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In this sabbatical, Paul (together with D Cleworth) pioneered the use of laser diffraction to follow sarcomere length changes in contracting striated muscle.3 He not only used to talk about this period with joy but also mentioned how he lost a single muscle fiber in an earthquake while dissecting. Critical sarcomere extension required to recruit a decaying component of extra force during stretch in tetanic contractions of frog skeletal muscle fibers. In the 1960s and 1970s, Paul's research interests shifted back to striated muscle, with published work on both cardiac and skeletal muscle with several well-cited papers (eg Ref. [2]), but from the end of the 1970s his main field of interest was skeletal muscle. The work of Paul Edman at UCLA was a starting point for his interest in the functional variability between different sarcomeres and neighboring segments along a single muscle fiber. [Extracted from the article]

Aims: Nfix is a transcription factor belonging to the Nuclear Factor I (NFI) family comprising four members (Nfia, b, c, x). Nfix plays important roles in the development and function of several organs. In muscle development, Nfix controls the switch from embryonic to fetal myogenesis by promoting fast twitching fibres. In the adult muscle, following injury, lack of Nfix impairs regeneration, inducing higher content of slow‐twitching fibres. Nfix is expressed also in the heart, but its function has been never investigated before. We studied Nfix role in this organ. Methods: Using Nfix‐null and wild type (WT) mice we analyzed: (1) the expression pattern of Nfix during development by qPCR and (2) the functional alterations caused by its absence, by in vivo telemetry and in vitro patch clamp analysis. Results and Conclusions: Nfix expression start in the heart from E12.5. Adult hearts of Nfix‐null mice show a hearts morphology and sarcomeric proteins expression similar to WT. However, Nfix‐null animals show tachycardia that derives form an intrinsic higher beating rate of the sinus node (SAN). Molecular and functional analysis revealed that sinoatrial cells of Nfix‐null mice express a significantly larger L‐type calcium current (Cacna1d + Cacna1c). Interestingly, downregulation of Nfix by sh‐RNA in primary cultures of neonatal rat ventricular cardiomyocytes induced a similar increase in their spontaneous beating rate and in ICaL current. In conclusion, our data provide the first demonstration of a role of Nfix that, increasing the L‐type calcium current, modulates heart rate. [ABSTRACT FROM AUTHOR]

Aim: General anesthesia can induce cognitive deficits in both humans and rodents, correlating with pathological alterations in the hippocampus. However, whether general anesthesia affects olfactory behaviors remains controversial as clinical studies have produced inconsistent results. Therefore, we aimed to investigate how olfactory behaviors and neuronal activity are affected by isoflurane exposure in adult mice. Methods: The olfactory detection test, olfactory sensitivity test, and olfactory preference/avoidance test were used to examine olfactory function. In vivo electrophysiology was performed in awake, head‐fixed mice to record single‐unit spiking and local field potentials in the olfactory bulb (OB). We also performed patch‐clamp recordings of mitral cell activity. For morphological studies, immunofluorescence and Golgi–Cox staining were applied. Results: Repeated exposure to isoflurane impaired olfactory detection in adult mice. The main olfactory epithelium, the first region exposed to anesthetics, displayed increased proliferation of basal stem cells. In the OB, a crucial hub for olfactory processing, repeated isoflurane exposure increased the odor responses of mitral/tufted cells. Furthermore, the odor‐evoked high gamma response was decreased after isoflurane exposure. Whole‐cell recordings further indicated that repeated isoflurane exposure increased the excitability of mitral cells, which may be due to weakened inhibitory input in isoflurane‐exposed mice. In addition, elevated astrocyte activation and glutamate transporter‐1 expression in the OB were observed in isoflurane‐exposed mice. Conclusions: Our findings indicate that repeated isoflurane exposure impairs olfactory detection by increasing neuronal activity in the OB in adult mice. [ABSTRACT FROM AUTHOR]

Aim: Histidine‐containing dipeptides (HCDs) are pleiotropic homeostatic molecules with potent antioxidative and carbonyl quenching properties linked to various inflammatory, metabolic, and neurological diseases, as well as exercise performance. However, the distribution and metabolism of HCDs across tissues and species are still unclear. Methods: Using a sensitive UHPLC–MS/MS approach and an optimized quantification method, we performed a systematic and extensive profiling of HCDs in the mouse, rat, and human body (in n = 26, n = 25, and n = 19 tissues, respectively). Results: Our data show that tissue HCD levels are uniquely produced by carnosine synthase (CARNS1), an enzyme that was preferentially expressed by fast‐twitch skeletal muscle fibres and brain oligodendrocytes. Cardiac HCD levels are remarkably low compared to other excitable tissues. Carnosine is unstable in human plasma, but is preferentially transported within red blood cells in humans but not rodents. The low abundant carnosine analogue N‐acetylcarnosine is the most stable plasma HCD, and is enriched in human skeletal muscles. Here, N‐acetylcarnosine is continuously secreted into the circulation, which is further induced by acute exercise in a myokine‐like fashion. Conclusion: Collectively, we provide a novel basis to unravel tissue‐specific, paracrine, and endocrine roles of HCDs in human health and disease. [ABSTRACT FROM AUTHOR]

Chemically skinned fibres allow the study of human muscle contractile function in vitro. A particularly important parameter is specific force (SF), that is, maximal isometric force divided by cross‐sectional area, representing contractile quality. Although SF varies substantially between studies, the magnitude and cause of this variability remains puzzling. Here, we aimed to summarize and explore the cause of variability in SF between studies. A systematic search was conducted in Medline, Embase and Web of Science databases in June 2020, yielding 137 data sets from 61 publications which studied healthy, young adults. Five‐fold differences in mean SF data were observed. Adjustments to the reported data for key methodological differences allowed between‐study comparisons to be made. However, adjustment for fibre shape, swelling and sarcomere length failed to significantly reduce SF variance (I2 = 96%). Interestingly, grouping papers based on shared authorship did reveal consistency within research groups. In addition, lower SF was found to be associated with higher phosphocreatine concentrations in the fibre activating solution and with Triton X‐100 being used as a skinning agent. Although the analysis showed variance across the literature, the ratio of SF in single fibres containing myosin heavy chain isoforms IIA or I was found to be consistent across research groups. In conclusion, whilst the skinned fibre technique is reliable for studying in vitro force generation of single fibres, the composition of the solution used to activate fibres, which differs between research groups, is likely to heavily influence SF values. [ABSTRACT FROM AUTHOR]

The paper by Pedersen et al1 published in this issue of I Acta Physiologica i provides compelling evidence that ClC-1 Cl SP - sp channels are important not only in the regulation of skeletal muscle excitability in mammals,2 but also in modulating the threshold for signal transmission at the neuromuscular junction (NMJ). In turn, the action potential in the muscle fibre triggers a series of events that cause the fibre to contract.4 Therefore, if the action potentials reaching the neuron terminal at an NMJ do not translate consistently into to an equivalent number of action potentials at the surface of the muscle fibre, the muscle fibre will not contract as it should, resulting in muscle weakness. Generally, the NMJs of twitch muscles in vertebrates are robust all-or-none excitatory synapses that display a high safety margin under normal physiological conditions to ensure that each action potential at the neuron terminal results in the generation of one action potential in the surface membrane of the muscle fibre. [Extracted from the article]

Aim: We aimed to investigate the inter‐individual variability in redox and physiological responses of antioxidant‐deficient subjects after antioxidant supplementation. Methods: Two hundred individuals were sorted by plasma vitamin C levels. A low vitamin C group (n = 22) and a control group (n = 22) were compared in terms of oxidative stress and performance. Subsequently, the low vitamin C group received for 30 days vitamin C (1 g) or placebo, in randomized, double‐blind, crossover fashion, and the effects were examined through a mixed‐effects model, while individual responses were calculated. Results: The low vitamin C group exhibited lower vitamin C (−25 μmol/L; 95%CI[−31.7, −18.3]; p < 0.001), higher F2‐isoprostanes (+17.1 pg/mL; 95%CI[6.5, 27.7]; p = 0.002), impaired VO2max (−8.2 mL/kg/min; 95%CI[−12.8, −3.6]; p < 0.001) and lower isometric peak torque (−41.5 Nm; 95%CI[−61.8, −21.2]; p < 0.001) compared to the control group. Regarding antioxidant supplementation, a significant treatment effect was found in vitamin C (+11.6 μmol/L; 95%CI[6.8, 17.1], p < 0.001), F2‐isoprostanes (−13.7 pg/mL; 95%CI[−18.9, −8.4], p < 0.001), VO2max (+5.4 mL/kg/min; 95%CI[2.7, 8.2], p = 0.001) and isometric peak torque (+18.7; 95%CI[11.8, 25.7 Nm], p < 0.001). The standard deviation for individual responses (SDir) was greater than the smallest worthwhile change (SWC) for all variables indicating meaningful inter‐individual variability. When a minimal clinically important difference (MCID) was set, inter‐individual variability remained for VO2max, but not for isometric peak torque. Conclusion: The proportion of response was generally high after supplementation (82.9%–95.3%); however, a few participants did not benefit from the treatment. This underlines the potential need for personalized nutritional interventions in an exercise physiology context. [ABSTRACT FROM AUTHOR]

Many neuromuscular disorders can have a differential impact on a specific myofibre type, forming the central premise of this review. The many different skeletal muscles in mammals contain a spectrum of slow‐ to fast‐twitch myofibres with varying levels of protein isoforms that determine their distinctive contractile, metabolic, and other properties. The variations in functional properties across the range of classic 'slow' to 'fast' myofibres are outlined, combined with exemplars of the predominantly slow‐twitch soleus and fast‐twitch extensor digitorum longus muscles, species comparisons, and techniques used to study these properties. Other intrinsic and extrinsic differences are discussed in the context of slow and fast myofibres. These include inherent susceptibility to damage, myonecrosis, and regeneration, plus extrinsic nerves, extracellular matrix, and vasculature, examined in the context of growth, ageing, metabolic syndrome, and sexual dimorphism. These many differences emphasise the importance of carefully considering the influence of myofibre‐type composition on manifestation of various neuromuscular disorders across the lifespan for both sexes. Equally, understanding the different responses of slow and fast myofibres due to intrinsic and extrinsic factors can provide deep insight into the precise molecular mechanisms that initiate and exacerbate various neuromuscular disorders. This focus on the influence of different myofibre types is of fundamental importance to enhance translation for clinical management and therapies for many skeletal muscle disorders. [ABSTRACT FROM AUTHOR]

Aim: Enhanced cardiac sympathetic afferent reflex (CSAR) promotes sympathetic hyperactivation in chronic heart failure (CHF). Salusin‐β is a torsin family 2 member A (TOR2A) gene product and a cardiovascular active peptide closely associated with cardiovascular diseases. We aimed to determine the roles of salusin‐β in the paraventricular nucleus (PVN) in modulating enhanced CSAR and sympathetic hyperactivation in rats with CHF induced by coronary artery ligation and elucidate the underlying molecular mechanisms. Methods: CSAR was evaluated based on the responses of mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) to the epicardial administration of capsaicin in rats under anesthesia. Results: Salusin‐β protein expression was upregulated in the PVN of the CHF compared with sham‐operated rats. Salusin‐β microinjection into the PVN dose‐dependently increased MAP and RSNA and enhanced CSAR, while anti‐salusin‐β IgG exerted opposite effects. The effect of salusin‐β was inhibited by reactive oxygen species (ROS) scavenger or NAD(P)H oxidase inhibitor but promoted by superoxide dismutase inhibitor. The effect of anti‐salusin‐β IgG was interdicted by nitric oxide (NO) synthase inhibitor. Furthermore, chronic salusin‐β gene knockdown in PVN attenuated CSAR, reduced sympathetic output, improved myocardial remodeling and cardiac function, decreased NAD(P)H oxidase activity and ROS levels, and increased NO levels in the CHF rats. Conclusion: Increased salusin‐β activity in the PVN contributes to sympathetic hyperactivation and CSAR in CHF by inhibiting NO release and stimulating NAD(P)H oxidase‐ROS production. Reducing endogenous central salusin‐β expression might be a novel strategy for preventing and treating CHF in the future. [ABSTRACT FROM AUTHOR]

Aim: The purpose of this study was to 1. investigate if glucose tolerance is affected after one acute bout of different types of exercise; 2. assess if potential differences between two exercise paradigms are related to changes in mitochondrial function; and 3. determine if endurance athletes differ from nonendurance‐trained controls in their metabolic responses to the exercise paradigms. Methods: Nine endurance athletes (END) and eight healthy nonendurance‐trained controls (CON) were studied. Oral glucose tolerance tests (OGTT) and mitochondrial function were assessed on three occasions: in the morning, 14 h after an overnight fast without prior exercise (RE), as well as after 3 h of prolonged continuous exercise at 65% of VO2max (PE) or 5 × 4 min at ~95% of VO2max (HIIT) on a cycle ergometer. Results: Glucose tolerance was markedly reduced in END after PE compared with RE. END also exhibited elevated fasting serum FFA and ketones levels, reduced insulin sensitivity and glucose oxidation, and increased fat oxidation during the OGTT. CON showed insignificant changes in glucose tolerance and the aforementioned measurements compared with RE. HIIT did not alter glucose tolerance in either group. Neither PE nor HIIT affected mitochondrial function in either group. END also exhibited increased activity of 3‐hydroxyacyl‐CoA dehydrogenase activity in muscle extracts vs. CON. Conclusion: Prolonged exercise reduces glucose tolerance and increases insulin resistance in endurance athletes the following day. These findings are associated with an increased lipid load, a high capacity to oxidize lipids, and increased fat oxidation. [ABSTRACT FROM AUTHOR]

Aim: This study mapped the spatiotemporal positions and connectivity of Onecut3+ neuronal populations in the developing and adult mouse brain. Methods: We generated fluorescent reporter mice to chart Onecut3+ neurons for brain‐wide analysis. Moreover, we crossed Onecut3‐iCre and Mapt‐mGFP (Tau‐mGFP) mice to visualize axonal projections. A dual Cre/Flp‐dependent AAV construct in Onecut3‐iCre cross‐bred with Slc17a6‐FLPo mice was used in an intersectional strategy to map the connectivity of glutamatergic lateral hypothalamic neurons in the adult mouse. Results: We first found that Onecut3 marks a hitherto undescribed Slc17a6+/Vglut2+ neuronal cohort in the lateral hypothalamus, with the majority expressing thyrotropin‐releasing hormone. In the adult, Onecut3+/Vglut2+ neurons of the lateral hypothalamus had both intra‐ and extrahypothalamic efferents, particularly to the septal complex and habenula, where they targeted other cohorts of Onecut3+ neurons and additionally to the neocortex and hippocampus. This arrangement suggests that intrinsic reinforcement loops could exist for Onecut3+ neurons to coordinate their activity along the brain's midline axis. Conclusion: We present both a toolbox to manipulate novel subtypes of hypothalamic neurons and an anatomical arrangement by which extrahypothalamic targets can be simultaneously entrained. [ABSTRACT FROM AUTHOR]

Aim: Accurate evaluation of glomerular filtration rate (GFR) is crucial in Oncology as drug eligibility and dosing depend on estimates of GFR. However, there are no clear guidelines on the optimal method of determining kidney function in patients with cancer. We aimed to summarize the evidence on estimation of kidney function in patients with cancer. Methods: We searched PubMed for literature discussing the performance of GFR estimating equations in patients with malignancy to create a table of the evidence for creatinine‐ and cystatin c‐based equations. We further reviewed novel estimation techniques such as panel eGFR, real‐time measured GFR, and functional magnetic resonance imaging. Results: The commonly used GFR estimating equations were derived from populations of patients without cancer. These equations may be less applicable in Oncology due to severe sarcopenia, inflammation, and other physiologic changes in patients with cancer. The Cockcroft‐Gault equation currently dominates in clinical Oncology despite significant limitations and accumulating evidence for use of the CKD‐EPICr formula. Additional considerations in the practice of Oncology include a recently developed equation (CamGFRv2, also called the Janowitz formula) and the use of cystatin c‐based equations to overcome some of the barriers to accurate GFR estimation based on creatinine alone. Conclusion: Overall, we suggest using the CKD‐EPI equations (either cystatin c or creatinine‐based) among patients with cancer in routine clinical practice and measured GFR for patients at a critical threshold for treatment decisions. [ABSTRACT FROM AUTHOR]

Save imaging techniques like MRI and PET scanning are of great advantage here and these techniques also play an important role in refining animal experiments.20 Nevertheless, it is extremely difficult to eliminate all animal models from physiological research. Recent 3D tissue models of human colonic epithelium have been developed which will improve the mechanistic understanding of human intestinal function.11 The possibility of obtaining the information required through ethical research on human subjects or human tissue should be considered. Factors that confound the prediction of renal medullary oxygenation and risk of acute kidney injury from measurement of bladder urine oxygen tension. In recent years, most of the manuscripts published as a regular paper in ACTA Physiologica are based on animal experiments. [Extracted from the article]

The global north is facing an unprecedented rise in the prevalence of neurodegenerative diseases. The increasing incidence of Parkinson's disease is being referred to as a pandemic. The reason for the enormous increase is only partly understood. Lifestyle factors are known to play a role, but they alone cannot account for the surge. One factor that—although being recognized as important—has not been explored in detail so far is the influence of circadian rhythms. Sleep and circadian rhythm disruption are known as key factors in neurodegeneration, and their occurrence during early disease stages suggests a causal role in the pathogenesis. Isolated rapid eye movement (REM) sleep behavior disorder (iRBD) has been identified as a prodromal state of α‐synucleinopathies, such as Parkinson's disease, Lewy body dementia, and multiple system atrophy offering a window for insights into the early development of these diseases. Even though REM sleep is the sleep state most pronounced, driven and modulated by the circadian timing system, specific circadian abnormalities have not been described in iRBD. Novel experimental and clinical approaches exploiting the molecular circuitry underlying circadian timekeeping hold promise to disentangle some of the pathophysiologic mechanisms of α‐synucleinopathies. In this review, we summarize current knowledge on sleep and circadian rhythm disruptions in α‐synucleinopathies with an emphasis on molecular aspects and therapeutic potentials. These insights might contribute to our understanding of the pathogenesis of neurodegenerative diseases and may allow therapeutic interventions addressing the disturbed circadian system at the early stage of disease. [ABSTRACT FROM AUTHOR]

Extreme environments are powerful drivers of physiological adaptation. Naked mole‐rats offer an informative example of this relationship as they putatively encounter intermittent hypoxia and hypercapnia in their subterranean habitat. This has presumably driven the evolution of a suite of cellular and physiological adaptations that enable life in these conditions. Recently, my laboratory and others have begun to examine physiological responses to environmental hypoxia and hypercapnia in naked mole‐rats, and the underlying cellular and molecular mechanisms that differentiate the responses of this species from those of other small mammals. Prominent among these adaptations are a robust hypoxic metabolic response and blunted ventilatory responses to hypoxia and hypercapnia. These responses are mediated in part by modifications to the central nervous system signaling pathways that sense and communicate changes in environmental gas levels and initiate and maintain downstream physiological responses. For example, naked mole‐rats retain the signaling architecture necessary for "normal" ventilatory responses to hypoxia and hypercapnia; however, the underlying signaling pathways are muted, resulting in reduced, or even the absence of, sensitivity to otherwise powerful environmental stimuli. Herein, I discuss what we have learned about the manifestation and control of ventilatory and metabolic responses to hypoxia and hypercapnia in naked mole‐rats. I also highlight areas where additional work is warranted and consider the implications of what we have learned for the ecophysiology of a species that thrives in conditions that are deleterious or lethal to most adult mammals. [ABSTRACT FROM AUTHOR]

Data integration, data sharing, and standardized analyses are important enablers for data‐driven medical research. Circadian medicine is an emerging field with a particularly high need for coordinated and systematic collaboration between researchers from different disciplines. Datasets in circadian medicine are multimodal, ranging from molecular circadian profiles and clinical parameters to physiological measurements and data obtained from (wearable) sensors or reported by patients. Uniquely, data spanning both the time dimension and the spatial dimension (across tissues) are needed to obtain a holistic view of the circadian system. The study of human rhythms in the context of circadian medicine has to confront the heterogeneity of clock properties within and across subjects and our inability to repeatedly obtain relevant biosamples from one subject. This requires informatics solutions for integrating and visualizing relevant data types at various temporal resolutions ranging from milliseconds and seconds to minutes and several hours. Associated challenges range from a lack of standards that can be used to represent all required data in a common interoperable form, to challenges related to data storage, to the need to perform transformations for integrated visualizations, and to privacy issues. The downstream analysis of circadian rhythms requires specialized approaches for the identification, characterization, and discrimination of rhythms. We conclude that circadian medicine research provides an ideal environment for developing innovative methods to address challenges related to the collection, integration, visualization, and analysis of multimodal multidimensional biomedical data. [ABSTRACT FROM AUTHOR]

Aim: The aim of this study is to investigate associations between the physical activity (PA) spectrum (sedentary behavior to exercise) and tissue‐specific insulin resistance (IR). Methods: We included 219 participants for analysis (median [IQR]: 61 [55; 67] years, BMI 29.6 [26.9; 32.0] kg/m2; 60% female) with predominant muscle or liver IR, as determined using a 7‐point oral glucose tolerance test (OGTT). PA and sedentary behavior were measured objectively (ActivPAL) across 7 days. Context‐specific PA was assessed with the Baecke questionnaire. Multiple linear regression models (adjustments include age, sex, BMI, site, season, retirement, and dietary intake) were used to determine associations between the PA spectrum and hepatic insulin resistance index (HIRI), muscle insulin sensitivity index (MISI) and whole‐body IR (HOMA‐IR, Matsuda index). Results: In fully adjusted models, objectively measured total PA (standardized regression coefficient β = 0.17, p = 0.020), light‐intensity PA (β = 0.15, p = 0.045) and moderate‐to‐vigorous intensity PA (β = 0.13, p = 0.048) were independently associated with Matsuda index, but not HOMA‐IR (p > 0.05). A higher questionnaire‐derived sport index and leisure index were associated with significantly lower whole‐body IR (Matsuda, HOMA‐IR) in men but not in women. Results varied across tissues: more time spent sedentary (β = −0.24, p = 0.045) and a higher leisure index (β = 0.14, p = 0.034) were respectively negatively and positively associated with MISI, but not HIRI. A higher sport index was associated with lower HIRI (β = −0.30, p = 0.007, in men only). Conclusion: While we confirm a beneficial association between PA and whole‐body IR, our findings indicate that associations between the PA spectrum and IR seem distinct depending on the primary site of insulin resistance (muscle or liver). [ABSTRACT FROM AUTHOR]

Exactly 50 years ago, I was a post‐doc in the laboratory of Olov Lindberg in Stockholm measuring fatty acid oxidation by mitochondria isolated from thermogenic brown adipose tissue, when we noticed a curious nonlinearity in the respiration rate. This initiated a convoluted chain of experiments revealing that the mitochondria were textbook demonstrations of the then novel and highly controversial "chemiosmotic hypothesis" of Peter Mitchell and that thermogenesis was regulated by a proton short‐circuit, mediated by a 32 kDa "uncoupling protein," UCP1, activated by fatty acid. This review is a personal account of the research into the bioenergetics of isolated brown adipocytes and isolated mitochondria, which led, after fifteen years of investigation, to what is still accepted as the "canonical" UCP1‐mediated mechanism of nonshivering thermogenesis, uniting whole animal physiology with mitochondrial bioenergetics. [ABSTRACT FROM AUTHOR]

Aim: Although calcium‐sensing receptor (CaSR) and transient receptor potential vanilloid 4 (TRPV4) channels are functionally expressed on macrophages, it is unclear if they work coordinately to mediate macrophage function. The present study investigates whether CaSR couples to TRPV4 channels and mediates macrophage polarization via Ca2+ signaling. Methods: The role of CaSR/TRPV4/Ca2+ signaling was assessed in lipopolysaccharide (LPS)‐treated peritoneal macrophages (PMs) from wild‐type (WT) and TRPV4 knockout (TRPV4 KO) mice. The expression and function of CaSR and TRPV4 in PMs were analyzed by immunofluorescence and digital Ca2+ imaging. The correlation factors of M1 polarization, CCR7, IL‐1β, and TNFα were detected using q‐PCR, western blot, and ELISA. Results: We found that PMs expressed CaSR and TRPV4, and CaSR activation‐induced marked Ca2+ signaling predominately through extracellular Ca2+ entry, which was inhibited by selective pharmacological blockers of CaSR and TRPV4 channels. The CaSR activation‐induced Ca2+ signaling was significantly attenuated in PMs from TRPV4 KO mice compared to those from WT mice. Moreover, the CaSR activation‐induced Ca2+ entry via TRPV4 channels was inhibited by blocking phospholipases A2 (PLA2)/cytochromeP450 (CYP450) and phospholipase C (PLC)/Protein kinase C (PKC) pathways. Finally, CaSR activation promoted the expression and release of M1‐associated cytokines IL‐1β and TNFɑ, which were attenuated in PMs from TRPV4 KO mice. Conclusion: We reveal a novel coupling of the CaSR and TRPV4 channels via PLA2/CYP450 and PLC/PKC pathways, promoting a Ca2+‐dependent M1 macrophage polarization. Modulation of this coupling and downstream pathways may become a potential strategy for the prevention/treatment of immune‐related disease. [ABSTRACT FROM AUTHOR]

Arterial hypertension is a serious medical condition that significantly increases the risks of heart, brain, kidney, and other diseases affecting 1.28 billion adults worldwide. The authors employed an animal model with wild-type (Floxed) and CD-specific deletion of renin (CD renin KO) mice. In animals, immunofluorescence analysis of renal sections from mice after 5 days of acute pyelonephritis revealed apical plasma membrane targeting of AQP2 and pS256-AQP2 in inner medullary collecting ducts. CD renin KO mice had blunted HK-induced intrarenal renin response and developed impaired kaliuresis and elevated plasma potassium level. [Extracted from the article]

Aim: Nonalcoholic steatohepatitis (NASH) is a severe form of nonalcoholic fatty liver disease (NAFLD) and lacks effective treatment options. Heme oxygenase‐1 (HO‐1) is a critical defense against oxidative stress and inflammation in the liver injury. This study aims to investigate the protective role and underlying mechanisms of HO‐1 in NASH pathogenesis. Methods: The hepatocyte‐specific HO‐1 knockout (HO‐1HEPKO) mice on a C57BL/6J background (HO‐1fl/fl/Alb‐Cre) were generated and fed a high‐fat/western‐style diet (HFD) or methionine‐choline‐deficient diet (MCD). Changes in mitochondrial ultrastructure were observed by transmission electron microscopy and confocal microscopy. A mitochondrial PCR array was used to identify the crucial genes associated with mitochondrial dysfunction. Results: Hepatocyte‐specific HO‐1HEPKO mice developed steatohepatitis with severe steatosis, ballooning, and necroinflammation. Dysregulated hepatic expression of mitochondria‐related proteins, including DRP1, Tomm20, MFN1 and MFN2 were detected in NASH animals. Ultrastructural mitochondrial damage was observed in HO‐1HEPKO mice. Mitochondrial dysfunction was recapitulated in HO‐1‐knockdown cells in vitro, as evidenced by decreased membrane potential, reduced ATP content, and mtDNA damage. Conversely, HO‐1 overexpression restored these changes in vitro. Mechanistically, HO‐1 deficiency reduced the inhibitory effect on Tomm20, leading to mitochondrial dysfunction, and thereby causing steatohepatitis. Conclusions: HO‐1 attenuates diet‐induced steatohepatitis by preventing mitochondrial dysfunction, indicating that HO‐1 may constitute a potential therapeutic target for NASH. [ABSTRACT FROM AUTHOR]

Aims: We aimed to investigate the tolerability, safety, and effectiveness of enhanced external counterpulsation therapy (EECP) versus individual shear rate therapy (ISRT) in patients with lower extremity atherosclerotic disease (LEAD). Methods: Eighteen patients (age: 73.1 ± 6 years) underwent EECP and ISRT, each daily over five consecutive days in a cross‐over design with a 1 week resting period in between the two regimens. A quality‐of‐life questionnaire was used to assess the therapy experience. Oxygen saturation (SO2), relative hemoglobin amount (rHb) and blood flow (Flow) in the capillary‐venous‐system (microcirculation) of the skin were monitored continuously during all therapy sessions using the micro‐lightguide spectrophotometer, also known as oxygen to see (O2C). The effects of EECP and ISRT on the renal function and skeletal muscles were evaluated using serial blood and urine tests. Results: EECP therapy had to be terminated early before the end of the 5th session in 10 patients (55.6%) because of discomfort. Four patients (22.2%) experienced signs of critical limb ischaemia under EECP. The total score of the quality‐of‐life questionnaire was significantly higher (= better tolerated) post‐ISRT compared with EECP. Microcirculation monitoring revealed that ISRT significantly increased the SO2, blood flow and rHb during the therapy. All three parameters remained significantly increased in the observation period after ISRT. The serum levels of creatin kinase and myoglobin increased significantly under EECP. Conclusions: ISRT significantly improves tolerability, safety, and effectiveness over EECP in patients with LEAD. [ABSTRACT FROM AUTHOR]

Aim: Tissue hypoxia is an early key feature of acute kidney injury. Assessment of renal oxygenation using magnetic resonance imaging (MRI) markers T2 and T2* enables insights into renal pathophysiology. This assessment can be confounded by changes in the blood and tubular volume fractions, occurring upon pathological insults. These changes are mirrored by changes in kidney size (KS). Here, we used dynamic MRI to monitor KS for physiological interpretation of T2* and T2 changes in acute pathophysiological scenarios. Methods: KS was determined from T2*, T2 mapping in rats. Six interventions that acutely alter renal tissue oxygenation were performed directly within the scanner, including interventions that change the blood and/or tubular volume. A biophysical model was used to estimate changes in O2 saturation of hemoglobin from changes in T2* and KS. Results: Upon aortic occlusion KS decreased; this correlated with a decrease in T2*, T2. Upon renal vein occlusion KS increased; this negatively correlated with a decrease in T2*, T2. Upon simultaneous occlusion of both vessels KS remained unchanged; there was no correlation with decreased T2*, T2. Hypoxemia induced mild reductions in KS and T2*, T2. Administration of an X‐ray contrast medium induced sustained KS increase, with an initial increase in T2*, T2 followed by a decrease. Furosemide caused T2*, T2 elevation and a minor increase in KS. Model calculations yielded physiologically plausible calibration ratios for T2*. Conclusion: Monitoring KS allows physiological interpretation of acute renal oxygenation changes obtained by T2*, T2. KS monitoring should accompany MRI‐oximetry, for new insights into renal pathophysiology and swift translation into human studies. [ABSTRACT FROM AUTHOR]

The attained 30-fold coverage allowed for direct estimation of Denisovan heterozygosity, which showed that genetic diversity among the Denisovans was surprisingly low.[6] The application of a single-stranded DNA library also enabled the high-coverage genome sequencing of a Neanderthal woman from Siberia in 2014. Further studies involving other available archaic genomes and 25 modern genomes detected several gene flow events among Neanderthals, Denisovans and anatomically modern humans, thus giving evidence for possible common ancestry between hominins in the Late Pleistocene. The full mitochondrial genome was sequenced in 2008, while in 2010 appeared the first draft of the Neanderthal genome, followed by high coverage Neanderthal genome in 2014. [Extracted from the article]

The most in-depth study is probably the report by Andrei et al from 2016.5 They observed a TRPA1 immunolabelling on freshly isolated mouse cardiomyocytes and modifications of Ca SP 2+ sp -transient of these cells after the application of allyl isothiocyanate (AITC), the classical TRPA1 agonist, given that TRPA1 is permeable to cations (five times more permeable for Ca SP 2+ sp than for Na SP + sp ).6 The same group has further demonstrated that TRPA1 is involved in cardiomyocytes contraction7 and protects against ischemia-induced cardiomyocytes death,8 highlighting physiological and pathological implication in cardiomyocytes. In the specific case of TRPA1, few studies have reported the presence of a functional channel in mouse cardiomyocytes as well as TRPA1 protein revealed by western blot. Some of their detection of TRPA1 or TRPV1 are probably questionable as extensively discussed in the paper by Hoebart et al.1 Commercially available antibodies against TRPA1 are often not really efficient or specific as recently reported.15 At the functional level, a whole-cell NSC current can be composed of several entities carried by many supporting molecules. [Extracted from the article]

In the current issue of I Acta Physiologica i , two papers are published investigating the development of arterial hypertension under conditions of pathological loss of free water in two disease models. Aestivation motifs explain hypertension and muscle mass loss in mice with psoriatic skin barrier defect. The contribution of a tentative aestivation versus natriuresis component to CKD-induced hypertension might therefore increase with higher ratio of salt to water intake and minimize for isotonic intake. Aestivation motifs explain hypertension and muscle mass loss in mice with psoriatic skin barrier defect. [Extracted from the article]

Aim: Investigate whether juvenile exercise could induce a long‐term muscle memory, boosting the effects of exercise in adults. Methods: We devised a 5‐week climbing exercise scheme with food reward administered to male juvenile rats (post‐natal week 4–9). Subsequently, the animals were subjected to 10 weeks of detraining (week 9–19) without climbing and finally retraining during week 19–21. Results: The juvenile exercise increased fiber cross‐sectional area (fCSA) by 21% (p = 0.0035), boosted nuclear accretion by 13% (p = 0.057), and reduced intraperitoneal fat content by 28% (p = 0.007) and body weight by 9% (p = 0.001). During detraining, the fCSA became similar in the animals that had been climbing compared to naive controls, but the elevated number of myonuclei induced by the climbing were maintained (15%, p = 0.033). When the naive rats were subjected to 2 weeks of adult exercise there was little effect on fCSA, while the previously trained rats displayed an increase of 19% (p = 0.0007). Similarly, when the rats were subjected to unilateral surgical overload in lieu of the adult climbing exercise, the increase in fCSA was 20% (p = 0.0039) in the climbing group, while there was no significant increase in naive rats when comparing to the contralateral leg. Conclusion: This demonstrates that juvenile exercise can establish a muscle memory boosting the effects of adult exercise. The juvenile climbing exercise with food reward also led to leaner animals with lower body weight. These differences were to some extent maintained throughout the adult detraining period in spite of all animals being fed ad libitum, indicating a form of body weight memory. [ABSTRACT FROM AUTHOR]

Aims: Diets containing high‐fat and high sugar (HFHS) lead to overweight/obesity. Overweight/obesity increases the risk of infertility, and of the pregnant mother and her child for developing metabolic conditions. Overweight/obesity has been recreated in mice, but most studies focus on the effects of chronic, long‐term HFHS diet exposure. Here, we exposed mice to HFHS from 3 weeks prior to pregnancy with the aim of determining impacts on fertility, and gestational and neonatal outcomes. Methods: Time‐domain NMR scanning was used to assess adiposity, glucose, and insulin tolerance tests were employed to examine metabolic physiology, and morphological and proteomic analyses conducted to assess structure and nutrient levels of maternal organs and placenta. Results: Fertility measures of HFHS dams were largely the same as controls. HFHS dams had increased adiposity pre‐pregnancy, however, exhibited exacerbated lipolysis/hyper‐mobilization of adipose stores in late pregnancy. While there were no differences in glucose or insulin tolerance, HFHS dams were hyperglycemic and hyperinsulinemic in pregnancy. HFHS dams had fatty livers and altered pancreatic islet morphology. Although fetuses were hyperglycemic and hyperinsulinemic, there was no change in fetal growth in HFHS dams. There were also reductions in placenta formation. Moreover, there was increased offspring loss during lactation, which was related to aberrant mammary gland development and milk protein composition in HFHS dams. Conclusions: These findings are relevant given current dietary habits and the development of maternal and offspring alterations in the absence of an increase in maternal weight and adiposity during pregnancy, which are the current clinical markers to determine risk across gestation. [ABSTRACT FROM AUTHOR]

In this case, Southern was not a direction, but a scientist: Edwin M. Southern. I Acta Physiologica i caters to a wide variety of research efforts related to physiology - itself one of if not the most diverse discipline of biomedical research. He established a method to split up DNA via electrophoresis and detect specific sequences by hybridization - the Southern blot.4 Methods for detecting specific proteins5 and RNA sequences6 were established soon after, based on the same principle. Subsequently, searching recent I Acta Physiologica i publications for references to "blotting" should provide a reliable and lower bound estimate of the prominence of "blotting" in biomedicine. [Extracted from the article]

Scientific data output, as measured by the number of peer-reviewed publications, is growing exponentially. 17 McDougle M, Quinn D, Diepenbroek C, Singh A, de la Serre C, de Lartigue G. Intact vagal gut-brain signalling prevents hyperphagia and excessive weight gain in response to high-fat high-sugar diet. Scientific data output will continue to rise, rendering it impossible for any reader to honour all valuable publications in a given field of interest.2 Therefore, we use technology to aid and customize the information I allocation i process. Exercise-dependent increases in protein synthesis are accompanied by chromatin modifications and increased MRTF-SRF signalling. [Extracted from the article]

The skeletal muscle excitation-contraction coupling leading to tension development and increased mechanical stimuli change in concert, leaving it difficult to study their respective effect on molecular signalling and following adaptations. In conclusion, although the interest for mechanotransduction and deciphering relative effects of tension development and excitation in skeletal muscle has had a steep increase in later years, there is still work to do. Indeed, the present paper by Rindom et al1 is a valuable contribution to the field of mechanotransduction, as it establishes a better foundation to explore the complex network of skeletal muscle plasticity. [Extracted from the article]

22 Pontecorvi G, Bellenghi M, Ortona E, Carè A. microRNAs as new possible actors in gender disparities of Covid-19 pandemic. The recent global pandemic has lead to a surge in biomedical publications,1 which came at a time when a rather intense discussion on quality control for research results was already ongoing. In how far the COVID-19 pandemic has fostered sustainable extensive cooperation among biomedical disciplines and researchers worldwide (now more than ever easily connected through eg online meetings), remains to be seen. Covid-19 pandemic analogies to everyday life in medical sciences. [Extracted from the article]

Aim: Erythropoietin (EPO) is regulated by hypoxia‐inducible factor (HIF)‐2. In the kidney, it is produced by cortico‐medullary perivascular interstitial cells, which transdifferentiate into collagen‐producing myofibroblasts in response to injury. Inhibitors of prolyl hydroxylase domain (PHD) dioxygenases (HIF‐PHIs) activate HIF‐2 and stimulate kidney and liver EPO synthesis in patients with anemia of chronic kidney disease (CKD). We examined whether HIF‐PHIs can reactivate EPO synthesis in interstitial cells that have undergone myofibroblast transdifferentiation in established kidney fibrosis. Methods: We investigated Epo transcription in myofibroblasts and characterized the histological distribution of kidney Epo transcripts by RNA in situ hybridization combined with immunofluorescence in mice with adenine nephropathy (AN) treated with HIF‐PHI molidustat. Lectin absorption chromatography was used to assess liver‐derived EPO. In addition, we examined kidney Epo transcription in Phd2 knockout mice with obstructive nephropathy. Results: In AN, molidustat‐induced Epo transcripts were not found in areas of fibrosis and did not colocalize with interstitial cells that expressed α‐smooth muscle actin, a marker of myofibroblast transdifferentiation. Epo transcription was associated with megalin‐expressing, kidney injury molecule 1‐negative nephron segments and contingent on residual renal function. Liver‐derived EPO did not contribute to serum EPO in molidustat‐treated mice. Epo transcription was not associated with myofibroblasts in Phd2 knockout mice with obstructive nephropathy. Conclusions: Our studies suggest that HIF‐PHIs do not reactivate Epo transcription in interstitial myofibroblasts and that their efficacy in inducing kidney EPO in CKD is dependent on the degree of myofibroblast formation, the preservation of renal parenchyma and the level of residual renal function. [ABSTRACT FROM AUTHOR]

Aim & Methods: Extreme endurance exercise provides a valuable research model for understanding the adaptive metabolic response of older and younger individuals to intense physical activity. Here, we compare a wide range of metabolic and physiologic parameters in two cohorts of seven trained men, age 30 ± 5 years or age 65 ± 6 years, before and after the participants travelled ≈3000 km by bicycle over 15 days. Results: Over the 15‐day exercise intervention, participants lost 2–3 kg fat mass with no significant change in body weight. V̇O2max did not change in younger cyclists, but decreased (p = 0.06) in the older cohort. The resting plasma FFA concentration decreased markedly in both groups, and plasma glucose increased in the younger group. In the older cohort, plasma LDL‐cholesterol and plasma triglyceride decreased. In skeletal muscle, fat transporters CD36 and FABPm remained unchanged. The glucose handling proteins GLUT4 and SNAP23 increased in both groups. Mitochondrial ROS production decreased in both groups, and ADP sensitivity increased in skeletal muscle in the older but not in the younger cohort. Conclusion: In summary, these data suggest that older but not younger individuals experience a negative adaptive response affecting cardiovascular function in response to extreme endurance exercise, while a positive response to the same exercise intervention is observed in peripheral tissues in younger and older men. The results also suggest that the adaptive thresholds differ in younger and old men, and this difference primarily affects central cardiovascular functions in older men after extreme endurance exercise. [ABSTRACT FROM AUTHOR]

Aims: Dietary inorganic phosphate (Pi) modulates renal Pi reabsorption by regulating the expression of the NaPi‐IIa and NaPi‐IIc Pi transporters. Here, we aimed to clarify the role of several Pi‐regulatory mechanisms including parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23) and inositol hexakisphosphate kinases (IP6‐kinases) in the acute regulation of NaPi‐IIa and NaPi‐IIc. Methods: Wildtype (WT) and PTH‐deficient mice (PTH‐KO) with/without inhibition of FGF23 signalling were gavaged with Pi/saline and examined at 1, 4 and 12 h. Results: Pi‐gavage elevated plasma Pi and decreased plasma Ca2+ in both genotypes after 1 h Within 1 h, Pi‐gavage decreased NaPi‐IIa abundance in WT and PTH‐KO mice. NaPi‐IIc was downregulated 1 h post‐administration in WT and after 4 h in PTH‐KO. PTH increased after 1 h in WT animals. After 4 h Pi‐gavage, FGF23 increased in both genotypes being higher in the KO group. PTHrp and dopamine were not altered by Pi‐gavage. Blocking FGF23 signalling blunted PTH upregulation in WT mice and reduced NaPi‐IIa downregulation in PTH‐KO mice 4 h after Pi‐gavage. Inhibition of IP6‐kinases had no effect. Conclusions: (1) Acute downregulation of renal Pi transporters in response to Pi intake occurs also in the absence of PTH and FGF23 signalling, (2) when FGF23 signalling is blocked, a partial contribution of PTH is revealed, (3) IP6 kinases, intracellular Pi‐sensors in yeast and bacteria, are not involved, and (4) Acute Pi does not alter PTHrp and dopamine. Thus, signals other than PTH, PTHrp, FGF23 and dopamine contribute to renal adaption. [ABSTRACT FROM AUTHOR]

Neither the total cell number nor the percentage of Pax-7-positive cells was changed by the treatment of Yoda 1 at a concentration which would activate most Piezo1 channels. Screened from ~3.25 million compounds with an unbiased cell-based fluorescence assay, Yoda1 with its specificity against Piezo1 has been widely tested.4 Many studies have convincingly demonstrated that Yoda1 is highly selective for the Piezo1 channel. Mechanical signals generated from the cellular environment initiate and promote myogenesis during which satellite cells (SC cells), the muscle precursor cells, are directed to proliferate, differentiate and migrate with temporal and spatial patterns. [Extracted from the article]

Applications of Artificial Intelligence (AI) have become part of our life. The reason for the success here was the tremendously improved computing power and the availability of graphics processing units (GPUs) that have an intrinsic architecture ideally suited for artificial neuronal network applications. [Extracted from the article]

Aim: To describe ribosome biogenesis during resistance training, its relation to training volume and muscle growth. Methods: A training group (n = 11) performed 12 sessions (3‐4 sessions per week) of unilateral knee extension with constant and variable volume (6 and 3‐9 sets per session respectively) allocated to either leg. Ribosome abundance and biogenesis markers were assessed from vastus lateralis biopsies obtained at baseline, 48 hours after sessions 1, 4, 5, 8, 9 and 12, and after eight days of de‐training, and from a control group (n = 8). Muscle thickness was measured before and after the intervention. Results: Training led to muscle growth (3.9% over baseline values, 95% CrI: [0.2, 7.5] vs. control) with concomitant increases in total RNA, ribosomal RNA, upstream binding factor (UBF) and ribosomal protein S6 with no differences between volume conditions. Total RNA increased rapidly in response to the first four sessions (8.6% [5.6, 11.7] per session), followed by a plateau and peak values after session 8 (49.5% [34.5, 66.5] above baseline). Total RNA abundance was associated with UBF protein levels (5.0% [0.2, 10.2] per unit UBF), and the rate of increase in total RNA levels predicted hypertrophy (0.3 mm [0.1, 0.4] per %‐point increase in total RNA per session). After de‐training, total RNA decreased (−19.3% [−29.0, −8.1]) without muscle mass changes indicating halted biosynthesis of ribosomes. Conclusion: Ribosomes accumulate in the initial phase of resistance training with abundances sensitive to training cessation and associated with UBF protein levels. The average accumulation rate predicts muscle training‐induced hypertrophy. [ABSTRACT FROM AUTHOR]

Aim: Defects in hepatic glycogen synthesis contribute to post‐prandial hyperglycaemia in type 2 diabetic patients. Chromogranin A (CgA) peptide Catestatin (CST: hCgA352‐372) improves glucose tolerance in insulin‐resistant mice. Here, we seek to determine whether CST induces hepatic glycogen synthesis. Methods: We determined liver glycogen, glucose‐6‐phosphate (G6P), uridine diphosphate glucose (UDPG) and glycogen synthase (GYS2) activities; plasma insulin, glucagon, noradrenaline and adrenaline levels in wild‐type (WT) as well as in CST knockout (CST‐KO) mice; glycogen synthesis and glycogenolysis in primary hepatocytes. We also analysed phosphorylation signals of insulin receptor (IR), insulin receptor substrate‐1 (IRS‐1), phosphatidylinositol‐dependent kinase‐1 (PDK‐1), GYS2, glycogen synthase kinase‐3β (GSK‐3β), AKT (a kinase in AKR mouse that produces Thymoma)/PKB (protein kinase B) and mammalian/mechanistic target of rapamycin (mTOR) by immunoblotting. Results: CST stimulated glycogen accumulation in fed and fasted liver and in primary hepatocytes. CST reduced plasma noradrenaline and adrenaline levels. CST also directly stimulated glycogenesis and inhibited noradrenaline and adrenaline‐induced glycogenolysis in hepatocytes. In addition, CST elevated the levels of UDPG and increased GYS2 activity. CST‐KO mice had decreased liver glycogen that was restored by treatment with CST, reinforcing the crucial role of CST in hepatic glycogenesis. CST improved insulin signals downstream of IR and IRS‐1 by enhancing phospho‐AKT signals through the stimulation of PDK‐1 and mTORC2 (mTOR Complex 2, rapamycin‐insensitive complex) activities. Conclusions: CST directly promotes the glycogenic pathway by (a) reducing glucose production, (b) increasing glycogen synthesis from UDPG, (c) reducing glycogenolysis and (d) enhancing downstream insulin signalling. [ABSTRACT FROM AUTHOR]

Using this technique, discrete electrically-evoked Ca SP 2+ sp release events termed Ca SP 2+ sp spikes were recorded, supporting the notion that propagation of these events leads to the Ca SP 2+ sp transient. The cardiomyocyte firestarter - RyR clusters ignite their neighbours after augmentation of Ca2+ release by -stimulation Adrenergic stimuli augment cardiomyocyte contraction through modulation of intracellular Ca SP 2+ sp transient properties. Ca SP 2+ sp release at RyR clusters gives rise to Ca SP 2+ sp sparks, which summate in space and time to produce the cell-wide Ca SP 2+ sp transient. [Extracted from the article]

Aim: The current study investigated the impact of maternal obesity on placental phenotype in relation to fetal growth and sex. Methods: Female C57BL6/J mice were fed either a diet high in fat and sugar or a standard chow diet, for 6 weeks prior to, and during, pregnancy. At day 19 of gestation, placental morphology and mitochondrial respiration and dynamics were assessed using high‐resolution respirometry, stereology, and molecular analyses. Results: Diet‐induced maternal obesity increased the rate of small for gestational age fetuses in both sexes, and increased blood glucose concentrations in offspring. Placental weight, surface area, and maternal blood spaces were decreased in both sexes, with reductions in placental trophoblast volume, oxygen diffusing capacity, and an increased barrier to transfer in males only. Despite these morphological changes, placental mitochondrial respiration was unaffected by maternal obesity, although the influence of fetal sex on placental respiratory capacity varied between dietary groups. Moreover, in males, but not females, maternal obesity increased mitochondrial complexes (II and ATP synthase) and fission protein DRP1 abundance. It also reduced phosphorylated AMPK and capacity for lipid synthesis, while increasing indices of oxidative stress, specifically in males. In females only, placental mitochondrial biogenesis and capacity for lipid synthesis, were both enhanced. The abundance of uncoupling protein‐2 was decreased by maternal obesity in both fetal sexes. Conclusion: Maternal obesity exerts sex‐dependent changes in placental phenotype in association with alterations in fetal growth and substrate supply. These findings may inform the design of personalized lifestyle interventions or therapies for obese pregnant women. [ABSTRACT FROM AUTHOR]

Aim: In the mammalian circadian clock, the CLOCK/BMAL1 heterodimer binds to E‐box enhancer elements in the promoters of its target genes to activate transcription. The classical Clock mice, the first circadian mouse mutant discovered, are behaviourally arrhythmic. In this mutant, CLOCK lacks a 51 amino acid domain corresponding to exon 19 (CLOCKΔ19), which is required for normal transactivation. While the importance of this CLOCK domain for circadian rhythms is well established, the exact molecular mechanism is still unclear. Methods: Using CRISPR/Cas9 technology, we created a CLOCK knockout – CLOCK rescue system in human circadian reporter cells and performed systematic mutational scanning to assess the functionality of individual amino acids within the CLOCK exon 19‐domain. Results: CLOCK knockout cells were arrhythmic, and circadian rhythms could be rescued by introducing wild‐type CLOCK, but not CLOCKΔ19. In addition, we identified several residues, whose mutation failed to rescue rhythms in CLOCK knockout cells. Many of these are part of the hydrophobic binding interface of the predicted dimer of the CLOCK exon 19‐domain. Conclusion: Our data not only indicate that CLOCK/BMAL1 oligomerization mediated by the exon 19‐domain is important for circadian dynamics but also suggest that the exon 19‐domain provides a platform for binding coactivators and repressors, which in turn is required for normal circadian rhythms. [ABSTRACT FROM AUTHOR]

Aim: To develop a method for direct measurement of the fluorescent d‐glucose analogue 2‐NBDG transport across the plasma membrane of single skeletal muscle fibres and derive the theoretical framework for determining the kinetic parameters for d‐glucose transport under basal conditions. Methods: A novel method is described for measuring free 2‐NBDG transport across plasma membrane of single rat muscle fibres at rest. The 2‐NBDG uptake was >90% suppressed by 100 µM cytochalasin B in both fast‐twitch and slow‐twitch fibres, indicating that the 2‐NBDG transport is GLUT‐mediated. Fibres were identified as fast‐twitch or slow‐twitch based on the differential sensitivity of their contractile apparatus to Sr2+. Results: The time course of 2‐NBDG uptake in the presence of 50 µM 2‐NBDG follows a one‐phase exponential plateau curve and is faster in fast‐twitch (rate constant 0.053 ± 0.0024 s‐1) than in slow‐twitch fibres (rate constant 0.031 ± 0.0021 s‐1). The rate constants were markedly reduced in the presence of 20 mM d‐glucose to 0.0082 ± 0.0004 s‐1 and 0.0056 ± 0.0002 s‐1 in fast‐twitch and slow‐twitch fibres respectively. 2‐NBDG transport was asymmetric, consistent with GLUT1 being the major functional GLUT isoform transporting 2‐NBDG in muscle fibres at rest. The parameters describing the transport kinetics for both 2‐NBDG and d‐glucose (dissociation constants, Michaelis–Menten constants, maximal rates of uptake and outflow) were calculated from the measurements made with 2‐NBDG. Conclusion: Free 2‐NBDG and d‐glucose transport across the plasma membrane of single rat muscle fibres at rest is fast, conclusively showing that the rate‐limiting step in d‐glucose uptake in skeletal muscle is not necessarily the GLUT‐mediated transport of d‐glucose. [ABSTRACT FROM AUTHOR]

Aims: It is unknown how β‐adrenergic stimulation affects calcium dynamics in individual RyR2 clusters and leads to the induction of spontaneous calcium waves. To address this, we analysed spontaneous calcium release events in green fluorescent protein (GFP)‐tagged RyR2 clusters. Methods: Cardiomyocytes from mice with GFP‐tagged RyR2 or human right atrial tissue were subjected to immunofluorescent labelling or confocal calcium imaging. Results: Spontaneous calcium release from single RyR2 clusters induced 91.4% ± 2.0% of all calcium sparks while 8.0% ± 1.6% were caused by release from two neighbouring clusters. Sparks with two RyR2 clusters had 40% bigger amplitude, were 26% wider, and lasted 35% longer at half maximum. Consequently, the spark mass was larger in two‐ than one‐cluster sparks with a median and interquartile range for the cumulative distribution of 15.7 ± 20.1 vs 7.6 ± 5.7 a.u. (P <.01). β2‐adrenergic stimulation increased RyR2 phosphorylation at s2809 and s2815, tripled the fraction of two‐ and three‐cluster sparks, and significantly increased the spark mass. Interestingly, the amplitude and mass of the calcium released from a RyR2 cluster were proportional to the SR calcium load, but the firing rate was not. The spark mass was also higher in 33 patients with atrial fibrillation than in 36 without (22.9 ± 23.4 a.u. vs 10.7 ± 10.9; P =.015). Conclusions: Most sparks are caused by activation of a single RyR2 cluster at baseline while β‐adrenergic stimulation doubles the mass and the number of clusters per spark. This mimics the shift in the cumulative spark mass distribution observed in myocytes from patients with atrial fibrillation. [ABSTRACT FROM AUTHOR]

Aim: Adaptive mechanisms in spinal circuits are likely involved in homeostatic responses to maintain motor output in amyotrophic lateral sclerosis. Given the role of Renshaw cells in regulating the motoneuron input/output gain, we investigated the modulation of heteronymous recurrent inhibition. Methods: Electrical stimulations were used to activate recurrent collaterals resulting in the Hoffmann reflex depression. Inhibitions from soleus motor axons to quadriceps motoneurons, and vice versa, were tested in 38 patients and matched group of 42 controls. Results: Compared with controls, the mean depression of quadriceps reflex was larger in patients, while that of soleus was smaller, suggesting that heteronymous recurrent inhibition was enhanced in quadriceps but reduced in soleus. The modulation of recurrent inhibition was linked to the size of maximal direct motor response and lower limb dysfunctions, suggesting a significant relationship with the integrity of the target motoneuron pool and functional abilities. No significant link was found between the integrity of motor axons activating Renshaw cells and the level of inhibition. Enhanced inhibition was particularly observed in patients within the first year after symptom onset and with slow progression of lower limb dysfunctions. Normal or reduced inhibitions were mainly observed in patients with motor weakness first in lower limbs and greater dysfunctions in lower limbs. Conclusion: We provide the first evidence for enhanced recurrent inhibition and speculate that Renshaw cells might have transient protective role on motoneuron by counteracting hyperexcitability at early stages. Several mechanisms likely participate including cortical influence on Renshaw cell and reinnervation by slow motoneurons. [ABSTRACT FROM AUTHOR]