT1, frequently used to block free fatty acids from entering the mitochondria via CPT1. Although the clinical use of etomoxir has ceased because of adverse effects, the CPT1 inhibitor perhexiline can sensitize breast cancer cells to paclitaxel, and other CPT1 inhibitors are currently being investigated for use in cancer therapy. The CPT1A inhibitor ST1326 has been shown to cause cell growth arrest, mitochondrial damage, and apoptosis in AML cells in a dose- and time-dependent manner. Another novel FAO inhibitor, avocatin B, which is derived from avocados, decreased NADPH levels that were increased by FAO through acetyl-CoA and NADH production, inducing ROS-dependent cell death in AML cells,94. Finally, the fatty acid synthase inhibitor orlistat has induced apoptosis in leukemic cells.FAO inhibition disrupts metabolic homeostasis, increases ROS levels, and induces expression of the integrated stress response mediator ATF4 in AML cells, all of which contribute to apoptosis. AYNE reduced mitochondrial respiration by altering FAO, which led to reduced ATP production in AML cells, even though AYNE also moderately upregulated glycolysis. In a mouse model, pharmacological inhibition of VLCAD with AYNE significantly reduced the repopulation potential of leukemia cells and was well tolerated. Notably, normal HSCs compensate for this reduced replicative capacity through glycolysis which maintains their ATP levels and thus their viability,96. These findings demonstrate the importance of focusing on the specific metabolic vulnerabilities of residual AML cells and LSCs that survive chemotherapy-induced stress. Unfortunately, only a few FAO inhibitors have advanced from preclinical to clinical studies [The intramitochondrial FAO enzyme very long-chain acyl-CoA dehydrogenase (VLCAD) is critical in supporting both FAO and OXPHOS in AML cells and LSCs. Recently, preclinical studies have demonstrated the antileukemia activity of a novel small-molecule VLCAD inhibitor, a polyhydroxylated fatty alcohol with a terminal alkyne (AYNE) studies .. This compensatory induction of glycolysis is a sustained source of ATP to AML cells and, in turn, induces substantial lactate production. Similarly, FAO-deficient Abcb11-knockout mice exhibited high FABP4 and CD36 expression and free fatty acid uptake. In sum, it is clear that FAO inhibition initiates several different adaptive mechanisms that promote AML cell survival in the BM microenvironment.Because AML is heterogeneous and multiclonal, blocking only one part of this complex metabolic system may allow residual cells to adapt metabolically. For instance, it has been shown that BM-derived stromal cells, including adipocytes, diminish the antileukemia effects of FAO inhibitors in AML cells by increasing glycolysis and glucose and free fatty acid uptake. For example, FAO and OXPHOS are increased in cytarabine-resistant AML cells; FAO inhibition with etomoxir induced a metabolic shift from high to low OXPHOS, sensitizing the cells to cytarabine. Similarly, the combination of avocatin B and cytarabine synergized to enhance ROS production and induce apoptosis in AML cells co-cultured with BM adipocytes. The role of avocatin B in apoptosis induction was attributed to activation of endoplasmic reticulum stress-induced ATF4. These findings suggest that AML cells treated with cytarabine exhibit increased dependence on FAO, which may account for the synergism of cytarabine and FAO inhibitors.For this reason, treatment options based on combination regimens have been tested. Although FAO inhibition alone can trigger compensatory activation of other metabolic pathways, FAO inhibitors can also synergize with conventional antitumor agents such as paclitaxel and FAO for survival. Compared to healthy HSCs, AML cells and LSCs are more susceptible to mitochondrial stress because their respiratory chain reserve capacity is lower. These characteristic differences in the metabolism of AML cells and their normal hematopoietic-cell counterparts represent a specific vulnerability of leukemia cells and therefore are drawing a great deal of attention as targets for AML therapy. The results of studies in preclinical models using agents that target fatty acid metabolism have been encouraging.AML cells and LSCs both strongly depend on the production of mitochondrial biomass and on OXPHOSin vivo studies and clinical trials have shown that the use of metabolic inhibitors alone is ineffective both because of their narrow therapeutic window and because of these adaptive mechanisms. Alternatively, inhibition of FAO and other metabolic mechanisms along with conventional chemotherapy or targeted therapy may synergistically eradicate chemotherapy-resistant AML cells present in the BM.Although they are vulnerable to the targeting of their metabolic pathways, AML cells, drawing on microenvironmental factors, can adapt to metabolic stress by activating metabolic bypass processes. Several In conclusion, understanding AML cell metabolism in the specific context of the BM microenvironment is crucial to improving therapies for AML. Because characteristics of the BM microenvironment enable the acquisition of resistance to OXPHOS and FAO inhibitors, drug combination strategies that interfere with these adaptations are needed. A more comprehensive understanding of the mechanisms of AML cell metabolism in future studies may reveal new treatment options targeting OXPHOS and FAO, enhance the efficacy of chemotherapeutic agents that target related pathways, reduce the toxicity of these agents, and enable the translation of new combinations of agents into clinically applicable treatment strategies.


Action potential records obtained using intracellular microelectrodes demonstrated that EMPA reduced the action potential duration at 30%, 50%, and 90% repolarization and arrhythmogenic events in rat and human cardiomyocytes under normoxia and hypoxia. Analysis of Ca2+ transients using Fura-2-AM and contractility kinetics showed that EMPA increased Ca2+ transient amplitude and decreased the half-time to recover Ca2+ transients and relaxation time in rat neonatal cardiomyocytes. We also observed that the combination of EMPA with the Na+/H+ exchanger isoform 1 (NHE1) inhibitor cariporide (10 µM) exerted a more pronounced effect on Ca2+ transients and contractility than either EMPA or cariporide alone. Besides, EMPA, but not cariporide, increased phospholamban phosphorylation at serine 16. Collectively, our data reveal that EMPA reduces arrhythmogenic events, decreases the action potential duration in rat neonatal and human cardiomyocytes under normoxic or hypoxic conditions, and improves cytosolic calcium handling at least partially independent of NHE1. Moreover, we provided further evidence that SGLT2 inhibitor-mediated cardioprotection may be partly attributed to its cardiomyocyte electrophysiological effects.The antidiabetic agent class of sodium-glucose cotransporter 2 (SGLT2) inhibitors confer unprecedented cardiovascular benefits beyond glycemic control, including reducing the risk of fatal ventricular arrhythmias. However, the impact of SGLT2 inhibitors on the electrophysiological properties of cardiomyocytes exposed to stimuli other than hyperglycemia remains elusive. This investigation tested the hypothesis that the SGLT2 inhibitor empagliflozin (EMPA) affects cardiomyocyte electrical activity under hypoxic conditions. Rat neonatal and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes incubated or not with the hypoxia-mimetic agent CoCl Cardiovascular outcome trials indicated that gliflozins induce a striking reduction in death risk from cardiovascular causes and heart failure hospitalization in patients with heart failure regardless of the presence5 or absence8 of type 2 diabetes. Numerous mechanisms have been proposed to explain the cardiovascular benefits of SGLT2 inhibitors supporting the concept that these agents act in a multifactorial multiorgan-beneficial fashion13. Notably, in the trials, the cardioprotection conferred by the gliflozins occurred early, and these benefits continued throughout the studies. Moreover, clinical trial data and meta-analysis revealed that SGLT2 inhibitors might reduce the risk of ventricular arrhythmias and sudden cardiac death in heart failure patients16. However, to the best of our knowledge, the effects of SGLT2 inhibitors on the cardiomyocyte electrophysiological properties, especially in response to stimuli other than hyperglycemia, have not been fully elucidated.Sodium-glucose cotransporter 2 (SGLT2) inhibitors, also known as gliflozins, are a novel class of antidiabetic agents that limit the reabsorption of glucose in the renal proximal tubule, leading to glycosuria and decreased hyperglycemia18, which may be associated with increased intracellular concentrations of sodium ([Na+]i) and calcium ([Ca2+]i) in the myocardium19. Elevated levels of [Ca2+]i induce the spontaneous release of calcium from the sarcoplasmic reticulum (SR), leading to the generation of a transient inward current responsible for delayed afterdepolarizations (DADs)20. In this regard, Janse and colleagues have reported that increased spontaneous calcium release from SR observed in isolated myocytes is associated with ventricular tachycardia in rabbits with heart failure21. Furthermore, a reduction in [Ca2+]i secondary to a decrease in [Na+]i is related to the decline in spontaneous calcium release from SR and arrhythmic events24.Ventricular arrhythmias can occur after acute ischemia due to the prolongation of action potential duration (APD)+ binding site of the Na+/H+ exchanger isoform 1 (NHE1)25, demonstrating that the direct cardiac effects of the gliflozins may be mediated via SGLT2-independent pathways. Cardiac NHE1 activity is relatively quiescent under basal conditions26, but it is stimulated by the intracellular acidification that occurs during myocardial ischemia resulting in a reversal of the Na+/Ca2+ exchanger and accumulation of calcium in the cytosol28. Thus, SGLT2 inhibitor-mediated NHE1 inhibition would act to lessen [Ca2+]i, thereby improving cytosolic calcium handling. However, it remains to be determined whether the effects of SGLT2 inhibitors on cytosolic calcium handling can be attributed to NHE1.Molecular docking studies revealed that SGLT2 inhibitors have high affinity to the extracellular Na2+ transient, contraction-relaxation waves, and on posttranslational modulation of phospholamban, a key regulator of SR Ca+ sequestration29.Based on these observations, the present study tested the hypothesis that empagliflozin (EMPA) affects cardiac electrical activity in non-hyperglycemic cardiomyocytes. More specifically, we investigated the direct impact of EMPA treatment on action potential recordings and spontaneous arrhythmogenic events in rat and human cardiomyocytes subjected to hypoxia. Furthermore, we investigated the effect of EMPA in association with the NHE1 inhibitor cariporide on cardiomyocytes CaP < 0.001; APD50: 150 ± 3 vs. 131 ± 3 ms, P < 0.001; APD90: 292 ± 4 vs. 259 ± 5 ms, P < 0.001)   and hypoxia   and hypoxia  . Figure 2+]i transients from cells subjected to normoxia and hypoxia. Baseline Ca2+ levels were similar in all groups in normoxic  and hypoxic  conditions. Compared with vehicle treatment, 24 h of EMPA exposure significantly increased the amplitude and reduced the half-time for the recovery of [Ca2+]i transients in cardiomyocytes subjected to normoxia and hypoxia  and total phospholamban (PLN) as well as the ratio of PS16-PLN to total PLN. As seen in Fig. To explore additional mechanisms underlying the effects of EMPA on cytosolic Ca2+]i transients. Moreover, EMPA increased cardiomyocyte relaxation kinetics under either normoxia or chemical hypoxia. Notably, the effects of the combination of EMPA with the NHE1 inhibitor cariporide on calcium transient and cardiomyocyte contractility were more pronounced than those of either EMPA or cariporide alone.This study provides novel experimental evidence that EMPA directly affects the electrophysiological properties of cardiomyocytes under non-hyperglycemic conditions. More specifically, we found that EMPA reduces arrhythmogenic events in rat and human cardiomyocytes under hypoxia, demonstrated by a decrease in the percentage of cells with irregular beating rhythms and/or absence of DADs or triggered activity in the electrical potential records. These anti-arrhythmogenic effects were accompanied by a decline in the action potentials' duration and a decrease in the half-time for the recovery of i49, we investigated whether EMPA affects Ca2+ transients. We observed that EMPA treatment increased the amplitude and reduced the half-time for the recovery of [Ca2+]i transients and the relaxation time under hypoxic and normoxic conditions. In support of our results, Azam and colleagues showed that EMPA mitigated cardiac Ca2+ dysregulation in an ex vivo model of ischemia–reperfusion50. In addition, EMPA has also been reported to increase the Ca2+ transient amplitude in failing ventricular cardiomyocytes of the transverse aortic constriction (TAC) heart failure model and healthy cells42. Notably, we observed a significant increase of PLN phosphorylation at serine 16 residue, a common regulatory mechanism for upregulating SERCA activity51. The finding that SERCA2 overexpression in rabbit cardiomyocytes is associated with lower after-contraction events52 further supports that the observed improvement of Ca2+ decay kinetics and potential protection against SR Ca2+ leak may be a relevant mechanism accounting for decreased DAD-like events. It is worth mentioning that Ca2+ decay acceleration induced by EMPA might not only contribute to protecting cardiomyocytes from arrhythmogenic events, but it might also be a vital component accounting for the improvement of cardiac relaxation ability observed in experimental and clinical SGLT2 inhibitor studies55.Changes in the morphology of action potentials can lead to arrhythmias and, in more severe cases, sudden cardiac death2+ transients. One potential explanation to reconcile these results is that EMPA may induce posttranslational modification of contractile proteins and consequently modulate the sensitivity of these proteins to calcium. This hypothesis arises from the Pabel and colleagues' study, which showed that EMPA acutely reduced the diastolic tension of isolated cardiac muscles from patients with preserved ejection fraction (HFpEF) without affecting Ca2+ transient parameters53. These authors observed that the phosphorylation pattern of contractile proteins was rescued by acute EMPA treatment in human and rat HFpEF samples. Interestingly, the increase in protein phosphorylation induced by EMPA was observed for some contractile proteins, even in non-failing human and murine hearts53. Consistent with Pabel's study, Kojin and colleagues found that acute EMPA treatment of heart tissue from obese rats, HFpEF rats, and HFpEF patients was associated with an improvement of passive cardiomyocyte stiffness and an increase of phosphorylation status of different contractile proteins at consensus sites for proteins kinase A (PKA) and G (PKG), including the cardiac troponin I phosphorylation at serine 23/24 residues56. As demonstrated by Rao and colleagues, phosphorylation of cardiac troponin I at serine 23/24 is associated with decreased interaction between cardiac troponin I and C, reduced contractile kinetics, and acceleration of the slow phase of relaxation57. Accordingly, we observed an increase of phospholamban phosphorylation at serine 16 residue, a target for PKA/PKG cascades51, suggesting that EMPA may upregulate signaling pathways involved in the modulation of contractile machinery kinetics under our experimental model and conditions. Such modulation of contractile proteins not only explains why we observed a neutral effect of EMPA in the contraction phase but also would cooperate with faster Ca2+ decay kinetics to improve diastolic function observed in EMPA-treated cardiomyocytes.Intriguingly, we noted that EMPA-treated cardiomyocytes did not accelerate contraction phase kinetics, albeit presenting higher and faster Ca2+ transients, particularly in Ca2+ decay kinetics, may involve the potential modulation of NHE1 by SGLT2i60. The inhibition of NHE1 by EMPA has been postulated to decrease [Na+]i58 and thus change chemical gradients for Na+:Ca2+ exchange between cytosol and mitochondria and between cytosol and extracellular fluid28. However, Chung and colleagues61 found that EMPA, over a wide range of concentrations, does not inhibit NHE1 activity in isolated rat ventricular cardiomyocytes or Langendorff-perfused beating mouse hearts61. In this controversial scenario, we evaluated how Ca2+ transients would be affected by the NHE1 inhibitor cariporide alone or in combination with EMPA. Unexpectedly, acute treatment with cariporide did not affect diastolic Ca2+ levels  but enhanced Ca2+ transient peaks and improved Ca2+ decay and relaxation kinetics. Similarly, Li et al.62 observed that cariporide induced higher Ca2+ transients in isolated cardiomyocytes from healthy adult mice without affecting diastolic Ca2+ levels. This controversy might be explained by the simultaneous effects of cariporide on lowering [Na+]i and pHi. As postulated by Mattiazzi and colleagues, acidosis might affect Ca2+ handling and, thus, contraction-relaxation dynamics via protein phosphatase-1 inhibition, leading to increased phosphorylation of different proteins, including Ca2+/calmodulin-dependent kinase II (CaMKII) and PLN at threonine 17 residue63. Regardless of the underlying mechanism, we noticed that EMPA-mediated Ca2+ decay and relaxation kinetics improvements persisted even in cariporide-treated cells. Albeit an inhibition of NHE1 by EMPA may not be ruled out in the present study, our results suggest that SGLT2 inhibitors might also recruit an additional NHE1-independent mechanism, which is supported by the observation of EMPA-induced increased PS16-PLN.An additional consideration about the effects of EMPA on Ca2+]i transient recovery, and improving relaxation kinetics, at least in part by an NHE1-independent pathway. Therefore, our study provides further evidence that the cardioprotection conferred by SGLT2 inhibitors may partially be attributed to its cardiomyocyte electrophysiological effects.In summary, the present study demonstrates that EMPA influences electrical properties in rat and human cardiomyocytes, reducing arrhythmogenic events, enhancing . After that, the cells were preplated in 100-mm culture dishes for 45 min to reduce the fibroblast content. The supernatant, after preplating, was centrifuged at 230×g for 5 min. Cells were resuspended in a culture medium with 1% bromodeoxyuridine (BrdU), counted, plated on laminin- or fibronectin-coated culture surfaces depending on the experiment, and incubated at 37 °C in a humidified atmosphere containing 5% CO2.Ventricular cardiomyocytes were obtained from 1- to 2-day-old neonatal Wistar rat hearts (n = 137) as previously described2)  (added to new culture medium) for 4 h. Then, the cells were treated with vehicle  or 1 µM EMPA  (added to new culture medium) for 24 h. The EMPA concentration (1 µM) was in the range of the typical plasma concentration during the 25 mg q.d. oral treatment of patients65. After that, experiments were conducted to analyze electrical potentials, mRNA , protein expression, Ca2+ transients, and contractility. For Ca2+ transients and contractility experiments, cells were incubated with the NHE1 inhibitor cariporide (10 µM) for 20 min in combination or not with EMPA. This concentration was chosen based on previous studies which show that 10 µM cariporide effectively inhibits NHE1-mediated Na+/H+ exchange in ventricular myocytes isolated from different species66. The study design is depicted in Supplementary Fig. After 2 days, the culture medium was changed. Four days after cell plating, flow cytometric analyses were performed to evaluate the efficiency of the cardiomyocyte isolation protocol . Hypoxia conditions were chemically induced using 200 μM cobalt chloride . Then, the cells were cultured on a murine embryonic feeder layer in DMEM/F12, GlutaMAX supplement, with 20% KSR, 1% penicillin–streptomycin, 100 µM NEEA, 0.1 mM β-mercaptoethanol, 10 ng/ml of bFGF. After confirming pluripotent state67, the iPSCs were maintained as feeder-free culture by using 1% Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Thermo Fisher Scientific) in StemFlex medium (Thermo Fisher Scientific) with 1% penicillin–streptomycin.All procedures were performed in accordance with relevant guidelines and regulations and approved by the National Institute of Cardiology ethics review board under number 27044614.3.0000.5272. The iPSCs used in this study were obtained from reprogramming erythroblasts derived from a healthy individual, as described previously68. iPSCs were dissociated with TrypLE™ Express Enzyme (Thermo Fisher Scientific), and 4 × 105 iPSCs were seeded in each well of a 48-well plate coated with 1% Geltrex™ and cultured for 2 days. The Wnt signaling was activated on day 0 of the protocol by treatment with 9 μM of CHIR99021 (Tocris) diluted in basal medium (RPMI 1640 supplemented with B-27 without insulin (Thermo Fisher Scientific)). On days 1 and 2, cells were maintained in basal medium. On days 3 and 4, cells were treated, respectively, with 10 μM and 5 μM of XAV939 (Tocris) for Wnt inhibition. On day 5, XAV939 was removed from the medium, and, finally, on day 7, the medium was changed for RPMI plus B-27 with insulin (Thermo Fisher Scientific). iPSC-derived cardiomyocytes were cultured until day 24 with medium changes every 3 days. Lactic acid 4 μM (Sigma-Aldrich) in RPMI 1640 with insulin without glucose/B-27 was added to the cell culture media at differentiation day 24 and maintained for 6 days with medium changes every 2 days. From day 28, iPSC-derived cardiomyocytes were dissociated with trypsin–EDTA, and 5 × 105 cells were transferred in the central region of the 35-mm plate coated with matrigel. From day 30, cell cultures were subjected to normoxia or chemical hypoxia induced by 250 µM CoCl2 for 4 h, as previously established by our laboratory69. Then, the cells were treated with vehicle (DMSO) or 1 µM EMPA for 24 h. After that, cells were used for action potential recordings.The differentiation protocol of iPSCs into cardiomyocytes was based on the modulation of Wnt pathway signaling described by Lian and colleagues5 cardiomyocytes derived from neonatal rats (n = 3 for each of 3 independent experiments) or iPSCs (n = 3 for each of 2 cardiac differentiation), respectively, were transferred to the stage of an inverted microscope . The action potential recording method was adapted from a previously described protocol67. Cells were superfused with Tyrode's solution containing 150.8 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 11 mM D-glucose, and 10 mM HEPES (pH 7.4 adjusted with NaOH). The superfusion was carried out at 37.0 ± 0.5 °C using a temperature controller (Harvard Apparatus) saturated with oxygen at a perfusion flow rate of 0.5 ml/min (Minipuls 3). The transmembrane potential was recorded using glass microelectrodes (40–80 MΩ DC resistance) filled with 2.7 M KCl connected to a microelectrode amplifier . Amplified signals were digitized (1440 Digidata A/D interface) and stored on a computer for future analysis using LabChart 7.3 software (ADInstruments). Paced (1 Hz) and/or spontaneous action potentials records were acquired. The following parameters were recorded: resting membrane potential (RMP), action potential amplitude (APA), dV/dtmax, and APD at 30, 50, and 90% repolarization . For the spontaneous action potentials records, beating frequency was also evaluated . Measures were obtained automatically. In rat cardiomyocytes, APDs were corrected by the beat rate. In human iPSC-derived cardiomyocytes, action potential recordings were analyzed from ventricle-like cells, classified by the following parameters: dV/dtmax > 10 V/s and APD30/APD90 > 0.370. The starting point of the action potential was determined by the software as a 10% variation in voltage from the maximum diastolic potential.The 35-mm laminin- or matrigel-coated plates containing 5 × 1064 with some modifications. Briefly, 3 × 105 cardiomyocytes derived from neonatal rats were plated on laminin-coated glass coverslips  inside 60 mm culture plates (n = 3). Cells were moved to a fresh medium supplemented with 3 μM Fura-2 AM  and incubated for 15 min at 37 °C. The medium was refreshed again with a medium free of Fura-2 AM, and the coverslip was transferred to a recording chamber. The cardiomyocytes were visualized using a microscope  equipped with a MyoCam-S camera . All the recordings were performed under electrical stimulation at 1 Hz for 4 ms and 10 V . The range in fluorescence emission wavelength was 340–380 nm, and the fluorescence signal was collected with a photomultiplier tube via the × 40 oil objective during continuous excitation at 510 nm with a 75-W Xenon lamp. The recordings were assessed by an expert blinded to the experimental groups. Data were processed and analyzed using IonWizard . The raw traces were filtered to reduce noise, and representative peaks from each cell were selected. Measurements of [Ca2+]i transient amplitude, time to peak, and time to 50% relaxation (DT50) were performed. Mean curves of [Ca2+]i transients for each experimental group were created by aligning the fluorescence signals of each cell recorded and calculated by the average of the fluorescence per timepoint.Calcium transient recording experiments were performed as previously described4 cells/well) were seeded onto laminin-coated 96-well plates (n = 3). Cells were incubated in a thermostatic chamber (21% O2 and 5% CO2 at 37 °C) to maintain physiologic conditions. The cardiomyocytes derived from neonatal rats were visualized using EVOS Cell Imaging System (ThermoFisher Scientific), equipped with a 20 × objective. Cells were allowed to stabilize for at least 10 min before any recordings. All the recordings were performed under electrical stimulation at 1 Hz for 20 ms and 20 V  and obtained as previously described71. Movie images of beating cardiomyocytes were acquired with a duration of 10 s for each position, at 30 frames per second, 2048 × 1536 pixel resolution, and a pixel size of 0.307 μm/pixel with a depth of 8 bits. Data were processed and analyzed using Contractionwave, an open-source software for large-scale analysis of cardiomyocyte contraction72, allowing us to determine the contraction and relaxation times .To measure contractile properties, cardiomyocytes derived from neonatal rats . Subsequently, the cells were lysed in RIPA lysis buffer (10 ×) containing protease inhibitors  and phosphatase inhibitor cocktails 2 and 3 (1:100 dilution). The cell lysate was mixed by vortexing for 30 s and centrifuged at 15,800×g for 15 min at 4 °C. The supernatant was isolated and stored at − 80 °C. Protein concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific) following the manufacturer's instructions. Then, samples of the cardiomyocyte proteins were solubilized in the Laemmli sample buffer and separated by SDS-PAGE using 7.5% polyacrylamide gels. For immunoblotting, proteins were transferred to PVDF membranes  and incubated with a blocking solution containing 5% skim milk or BSA and 0.1% Tween 20 in PBS (pH 7.4) for 1 h to block nonspecific antibody binding, followed by overnight (4 °C) incubation with specific monoclonal antibodies against HIF-1α (D2U3T) , phospholamban (PLN) [2D12] , actin  or a polyclonal antibody against PLN (phospho S16) . The PVDF membranes were washed five times in a blocking solution and incubated for 1 h at room temperature with the horseradish peroxidase-conjugated immunoglobulin secondary antibody . After washing five times with blocking solution and twice in PBS (pH 7.4), the PVDF membranes were incubated for 1 min with an enhanced chemiluminescence detection (ECL) system  for visualization of the bound antibodies. The visualized bands were digitized using ImageScanner III (Cytiva) and quantified using ImageJ software . The unedited gel images are included within the supplementary material of the manuscript.Cardiomyocytes were seeded onto 35-mm laminin-coated plates at 10t-test or Mann–Whitney test. For analyses of proportions, Fisher's exact test was used. For calcium transients, contractility, and immunoblot analyses, statistical significance was determined using one-way ANOVA followed by a post hoc Tukey analysis or Kruskal–Wallis test, both with multiple comparisons with 95% confidence. A P value < 0.05 was used to indicate significance. All statistical analyses were performed using GraphPad Prism version 8.0 .The results are reported as the mean ± standard error of the mean (SEM). Data normality was tested (Shapiro–Wilk test or Kolmogorov–Smirnov test). For the analysis of action potential, statistical significance was determined using unpaired Supplementary Information.