Constitutive Deletion Research Articles

Meis transcription factors regulate cardiac conduction system development and adult function.

The Cardiac Conduction System (CCS) is progressively specified during development by interactions among a discrete number of Transcriptions Factors that ensure its proper patterning and the emergence of its functional properties. Meis genes encode homeodomain transcription factors (TFs) with multiple roles in mammalian development. In humans, Meis genes associate with congenital cardiac malformations and alterations of cardiac electrical activity, however the basis for these alterations has not been established. Here we studied the role of Meis transcription factors in cardiomyocyte development and function during mouse development and adult life. We studied Meis1 and Meis2 conditional deletion mouse models that allowed cardiomyocyte-specific elimination of Meis function during development and inducible elimination of Meis function in cardiomyocytes of the adult CCS. We studied cardiac anatomy, contractility and conduction. We report that Meis factors are global regulators of cardiac conduction, with a predominant role in the CCS. While constitutive Meis deletion in cardiomyocytes led to congenital malformations of the arterial pole and atria, as well as defects in ventricular conduction, Meis elimination in cardiomyocytes of the adult CCS produced sinus node dysfunction and delayed atrio-ventricular conduction. Molecular analyses unraveled Meis-controlled molecular pathways associated with these defects. Finally, we studied in transgenic mice the activity of a Meis1 human enhancer related to an SNP associated by GWAS to PR elongation and found that the transgene drives expression in components of the atrio-ventricular conduction system. Our study identifies Meis TFs as essential regulators of the establishment of cardiac conduction function during development and its maintenance during adult life. In addition, we generated animal models and identified molecular alterations that will ease the study of Meis-associated conduction defects and congenital malformations in humans.

A Constitutive Heterochromatic Region Shapes Genome Organization and Impacts Gene Expression in Neurospora crassa.

Organization of the eukaryotic genome is essential for proper function, including gene expression. In metazoans, chromatin loops and Topologically Associated Domains (TADs) organize genes into transcription factories, while chromosomes occupy nuclear territories in which silent heterochromatin is compartmentalized at the nuclear periphery and active euchromatin localizes to the nucleus center. A similar hierarchical organization occurs in the fungus Neurospora crassa where its seven chromosomes form a Rabl conformation typified by heterochromatic centromeres and telomeres independently clustering at the nuclear membrane, while interspersed heterochromatic loci aggregate across Megabases of linear genomic distance to loop chromatin in TAD-like structures. However, the role of individual heterochromatic loci in normal genome organization and function is unknown. We examined the genome organization of a Neurospora strain harboring a ~47.4 kilobase deletion within a temporarily silent, facultative heterochromatic region, as well as the genome organization of a strain deleted of a 110.6 kilobase permanently silent constitutive heterochromatic region. While the facultative heterochromatin deletion minimally effects local chromatin structure or telomere clustering, the constitutive heterochromatin deletion alters local chromatin structure, the predicted three-dimensional chromosome conformation, and the expression of some genes, which are qualitatively repositioned into the nucleus center, while increasing Hi-C variability. Our work elucidates how an individual constitutive heterochromatic region impacts genome organization and function. Specifically, one silent region indirectly assists in the hierarchical folding of the entire Neurospora genome by aggregating into the "typical" heterochromatin bundle normally observed in wild type nuclei, which may promote normal gene expression by positioning euchromatin in the nucleus center.

Constitutive Pleiotrophin Deletion Results in a Phenotype with an Altered Pancreatic Morphology and Function in Old Mice.

Pleiotrophin (PTN) is crucial for embryonic development and pancreas organogenesis as it regulates metainflammation, metabolic homeostasis, thermogenesis, and glucose tolerance. Pleiotrophin deletion is associated with a lipodystrophic phenotype in which adipose tissue plasticity is altered in late life. This study explored the impact of pleiotrophin deletion on pancreatic morphology and function in later life. We analyzed glucose tolerance and circulating parameters on female wild-type (Ptn+/+) and knock-out (Ptn-/-) mice. At 9 and 15 months, we conducted morphometric analyses of pancreatic islets and evaluated the levels of insulin, glucagon, somatostatin, glucose transporter 2 (GLUT2), vesicle-associated membrane protein 2 (VAMP2), and synaptosome-associated protein 25 (SNAP25) via immunofluorescence. The effect of PTN on glucose-stimulated insulin secretion (GSIS) was evaluated in INS1E cells and isolated islets. Ptn-/- mice showed hyperinsulinemia, impaired glucose tolerance, and increased homeostatic model assessment for insulin resistance (HOMA-IR) with age. While Ptn+/+ islets enlarge with age, in Ptn-/- mice, the median size decreased, and insulin content increased. Vesicle transport and exocytosis proteins were significantly increased in 9-month-old Ptn-/- islets. Islets from Ptn-/- mice showed impaired GSIS and decreased cell membrane localization of GLUT2 whereas, PTN increased GSIS in INS1E cells. Ptn deletion accelerated age-related changes in the endocrine pancreas, affecting islet number and size, and altering VAMP2 and SNAP25 levels and GLUT2 localization leading to impaired GSIS and insulin accumulation in islets.

The small noncoding RNA Vaultrc5 is dispensable to mouse development.

Vault RNAs (vtRNAs) are evolutionarily conserved small noncoding RNAs transcribed by RNA polymerase III. Vault RNAs were initially described as components of the vault particle, but have since been assigned multiple vault-independent functions, including regulation of PKR activity, apoptosis, autophagy, lysosome biogenesis, and viral particle trafficking. The full-length transcript has also been described as a noncanonical source of miRNAs, which are processed in a DICER-dependent manner. As central molecules in vault-dependent and independent processes, vtRNAs have been attributed numerous biological roles, including regulation of cell proliferation and survival, response to viral infections, drug resistance, and animal development. Yet, their impact to mammalian physiology remains largely unexplored. To study vault RNAs in vivo, we generated a mouse line with a conditional Vaultrc5 loss-of-function allele. Because Vaultrc5 is the sole murine vtRNA, this allele enables the characterization of the physiological requirements of this conserved class of small regulatory RNAs in mammals. Using this strain, we show that mice constitutively null for Vaultrc5 are viable and histologically normal but have a slight reduction in platelet counts, pointing to a potential role for vtRNAs in hematopoiesis. This work paves the way for further in vivo characterizations of this abundant but mysterious RNA molecule. Specifically, it enables the study of the biological consequences of constitutive or lineage-specific Vaultrc5 deletion and of the physiological requirements for an intact Vaultrc5 during normal hematopoiesis or in response to cellular stresses such as oncogene expression, viral infection, or drug treatment.

Abstract Or105: Constitutive Deletion of the Obscurin Ig58/59 Domains Induces Structural and Electrophysiological Remodeling in Atria

Introduction: Obscurin is a giant cytoskeletal protein that supports muscle development, tethers intracellular compartments and regulates contraction. In the ObscnΔIg58/59 mouse model, expressing obscurin lacking Immunoglobulin (Ig) domains 58 and 59, aging males exhibit irregular heart rhythm with prominent atrial fibrillation, atrial enlargement, and progressive remodeling of the ventricles. A mechanistic basis for the emergence of arrhythmia early on could not be identified in ObscnΔIg58/59 ventricles, suggesting that the atria are preferentially impacted by deletion of the obscurin Ig58/59 module prior to the ventricles. Hypothesis: We hypothesize that Ig58/59 deletion elicits unique structural, electrical, and functional consequences in the atria preceding ventricular maladaptation. Aims: I. Examine the structural organization of ObscnΔIg58/59 atria II. Assess Ca 2+ handling in isolated atrial cardiomyocytes and determine molecular alterations. Methods: Electron microscopy and super resolution microscopy were utilized to visualize sarcomeric ultrastructure and the transverse-axial tubule (TAT) network, respectively. Ca 2+ handling activity and synchrony were also resolved, while Phos-Tag gels were used to quantify T-cap phospho-species. Results: ObscnΔIg58/59 atria exhibited misalignment of Z-disks. Spontaneous and stimulated Ca 2+ cycling behavior were [AB1] differentially disrupted in atrial cardiomyocytes in 6- and 12-month ObscnΔIg58/59 males. Relatedly, atrial cells showed an age-dependent deterioration in TAT architecture. Finally, as a function of aging, ObscnΔIg58/59 atria displayed alterations in the expression and phosphorylation of T-cap, a Z-disk protein implicated in the integration of t-tubules with the sarcomeric cytoskeleton. Conclusions: The structural and electrical alterations that arise in ObscnΔIg58/59 atria precede ventricular dysfunction and coincide with the emergence of atrial fibrillation. Collectively, our work indicates that the atria are principally affected by Ig58/59 elimination, instigating arrhythmias and morphological alterations with age. The ObscnΔIg58/59 mouse model has thus emerged as a proxy for atrial cardiomyopathy.

PKMζ alters oxycodone-taking in a dose- and sex-dependent manner

Opioid use disorder involves disruptions to glutamate homeostasis and dendritic spine density in the reward system. PKMζ is an atypical isoform of protein kinase C that is expressed exclusively in neurons and plays a role in postsynaptic glutamate signaling and dendritic spine maturation. As opioid use leads to alterations in glutamate transmission and dendritic spine density, we hypothesized that PKMζ deletion would alter opioid-taking behaviors. The current study examined two doses of oxycodone self-administration in male and female mice with constitutive deletion of PKMζ compared to wildtype controls. At a dose of 0.25 mg/kg/infusion, PKMζ deletion significantly potentiated oxycodone self-administration in both male and female mice. However, increases in motivation for oxycodone, as indicated by increased breakpoint on a progressive ratio schedule, were only seen in male PKMζ knockout mice and not females. When we examined a lower dose of oxycodone, 0.125 mg/kg/infusion, PKMζ knockout led to increases in oxycodone self-administration only in female mice. Additionally, female PKMζ knockout mice exhibited higher breakpoints on a progressive ratio schedule at this dose compared to all other groups. In addition to the self-administration studies, we also examined locomotor sensitization in response to experimenter administered oxycodone. PKMζ KO decreased oxycodone induced locomotion in males and potentiated oxycodone sensitization in females. Together, these results suggest that PKMζ acts to dampen oxycodone taking in both sexes, but females may be more sensitive to its effects.

Connexin-45 is expressed in mouse lymphatic endothelium and required for lymphatic valve function.

The expression and functional relevance of the gap junction molecule connexin-45 (Cx45; GJC1) in lymphatic endothelium were not previously known. We found that Cx45 was expressed widely in the endothelium of murine lymphatics, in both valve and nonvalve regions. Cell-specific deletion of Cx45, driven by a constitutive Cre line (Lyve1-Cre) or an inducible Cre line (Prox1-CreERT2), compromised the function of lymphatic valves, as assessed by physiological tests (back leak and closure) of isolated, single-valve vessel segments. The defects were comparable to those previously reported for loss of Cx43, and as with Cx43, deletion of Cx45 resulted in shortening or increased asymmetry of lymphatic valve leaflets, providing an explanation for the compromised valve function. In contrast with Cx43, lymphatic endothelial cell-specific (LEC-specific) deletion of Cx45 did not alter the number of valves in mesenteric or dermal lymphatic networks or the expression patterns of the canonical valve-associated proteins PROX1, ITGA9, or CLAUDIN5. Constitutive deletion of Cx45 from LECs resulted in increased backflow of injected tracer in popliteal networks in vivo and compromised the integrity of the LEC permeability barrier in a subset of collecting vessels. These findings provide evidence for an unexpected role of Cx45 in the development and maintenance of lymphatic valves.

Cardiomyocyte PANX1 Controls Glycolysis and Neutrophil Recruitment in Hypertrophy.

PANX1 (pannexin 1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation, blood pressure regulation, and myocardial infarction. However, the possible role of PANX1 in cardiomyocytes in the progression of heart failure has not yet been investigated. We generated a novel mouse line with constitutive deletion of PANX1 in cardiomyocytes (Panx1MyHC6). PANX1 deletion in cardiomyocytes had no effect on unstressed heart function but increased the glycolytic metabolism and resulting glycolytic ATP production, with a concurrent decrease in oxidative phosphorylation, both in vivo and in vitro. In vitro, treatment of H9c2 (H9c2 rat myoblast cell line) cardiomyocytes with isoproterenol led to PANX1-dependent release of ATP and Yo-Pro-1 uptake, as assessed by pharmacological blockade with spironolactone and siRNA-mediated knockdown of PANX1. To investigate nonischemic heart failure and the preceding cardiac hypertrophy, we administered isoproterenol, and we demonstrated that Panx1MyHC6 mice were protected from systolic and diastolic left ventricle volume increases as a result of cardiomyocyte hypertrophy. Moreover, we found that Panx1MyHC6 mice showed decreased isoproterenol-induced recruitment of immune cells (CD45+), particularly neutrophils (CD11b+ [integrin subunit alpha M], Ly6g+ [lymphocyte antigen 6 family member G]), to the myocardium. Together, these data demonstrate that PANX1 deficiency in cardiomyocytes increases glycolytic metabolism and protects against cardiac hypertrophy in nonischemic heart failure at least in part by reducing immune cell recruitment. Our study implies PANX1 channel inhibition as a therapeutic approach to ameliorate cardiac dysfunction in patients with heart failure.

IRF8 configures enhancer landscape in postnatal microglia and directs microglia specific transcriptional programs.

Microglia are innate immune cells in the brain. Transcription factor IRF8 is highly expressed in microglia. However, its role in postnatal microglia development is unknown. We demonstrate that IRF8 binds stepwise to enhancer regions of postnatal microglia along with Sall1 and PU.1, reaching a maximum after day 14. IRF8 binding correlated with a stepwise increase in chromatin accessibility, which preceded the initiation of microglia-specific transcriptome. Constitutive and postnatal Irf8 deletion led to a loss of microglia identity and gain of disease-associated microglia-like genes. Combined analysis of scRNA-seq and scATAC-seq revealed a correlation between chromatin accessibility and transcriptome at a single-cell level. IRF8 was also required for microglia-specific DNA methylation patterns. Lastly, in the 5xFAD model, constitutive and postnatal Irf8 deletion reduced the interaction of microglia with Aβ plaques and the size of plaques, lessening neuronal loss. Together, IRF8 sets the epigenetic landscape, which is required for postnatal microglia gene expression.

Loss of Toll-like receptor 9 protects from hepatocellular carcinoma in murine models of chronic liver disease

Background & aimsToll-like receptor 9 (Tlr9) is a pathogen recognition receptor detecting unmethylated DNA derivatives of pathogens and damaged host cells. It is therefore an important modulator of innate immunity. Here we investigated the role of Tlr9 in fibrogenesis and progression of hepatocellular carcinoma in chronic liver disease. Materials and methodsWe treated mice with a constitutive deletion of Tlr9 (Tlr9−/−) with DEN/CCl4 for 24 weeks. As a second model, we used hepatocyte-specific Nemo knockout (NemoΔhepa) mice and generated double knockout (NemoΔhepaTlr9−/−) animals. ResultsWe show that Tlr9 is in the liver primarily expressed in Kupffer cells, suggesting a key role of Tlr9 in intercellular communication during hepatic injury. Tlr9 deletion resulted in reduced liver fibrosis as well as tumor burden. We observed down-regulation of hepatic stellate cell activation and consequently decreased collagen production in both models. Tlr9 deletion was associated with decreased apoptosis and compensatory proliferation of hepatocytes, modulating the initiation and progression of hepatocarcinogenesis. These findings were accompanied by a decrease in interferon-β and an increase in chemokines having an anti-tumoral effect. ConclusionsOur data define Tlr9 as an important receptor involved in fibrogenesis, but also in the initiation and progression of hepatocellular carcinoma during chronic liver diseases.

Thalamocortical mGlu8 Modulates Dorsal Thalamus Excitatory Transmission and Sensorimotor Activity.

Metabotropic glutamate receptor 8 (mGlu8) is a heterogeneously expressed and poorly understood glutamate receptor with potential pharmacological significance. The thalamic reticular nucleus (TRN) is a critical inhibitory modulator of the thalamocortical-corticothalamic (TC-CT) network and plays a crucial role in information processing throughout the brain, is implicated in a variety of psychiatric conditions, and is also a site of significant mGlu8 expression. Using both male and female mice, we determined via fluorescent in situ hybridization that parvalbumin-expressing cells in the TRN core and shell matrices (identified by spp1+ and ecel1+ expression, respectively), as well as the cortical layers involved in CT signaling, express grm8 mRNA. We then assayed the physiological and behavioral impacts of perturbing grm8 signaling in the TC circuit through conditional (adeno-associated virus-CRE mediated) and cell-type-specific constitutive deletion strategies. We show that constitutive parvalbumin grm8 knock-out (PV grm8 knock-out) mice exhibited (1) increased spontaneous excitatory drive onto dorsal thalamus relay cells and (2) impaired sensorimotor gating, measured via paired-pulse inhibition, but observed no differences in locomotion and thigmotaxis in repeated bouts of open field test (OFT). Conversely, we observed hyperlocomotive phenotypes and anxiolytic effects of AAV-mediated conditional knockdown of grm8 in the TRN (TRN grm8 knockdown) in repeated OFT. Our findings underscore a role for mGlu8 in regulating excitatory neurotransmission as well as anxiety-related locomotor behavior and sensorimotor gating, revealing potential therapeutic applications for various neuropsychiatric disorders and guiding future research endeavors into mGlu8 signaling and TRN function.

The small non-coding RNA Vaultrc5 is dispensable to mouse development.

Vault RNAs (vRNAs) are evolutionarily conserved small non-coding RNAs transcribed by RNA polymerase lll. Initially described as components of the vault particle, they have since also been described as noncanonical miRNA precursors and as riboregulators of autophagy. As central molecules in these processes, vRNAs have been attributed numerous biological roles including regulation of cell proliferation and survival, response to viral infections, drug resistance, and animal development. Yet, their impact to mammalian physiology remains largely unexplored. To study vault RNAs in vivo, we generated a mouse line with a conditional Vaultrc5 loss of function allele. Because Vaultrc5 is the sole murine vRNA, this allele enables the characterization of the physiological requirements of this conserved class of small regulatory RNAs in mammals. Using this strain, we show that mice constitutively null for Vaultrc5 are viable and histologically normal but have a slight reduction in platelet counts pointing to a potential role for vRNAs in hematopoiesis. This work paves the way for further in vivo characterizations of this abundant but mysterious RNA molecule. Specifically, it enables the study of the biological consequences of constitutive or lineage-specific Vaultrc5 deletion and of the physiological requirements for an intact Vaultrc5 during normal hematopoiesis or in response to cellular stresses such as oncogene expression, viral infection, or drug treatment.

Neuroinflammation and Lysosomal Abnormalities Characterise the Essential Role for Oxidation Resistance 1 in the Developing and Adult Cerebellum.

Loss-of-function mutations in the TLDc family of proteins cause a range of severe childhood-onset neurological disorders with common clinical features that include cerebellar neurodegeneration, ataxia and epilepsy. Of these proteins, oxidation resistance 1 (OXR1) has been implicated in multiple cellular pathways related to antioxidant function, transcriptional regulation and cellular survival; yet how this relates to the specific neuropathological features in disease remains unclear. Here, we investigate a range of loss-of-function mouse model systems and reveal that constitutive deletion of Oxr1 leads to a rapid and striking neuroinflammatory response prior to neurodegeneration that is associated with lysosomal pathology. We go on to show that neuroinflammation and cell death in Oxr1 knockouts can be completely rescued by the neuronal expression of Oxr1, suggesting that the phenotype is driven by the cell-intrinsic defects of neuronal cells lacking the gene. Next, we generate a ubiquitous, adult inducible knockout of Oxr1 that surprisingly displays rapid-onset ataxia and cerebellar neurodegeneration, establishing for the first time that the distinctive pathology associated with the loss of Oxr1 occurs irrespective of developmental stage. Finally, we describe two new homozygous human pathogenic variants in OXR1 that cause neurodevelopmental delay, including a novel stop-gain mutation. We also compare functionally two missense human pathogenic mutations in OXR1, including one newly described here, that cause different clinical phenotypes but demonstrate partially retained neuroprotective activity against oxidative stress. Together, these data highlight the essential role of Oxr1 in modulating neuroinflammatory and lysosomal pathways in the mammalian brain and support the hypothesis that OXR1 protein dosage may be critical for pathological outcomes in disease.

Effects of glucocorticoids on adipose tissue plasticity

Glucocorticoids (GCs) play an important role in metabolic adaptation, regulating carbohydrate-lipid homeostasis and the immune system. Because they also have anti-inflammatory and immunosuppressive properties, synthetic analogues of GCs have been developed and are widely used in the treatment of chronic inflammatory conditions and in organ transplantation. GCs are among the most commonly prescribed drugs in the world. However, long term and high GC doses can cause side effects such as GC-induced diabetes and lipodystrophy. In recent years, a large number of independent studies have reported the effects of constitutive and adipocyte-specific deletion of the GC receptor (GR) in mice under different diets and treatments, resulting in contrasting phenotypes. To avoid potential compensatory mechanisms associated with the constitutive adipocyte GR silencing during adipose tissue development, our team has generated an inducible mouse model of GR deletion specifically in the adipocyte (AdipoGR-KO). Using this mouse model, we were able to demonstrate the critical role of the adipocyte GR in GC-induced metabolic changes. Indeed, under conditions of hypercorticism, AdipoGR-KO mice showed an improvement in glucose tolerance and insulin sensitivity, as well as in lipid profile, despite a massive increase in adiposity. This result is explained by a densification of adipose tissue vascularization, highlighting the repressive role of adipocyte GR in the healthy expansion of this tissue. Our work has largely contributed to the demonstration of the important role of the adipocyte GR in the physiology and pathophysiology of the adipose tissue and its impact on energy homeostasis.

Characterization of a new arrhythmogenic cardiomyopathy mouse model due to filamin c haploinsufficiency

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Spanish Ministry of Universities FPU fellowship (FPU19/04044), Spanish Ministry of Science and Innovation Digital Transition call (TED2021-129774B-C22), EU HORIZON EIC Pathfinder Challenges 2022 Cardiogenomics call (Project 101115416). Background Filamin C (FLNC) is widely expressed in heart and skeletal muscle. It participates in cell junction and sarcomere assembly and function. We have previously shown that heterozygous FLNC truncating variants (FLNCtv) cause arrhythmogenic/dilated cardiomyopathy (ACM/DCM) in patients. Diverse studies have suggested haploinsufficiency as the potential mechanism of this disease. However, animal models of FLNCtv have only been reported to develop ACM/DCM in homozygosity. Purpose To develop a new model of ACM/DCM caused by a FLNCtv and study the progression of the disease in heterozygosity. Methods Two gRNAs flanking exon 15 of the Flnc gene and SpCas9 protein were microinjected into mouse zygotes to generate a mouse line with a constitutive deletion of Flnc exon 15 (FlncWt/Ex15del). mRNA and protein stability were determined by qRT-PCR and western blot, respectively. Cardiac function was analysed by ECG and echocardiography every 2 months, starting at 8 weeks of age (Fig. 1B). The mean ECG values were calculated using 3 time intervals and standard ECG waves. Results FlncWt/Ex15del mice showed a reduction in Flnc mRNA and protein similar to that observed in a human ACM/DCM patient bearing a similar mutation, suggesting that this FLNCtv causes haploinsufficiency. Both male and female FlncWt/Ex15del mice developed cardiac electrophysiological defects, including reduced amplitude of the QRS complex and the P-wave, notched R and/or S waves, and increased QT interval times, and these defects worsened with time. At 40 weeks of age, FlncWt/Ex15del mice showed some initial signs of left ventricular dilatation. Conclusions Mice carrying FLNCtv in heterozygosity develop electrophysiological changes resembling pathological features of ACM/DCM patients due to haploinsufficiency. The development of this pathological phenotype in heterozygosity, as opposed to previous models, paves the way for the development of new gene therapies using this model.

#187 Vasorin (VASN) commands podocyte quiescence and glomerular homeostasis. A novel FSGS mechanism?

Abstract Background and Aims Podocyte dysfunction and loss of quiescence contribute to focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CGN). Here, we characterized the role of Vasorin (VASN) in podocytes. Method We combined laser capture microdissection and deep RNA sequencing of human glomeruli, RNAscope-based in situ hybridization, immunostaining and mouse models with Vasn-driven fluorescent reporter, targeted Vasn gene deletion in podocytes and cultures of human podocytes. Results VASN mRNA is strongly expressed in healthy human and mouse glomeruli with a podocyte-specific pattern. Both global and podocyte-specific, constitutive or inducible gene deletion of Vasn in mice induced a nephrotic syndrome and kidney failure after day 14, with podocyte dedifferentiation and loss and severe FSGS lesions, indicating a fundamental and non-developmental role of VASN in podocyte homeostasis. Furthermore, we measured markedly decreased VASN expression in podocytes of patients with FSGS and ANCA-associated CGN but not with Minimal Change Disease (MCD) and Membranous Nephropathy (MN). We thus wondered whether VASN abundance could be a disease modifier for glomerulopathies. Mice with podocyte-specific partial heterozygous deletion of Vasn showed no evident baseline renal abnormalities. Still, they showed significantly more podocyte loss, albuminuria and kidney failure than wild-type mice when challenged by type 1 diabetes with glomerular hyperfiltration or CGN experimental models. Human podocytes transduced with lentiviral VASN-targeting shRNA (shVASN) displayed increased focal adhesion dynamics, resistance to oscillating mechanical stretches, enhanced migratory capacity and epithelial-mesenchymal transition (EMT) markers. Moreover, shVASN podocytes had a higher proliferative capacity, increased cell-cycle reentry and a relative S-phase slow-down. Likewise, transcriptomics analysis found the up-regulation of genes involved in cell metabolism, adhesion and EMT and the down-regulation of genes involved in the S to G2/M phase transition. In vivo, we confirmed that VASN deficiency increased podocyte proliferation in situ and urinary shedding. Conclusion These findings highlight the novel role of VASN as an essential master protein that maintains podocyte quiescence and homeostasis during health and kidney diseases while offering a potential new therapeutic target. VASN control mechanisms driving podocyte cell-cycle reentry and detachment and FSGS. Precise mechanisms of VASN functions in podocytes need further insights into VASN molecular partners.

Deletion of Myostatin Significantly and Preferentially Improves Muscle Function in a Sex-Dependent Manner in Both an Obese and Aging Mouse Model

Skeletal muscle mass and function are essential components of health and homeostasis. In addition to its role in balance and locomotion, skeletal muscle is the body’s largest metabolic reservoir, absorbing significant amounts of glucose and amino acids from the blood stream. The loss of muscle mass is associated with impaired glucose tolerance, threatens vascular health and contributes to cardiovascular disease in obese and aging (sarcopenic) populations, along with driving overall mortality. The myokine myostatin (GDF-8) is a potent negative regulator of skeletal muscle growth that is upregulated in humans and animal models of obesity and aging. Loss of myostatin is known to increase the number of glycolytic muscle fibers and has been shown to correct indices that govern cardiometabolic health in obesity and aging, improving endothelial function and glucose homeostasis, and protecting against renal injury and hypertension. However, these studies have largely been conducted using male mice, leaving the question regarding the effectiveness of myostatin deletion on skeletal muscle function, independent of sex, largely unexplored. As such, our hypothesis is that the deletion of myostatin will improve skeletal muscle function in both male and female mice, regardless of the disease phenotype being obesity or aging. Young (3-5 mo.) lean and obese mice were used, along with aged (18-24 mo.) mice. The obese db/db model was utilized as it is a well characterized model of hyperphagia that results in obesity and subsequent metabolic disease, including significant whole-body adiposity and reduced muscle mass. On this background we have crossed mice harboring constitutive genetic deletion of myostatin to reverse muscle loss. Thus, myostatin deletion was crossed onto the relevant backgrounds in combination with aging or obesity and included both sexes. Muscle function was assessed in all groups using in vivo plantarflexion. Young male mice demonstrated an ~20% increase in force production compared to the females. Results show that aging and obesity inhibited skeletal muscle function in both sexes, with female mice showing a lessened impact of obesity or aging. Myostatin deletion improved muscle function in male mice, returning both obesity-derived weakness and aging-induced sarcopenia back to levels of young control. However, female mice without myostatin experienced a relative gain-of-function, with force production significantly exceeding that of the young control. The implication of these results is that weight loss may be more effective in protecting muscle function in diseases in the male population whereas myostatin inhibition, either pharmaceutically or with prescription exercise, may result in greater improvements in the female sex comparatively. This research gratefully acknowledges support from the NIA (K01 AG064121), OCAST (HR21-045-1) and the Niblack Research Scholars Program. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cardiomyocyte Specific MLK3 Deletion Opposes LV Dysfunction and Cardiac Fetal Gene Re-Expression

Study Objective: To determine the cardiac myocyte (CM) and blood pressure-independent effects of Mixed lineage kinase 3 (MLK3) in the regulation of left ventricular (LV) function at baseline and in response to pathological stress. Background: MLK3 functions as a direct substrate effector of cGMP-dependent protein kinase 1α (PKG1α). Whole-body MLK3 deletion in mice increases cardiac dysfunction and remodeling after pressure overload and prevents the therapeutic effect of cGMP augmentation on LV function. However, MLK3 deletion also causes hypertension, which may confound these findings. Hypothesis: MLK3 opposes LV dysfunction through a blood pressure-independent function in the cardiac myocyte (CM). Methods: We generated mice harboring LoxP sites flanking the MAP3K11 gene encoding MLK3 (MLK3fl/fl) and crossed them with αMHC-Cre transgenic mice, enabling constitutive postnatal cardiac myocyte-specific MLK3 deletion (CMKO). Male and female 3- and 6-month-old MLK3 CMKO and control MLK3 intact (MLK3fl/fl Cre-) littermates were studied in the basal state. We also performed transaortic constriction (TAC) or sham surgery for 4 weeks on 3-month-old male mice. We assessed LV structure and function by organ mass, echocardiography, pressure volume loop analysis, qPCR, and immunoblot. In basal and TAC studies, we used age-matched αMHC-Cre x MLK3+/+ (non-floxed) mice as additional controls. Results: MLK3 gene excision in CMs was confirmed by PCR. By 6 months of age, MLK3 CMKO mice displayed: progressive reduction in LV ejection fraction; increase in LV internal diameter; and LV hypertrophy, indicating LV dysfunction and pathological remodeling, compared with littermate controls or with αMHC-Cre x MLK3+/+ non-floxed mice. This effect was more prominent in females than in males. LV fetal gene expression did not differ between genotypes at 3 months. At 6 months of age, however, the fetal genes nppa, RCAN, and CTGF were significantly upregulated in both male and female MLK3 CMKO mice, compared with MLK3 intact, indicating pathological remodeling. After 4-week TAC, MLK3 CMKO LVs developed more severe reduction in LV systolic function compared with MLK3 intact littermates as assayed by echocardiographic measures of LV ejection fraction; and invasive hemodynamic indices of preload recruitable stroke work, maximum LV pressure and LV developed pressure. The MLK3 CMKO LVs also demonstrated a more eccentric remodeling phenotype in which LV mass/tibia length, LV end diastolic and end systolic diameters increased more severely in CMKO mice, with corresponding reduced LV wall thicknesses. Finally, lung mass/tibia length was elevated in the MLK3 CMKO TAC mice compared to MLK3 intact TAC littermates, indicating overt heart failure. Conclusion: (1) MLK3 functions as a tonic inhibitor of LV dysfunction and pathological remodeling through a role in the CM, possibly through sex-specific mechanisms. (2) Intact MLK3 is required for normal LV compensation and concentric remodeling in response to pressure overload. These findings have important clinical implications, as drugs which activate PKG1 in heart failure improve outcomes but are limited by excess hypotension. As a myocardial PKG1 substrate which opposes pathological LV remodeling through blood pressure-independent mechanisms, MLK3 may represent a candidate therapeutic target for heart failure. NIH 1R01HL162919. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Urocortin 3 contributes to paracrine inhibition of islet alpha cells in mice.

Pancreatic alpha cell activity and glucagon secretion lower as glucose levels increase. While part of the decrease is regulated by glucose itself, paracrine signaling by their neighboring beta and delta cells also plays an important role. Somatostatin from delta cells is an important local inhibitor of alpha cells at high glucose. Additionally, urocortin 3 (UCN3) is a hormone that is co-released from beta cells with insulin and acts locally to potentiate somatostatin secretion from delta cells. UCN3 thus inhibits insulin secretion via a negative feedback loop with delta cells, but its role with respect to alpha cells and glucagon secretion is not understood. We hypothesize that the somatostatin-driven glucagon inhibition at high glucose is regulated in part by UCN3 from beta cells. Here, we use a combination of live functional Ca2+ and cAMP imaging as well as direct glucagon secretion measurement, all from alpha cells in intact mouse islets, to determine the contributions of UCN3 to alpha cell behavior. Exogenous UCN3 treatment decreased alpha cell Ca2+ and cAMP levels and inhibited glucagon release. Blocking endogenous UCN3 signaling increased alpha cell Ca2+ by 26.8 ± 7.6%, but this did not result in increased glucagon release at high glucose. Furthermore, constitutive deletion of Ucn3 did not increase Ca2+ activity or glucagon secretion relative to controls. UCN3 is thus capable of inhibiting mouse alpha cells, but, given the subtle effects of endogenous UCN3 signaling on alpha cells, we propose that UCN3-driven somatostatin may serve to regulate local paracrine glucagon levels in the islet instead of inhibiting gross systemic glucagon release.

Selenoprotein I is indispensable for ether lipid homeostasis and proper myelination

Selenoprotein I (SELENOI) catalyzes the final reaction of the CDP-ethanolamine branch of the Kennedy pathway, generating the phospholipids phosphatidylethanolamine (PE) and plasmenyl-PE. Plasmenyl-PE is a key component of myelin and is characterized by a vinyl ether bond that preferentially reacts with oxidants, thus serves as a sacrificial antioxidant. In humans, multiple loss-of-function mutations in genes affecting plasmenyl-PE metabolism have been implicated in hereditary spastic paraplegia (HSP), including SELENOI. Herein, we developed a mouse model of nervous system-restricted SELENOI deficiency that circumvents embryonic lethality caused by constitutive deletion and recapitulates phenotypic features of HSP. Resulting mice exhibited pronounced alterations in brain lipid composition, which coincided with motor deficits and neuropathology including hypomyelination, elevated reactive gliosis, and microcephaly. Further studies revealed increased lipid peroxidation in oligodendrocyte lineage cells and disrupted oligodendrocyte maturation both in vivo and in vitro. Altogether, these findings detail a critical role for SELENOI-derived plasmenyl-PE in myelination that is of paramount importance for neurodevelopment.