Journal of Cellular Physiology

Muscle satellite cells (MuSCs) are muscle-resident stem cells that are responsible for myofiber regeneration. Although the importance of calcium ions (Ca2+) in muscle physiology has been well established, the mechanism by which Ca2+ mobilization governs MuSC function remains poorly understood. In this study, we aimed to systematically characterize Ca2+ dynamics in MuSCs and to define the mechanisms regulating these signals during muscle regeneration. By employing modified protocols for mouse MuSC isolation and Ca2+ measurement, we observed spontaneous Ca2+ fluctuations in MuSCs isolated from regenerating muscle after cardiotoxin-induced myofiber injury. Our detailed analysis using chemical Ca2+ indicators and a genetically encoded Ca2+ indicator revealed that the frequency and amplitude of Ca2+ fluctuations increased significantly during the activated and proliferative stages of MuSCs in muscle regeneration. This effect was more pronounced in MuSCs isolated from dystrophic and aged mice. Mechanistically, these Ca2+ fluctuations were at least partially mediated by mechanosensitive ion channels, including PIEZO1 and TRPM7, which promote MuSC migration. Collectively, our findings demonstrate that Ca2+ fluctuations through mechanosensitive ion channels act as a key regulator of MuSC activation during muscle regeneration and may provide new insights into the role of Ca2+ influx in muscle biology and the pathogenesis of muscle diseases.

Understanding what is requisite for attaining a biomedical faculty career is crucial for guiding trainees preparing for these roles. For nearly two decades, we have collected accounts of biomedical training and career transitions from a large cohort through annual in-depth interviews and tracking of competencies and achievements. This paper elucidates the common and varied credentials of 40 who entered research-intensive faculty careers (RIFCs). Participants completed PhDs and postdocs in a range of research-intensive institutional settings. Developing research independence and a niche were essential to RIFC attainment, and mentors played a crucial role in this development. Counter to common assumptions, high-prestige publications and grants were not in and of themselves necessary for RIFC attainment. Our findings can aid RIFC aspirants and mentors who guide them.

Seismic shifts within academia over the last several decades have seen the growth of biomedical PhD recipients alongside the relative stagnation of tenure-track research-intensive faculty careers (RIFCs). This hypercompetitive academic job market has prompted interest in the paths of those who attain RIFCs. Understanding what drives recent biomedical PhDs to make their career decisions and persist toward them requires a clear picture of how career perceptions, motivations, and intentions develop and crystallize over time. Using annual in-depth interviews across nearly two decades, this report explores the evolution of career thinking and differentiation among 40 who attained a RIFC from diverse starting points to their attainment of a RIFC. Participants strategies for navigating early scientific experiences were patterned by their varied educational and socioeconomic backgrounds. Nearly half of participants did not start with or maintain stable interest in RIFCs, exhibiting changes in both PhD and postdoctoral phases. Participants highlighted six drivers toward RIFCs including desire for independence/autonomy and contributing to knowledge/health. Our results are instructive for trainees and mentors guiding career exploration and differentiation.

IntroductionThe mitochondrial citrate carrier (CiC), which mediates the transport of citrate across mitochondria, has been implicated in various diseases, but its role in kidney tubules is unclear. Here, we unraveled a novel role of CiC in tubular metabolism in the context of antibiotics-induced acute tubular injury (ATI). MethodsATI was induced by administration of vancomycin and gentamycin for 48 hours in mice (V+G-ATI). Tubular-specific CiC knockout (KO) was induced by adeno-associated virus (AAV) serotype 9 encoding Cre recombinase driven by KSP promoter (AAV9-Ksp-Cre) injection. Unbiased proteomic and metabolomic analyses were performed in CiC KO mouse kidneys. We performed in vivo 13C metabolic flux analysis to elucidate metabolic alterations in ATI and the effect of CiC KO. ResultsIn this study, V+G-induced ferroptosis, oxidative damage, and extensive ATI in mice were alleviated by CiC KO. Metabolic reprogramming induced by CiC KO increased mitochondrial TCA cycle intermediates, including alpha ketoglutarate (AKG), and elevated levels of the endogenous antioxidant glutathione (GSH). Supplementation with AKG or GSH attenuated V+G-ATI in mice. Tracking of the 13C pyruvate / lactate revealed an increased flux of glucose oxidation pathway in V+G-ATI. Interestingly, tubular-specific CiC KO expands the effective TCA cycle pool reserve space, which may contribute to mitigation of ROS. The beneficial metabolic alteration in CiC KO requires AKG and glutamate, as simultaneous inhibition of mitochondrial transporters of AKG and glutamate attenuated the cytoprotective effects of CiC KO against antibiotic-induced oxidative damage. ConclusionsThis is the first study to demonstrate the role of mitochondrial CiC in kidney tubular epithelial cells, showing that it induces metabolic alterations that protect against antibiotic-induced ATI.

Interleukin-6 (IL-6), produced by skeletal muscle and extramuscular tissues, regulates skeletal muscle function through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. However, the interaction between intrinsic (locally produced) IL-6 and extrinsic (circulating) IL-6 in skeletal muscle remains unclear. We investigated whether and how intrinsic expression of IL-6 in cultured primary human myoblasts influences their response to extrinsic stimulation with recombinant human IL-6 (rhIL-6). Using gene silencing, we found that suppression of intrinsic IL-6 enhanced rhIL-6-induced phosphorylation of STAT1 and STAT3. Silencing STAT3 also increased rhIL-6-induced STAT1 phosphorylation, but silencing STAT1 had no effect on STAT3 phosphorylation. Pretreatment of myoblasts with neutralising anti-IL-6 antibodies increased phosphorylation of STAT1 and STAT3 induced by 50 ng/mL rhIL-6, whereas pretreatment with 5 ng/mL rhIL-6 reduced this response. Despite increased JAK/STAT signalling, IL-6 silencing decreased glucose and oleic acid uptake and oxidation under both basal and rhIL-6-stimulated conditions. Collectively, our results imply that intrinsic IL-6 restrains activation of the JAK/STAT pathway by extrinsic IL-6, but acts synergistically with it to promote myoblast energy metabolism.

Scientific supervision is central to the experience of early-career researchers (ECRs), yet its role in shaping wellbeing and retention remains underexamined from the ECR perspective. We analyzed 2,604 anonymous survey responses from predoctoral, postdoctoral and former researchers across 65 countries. Overall, 76% of respondents reported that their supervisors attitude had a moderate or severe impact on mental health. Although most entered academia for vocational reasons, negative experiences with supervisors were among the most frequently reported reasons for leaving among former researchers (48%), comparable to job insecurity and financial instability. Harm was most often associated with poor communication, disregard for wellbeing, micromanagement and competitiveness. In contrast, ECRs valued supportive rather than boss-like supervision, regular communication, realistic expectations and respect for personal time. These findings identify supervisory behavior as a major and modifiable determinant or ECRs wellbeing and retention, and highlight the need for stronger institutional accountability, mentor training and funding incentives that recognize mentorship as a core component of research culture.

Increased mechanical loading induces skeletal muscle growth and, at the ultrastructural level, promotes myofibrillogenesis and the radial growth of myofibrils. However, the mechanisms regulating these ultrastructural adaptations are not known. Here, we sought to determine whether the mechanistic target of rapamycin complex 1 (mTORC1) regulates these processes. To accomplish this, muscle-specific, tamoxifen-inducible raptor knockout (iRAmKO) mice were used to inhibit signaling through mTORC1, and growth was induced with a model of chronic mechanical overload (MOV). Using a next-generation fluorescence imaging pipeline for ultrastructural analyses, we found that mTORC1 is a critical regulator of the myofibrillogenesis and radial growth of myofibrils that occur in response to MOV. Together with other recent advances in the field, we propose a model in which mTORC1 acts as a gatekeeper that permits the retention, rather than the synthesis, of proteins that drive the ultrastructural adaptations.

Aortic dissection is life-threatening due to continued loss of medial integrity that may culminate in secondary rupture within hours to days. While pre-existing defects or hemodynamic loads compound structural deterioration of the aorta, pathological progression from symptomatic dissection channel to lethal transmural tear is poorly understood. We examined the structure of referent and acutely dissected ascending aortas by microscopy. Elastic, collagen, and cellular components of non-dissected media were intricately interconnected. Medial damage in dissection lesions was traced from ingress to central to peripheral areas. Entry tears broke cleanly through successive laminae leading to cavernous false lumens in which medial structure was destroyed. Nearby laminae with widening between flanking elastic lamellae (termed minor delaminations) were filled with blood and showed severe medial damage. Farther laminae without delamination but containing red blood cells (termed blood extravasation) displayed moderate medial damage. More distant, non-delaminated laminae with accumulation of albumin but not red blood cells (termed plasma extravasation) exhibited mild medial damage. Varying medial hemorrhage with scattered sloughing of laminae was observed along the entire false lumen. We conclude that hydraulic fracturing of residual dissected media by pressurized blood via communications from the false lumen contributes to further structural weakening of the aortic wall.

BackgroundCalcific aortic valve disease (CAVD) is the most common valvular heart disease in developed countries, yet no pharmacological therapy is available to slow or halt its progression. CAVD is driven by progressive calcification of aortic valve leaflets, in which myeloid cells play a central role. While macrophages have been implicated in CAVD pathogenesis, the contribution of their precursors, monocytes, remains poorly understood. We hypothesized that circulating monocytes acquire a pro-calcific and pro-inflammatory phenotype contributing to valve remodelling and CAVD progression. MethodsWe profiled circulating CD14+ monocytes from healthy volunteers (Vol), patients with CAVD, and without CAVD (NCAVD). Peripheral blood mononuclear cells (PBMCs) were isolated, and monocyte subpopulations were phenotyped by flow cytometry. Transcriptome profiling by RNA sequencing identified disease-associated gene signatures, which were validated by RT-qPCR. The CD14+ monocyte secretome was analysed using multiplex assays. Functional ability of CAVD-derived CD14+ monocytes to induce myofibroblastic transdifferentiation (MT) and osteoblastic differentiation (OD) of human valvular interstitial cells (VICS) was evaluated by immunocytochemistry and quantitative o-cresolphthalein complexone assays. ResultsIn PBMCs, CAVD monocytes displayed a subpopulation shift, with an increased proportion of CD14CD16- classical monocytes and a reduced CD14CD16 non-classical monocyte levels. In CD14+ monocytes, transcriptomic analysis revealed upregulation of inflammation-related (PDK4) and calcification-related (ATP2B1) genes, alongside downregulation of immunomodulatory genes (DDR1, IKBKE). Secretome analysis showed reduced production of immunomodulatory and anti-osteoblastogenic cytokines (IL-4, CCL3) while promoting gene expression of factors promoting MT and OD in VICS. These alterations were associated with a marked monocyte-induced increase in SMA and OPN expression in VICS and a two-fold increase in calcification. ConclusionWe demonstrate for the first time that circulating monocytes from patients with CAVD exhibit enhanced pro-inflammatory and pro-calcific properties that may contribute to CAVD progression. Additionally, we identify dysregulated gene sets within these monocytes that represent potential novel therapeutic targets for CAVD.

Osteocytes and adipocytes represent cells with disparate functions. Osteocytes regulate bone metabolism (remodeling) and bone homeostasis, while adipocytes regulate energy metabolism and energy storage. Here, we demonstrate that osteocyte phenotype consists of adipocytic features which are under control of peroxisome proliferator-activated receptor gamma (PPARG), a master regulator of adipocyte differentiation and function. Using a mouse model with osteocyte-specific deletion of PPARG (OT{gamma}KO) and osteocyte cellular model of MLO-Y4 cells edited with CRISPR/Cas9 for PPARG deficiency, we are demonstrating that under PPARG control osteocytes produce and secrete adiponectin (ADIPOQ), and they are equipped in adipocyte-specific mechanisms for lipid-storage and their metabolism. Under PPARG, osteocytes accumulate lipid droplets which correlate with their capability to cover up to 20% of energy requirements from fatty acids metabolism. Although osteocytes like osteoblasts mainly express perilipin 2 (Plin2), however similarly to adipocytes, lipid droplets accumulation is associated with expression of perilipin 1 (Plin1) under PPARG control. Similarly, lipids accumulation and metabolism involve adipocyte-specific activities including fatty acids binding protein 4 (Fabp4), hormone-specific lipase (Hsl) and adipocyte-specific triglyceride lipase (Atgl), which expression are under PPARG control. These studies provide a new understanding of osteocyte biology which include adipocyte-like endocrine and lipid metabolism features probably reflecting an adaptation to their unique localization and a need for a maintenance of functional fitness in these conditions. They deepen our comprehension of the crossroads of osteocyte and adipocyte function and underscore the therapeutic potential of targeting common molecular pathways in both cell types for managing metabolic disorders and skeletal diseases.

Regulation of water permeability in the collecting duct is important for osmoregulatory acclimation in teleost fish. In hyperosmotic environments such as seawater (SW), the teleost kidney functions as a site of divalent ion excretion. The collecting ducts reabsorb Na+, Cl-, and water, thereby reducing urine volume and producing small amounts of isotonic urine with high concentrations of divalent ions. In hypoosmotic environments such as freshwater (FW) or low-salinity brackish water (BW), the kidney produces large volumes of hypotonic urine and serves as a site of water excretion; under these conditions, the collecting ducts reabsorb Na+ and Cl- but not water. To identify aquaporins (Aqps) involved in regulating water permeability in the collecting ducts of teleosts, we analyzed renal Aqp expression in a euryhaline marine fish, the Japanese pufferfish (Takifugu rubripes), which possesses 16 Aqp genes in its genome, seven of which (Aqp1aa, 1ab, 3a, 4a, 7, 8bb, and 11a) are expressed in the kidney. Quantitative RT-PCR analysis showed that Aqp1aa and Aqp4a were highly expressed in collecting duct tissues, and that Aqp1aa expression was markedly reduced in fish acclimated to BW. Immunohistochemistry revealed apical localization of Aqp1aa and basolateral localization of Aqp4 in collecting duct cells, with apical Aqp1aa downregulated in BW. These results suggest that Aqp1aa and Aqp4 mediate water reabsorption in SW and that downregulation of Aqp1aa contributes to hypotonic urine production in BW. NEW & NOTEWORTHYRegulation of water permeability in the collecting duct is important for osmoregulation in teleost fish. Expression analyses of aquaporins (Aqps) in the marine pufferfish Takifugu rubripes showed that Aqp1aa and Aqp4a are highly expressed in the collecting duct and localized to the apical and basolateral membranes, respectively. Renal Aqp1aa expression was markedly reduced in fish acclimated to hypoosmotic brackish water. These results indicate that collecting duct water permeability is regulated by Aqp1aa expression.

Mitochondrial homeostasis, governed by the balance between biogenesis and mitophagy, is essential for steroidogenesis in adrenocortical cells. While the requirement of active mitochondria for steroid synthesis is well-established, the hormonal regulation of genes governing mitochondrial function remains poorly understood. This study investigated whether angiotensin II (Ang II) and the cAMP/PKA pathway modulate the expression of key regulatory factors involved in mitochondrial biogenesis and redox status in the human adrenocortical H295R cell line. Using real-time qPCR and Western blot, we show that Ang II and 8Br-cAMP --a permeant analogue of cAMP-- modulate NRF-1, Nrf2, UCP2, and ANT1 impacting on mitochondrial biogenesis, antioxidant defense, and respiratory activity. These molecular changes correlated with increased mitochondrial membrane polarization, as confirmed by MitoTracker red staining. Interestingly, Ang II stimulation promoted a time-dependent increase in TFAM levels, a key transcription factor in mitochondria, which correlates with the increase in mitochondrial DNA (mtDNA) content. The rate of oxygen consumption (OCR) and mitochondrial parameters were determined, with results showing that Ang II led to a significant increase in basal and maximum respiration, ATP production, and proton leak. These findings suggest that hormone stimulation favors mitochondrial activity, thereby enhancing the bioenergetic capacity of adrenocortical cells. Furthermore, treatment with the uncoupler CCCP triggered a retrograde signaling response, upregulating nuclear-encoded mitochondrial genes to counteract mitochondrial membrane depolarization. Our findings demonstrate for the first time that hormonal signals directly modulate the mitochondrial genetic program in H295R human adrenocortical cells, optimizing the bioenergetic platform required for efficient steroidogenic function.

We investigated effects of three aerobic exercise interventions, varying in amount and intensity with durations of 8-9-months on small RNA (smRNA) expression and regulatory pathways in skeletal muscle and plasma from 120 participants. Using untargeted smRNA sequencing focused on miRNAs and piRNAs, adjusting for demographics and bodyweight, we identified 124 muscle smRNAs altered by exercise amount and 15 by intensity, and 47 plasma smRNAs altered by intensity and one by amount. These smRNAs were enriched in metabolic, transcriptional, translational, and cell cycle pathways. Exercise-induced changes in several smRNAs-six from muscle and five from plasma-and exercise-induced reduction in body weight, aligned with improvement in insulin sensitivity (p<0.05). These findings demonstrate tissue-specific regulation of smRNAs by exercise and identify potential candidates for exercise mimetics to modulate muscle insulin sensitivity.

The heart maintains systemic perfusion through the coordinated function of its four chambers: the left and right atria and ventricles. Each chamber has distinct structural, functional, and molecular properties tailored to its role in circulation, which may result in chamber-specific differences in myofilament structure and regulation between atria and ventricles. To test this hypothesis, we employed muscle mechanics and X-ray diffraction to investigate functional and structural differences in porcine left atrial (LA) and left ventricular (LV) tissue. Here, we report the first X-ray diffraction study of atrial tissue, demonstrating that under resting conditions, myosin filaments in LA adopted a more ON-like, structurally distinct configuration compared with those in LV. Under contracting conditions, LV generated greater force and exhibited higher sinusoidal stiffness than LA across multiple calcium concentrations. LA showed faster kTR than in LV, with no calcium-dependence, in contrast to the calcium-dependence of kTR seen in LV. Structurally, the distinct myosin head configuration seen in the relaxed LA persisted during contraction. Furthermore, using the troponin inhibitor MYK-7660 to inhibit active contraction, we showed that, unlike LV, LA showed no direct calcium-dependent thick filament activation, reconciling discrepancies between fast rat and slow porcine ventricular myocardium regarding calciums role in thick filament regulation. Altogether, our study reveals that LA myosin filaments adopt a molecular architecture and regulatory mechanism distinct from their LV counterparts, suggesting that myosin filament structure and regulation have evolved differently to meet the unique functional demands of each cardiac chamber. Moreover, atrial disease is often associated with cardiomyopathy-related genetic variants, highlighting the atrial myocardium as an important therapeutic target and understanding atrial-specific regulatory mechanisms provides new insights into therapeutic strategies for atrial diseases.

Course-based undergraduate research experiences (CUREs) can expand undergraduates access to research and motivate students to stay in science. Yet, little research has examined how CURE instruction shapes student motivation. We leveraged a motivation-related characterization of non-content talk of 48 CURE and non-CURE instructors to predict the motivation-related outcomes of 462 students. We fit a series of multi-level models (MLM) in which we regressed students post-course scientific self-efficacy, task values, scientific identity, and science-related intentions onto instructors self-efficacy and task values-related talk, controlling for students pre-course levels. We also fit an MLM to explore whether instructors relationship-building talk (immediacy talk) was associated with students rapport with their instructor. Instructors self-efficacy talk did not affect students self-efficacy, and instructors immediacy talk had a marginally positive but non-significant association with students rapport ratings. Instructors task values talk positively influenced students scientific identity and some but not all of their task values. Instructors task values talk also positively influenced students intentions to pursue a science career, but not graduate education or research careers. Collectively, these results suggest that instructors task values talk may underpin some of the motivational effects of CURE instruction, but that task values talk need not be limited to CUREs. HIGHLIGHTWe examine whether instructor talk predicts students motivational outcomes in CURE and non-CURE lab courses. Self-efficacy talk had no effect on student self-efficacy. Task values talk positively affected students science identity and career intentions, and some value beliefs. Immediacy talk was marginally related to student-instructor rapport.

Graduate-level genomics courses require students to integrate dense material across subfields, concepts and methods. In modular, multi-instructor courses, students may struggle because the coherence between lectures can be difficult to navigate, while the course structure may be visible to instructors. We evaluated a 2025 navigation redesign of BIO322, a graduate genomics course at the Norwegian University of Life Sciences, while preserving course content, multi-instructor teaching, modular organization and assessment framework. The redesign includes introducing a standardized self-learning guide, expanded syllabus, enriched online quiz feedback, and added support for a final group research proposal. Using anonymized course evaluation scores from 2021-2025 and aggregated learning management system access data from 2023-2025, we examined student experience and resource use. In 2025, five of six course evaluation items reached their highest observed BIO322 scores, while one, lecture-specific score remained within the previous range. The consolidated self-learning guide was accessed by nearly all students, whereas access to optional readings declined across the course sequence, despite comparatively stable page views per accessing student. These course-level findings are consistent with improved perceived navigability following the introduction of standardized learning support. However, some students continued to report difficulty identifying priorities and connections among course components, indicating that challenges in perceived course coherence remained for part of the cohort despite the redesign. Practitioner PointsO_LIMaking course structure explicit may improve students perceived navigability in multi-instructor graduate genomics courses. C_LIO_LIA centralized self-learning guide can broaden access to preparatory guidance without changing core course content or assessment. C_LIO_LIOptional learning supports may be used unevenly, so resource availability should not be assumed to translate into uniform resource access. C_LI

The activation of thermogenesis in brown adipose tissue (BAT) represents a pivotal target for ameliorating disorders of glucose and lipid metabolism. This study sought to elucidate the regulatory effects of quercetin on thermogenesis and glucose-lipid metabolism within brown adipocytes, alongside its underlying molecular mechanisms. The findings demonstrated that quercetin markedly upregulated the expression of uncoupling protein 1 (UCP1), a critical thermogenic protein in brown adipocytes, thereby enhancing cellular thermogenic capacity and effectively mitigating glucose and lipid metabolism disorders. Subsequent mechanistic investigations confirmed that quercetin activated the COX2-PGE2-EP4-UCP1 signaling axis by augmenting the stability of cyclooxygenase 2 (COX2) protein, thus mediating its thermogenic-promoting and metabolism-improving effects. This study identifies quercetin as a potential therapeutic agent for the improvement of glucose and lipid metabolism disorders, uncovers a novel molecular mechanism through which quercetin regulates brown adipocyte thermogenesis, and provides a theoretical and experimental foundation for the application of quercetin in the prevention and treatment of obesity and related metabolic diseases.

The adult skeletal muscle regenerates robustly upon injury, but this regenerative capacity rapidly declines with age. In this study, we identify the lanthionine synthetase C-Like (LanCL) proteins, mammalian homologs of the bacterial peptide cyclase LanC, as positive regulators of muscle regeneration in middle-aged mice. In a barium chloride-induced injury model, we found the protein levels of LanCL1 and LanCL2 to increase during an early phase of regeneration in middle-aged (12-month-old) but not young adult (4-month-old) mice. Utilizing a mouse line lacking all three LanCL proteins (LanCL triple KO or LTKO), we examined a potential role of LanCL in injury-induced muscle regeneration. Consistent with an age-dependent function of LanCL, we observed a delayed regeneration of the tibialis anterior (TA) muscle after injury, as reflected by reduced sizes of regenerating myofibers in middle-aged (but not young) LTKO compared to age-matched WT mice. Although the pool size of quiescent satellite cells (Pax7+) was comparable between 12-month-old LTKO and WT muscles without injury, the number of Pax7+ cells was significantly higher in regenerating LTKO muscles at day 5 after injury, accompanied by drastically decreased numbers of MyoD+ and MyoG+ cells, as well as increased numbers of proliferating cells. In addition, we detected elevated expression of pro-inflammatory cytokines in regenerating LTKO muscles, while the number of macrophages was similar comparing LTKO and WT muscles. Taken together, our observations suggest that in aging muscles LanCLs are important for proper timing of inflammation resolution and regeneration upon injury. New & NoteworthyPhysiological roles of the mammalian homologs of bacterial LanC, LanCLs, are poorly understood. Our work uncovers a function of LanCLs in post-injury regeneration of aging skeletal muscles. Middle-aged LanCL triple KO mice displayed a delay in satellite cell differentiation and regenerative myofiber formation, as well as persistent inflammatory cytokine expression, suggesting that LanCLs may have an age-dependent role in modulating inflammation in the injured muscles to facilitate regeneration.

The tight regulation of iron homeostasis is of great importance for cellular health. An increase in intracellular iron levels results in the formation of free radicals, which damages macromolecules and membranes, eventually resulting in cell death by Ferroptosis. Recently, we showed that patients with mutations in VPS41 display a severe neurodegenerative phenotype with iron deposition in the brain. VPS41 is well known as subunit of the HOPS complex required for fusion of late endosomes and autophagosomes with lysosomes. However, VPS41 has also been identified as inhibitor of Ferroptosis and regulator of redox homeostasis. How VPS41 exerts these functions and if these are dependent on the HOPS complex is unknown. Here we show that depletion of VPS41 results in increased intracellular iron levels, ROS formation and mitochondrial fission. Our findings indicate an important role for VPS41 in the regulation of iron homeostasis and mitochondrial fission and suggest Ferroptosis as a possible cause for neurodegeneration in VPS41 patients.

Study questionDoes the human placenta utilize the creatine phosphagen system for energy homeostasis during development? Summary answerComponents of the creatine (Cr)-creatine kinase (CK)-phosphocreatine (PCr) system are dynamically expressed by the trophoblast and mesenchymal compartments throughout gestation wherein creatine kinase is required for cellular ATP metabolism, cell cycle, and proliferation of trophoblast cells. What is known alreadyThe Cr-CK-PCr system maintains ATP homeostasis in tissues with high energy demand and is required for proliferation, migration, and invasion of tumor cells. The term human placenta can synthesize and transport creatine locally. Early placental development involves trophoblast proliferation, an event requiring ATP, but the role of the creatine phosphagen system during early placental development remains unknown. Study design, size, durationWe performed immunohistochemistry (IHC) and immunofluorescence (IF) for different components (biosynthesis, transport, utilization) of the Cr-Ck-PCr system in human placentae (n=3/group) across gestation including first trimester, second trimester, and term. Using primary human trophoblast stem cells (hTSCs) and trophoblast organoids (TO), we determined the role of the creatine phosphagen system in trophoblast growth by functional inhibition of creatine kinase. Participants/materials, setting, methodsIHC/IF were performed in human placentae across gestation for proteins involved in biosynthesis (AGAT and GAMT), transport (SLC6A8, SLC22A15, and SLC6A13) and utilization (CKB and CKMT1) of creatine to determine the presence of the creatine phosphagen system locally in the placenta. For delineating the functional importance of this system in placental development, cyclocreatine (cCr), a creatine analogue, was used for functional inhibition of CK. Primary hTSCs were culture in medium containing 0 (control), 1, 10, 20 mM cCr for 48 hours followed by analysis of cell growth (cell count), cell cycle (EdU incorporation assay), apoptosis (Annexin V/PI flow cytometry), energy metabolism (Sea horse mito-stress and glycolytic stress tests), and gene expression (qPCR). Primary TO were also treated with 20mM cCr for 6 days in vitro to determine the role of Cr-CK-PCr system in placental development. Main results and the role of chanceAGAT localized to the fetal villous mesenchyme, while GAMT was broadly expressed in the trophoblast and fetal mesenchyme compartments across gestation. CKB localized primarily to fetal mesenchyme with strongest expression at term. CKMT1 was broadly expressed in all trophoblast subtypes. SLC6A8 was abundant in early syncytiotrophoblast but absent at term, where its expression shifted to fetal blood vessels. SLC22A15 was expressed in the endothelial cells of fetal capillaries across gestation. In primary hTSCs, cyclocreatine (20mM) treatment reduced proliferation (P<0.001), decreased expression of trophoblast epithelial marker EGFR (P<0.05), induced G0/G1 and G2/M arrests (P<0.0001), enhanced early and late apoptosis (P<0.0001), and downregulated GPX8 expression (P<0.05). Seahorse analysis revealed marked reductions (P<0.01) in mitochondrial (basal, maximal, and ATP-linked) and glycolytic (rate, capacity, and reserve) function compared to controls. In primary human TO, cyclocreatine treatment reduced the growth of organoids (P<0.05) as well the expression of EGFR (P<0.05). Large scale dataN/A Limitations, reasons for cautionFurther experiments assessing apoptosis, cellular stress and redox imbalance may provide more mechanistic role of the creatine phosphagen system in trophoblast metabolism and function. Since the functional role of the Cr-CK-PCr system was investigated in vitro, findings of this study should be taken with caution for implications of in vivo placental development. Nevertheless, reproducible results of reduced growth of trophoblast cells using both 2D and 3D cultures is highly suggestive of the importance of the creatine phosphagen system in early placental development. Wider implications of the findingsThis study provides foundational knowledge that the placenta contains the creatine phosphagen system, known for ATP homeostasis, and that this system ensures proper cell division, survival and placental development. Dysregulation of components of Cr-CK-PCr system in placenta has been observed in pregnancy disorders such as preeclampsia and fetal growth restriction warranting continued investigation into mechanisms and potential remediation using creatine supplementation. Stem cells share similar metabolic features so findings of this study can be implicated in other stem cells models as well. Study funding/competing interest(s)This work was supported by CIRM EDUC4-12804 Interdisciplinary Stem Cell Training Grant and a Lalor Foundation Postdoctoral Fellowship awarded to NS, and by the California Institute for Regenerative Medicine (DISC0-13757) and the National Institute of Child Health and Human Development (R01-HD096260) award to FS. The authors have no competing interest to declare.