WT Littermates Research Articles

Abstract P1087: Cd36-retinoic Acid-PPARδ Signaling Axis Modulates Cardiomyocytes’ Ability To Proliferate In Newly Born Neonatal Mice

Background: A pertinent question is why only a subset of fetal/neonatal cardiomyocytes (CMs) spontaneously proliferate. Using single-cell RNAseq, we previously showed a specific population of CMs that is more responsive to cell cycle induction (Abouleisa et al., Circulation, 2022). This population is characterized by expression of the transmembrane scavenger receptor, CD36 (fatty acid (FA) internalization receptor). Here, we aim to investigate the mechanism by which CD36 modulates CMs decision to enter into the cell cycle in neonatal P1 CMs. Methods and Results: Our single-cell RNAseq from primary CMs isolated from P1 hearts demonstrated that CD36 is specifically expressed in the spontaneously proliferating CMs and the CMs that respond to the exogenous cell cycle stimulation. CMs isolated from P1 CD36 knockout mice (CD36 KO ) showed 50% less proliferation capacity compared to WT P1 CMs. Interestingly, CD36 KO mice are born with smaller hearts which contain fewer CMs compared with their WT littermates. Furthermore, compared with WT littermates, which completely regenerate the heart apex following apical resection at P1, CD36 KO showed minimal regenerative capacity. Surprisingly, CD36 KO CMs didn’t show significant disruption in FA oxidation or glycolysis; however, bulk RNAseq data from the P1 CD36 KO hearts showed significant downregulation in the expression of retinoic acid (RA)-dependent signaling genes (e.g., PPARδ and RXRa) and a significant increase in PPARα expression. Confirming a causal role of CD36 in CM proliferation, we found that Cd36 is crucial in regulating the entry and interactions of RA or retinoid precursors in CMs. The CD36 + FACS-sorted CMs express significantly higher levels of PPARδ, RXRa, and FABP5 and lower levels of PPARα, which are crucial in regulating the response to cell proliferation. Furthermore, small molecule activation of PPARδ doubled cell cycle entry in P1 CMs and completely restored proliferation in CD36 KO CMs. These data suggest that CD36 is essential to prime CMs proliferation during development by regulating RA/PPARδ/RXR transcriptional signaling. Conclusions: These findings suggest that the CD36-RA-PPARδ signaling axis modulates the CMs’ ability of P1 to spontaneously enter the cell cycle.

Abstract P1181: Targeted Loss Of Sarcolemmal Membrane Associated Protein Isoform 3 (slmap3) In Cardiac Progenitors Stunts The Embryonic Growth Of Myocardium

Sarcolemmal membrane associated proteins (SLMAPs) belong to the superfamily of tail anchored membrane proteins known to regulate variety of functions including vesicle transport. SLMAP3 is the largest isoform which is ubiquitously expressed and has been linked to Brugada syndrome and sodium channel activity. SLMAP3 is expressed during cardiogenesis, and we examined its role by nullifying its expression in mouse cardiac progenitors using the Nkx2.5-cre-lox system. Hearts from SLMAP3-knockout (KO) mouse embryos at different stages of cardiogenesis, e9.5, e12.5 and e16.5, were smaller (p<0.05) than wildtype (Wt) littermates. Histological analysis of heart wall measurements from the left ventricles revealed thinner walls in SLMAP3-KO from e9.5 (-25.16%, p<0.05), e12.5 (-29.36%, p<0.05) and e16.5 (-40.27%, p<0.05) compared to Wt although normal chamber formations was observed at these developmental stages. To evaluate the potential reasons for the early difference in cardiac growth, we examined myocardial cell numbers and size with markers of proliferation and hypertrophy. Immunofluorescent analysis of e12.5 hearts with proliferative markers (pH3, Ki67) was not significantly (-5%, p>0.05) altered in KO hearts while cardiomyocyte size differences assessed by immunofluorescence with anti-Troponin C, revealed that KO cardiomyocytes were significantly (-19.4%, p<0.05) smaller than Wt. However, the phospho-to total ratios of AKT1(1.45%, p>0.05) and MTOR1(-12%, p>0.05) were not significantly altered in KO hearts when compared to Wt. SLMAP3 has been implicated in Hippo signaling via the STRIPAK (striatin interacting phosphatase and kinase) complex to impact YAP/TAZ and cell growth. Western blot analysis of e12.5 Wt and KO hearts showed no change in phosphorylation of MST1/2(-6.64%, p>0.05) or YAP-Ser 127(-8.84%, p>0.05) ruling out involvement of Hippo signaling. Interestingly, the expression of the other SLMAP isoforms, SLMAP1 and SLMAP2 remained unaffected during cardiac development in SLMAP3 KO hearts. Thus, SLMAP expression is critical for normal development since the loss of SLMAP3 isoform leads to stunted growth of the embryonic heart. Supported by CIHR.

Abstract P2013: Cardiac Remodeling, Amyloidosis, Neurotrophic Signaling, And Innervation Impairment In The Tg2576 Model Of Alzheimer's Disease

Background: In Alzheimer's disease (AD), the characteristic cerebral amyloid β (Aβ) deposition and tau pathology are accompanied by a progressive loss of the two main neuromodulators, NGF and BDNF. This AD-mediated derangement of the brain's neuro-signaling pathway may extend to the periphery and, along with potential peripheral amyloid deposition, induce peripheral nervous system impairment, thus affecting other organs, including the heart. However, whether and how AD pathology affects cardiac physiology, neurotrophins, innervation, and heart amyloidosis is still unclear. Method: Here, we describe for the first time that cardiac remodeling and neuro-signaling dysregulation are present in the hearts of Tg2576 mice, a widely used model of AD and cerebral amyloidosis. Results: Echocardiographic analysis showed significant deterioration of left ventricle function, as demonstrated by a decline of both ejection fraction and fraction shortening in 12-month-old Tg2576 mice compared to age-matched WT littermates. The transgenic mice hearts displayed an increase in interstitial fibrosis, with an accumulation of amyloid aggregates, including Aβ, associated with severe cardiac nervous system dysfunction. Tg2576 mice exhibited a depletion in cardiac nerve fiber density, both adrenergic (stained with tyrosine hydroxylase- TH) and regenerating nerve terminals (labeled with GAP-43), compared to the WT mice. This myocardial denervation was accompanied by a decline in NGF and BDNF protein expression as well as GAP-43 expression in both the brain and heart of Tg2576 mice. Likewise, human neuroblastoma cells (SH-SY5Y) and human cardiomyocytes (AC16) challenged with human Aβ-40 or, Aβ-42 oligomers showed significant downregulation of both BDNF and GAP-43 activity. Conclusion: Overall, this study highlights the harmful impact of cerebral amyloidosis on the cardiac nervous system and heart physiology, providing potential therapeutic targets, such as neurotrophic factors, to prevent or limit AD-mediated degenerative mechanisms in the cardiovascular system.

Abstract GS.103: Human Cardiomyopathy Disease Variant In Formin Homology 2 Domain Containing 3 (FHOD3) Protein Revealed Novel Early Autophagy Dysregulation And Mitochondrial Dysfunction

Introduction: The FHOD3-V1151I variant has been identified by multiple Genome Wide Association Studies to be associated with hypertrophic cardiomyopathy (HCM). FHOD3 regulates actin filament formation which is central to key cardiomyocyte processes including autophagy. The precise role of autophagy in cardiomyopathy is an emerging area of research but poorly characterized. We hypothesize that V1151I may perturb FHOD3-dependent cardiac autophagy regulation. Methods: The homologous V1151I variant was introduced into mice using CRISPR. Cardiac function was studied by echocardiography, cardiac mitochondrial function measured by Seahorse (Agilent) and oil red O staining of lipid droplets in cardiac sections. Cultured H9C2 cardiomyoblasts were transfected with FHOD3-V1151V (WT) or V1151I (variant) expression plasmids and immunoprecipitation and immunofluorescence were performed to assess protein interaction and localization. Phosphatidylinositol 3-phosphate (PI3P, Echelon) was used for the rescue experiments. Results: Heart tissue from FHOD3-V1151I variant mice (n=7) showed a 5-fold (p<0.0001) increase in lipid droplet accumulation and impaired mitochondrial respiration and ATP production (p<0.0001), while cardiac structure and function assessed by echocardiography was similar to V1151V WT littermate control hearts (n=7). A bioinformatics analysis predicted FHOD3 interaction with PFN4 which we confirmed by co-immunoprecipitation in addition to ATG16, a regulator of autophagic vesicle membrane biogenesis. The FHOD3-PFN4-ATG16 interaction was however lost in FHOD3-V1151I hearts. Immunofluorescence of H9C2 cells expressing FHOD3-V1151I revealed that the variant disrupts interaction of LC3 with PI3P, a hallmark of early autophagy. The disrupted LC3-PI3P interaction seen with the V1151I variant phenocopies cells treated with early autophagy inhibitors 3-MA or spautin, but not late autophagy inhibitor chloroquine. Furthermore, exogenous PI3P restored the molecular interaction and autophagy dysregulation. Conclusions: We demonstrate for the first time that early autophagy dysregulation underlies energetic defects observed in cardiomyopathy, and is reversible, thus providing a novel potential therapeutic target.

Abstract P3050: Cpne5 Is Necessary For Normal Murine Sinoatrial Node Function In Vivo

Cpne5 ( Copine 5 ) is a Ca 2+ -dependent phospholipid binding protein implicated in membrane trafficking but has no known role within the heart to date. In single-cell RNA-sequencing of the developing mouse heart, we previously found Cpne5 gene expression to be highly enriched in the cardiac conduction system (CCS). Further, variants in CPNE5 were identified in 6 independent GWAS related to heart rate variability in humans. We therefore hypothesized that Cpne5 is necessary for normal CCS function through Ca 2+ -dependent membrane trafficking of ion channels. Whole mount immunostaining with 3D volumetric imaging (iDISCO+) of intact WT hearts demonstrated robust and specific protein expression of Cpne5 throughout all CCS components, including the sinoatrial node (SAN) and SAN-transitional cells. Next, to assess the necessity of Cpne5 within the CCS, systemic knockout mice (Cpne5KO) were generated using CRISPR/Cas9. Successful deletion of Cpne5 in CRISPR-generated KO mice was confirmed by PCR and, at the protein level, immunofluorescence. Homozygous Cpne5KO mice were viable, showed normal growth and allele frequencies segregated in a normal Mendelian fashion. No gross anatomic defects of the CCS were observed in Cpne5KO mice and transthoracic echocardiogram studies showed a slight reduction in left ventricular fractional shortening but otherwise a structurally normal heart. Notably, while surface electrocardiogram (ECG) demonstrated normal intervals (PR, QRS, QTc), Cpne5KO mice exhibited pronounced sinus arrhythmia, with notable sinus pauses, sinus bradycardia, and increased heart rate variability as compared to WT littermate controls on long term telemetry. Systemic loss-of-function of Cpne5 in mice resulted in marked sinus node dysfunction, consistent with human GWAS data, implicating a novel role for Cpne5 in CCS function and/or development. Additional phenotyping and molecular studies, including CPNE5 deletion in human induced-pluripotent cell-derived SAN-like cells, are ongoing to further elucidate its underlying mechanism.

Abstract P1152: Molecular Regulation Of Ferroptosis In The Type 1 Diabetic Heart

Background/Hypothesis: The molecular mechanisms underpinning ferroptosis, a newly emerged mechanism of myocardial cell death, remains unclear in the T1DM heart. Although the targeted deletion of matrix metalloproteinase-9 (MMP9) is cardioprotective, the role of MMP9 in T1DM-induced cardiac ferroptosis is unknown. We hypothesize that the lack of MMP9 mitigates ferroptosis in the T1DM heart. Methods/Results: We randomly selected 15-week-old male Ins2 +/- Akita (T1DM) and age and gender-matched Ins2 +/+ littermate WT mice to examine T1DM-induced cardiac ferroptosis. We used our novel Akita/MMP9KO (DKO) mice to investigate the specific effect of MMP9 deficiency on T1DM-induced cardiac ferroptosis. Following the guidelines on myocardial cell death , we evaluated ferroptosis in the heart (LV) of WT, Akita, DKO, and MMP9KO mice. The comparison of protein levels in (i) Akita vs. WT and (ii) Akita vs. DKO (n=3-6) revealed that the lack of MMP9 prevents T1DM-induced upregulation of iron import [Transferrin Receptor (TFR): (i) +2.44-fold, (ii) -1.78-fold], downregulation of iron export [Ferroportin-1: (i) -3.20-fold, (ii) +2.20-fold], amplification of lipid peroxidation via a decrease in Glutathione Peroxidase-4 (GPX4) [(i) -1.95-fold, (ii) +2.01-fold], antioxidants [ {Superoxide dismutase-1 (SOD1): (i) -1.1-fold, (ii) +1.00-fold} and SOD2 {(i) -1.58-fold, (ii) +1.01-fold}], and an increase in Lysophosphatidylcholine Acyltransferase-3 (LPCAT3) [(i) +3.14-fold, (ii) -2.09-fold] in the heart. Conclusion: We reveal the mechanisms by which MMP9 deficiency mitigates ferroptosis in the T1DM heart (Figure 1). This is the first report of MMP9 role in cardiac ferroptosis.

Irisin protects against doxorubicin-induced cardiotoxicity by improving AMPK-Nrf2 dependent mitochondrial fusion and strengthening endogenous anti-oxidant defense mechanisms

Irisin, a new exercise-mediated myokine, plays an important role in cardiovascular diseases by regulating cell energy metabolism. The induction of mitochondrial dysfunction and oxidative stress are the crucial mechanisms involved in doxorubicin-induced cardiomyocyte damage and cardiac dysfunction, but the mitochondria-dependent protective mechanisms of irisin in DOX-impaired cardiomyocytes are poorly understood. In this study, we exposed mouse-FNDC5 (irisin-precursor)-knockout, FNDC5 transgenic mice and their WT littermates, as well as cultured neonatal rat cardiomyocytes to DOX at a dosage of 4 mg/kg (once a week for 4 weeks) in vivo and 2 μM in vitro, respectively, then investigated how irisin alleviated DOX-induced oxidative stress and myocardial injury. Irisin knockout worsened, while irisin overexpression attenuated DOX-induced mortality, body weight loss, myocardial atrophy, damage and oxidative stress, cardiac remodeling and dysfunction in mice. Exogenous irisin supplementation (20 nM) also relieved these DOX-induced damage in cardiomyocytes. Intriguingly, irisin activated AMPK-Nrf2 signaling axis, and then up-regulated the transcription and protein expression of the downstream target genes of Nrf2, including mitochondrial fusion-related genes (mitofusin 1/2 and Optic Atrophy Type 1) and endogenous anti-oxidant genes, to promote mitochondrial fusion, improve mitochondrial dynamics and mitochondrial function, and reduced oxidative stress damage in DOX-induced cardiomyocytes. These results suggest that irisin protects the hearts from DOX-induced cardiotoxicity by improving mitochondrial dynamics and strengthening the endogenous anti-oxidant system through an AMPK-Nrf2 axis dependent manner, thus reducing DOX–induced oxidative stress injury in cardiomyocytes.

Transcriptomic analysis reveals novel age-independent immunomodulatory proteins as a mode of cerebroprotection in P2X4 receptor knockout mice after ischemic stroke.

Identification of new potential drug target proteins and their plausible mechanisms for stroke treatment is critically needed. We previously showed that genetic deletion and short-term pharmacological inhibition of P2X4, a purinergic receptor for adenosine triphosphate (ATP), provides acute cerebroprotection. However, potential mechanisms remain unknown. Therefore, we employed RNA-Seq technology to identify the gene expression profiles and pathway analysis followed by qPCR validation of differentially expressed genes (DEGs). This analysis identified roles of DEGs in certain biological processes responsible for P2X4R-dependent cerebroprotection after stroke. We subjected both young and aged male and female global P2X4 receptor knock out (P2X4RKO) and littermate WT (WT) mice to ischemic stroke. After three days, mice were sacrificed, and total RNA was isolated using Trizol and subjected to RNA-Seq and NanoString-mediated qPCR. DESeq2, Gene Ontology (GO), and Ingenuity Pathway Analysis (IPA) were used to identify gene expression profiles and biological pathways. We found 2246 DEGs in P2X4R KO vs. WT tissue after stroke. Out of these DEGs, 1920 genes were downregulated and 325 genes were upregulated in P2X4R KO. GO/IPA analysis of the top 300 DEGs suggests an enrichment of inflammation and extracellular matrix component genes. qPCR validation of the top 30 DEGs revealed downregulation of two common age-independent genes in P2X4R KO mice: Interleukin-6 (Il-6), an inflammatory cytokine, and Cytotoxic T Lymphocyte-Associated Protein 2 alpha (Ctla2a), an immunosuppressive factor. These data suggest that P2X4R-mediated cerebroprotection after stroke is initiated by attenuation of immune modulatory pathways in both young and aged mice of both sexes.

191-OR: Deletion of the Type 2 Diabetes Candidate Gene SLC16A11 Reduces Peripheral Insulin Sensitivity in Mice

Background and Aims: Recent GWAS in large human populations identified the monocarboxylate transporter SLC16A11 as a candidate gene for T2D. While previous studies postulated that SLC16A11 plays a role in hepatic lipid and glucose metabolism, the existing data are controversial and exact mechanisms of how SLC16A11 affects T2D are missing. Here, we aimed at elucidating the role of SLC16A11 in the development of metabolic disease. Methods: HEK293 cell lines expressing murine and human SLC16A11 were established. Moreover, using CRISPR/Cas9, a SLC16A11 knockout mouse model was generated. Finally, SLC16A11 mRNA expression was measured in various insulin responsive tissues of ob/ob and db/db mice. Results: In HEK293 cells, substrates of the transporter were identified. mRNA expression of SLC16A11 was reduced in skeletal muscle of ob/ob and db/db mice (-69.8% and -22.8%; p<0.05) compared to littermate controls. SLC16A11 KO and WT littermate mice fed NCD or HFD did not differ in body weight and composition, energy expenditure or food intake. While basal glucose and insulin in SLC16A11 KO and WT littermates on a HFD were not different, glucose infusion rate during hyperinsulinemic euglycemic clamp was lower in SLC16A11 KO mice (SLC16A11 WT= 36.4±3 mg/kg/min; SLC16A11 KO= 24.7±2 mg/kg/min; P<0.01), indicating reduced whole-body insulin sensitivity. No changes were observed in hepatic glucose production, liver fat content or hepatic mitochondrial function using PINTA method. Instead, clamped 2-deoxy-glucose uptake was significantly reduced in skeletal muscle (-41%; p<0.05) of SLC16A11 KO, together with increased DAGs levels and PKCθ activation, indicating skeletal muscle insulin resistance. Conclusions: Our data show for the first time that in contrast to previous assumptions, SLC16A11 impairs skeletal muscle insulin sensitivity without affecting hepatic metabolism. Taking also human GWAS into account, we propose that SLC16A11 may be an interesting target for the treatment of T2D. Disclosure N.N.El-agroudy: None. T.Schumann: None. A.Kurzbach: None. G.Sancar: None. L.Sandforth: None. C.Herrmann: None. G.I.Shulman: Consultant; Novo Nordisk, DiCerna Pharmaceuticals, Inc., Bayer Consumer Care AG, Kriya Therapeutics, Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc., Other Relationship; AstraZeneca, Merck & Co., Inc., Janssen Research & Development, LLC, iMetabolic Biopharma Corporation, Maze Therapeutics, 89bio, Inc., Equator Therapeutics, Inc., Generian Pharmaceuticals, Fortress Biotech, Inc., OrsoBio, Aro Biotherapeutics Company, Stock/Shareholder; Levels Health, Inc. A.L.Birkenfeld: None.

Seizures exacerbate excitatory‐inhibitory imbalance and tau seeding effects in 5XFAD mice

AbstractBackgroundAlzheimer’s Disease (AD) neuropathology is largely driven by two pathological proteins, tau, and ß‐amyloid, both of which induce neuronal hyperexcitability and are thought to play a role in the high comorbidity between AD and epilepsy. Central to the progression of AD is the spread of tau along neuronal networks likely via neuronal activity. Our previous work suggests a bidirectional relationship between AD and epilepsy with seizures inducing AD pathology and vice versa (Gourmaud et al. Brain, 2021; awab268). Additionally, there is evidence of excitatory‐inhibitory (E:I) imbalance in AD reminiscent of E:I imbalances involved in epileptogenesis. Thus, we hypothesized that seizures can exacerbate E:I imbalance in AD models and that this enhanced hyperexcitability promotes the propagation of pathological tau.MethodWe utilized the Five times familial AD (5XFAD) model and an established pentylenetetrazol (PTZ) kindling method to induce seizures in these and WT littermates. To model pathological tau spread seen in AD, a cohort of animals also underwent intracerebral injection of human AD‐derived tau (AD‐tau) into the hippocampus and overlying cortex prior to seizure induction. Western blots were performed for the Cl− transporters, K+‐ Cl−cotransporter 2 (KCC2) and Na+‐K+‐Cl− cotransporter 1 (NKCC1), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPAR) subunits, GluA2 and GluA1, and GABAAR subunits α1 and α3, given their roles in E:I balance. AD‐tau injected mice were also examined by western blot for phospho‐tau AT8 [Ser202; Thr205] and phospho‐tau AT180 [Thr231] in sites proximal (hippocampus) and distal (frontal cortex) to injection site to determine the effects of seizures on tau propagation.ResultPTZ‐kindling enhanced the dysregulation of Cl‐ transporters, NKCC1 and KCC2 (p<0.05, n = 12‐19) and GluA2/GluA1 ratios (p<0.05, n = 12‐19) in 5XFAD mice, while 5XFAD mice showed decreased GABAARα1 (p<0.05, n = 12‐19). No significant changes were seen in the hippocampus for AT8 nor AT180, but seizure induction elevated AT8 levels in the frontal cortex across genotypes (p<0.05, n = 5‐9).ConclusionOur data suggest that seizures exacerbate E:I imbalance in 5XFAD mice and tau propagation in both WT and 5XFAD mice. Thus, drugs targeting mechanisms involved in E:I balance or controlling seizures with antiseizure medication shows therapeutic efficacy for AD patients with comorbid seizures.

B cells regulate luminal bile acid composition in the small intestine

Abstract Recent studies have demonstrated that bile acids (BA) influence immune cell homeostasis in the gut. However, whether and how, the host immune response can influence BA biochemistry is unknown. This is an important question to answer because small shifts in BA composition in the gut can have significant pathophysiological consequences. Previously, we have demonstrated that CD19 −/−mice, which have a defect in B cell activation and consequently an aberrant humoral immune response, develop a small intestine (SI) enteropathy that is associated with abnormal luminal BA composition. However, CD19 −/−mice are not B-cell-deficient and present with multiple aberrant immune phenotypes. Here, we used B cell-deficient JHminus;/minus; mice to more effectively define this interaction. Results of our experiments provide three key observations. First, J H−/−mice develop altered SI BA pools compared to their WT littermates. Second, adoptive transfer of WT B cells into neonatal J H−/−mice led to the development of normal BA pools and rescued mice from SI enteropathy. Third, adoptive transfer of B cells incapable of secreting IgA into neonatal J H−/−mice failed to restore normal bile acid composition and only moderately protected animals from developing SI enteropathy. Collectively, the results of our experiments clearly demonstrate that B cells regulate bile acid composition in the SI and that the synthesis of mucosal IgA may be particularly important for driving this effect.

Dopamine D4 receptor inhibit sodium chloride cotransporter in renal distal convoluted tubules

Background: Dopamine D4 receptor (D4R) is expressed in the distal convoluted tubule (DCT) and mice lacking the D4R have kidney-related hypertension. Hypothesis: The D4R negatively regulates the sodium chloride cotransporter (NCC) in vitro and in vivo. Methods: NCC activities were assessed in vivo on urinary sodium excretion and systolic blood pressures (SBP) responded to NCC inhibitor hydrochlorothiazide (HCTZ, 30mg/kg/day, IP) in Drd4 null ( Drd4 -/- ) mice and wild-type ( Drd4 +/+ ) littermates (female 20 weeks old). Total and phosphorylation/ubiquitination of renal NCC were determined. The protein abundance of NCC was measured in the cultured immortalized mouse renal distal-convoluted-tubule cells (mDCT) treated by the D4R agonist PD168077 (PD), D4R antagonist L-745,870(L) or Drd4 siRNA respectively. D4R-related interaction, internalization, ubiquitination, degradation and sodium transport of NCC were also determined in vitro. T-test was used for 2 group comparisons while one-way ANOVA, Holm-Sidak test for multi-group comparisons (P<0.05 was considered significant). Results: HCTZ produced a stronger natriuretic effect in Drd4 -/- mice than in WT littermates in initial 6-hrs (16.3±2.2 vs 13.1±3.1μmol/mg of Cr, n=8). SBP were higher in Drd4 -/- mice than Drd4 +/+ littermates at baseline (117.8±5.2 vs 104.1±6.3 mmHg, n=8, tail-cuff) but normalized by HCTZ on days 4-7. The renal protein abundances of NCC by immunoblotting (230.2±51.12, % of control, n=4-5) and immunofluorescence were greater but ubiquitinated-NCC levels (65.8±12.8, %, n=4) were lower in KO than in WT mice and mRNA levels of NCC (88.5±23.8, %) and phosphorylation-NCC (87.7±10.6, %) were not altered. In the mDCT cells, inhibition of D4R by its siRNA (1.5nM, 48 hrs) increased the protein expression of NCC (151.9±33.6, %, n=4). PD (24 hrs) decreased NCC protein abundance concentration-dependently blocked by L. Total NCC (72.1±18.1,%) was decreased but ubiquitinated-NCC was increased remarkably (265.8±114.1, %) by PD. There was a co-immunoprecipitation of D4R with NCC that was increased by PD. Colocalization of them was seen with confocal microscopy and confirmed by Fret. Within 1 hr, PD internalized NCC by membrane biotinlaytion-method and increased the colocalization of NCC with lysosomal-associated membrane protein 1. PD-induced NCC decrease was reversed by lysosomal inhibitor chloroquine. 1 hr PD also decreased total NCC and NCC-dependent-intracellular sodium transport (74±1, % of Veh, n=7), which was blocked by L. PD decreased NCC under CHX-chase time-dependently (33.7±21.3, %, 8 hrs, n=5) suggesting a reduced NCC half-life. Conclusion: D4R inhibits NaCl transport in renal DCT by reducing NCC activity and protein abundance and shortening NCC half-life with promoting its internalization, ubiquitination and consequent lysosomal degradation. This is the full abstract presented at the American Physiology Summit 2023 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.

The C-terminus of WNK1 kinase is evolutionarily conserved and mediates unique phase behavior

With-no-lysine 1 (WNK1) is a serine-threonine kinase with two isoforms, long (L)-WNK1 and kidney-specific (KS)-WNK1. L-WNK1 is an ancient kinase that is activated by hypertonic stress. Alternatively, KS-WNK1 emerged more recently, it is only expressed in the kidney, it is regulated by K+, and it lacks a functional kinase domain. Both WNK1 isoforms contain identical large C-term domains (CTD) with intrinsically disordered regions (IDRs) that drive the protein into discrete puncta. Our research has identified these puncta as membraneless biomolecular condensates that form via phase separation. In cells these condensates are visible at physiologic levels of hypertonic stress and at native levels of protein expression. In the kidney these condensates appear during K+ stress and are termed WNK bodies. We hypothesized that the large CTD of WNK1 mediates its phase behavior and that this function is evolutionarily conserved. To determine whether the CTD is required for condensate formation we performed live cell imaging studies of expressed mRuby2-tagged WNK1 constructs in cells. Full-length L-WNK1 was compared to two truncated constructs (1-494 or 1-1242), revealing that the CTD is required for condensate formation. To screen for the specific regions of the CTD that undergo phase separation, we used an optogenetic approach that mapped two areas within the WNK1 CTD that facilitate phase separation: (480-1242) and (1770-2126). Both regions were intrinsically disordered, exhibited low sequence complexity, and contained coiled-coil and prion-like domains- features typical of proteins that exhibit strong phase behavior. A bioinformatic analysis of human, C. elegan, and Drosophila WNK kinases revealed that these kinases shared nearly identical disorder tendency plots. A deeper analysis of CTD sequence complexity across 27 species ranging from humans to protists revealed that mammalian WNK kinases are enriched in prolines and serines, whereas invertebrates favor glutamine. Thus, the WNK CTD underwent a permissive Q-to-P/S amino acid shift during evolution, likely to preserve phase behavior. This conserved CTD has physiologic implications in the kidney as KS-WNK1 has an identical CTD and undergoes phase separation into WNK bodies during potassium deficiency. Mice that lack KS-WNK1 (KS-WNK1 KO) do not form WNK bodies during potassium deficiency and have a Gitelman-like phenotype with a 33% decrease in phospo-NCC [P = 0.01]. This decrease in pNCC correlated with decreased thiazide sensitivity—KO mice treated with HCTZ (25mg/kg IP) had no significant reduction in blood pressure, whereas WT littermates had a 4.5mmHg reduction in mean arterial pressure [P ≤ 0.05]. Based on these findings we conclude that during evolution KS-WNK1 conserved the intrinsically disordered WNK CTD to assemble WNK bodies to serve as protein scaffolds that enhance NCC activation during K+ stress. R01 DK098145, R01 DK119252, K08 DK118211 This is the full abstract presented at the American Physiology Summit 2023 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.

The role of Monocyte Chemoattractant Protein-Induced Protein-1 in the regulation of the immune responses to mycobacterial infection

Abstract Mycobacterium tuberculosis (Mtb) infection is mainly controlled by protective cell-mediated immunity. TNFα and other mediators, such as nitric oxide (NO), are crucial to the macrophage’s antimicrobial activity. The RNA-binding protein MCPIP1 is a zinc-finger protein with RNase activity that targets the 3’UTR region of cytokine’s mRNA for degradation like IL-6, IL-1β, and IL-12. Here, we aim to investigate the role of MCPIP1 during infection and its contribution to Mtb’s immune evasion. Our observations show that MCPIP1 increases after Mtb infection, correlating with disease progression. Similarly, global MCPIP1 KO (MCPIP1 −/−) mice showed lower lung bacterial burden than WT mice after 30 days. To address wheater the reduced number of bacteria was due to the macrophage’s absent MCPIP1 expression, we generated myeloid-specific MCPIP1 KO mice (M-MCPIP1 −/−). M-MCPIP1 −/−mice showed a similar reduction of bacterial growth as the MCPIP1 −/−mice compared with their WT littermates. Infection of bone marrow-derived macrophages confirmed that myeloid-MCPIP1 deficient cells had improved control of bacterial growth with increased IL-1β, IL-6, IL-12, iNOS, and TNFα. To evidence the mechanism by which M-MCPIP1 −/−macrophages contribute to Mtb growth control, we blocked TNFα and iNOS activity. Results showed that protection in M-MCPIP1 −/−is partly due to these molecule’s high expression, as the blockade of these mediators increased the bacterial burden. Our preliminary data suggest that the induction of MCPIP1 contributes to the bacteria’s immune evasion by targeting critical anti-TB molecules. Further experiments are needed to understand the mechanisms involved and consider MCPIP1 as a target for host-directed therapy for TB. R21AI151936 R21AI171734

Abstract 686: Glp-2 Predicts Cardiovascular Outcomes In Patients With Myocardial Infarction And Increases Atherosclerosis In Mice

Background: GLP-1 and GLP-2 (glucagon-like peptide-1/2) are incretin hormones that are co-secreted from intestinal L-cells in response to food. While GLP-1 is known to induce postprandial insulin secretion and to improve cardiovascular outcomes in patients with diabetes, GLP-2 is a local intestinal growth factor enhancing intestinal nutrient absorption. GLP-2 agonists are clinically used for the treatment of patients with short bowel syndrome. The relevance of GLP-2 beyond the gut is not well understood. The aim of this study was to investigate the role of GLP-2 for cardiovascular disease (CVD). Methods: Circulating GLP-2 levels were assessed at time of hospital admission in 1929 patients with myocardial infarction. The primary outcome of the study was the first occurrence of cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke (3-P-MACE). To induce atherosclerosis, Glp2r –/– or WT mice were injected with PCSK9 virus and fed a HCD for 12 weeks. Results: Kaplan-Meier survival plots and univariable cox regression analyses found GLP-2 to be associated with adverse outcome (3-P-MACE and all-cause mortality; log. GLP-2 values HR: 2.87; p<.0001). Further adjustment for age, sex, smoking, hypertension, hypercholesterinemia, previous CVD and diabetes mellitus did not affect this association (log. GLP-2 values HR: 2.66; p=0.0055). Receiver operating characteristic curve (ROC) analyses illustrated that GLP-2 is a strong indicator for early events, which proved to be superior to Troponin T and hs-CRP. To asses the functional role of GLP-2 in CVD in an experimental approach we injected Glp2r –/– or WT mice PCSK9 virus and fed these mice a HCD. After 12 weeks Glp2r –/– mice compared to WT littermates presented with a significant reduction in plaque volume and lesion size. While body weight and leukocyte numbers in various organs were unaffected, Glp2r –/– mice had significantly lower cholesterol levels. Conclusion: Circulating GLP-2 levels are independently associated with cardiovascular events in patients with myocardial infarction while inactivation of the GLP-2 system reduces atherosclerosis in mice. Future studies are needed to investigate whether the GLP-2 receptor might provide a novel therapeutic target for cardiovascular disease.

A Human STAT3 Gain of Function Variant Drives Th17 Expansion and IL-22-dependent Skin Inflammation in a Model of Psoriasiform Dermatitis

Patients with germline gain-of-function (GOF) variants in signal transducer and activator of transcription 3 (STAT3) present with early-onset polyautoimmunity, including psoriasis. STAT3 is a pleiotropic transcription factor that plays a role in the proliferation, survival, and function of many different cell types. In T cells, STAT3 is required for the polarization and function of Th17 cells and can inhibit development of peripheral Tregs. We hypothesized that STAT3 GOF variants enhance Th17 responses following an inflammatory stimulus, leading to autoimmune disease. To examine this, we generated a mouse model of STAT3 GOF using an amino acid substitution identified in patients (p.G421R). Aged STAT3 GOF mice develop spontaneous skin inflammation of the ears associated with increased neutrophil and T cell infiltration into the skin. The frequency of Th17 in the aged skin was also increased compared to age-matched WT littermates. In adult STAT3 GOF mice, Th17 polarization of naïve T cells was enhanced in vitro, indicating a potential for CD4 skewing toward Th17. To test if this would drive disease susceptibility in vivo, WT and STAT3 GOF mice were treated with topical imiquimod (IMQ). Following treatment, STAT3 GOF mice demonstrated increased ear swelling and an increased Th17 response in the skin and periphery. Adaptive lymphocytes mediated this response; however, CD8T cells and γδ T cells are not required. Analysis of T cell clonality by scRNA-seq revealed an increased frequency of expanded clones in untreated and IMQ-treated CD4 and CD8T cells of effector and exhausted subsets. Among the expanded clones, Il22 transcript was upregulated in STAT3 GOF IMQ-treated CD4s. IL-22 was then found to partially mediate the response to IMQ in STAT3 GOF. Using bone marrow chimeras, we identified a role for both radiosensitive and resistant cells in this inflammatory response. We also observed a competitive advantage for STAT3 GOF Th17 cells in the skin. Collectively, these data demonstrate that STAT3 GOF results in a cell-intrinsic skewing of T cells with a Th17 phenotype, suggesting a potential mechanism for the development of autoimmunity and a target for therapeutics in patients with STAT3 GOF syndrome.

Post-thymic Thpok-deficiency in CD4 T cells compromises control of Toxoplasma gondii infection

Abstract Toxoplasma gondii, an intracellular parasite, is estimated to chronically infect >25% of the world-wide human population. While generally kept in check by the host immune system, reactivation of infection can occur when individuals become immunocompromised, particularly with T cell deficiencies. Both CD4+ and CD8+ T cells play significant roles in controlling this parasite. The transcription factor Thpok, is critical for commitment of MHC Class II-restricted thymocytes to the CD4 lineage during thymic differentiation, but also plays a significant post-thymic role in shaping the effector function of mature CD4+ T cells. Based on these findings, we interrogated how mice with post-thymic deletion of Thpok manage a chronic infection using Toxoplasma gondii (“Toxo” hereafter). During acute infection, Thpok-deficient mice have equivalent expansion of Toxo-specific T cells compared to WT littermate controls, but these CD4 T cells have undergone “cytotoxic diversion”, upregulation of a CD8 transcriptional program and loss of CD4 helper function. During the chronic phase of infection Thpok-deficient mice die (~3 weeks p.i. vs >12 weeks for WT) and exhibit increased Toxo cysts in the brain compared to WT littermates, suggesting deficient control of the parasite. This death in the early chronic phase is not simply due to lack of CD4+ T cell help, as Class II-deficient mice survive significantly longer into the chronic phase of Toxo infection (~6 weeks, p.i.). This data indicates that Thpok-deficient CD4+ T cells are far worse at controlling Toxo than complete absence of CD4+ T cells. Studies are currently in progress to address the mechanism by which Thpok-deficiency dysregulates parasite control by CD4+ T cells. Intramural National Institutes of Health

Mutant and curli-producing E. coli enhance the disease phenotype in a hSOD1-G93A mouse model of ALS

The gut microbiome is a potential non-genetic contributing factor for Amyotrophic Lateral Sclerosis. Differences in gut microbial communities have been detected between ALS subjects and healthy controls, including an increase in Escherichia coli in ALS subjects. E. coli and other gram-negative bacteria produce curli proteins, which are functional bacterial amyloids. We examined whether long-term curli overexposure in the gut can exacerbate the development and progression of ALS. We utilized the slow-developing hSOD1-G93A mouse model of ALS with their C57BL/6J WT littermate controls, including males and females, with a total of 91 animals. These mice were on a normal chow diet and fed curli-producing or curli-nonproducing (mutant) E. coli in applesauce (vehicle) 3 times/week, from 1 through 7 months of age. Male hSOD1 mice demonstrated gradual slowing in running speed month 4 onwards, while females exhibited no signs of locomotive impairment even at 7 months of age. Around the same time, male hSOD1 mice showed a gradual increase in frequency of peripheral CD19+ B cells. Among the male hSOD1 group, chronic gut exposure to curli-producing E. coli led to significant shifts in α- and β-diversities. Curli-exposed males showed suppression of immune responses in circulation, but an increase in markers of inflammation, autophagy and protein turnover in skeletal muscle. Some of these markers were also changed in mutant E. coli-exposed mice, including astrogliosis in the brainstem and demyelination in the lumbar spinal cord. Overall, chronic overexposure to a commensal bacteria like E. coli led to distant organ pathology in our model, without the presence of a leaky gut at 6 months. Mechanisms underlying gut-distant organ communication are of tremendous interest to all disciplines.

Preclinical trials in Alzheimer's disease: Sample size and effect size for behavioural and neuropathological outcomes in 5xFAD mice.

5xFAD transgenic (TG) mice are used widely in AD preclinical trials; however, data on sample sizes are largely unaddressed. We therefore performed estimates of sample sizes and effect sizes for typical behavioural and neuropathological outcome measures in TG 5xFAD mice, based upon data from single-sex (female) groups. Group-size estimates to detect normalisation of TG body weight to WT littermate levels at 5.5m of age were N = 9-15 depending upon algorithm. However, by 1 year of age, group sizes were small (N = 1 -<6), likely reflecting the large difference between genotypes at this age. To detect normalisation of TG open-field hyperactivity to WT levels at 13-14m, group sizes were also small (N = 6-8). Cued learning in the Morris water maze (MWM) was normal in Young TG mice (5m of age). Mild deficits were noted during MWM spatial learning and memory. MWM reversal learning and memory revealed greater impairment, and groups of up to 22 TG mice were estimated to detect normalisation to WT performance. In contrast, Aged TG mice (tested between 13 and 14m) failed to complete the visual learning (non-spatial) phase of MWM learning, likely due to a failure to recognise the platform as an escape. Estimates of group size to detect normalisation of this severe impairment were small (N = 6-9, depending upon algorithm). Other cognitive tests including spontaneous and forced alternation and novel-object recognition either failed to reveal deficits in TG mice or deficits were negligible. For neuropathological outcomes, plaque load, astrocytosis and microgliosis in frontal cortex and hippocampus were quantified in TG mice aged 2m, 4m and 6m. Sample-size estimates were ≤9 to detect the equivalent of a reduction in plaque load to the level of 2m-old TG mice or the equivalent of normalisation of neuroinflammation outcomes. However, for a smaller effect size of 30%, larger groups of up to 21 mice were estimated. In light of published guidelines on preclinical trial design, these data may be used to provide provisional sample sizes and optimise preclinical trials in 5xFAD TG mice.

Sigmar1 ablation leads to lung pathological changes associated with pulmonary fibrosis, inflammation, and altered surfactant proteins levels.

Sigma1 receptor protein (Sigmar1) is a small, multifunctional molecular chaperone protein ubiquitously expressed in almost all body tissues. This protein has previously shown its cardioprotective roles in rodent models of cardiac hypertrophy, heart failure, and ischemia-reperfusion injury. Extensive literature also suggested its protective functions in several central nervous system disorders. Sigmar1's molecular functions in the pulmonary system remained unknown. Therefore, we aimed to determine the expression of Sigmar1 in the lungs. We also examined whether Sigmar1 ablation results in histological, ultrastructural, and biochemical changes associated with lung pathology over aging in mice. In the current study, we first confirmed the presence of Sigmar1 protein in human and mouse lungs using immunohistochemistry and immunostaining. We used the Sigmar1 global knockout mouse (Sigmar1-/-) to determine the pathophysiological role of Sigmar1 in lungs over aging. The histological staining of lung sections showed altered alveolar structures, higher immune cells infiltration, and upregulation of inflammatory markers (such as pNFκB) in Sigmar1-/- mice compared to wildtype (Wt) littermate control mice (Wt). This indicates higher pulmonary inflammation resulting from Sigmar1 deficiency in mice, which was associated with increased pulmonary fibrosis. The protein levels of some fibrotic markers, fibronectin, and pSMAD2 Ser 245/250/255 and Ser 465/467, were also elevated in mice lungs in the absence of Sigmar1 compared to Wt. The ultrastructural analysis of lungs in Wt mice showed numerous multilamellar bodies of different sizes with densely packed lipid lamellae and mitochondria with a dark matrix and dense cristae. In contrast, the Sigmar1-/- mice lung tissues showed altered multilamellar body structures in alveolar epithelial type-II pneumocytes with partial loss of lipid lamellae structures in the lamellar bodies. This was further associated with higher protein levels of all four surfactant proteins, SFTP-A, SFTP-B, SFTP-C, and SFTP-D, in the Sigmar1-/- mice lungs. This is the first study showing Sigmar1's expression pattern in human and mouse lungs and its association with lung pathophysiology. Our findings suggest that Sigmar1 deficiency leads to increased pulmonary inflammation, higher pulmonary fibrosis, alterations of the multilamellar body stuructures, and elevated levels of lung surfactant proteins.