Systemic Knockout Mice Research Articles

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.

PF219 THE LEDGF/P75 HOMOLOG HRP2 IS NOT IMPORTANT FOR THE INITIATION OF MLL‐REARRANGED LEUKEMIA

Background:HDGF‐related protein 2 or HRP2 is a member of the Hepatoma‐Derived Growth Factor‐related protein family. Through its N‐terminal PWWP domain it associates with methylated histone tails and functions as such as a chromatin reader. HRP2 also features an integrase binding domain (IBD), known for its interaction with cellular and viral proteins. Little is known about the cellular function of HRP2, yet it has been linked to the malignant hepatocellular carcinoma. HRP2 has mainly been studied in comparison with its paralog, Lens Epithelium Derived Growth Factor (LEDGF/p75), suggesting similar but not identical physiological roles for both proteins. Previous publications showed an important role for LEDGF/p75 in the development of MLL‐rearranged leukemia (MLL‐r). Interaction between the IBD domain of LEDGF/p75 and the N‐terminal part of an oncogenic MLL fusion protein results in aberrant expression of MLL target genes, which in turn are important for normal hematopoiesis.Aims:Since HRP2 is functionally related to LEDGF/p75 but its potential role in MLL‐r remains unknown, we aimed to investigate a potential role for HRP2 in MLL‐r leukemia.Methods:Protein‐protein interactions were investigated with immunoprecipitation and detected by western blot. Interactions were confirmed using recombinant proteins in Alpha Screen and NMR experiments. The role of HRP2 in normal hematopoiesis was investigated using a systemic HRP2 knockout mouse model. The role of HRP2 in mixed lineage leukemia was determined using lentiviral‐mediated knock down and rescue experiments in, and colony forming assay with human cell lines. In addition, we performed colony forming assay and bone marrow transplantation with primary mouse bone marrow cells, transduced with an MLL‐fusion containing vector.Results:We show for the first time that HRP2 directly interacts with MLL with similar affinities compared to LEDGF/p75. In addition, NMR analysis indicates a similar interface between MLL and either IBD domain. However, the HRP2‐MLL interaction occurs independently of MENIN, the stabilizing adaptor of the MLL‐LEDGF/p75 interaction. The systemic HRP2 knockout mice survived with wild type blood counts. Only upon serial plating of hematopoietic stem cells, a decrease in proliferation‐potential of HRP2 knockout cells was observed. Knocking down HRP2 in leukemic cell lines and primary murine MLL‐transformed cells resulted in a decreased transformation‐potential. In contrast, Hrp2 knockout cells from the systemic knock out mice were efficiently transformed by MLL fusions, suggesting that Hrp2 might be dispensable for the initiation of MLL‐rearranged leukemia.Summary/Conclusion:In summary, we report for the first time that HRP2 is able to interact with MLL, yet in a MENIN‐independent manner. However, our results suggest that HRP2 might not be important for the initiation of mixed lineage leukemia, pointing towards a different role for HRP2 and its homolog LEDGF/p75 in MLL‐r.

Identification of a role for Casz1 in the control of T helper differentiation and inflammation

Abstract While T helper (Th) cells constitute a critical arm of adaptive immune system and are important for host defense, unregulated expansion and imbalance in Th effector subsets contribute to inflammatory disorders. Here we show that Casz1, whose function is previously unknown in CD4+ T cells, determines the balance between various Th subsets. As systemic knockout mice are embryonically lethal, we generated conditional CD4 Casz1 knockout mice. Conditional deletion of Casz1 in CD4+ T cells significantly inhibits Th17 cell differentiation, but promotes Th1 differentiation. Loss of Casz1 in CD4+ T cells lowers susceptibility to experimental autoimmune encephalomyelitis, consistent with the reduced frequency of Th17 cells, despite an increase in Th1 cells. These results underscore the critical role of Casz1 in determining Th17 lineage differentiation in vivo. Transcriptome analyses of Casz1 deficient CD4+ T cells show a signature consistent with defective Th17 differentiation but enhanced Th1 differentiation. Taken together, these data reveal Casz1 as a new T helper cell regulator having important clinical implications for autoimmune inflammation.

Abstract 65: Soluble Angiotensin Converting Enzyme 2 as an Additional Source of Blood Pressure Regulation within the Kidney

Angiotensin Converting Enzyme 2 (ACE2) is a transmembrane carboxypeptidase that degrades angiotensin II (AngII) to Ang(1-7). This membrane-bound enzyme can be cleaved by metalloproteinases such as ADAM17, releasing the catalytically active ectodomain to generate soluble ACE2. The objective of this study is to determine if soluble ACE2, acting locally in the kidney, could modulate the blood pressure (BP) response to AngII infusion. In order to isolate the actions of ACE2 in the kidney from systemic tissues, we utilized kidney cross-transplantation. We studied male (129/SvEv x C57BL/6)F1 ACE2 knockout (KO) and wild-type (WT) mice. Kidney transplants were performed to generate 4 experimental groups: 1) WT, 2) kidney ACE2KO, 3) systemic ACE2KO, and 4) total ACE2KO. Mean arterial BPs (MAP) were measured with radiotelemetry at baseline and during a 2-week infusion of AngII (1ug/kg/min). Enzymatic activity was determined using an ACE2-specific quenched fluorescent substrate assay. Total ACE2KO mice had an enhanced hypertensive response to AngII compared to WT mice (152±6 vs.144±7 mmHg; p=0.03), consistent with our previous findings of enhanced susceptibility to AngII hypertension in mice globally deficient in ACE2. The presence of ACE2 in either the kidney or systemic tissues restored MAPs to levels similar to the WT group (kidney ACE2KO 140±7mmHg, systemic ACE2 KO 140±5 mmHg, p<0.01 vs. total KO). Serum ACE2 activity levels were decreased in systemic KO mice (40±35 RFU/ul/hr) but unaffected in kidney KO mice, compared to WT (153±25 vs. 126±26 RFU/ul/hr, respectively). However, despite genetic deletion from all kidney cells, kidney ACE2KO mice had similar urinary ACE2 activity compared to systemic ACE2KO (76±32 and 108±52 RFU/ul/hr, p=NS). Urinary ACE2 activity in WT mice was highest, 217±36 RFU/ul/hr, compared to total KO which was undetectable (p<0.001). Our studies demonstrate that restoration of ACE2 in either the kidney or systemic tissues is sufficient to abrogate the exaggerated hypertensive response to chronic AngII infusion. Soluble ACE2 delivered to the kidney may be an additional source of ACE2 to act within the kidney since the cells produce no ACE2 locally in kidney KO mice. Our studies highlight a possible role of soluble ACE2 in BP regulation.

Ferroxidase Hephaestin's Cell-Autonomous Role in the Retinal Pigment Epithelium

Hephaestin (Heph) is a ferroxidase protein that converts ferrous to ferric iron to facilitate cellular iron export by ferroportin. Many tissues express either Heph or its homologue, ceruloplasmin (Cp), but the retina expresses both. In mice, a combined systemic mutation of Heph and systemic knockout of Cp (Cp(-/-), Heph(sla/sla)) causes retinal iron accumulation and retinal degeneration, with features of human age-related macular degeneration; however, the role of Heph and Cp in the individual retinal cells is unclear. Herein, we used conditional knockout mice to study Heph's role in retinal pigment epithelial (RPE) and photoreceptor cells. Loss of both Heph and Cp from RPE cells alone results in RPE cell iron accumulation and degeneration. We found, however, that RPE iron accumulation in these conditional knockout mice is not as great as in systemic knockout mice. Photoreceptor-specific Heph knockout indicates that the additional iron in the RPE cells does not result from loss of ferroxidases in the photoreceptors, and Cp and Heph play minor roles in photoreceptors. Instead, loss of ferroxidases in other retinal cells causes retinal iron accumulation and transfer of iron to the RPE cells. Cp and Heph are necessary for iron export from the retina but are not essential for iron import into the retina. Thus, our studies, revise how we think about iron import and export from the retina.

Kidney in Hypertension

Regulation of blood pressure is a complex integrated response involving a variety of organ systems including the central nervous system (CNS), cardiovascular system, kidneys, and adrenal glands. These systems modulate cardiac output, fluid volumes, and peripheral vascular resistance, the key determinants of blood pressure. More than 40 years ago, Guyton and Coleman1 developed computer models of arterial pressure control, attempting to incorporate the known variables impacting blood pressure homeostasis. The conclusion of this analysis was that regulation of sodium excretion by the kidney and consequent effects on body fluid volumes made up the critical pathway determining the chronic level of intra-arterial pressure. Our own studies of the physiology of blood pressure regulation have focused on the renin-angiotensin (Ang) system (RAS) using genetically modified mouse models. Highly conserved through phylogeny, the RAS is an essential regulator of blood pressure and fluid balance. This biological system is a multienzymatic cascade in which angiotensinogen, its major substrate, is processed in a 2-step reaction by renin and Ang-converting enzyme (ACE), resulting in the sequential generation of Ang I and Ang II. Along with its importance in maintaining normal circulatory homeostasis, abnormal activation of the RAS can contribute to the development of hypertension and target organ damage. The importance of the RAS in clinical medicine is highlighted by the impressive efficacy of pharmacological agents that inhibit the synthesis or activity of Ang II.2–5 At the cellular level, responsiveness to Ang II is conferred by expression of Ang receptors. Ang receptors can be divided into 2 pharmacological classes: type 1 (AT1) and type 2, based on their differential affinities for various nonpeptide antagonists.6,7 Studies using these antagonists suggested that most of the classically recognized functions of the RAS are mediated by AT1 receptors. Gene targeting studies confirmed these conclusions.8 AT …