Tm Mice Research Articles

Dysregulation of cardiac CaMKII pathway is increased in aging and chronic inflammation

Chronic elevation of inflammatory mediators is common in frail older adults and predicts a host of adverse cardiovascular outcomes, including heart failure and sudden death. To date, few specific molecular mechanisms have been identified that connect chronic inflammation to age-related cardiomyopathies. Dysregulation in Ca2+and calmodulin-dependent protein kinase II (CaMKII) signaling has been implicated in promoting inflammatory responses in cardiomyopathies but is understudied in the context of aging. We compared myocardial CaMKII signaling in young (32 weeks) and old (104 weeks) wild type (WT) and interleukin 10 null mice (IL-10tm/tm), a model of chronic inflammation and frailty. Aged cohorts were treated with 0.6 mg of losartan. Hearts from mouse cohorts were assayed for total and autophosphorylated CaMKII (P-CaMKII) and the phosphorylation status of phospholamban (PLN) (downstream signaling pathway) using western blot techniques. We observed significant increases in total and activated, autophosphorylated, CaMKII (P-CaMKII) (2.4 ± 0.38 vs 1.2 ± 0.12 p< 0.02; 9.0 ± 1.9 vs 0.03 ± 0.01 p< 0.002), but decreases in the phosphorylation of PLN (0.6 ±0.3 vs 1.7 ± 0.3 p<0.03) at a validated CaMKII target site (T17) in old compared to young WT hearts. Similarly, we found increased P-CaMKII (6.9 ± 1.5 vs 0.7 ± 0.4 p< 0.004) and diminished PLN T17 phosphorylation (0.8 ± 0.2 vs 1.5 ± 0.2 p<0.02) in old compared to young IL-10tm/tm mice. Old IL-10tm/tm mouse hearts had less total CaMKII compared to old WT mouse hearts (1.1 ± 0.3 vs 2.9 ±0.2 p< 0.002). Four weeks treatment of old WT mice with Losartan was associated with reversal of observed age-related changes in the CaMKII signaling pathway. We observed decreases in total CaMKII, P-CaMKII (2.9 ± 0.2 vs.1.5 ± 0.2 p<0.001; 2.2 ± 0.9 vs 0.47 ± 0.1 p <0.03), and an increase in PLN T17 phosphorylation (0.55±0.3 vs 2.23 ± 0.4 p< 0.007). The effects of Losartan on the animals aging with chronic inflammation (old IL-10tm/tm) was less clear. We observed a losartan associated increase in total CaMKII (1.41 ± 0.02 vs 1.3 ± 0.02 p<0.02) but no impact on P-CaMKII or PLN T17 phosphorylation. We interpret our findings to suggest that changes in myocardial CaMKII signaling are a feature of aging that is affected by chronic inflammation.

Plasma levels of corticosterone, tumor necrosis factor receptor 1 and interleukin 6 are influenced by age, sex and chronic inflammation in mice treated with acute temperature stress.

Resiliency is the ability to respond to, adapt to and recover from stressors. Deterioration of resiliency in older adults has been hypothesized to be regulated by age-related changes in stress response systems, including the Hypothalamic Pituitary Adrenal (HPA) axis and the innate immune system response. Although age-related chronic inflammation is strongly related to lack of resiliency, the impact of chronic inflammation on acute stress response is unclear. Here we describe the impact of a five-hour exposure to cold temperature acute stressor, on immune and corticosterone response using older and younger IL-10tm/tm mice, a mouse model with chronic inflammatory pathway activation, and age and gender matched C57/Bl6 background control (WT) mice. Overall, mice exposed to 4°C for 5h had significantly higher plasma corticosterone levels compared to those that remained at room temperature (25°C), with the exception of the WT females. Cold stressed mice had lower plasma tumor necrosis factor receptor 1 (TNFR1) levels with varying significance, in all ages and phenotypes, with the exception of the old female WT mice. In contrast, the effects of cold stress on pro-inflammatory cytokine interleukin 6 (IL-6) levels were inconsistent and not significant, with the exception of the female IL-10tm/tm mice. In conclusion, these findings demonstrate that sex, age and chronic inflammatory pathway activation all influence corticosterone secretion and inflammatory processes in the face of acute cold stress.

Complement Deficiencies Result in Surrogate Pathways of Complement Activation in Novel Polygenic Lupus-like Models of Kidney Injury.

Lupus nephritis (LN) is a major contributor to morbidity and mortality in lupus patients, but the mechanisms of kidney damage remain unclear. In this study, we introduce, to our knowledge, novel models of LN designed to resemble the polygenic nature of human lupus by embodying three key genetic alterations: the Sle1 interval leading to anti-chromatin autoantibodies; Mfge8-/- , leading to defective clearance of apoptotic cells; and either C1q-/- or C3-/- , leading to low complement levels. We report that proliferative glomerulonephritis arose only in the presence of all three abnormalities (i.e., in Sle1.Mfge8 -/- C1q -/- and Sle1.Mfge8 -/- C3 -/- triple-mutant [TM] strains [C1q -/-TM and C3-/- TM, respectively]), with structural kidney changes resembling those in LN patients. Unexpectedly, both TM strains had significant increases in autoantibody titers, Ag spread, and IgG deposition in the kidneys. Despite the early complement component deficiencies, we observed assembly of the pathogenic terminal complement membrane attack complex in both TM strains. In C1q-/- TM mice, colocalization of MASP-2 and C3 in both the glomeruli and tubules indicated that the lectin pathway likely contributed to complement activation and tissue injury in this strain. Interestingly, enhanced thrombin activation in C3-/- TM mice and reduction of kidney injury following attenuation of thrombin generation by argatroban in a serum-transfer nephrotoxic model identified thrombin as a surrogate pathway for complement activation in C3-deficient mice. These novel mouse models of human lupus inform the requirements for nephritis and provide targets for intervention.

Genetic Deficiency of the Histamine H4-Receptor Reduces Experimental Colorectal Carcinogenesis in Mice

Colorectal cancer (CRC), a severe complication of inflammatory bowel diseases, is a common type of cancer and accounts for high mortality. CRC can be modeled in mice by application of the tumor promoter, azoxymethane (AOM), in combination with dextran sodium sulfate (DSS), which are able to induce colitis-like manifestations. Active colitis correlates with high mucosal concentrations of histamine, which, together with the histamine receptor subtype 4 (H4R), provide a pro-inflammatory function in a mouse colitis model. Here, we analyzed whether H4R is involved in the pathogenesis of AOM/DSS-induced CRC in mice. As compared to wild type (WT) mice, AOM/DSS-treated mice lacking H4R expression (TM) demonstrate ameliorated signs of CRC, i.e., significantly reduced loss of body weight, stiffer stool consistency, and less severe perianal bleeding. Importantly, numbers and diameters of tumors and the degree of colonic inflammation are dramatically reduced in TM mice as compared to WT mice. This is concomitant with a reduced colonic inflammatory response involving expression of cyclooxygenase 2 and the production of C-X-C motif chemokine ligand 1 (CXCL1) and CXCL2. We conclude that H4R is involved in the tumorigenesis of chemically-induced CRC in mice via cyclooxygenase 2 expression and, probably, CXCL1 and CXCL2 as effector molecules.

Abstract 439: Elevating 2-deoxy Adenosine Triphosphate Improves Contraction in a Mouse Model of Genetic Dilated Cardiomyopathy

Several studies have identified reduced tension and Ca 2+ sensitivity as key regulators that precipitate dilated cardiomyopathy (DCM). We are developing a novel therapy to increase tension using adeno-associated viral (AAV) vectors that increase intracellular 2 deoxy-ATP (dATP) via overexpression of the enzyme ribonucleotide reductase (RNR). We have reported that dATP substitution for ATP increases the magnitude and rate of contraction and that RNR overexpression increases intracellular dATP and enhances cardiac function. Here we use a multiscale approach to test the effect of dATP in a genetic model of dilated cardiomyopathy (DCM) with a missense mutation (D230N) in alpha-tropomyosin (Tm). Using recombinant proteins in the in vitro motility assay, we found that filament velocity was lower with D230N compared to WT Tm (2.03±0.04 vs. 2.44±0.04 μm/s). This deficit was corrected when dATP was substituted for ATP (2.90±0.04 μm/s). Next, we used transgenic D230N mice that show progressive ventricular dilation and systolic dysfunction. Intact and demembranated trabeculae from these mice have decreased tension and calcium sensitivity of force, and loss of length-dependent activation (LDA) compared to WT mice. Substitution of dATP for ATP, increased Ca 2+ sensitivity in D230N Tm myocardium (pCa 50 =5.48±0.02 vs. 5.57±0.02) and restored LDA. Complete replacement of ATP with dATP resulted in an ~70% increase in force at submaximal [Ca 2+ ] and force at submaximal Ca 2+ was significantly increased by replacement of only 5% of the nucleotide pool with dATP. For isolated myofibrils from D230N Tm and WT mice, tension at submaximal Ca 2+ (pCa=5.6) was lower in D230N mice compared to WT mice (51±4 vs. 75±8 mN/mm 2 ) and this deficit was improved with dATP (68±5 mN/mm 2 ). Similarly, the rate of activation in D230N myofibrils was slower than in WT mice (2.1±0.2 vs.3.3±0.2 s -1 ) and partially corrected with dATP (2.7±0.2 s -1 ). These results demonstrate that enhanced cross bridge binding, via increasing dATP, can recover lost contractile function in a thin filament mutation model of DCM. In ongoing studies, we are testing the ability of elevated dATP, via AAV- RNR, to protect against reduced systolic function and progressive ventricular dilation in 2-3 week old D230N mice .

Reduced type III neuregulin 1 expression does not modulate the behavioural sensitivity of mice to acute Δ9-tetrahydrocannabinol (D9-THC)

Mice with a mutation in the transmembrane domain of the schizophrenia risk gene, neuregulin 1 (Nrg1 TM HET), are more susceptible to the neuro-behavioural effects of Δ9-tetrahydrocannabinol (D9-THC), the principal psychoactive component in cannabis. However, NRG1 is transcriptionally complex with over 30 different isoforms, most of which carry a transmembrane domain, raising the question which NRG1 isoform(s) may contribute to this phenotype. Type III NRG1/Nrg1 is the most brain abundant isoform and brain studies have identified increased levels of type III NRG1 mRNA in humans carrying a NRG1 risk haplotype for schizophrenia.To investigate whether mice heterozygous for type III Nrg1 (i.e. knockout: type III Nrg1+/−) are more susceptible to the behavioural effects of acute doses of D9-THC, we injected male mice with either vehicle or D9-THC (3 or 10 mg/kg) before testing them for locomotion, anxiety, social interaction, and sensorimotor gating.Acute D9-THC led to reduced locomotion and reduced social interaction, but increased anxiety in mice. Furthermore, type III Nrg1 males displayed a robust deficit in sensorimotor gating and demonstrated reduced following during social interaction across drug conditions. However, they did not show a change in behavioural susceptibility to acute D9-THC compared to controls.These results reinforce the validity of type III Nrg1+/− mice for schizophrenia research and suggest that loss of function of type III Nrg1 may not be responsible for the exaggerated response to acute D9-THC observed in heterozygous Nrg1 TM mice. This highlights the importance of careful consideration of Nrg1 isoform type differences.

Lupus nephritis: the roles of C1q and C3 in preventing antibody mediated injury

Abstract Lupus nephritis (LN) is a common and serious complication of systemic lupus erythematosus (SLE). While defective clearance of apoptotic cells (AC) and immune complexes (IC) is implicated in LN pathogenesis, the precise way in which different complement components impact kidney protection and injury is unknown. To address this, we created C57BL/6 mice with defective clearance of AC (Mfge8 deficiency) and anti-chromatin antibodies (sle1 interval) that were also deficient in either C1q [C1q Triple mutant (C1qTM)] or C3 (C3TM). Both C1q and C3 TM mice spontaneously developed anti-DNA antibodies and glomerulonephritis associated with glomerular IgG deposits after the age of 6 months. To dissociate the effects of complement on B cells versus effects on the kidney, we examined the effects of antibody mediated kidney injury (nephrotoxic nephritis, NTN) in mice deficient in the clearance of AC with complement deficiency [double knockout (DKO) (Mfge8 C1q or Mfge8 C3) mice]. In NTN, 50% of C1q DKO and 25% of C3 DKO, but no Mfge8 KO, died after NTN induction and the surviving C1q DKO mice developed severe proteinuria. Surprisingly, both spontaneous and NTN-induced nephritis in C1q TM or C1q DKO had strong glomerular C3 and C3d staining, while strong staining for the membrane attack complex (MAC) was observed in C3 TM or C3 DKO mice. These findings indicate that: 1) C1q and C3 independently protect against kidney injury separate from their effects on B cell function 2) In the presence of excess AC and low C1q, antibody mediated kidney injury is associated with C3 activation, presumably by the lectin or alternative pathway and 3) In the presence of excess AC and low C3, antibodies can cause MAC activation by pathways that remain to be determined.

FRI-478 - Saroglitazar treatment prevents NASH, eliminates hepatocyte ballooning, and significantly improves serum LFTs, lipids and insulin resistance in DIAMOND(tm) mice compared to pioglitazone benchmark

FRI-478 - Saroglitazar treatment prevents NASH, eliminates hepatocyte ballooning, and significantly improves serum LFTs, lipids and insulin resistance in DIAMOND(tm) mice compared to pioglitazone benchmark

BLUNTED RESPONSE TO STRESS IN YOUNG IL-10TM/TM MICE: IMPLICATION FOR THE VULNERABILITY IN FRAILTY

AbstractThe hypothalamic pituitary adrenal axis (HPA axis) is a major neuroendocrine system for stress response and for regulating the immune system. Aged rats as well as older humans demonstrate progressive loss of control of the HPA axis, resulting in impaired diurnal rhythm and hypersecretion of glucocorticoids during times of stress. Higher levels and a blunted diurnal rhythm of cortisol have been observed in frail compared to more robust older adults. Understanding the factors underlying disturbed glucocorticoid secretion that precede age-related diseases and frailty are of considerable importance to prevent vulnerability and disability in late life. In order to study this system in vivo, we utilized a mouse model of chronic inflammation and as frailty and measured changes in plasma corticosterone and pro-inflammatory levels after acute cold stress. The plasma corticosterone level was determined 2 weeks before cold stimulation in 10 young (3 months), 10 old (21 months) C57BL/6J mice, and 10 young IL-10 tm/tm (3 months). 5 mice from each group were exposed to cold stress, the remainder served as controls and were not exposed to cold. The treatment groups were exposed to four degrees Celsius for 5 hours and the controls were kept in room temperature conditions at twenty five degrees Celsius. The results showed higher basal plasma corticosterone (P<0.01) and normal IL-6 and TNFR-1 levels in young IL-10tm/tm, compared to young and old C57BL/6J mice. However, there were unchanged corticosterone level and higher IL-6 and TNFR-1 levels (P=0.04, 0.02, respectively) in young IL-10tm/tm mice after acute cold stress compared to young wild type mice. Lower GRa mRNA expression in hippocampus was also observed in control and treated young IL-10tm/tm (P<0.01), compared the age-matched wild type mice. These findings provide initial evidence for the hypothesis that HPA axis dysfunction is through whole life of frail subjects and even earlier than changes in immune system.

Abstract 257: Determining the Potential Role of cTnT Isoform Switching in the Development of Early Childhood Tropomyosin-linked DCM

An oft noted component of sarcomeric DCM is the observation that patients within families carrying the same primary mutation exhibit significant phenotypic variability. This lack of a distinct link between genotype and phenotype has complicated clinical management. Recently two unrelated multigenerational families were identified with the tropomyosin (Tm) mutation Asp230Asn (D230N), exhibiting a striking “bimodal” distribution of severity. In these families, many children (&lt;1 year) with D230N Tm presented with a severe form of DCM that led to sudden, often fatal CHF, while adults developed a mild to moderate DCM in mid-life. Of note, children who survived the initial presentation often recovered significant systolic function into young adulthood. To explain this improvement, despite the persistence of D230N Tm, we hypothesized that the phenotype is modified by other thin filament (TF) isoforms. Thus we propose the age-dependent remodeling seen in children with D230N Tm is a result of temporal isoform switches involving a closely linked Tm binding partner cardiac Troponin T (cTnT). We have shown that D230N Tm leads to a more stable (rigid) filament primarily at the C-terminus of Tm near the Tm overlap, a crucial region for TF regulatory function that cTnT modulates. Myofilaments from D230N Tm mice exhibited a small decrease in calcium sensitivity of force development that was significantly reduced in the presence of cTnT1, supportive of a modulatory role. We assessed cardiac performance in our novel D230N Tm x cTnT1 double transgenic (DTg) mice, % FS was similarly reduced for D230N Tm and DTg mice at 2 months likely due to persistent endogenous expression of cTnT1. Divergent cardiac remodeling occurred at 4 months at which point DTg mice had significantly reduced % FS compared to D230N, indicating that additive exposure to cTnT1 is detrimental to the function of D230N hearts. Additionally, In vitro studies on non-failing and failing human heart tissue found that RNA levels of cTnT1 are significantly higher in failing hearts. Thus modulation by cTnT1 could be a more general mechanism for the progressive remodeling seen in heart failure.

Impaired mitochondrial degradation by autophagy in the skeletal muscle of the aged female interleukin 10 null mouse

Mitochondrial dysfunction, chronic inflammation and muscle aging are closely linked. Mitochondrial clearance is a process to dampen inflammation and is a critical pre-requisite to mitobiogenesis. The combined effect of aging and chronic inflammation on mitochondrial degradation by autophagy is understudied. In interleukin 10 null mouse (IL-10tm/tm), a rodent model of chronic inflammation, we studied the effects of aging and inflammation on mitochondrial clearance. We show that aging in IL-10tm/tm is associated with reduced skeletal muscle mitochondrial death signaling and altered formation of autophagosomes, compared to age-matched C57BL/6 controls. Moreover, skeletal muscles of old IL-10tm/tm mice have the highest levels of damaged mitochondria with disrupted mitochondrial ultrastructure and autophagosomes compared to all other groups. These observations highlight the interface between chronic inflammation and aging on altered mitochondrial biology in skeletal muscles.

11. Gene Therapy Mediated Increase in dATP Improves Cardiac Performance in Models of Systolic Heart Failure

Heart Failure results in more deaths and hospitalizations than almost any other single cause. We are exploring gene therapy treatment strategies that increase 2 deoxy-adenosine triphosphate (dATP) levels via cardiac specific expression of ribonucleotide reductase (RNR) in heart muscle. dATP is produced by RNR. In vitro studies show it increases the magnitude & rate of contraction in rodent, pig & failing human heart muscle without altering calcium handling or slowing relaxation. RNR expression in heart muscle increases intracellular dATP, cardiomyocyte contraction & cardiac performance in rodents. To do this we ligated a cDNA encoding both human RNR subunits to miniaturized cardiac specific enhancer/promoter portions of human cardiac troponin T and packaged the therapeutic “BB-R12” gene in AAV6. In the current study expanded the types of heart failure treated by BB-R12 by testing the effect of dATP on contractile function in a transgenic mouse model of familial dilated cardiomyopathy (DCM). These mice carry and phenocopy a missense mutation (D230N) in alpha-tropomyosin (Tm) that was identified in two DCM family cohorts that exhibit progressive ventricular dilation and loss of systolic function. We measured the effect of dATP on the Ca2+ sensitivity of force in demembranated trabeculae from young adult D230N & WT mice (3 mice & 8-10 trabeculae per group). pCa50 was decreased (lower Ca2+ sensitivity) for D230N Tm (5.47±0.01) compared to WT (5.59±0 0.03) mice, and substituting dATP for ATP rescued this defect (5.55±0.03). dATP also increased isometric force ~20% at pCa=5.8, the approximate level of intracellular Ca2+ seen in a cardiomyocyte twitch. We also measured force and the kinetics of activation & relaxation of isolated myofibrils. Force at pCa=5.6 decreased from 85±14 mN/mm2 in WT myofibrils to 52±7 mN/mm2 (p<0.05) in D230N Tm myofibrils. Importantly, the force deficit was improved to 72±14 mN/mm2 (p=0.47) with dATP. Similarly, the activation rate decreased in D230N Tm myofibrils (1.6±0.2 s-1) vs. WT mice (3.5±0.3 s-1), and this was partially corrected by dATP (2.5±0.4 s-1). dATP also corrected alterations in the early, slow relaxation phase of myofibrils from D230N Tm mice. In ongoing studies, we are systemically injecting 4-6 week old D230N Tm mice with AAV6-BB-R12 to determine how this affects cardiac function acutely, as well as the time course of reduced systolic function, progressive ventricular dilation & wall remodeling. In related studies, we found AAV6-BB-R12 rescues cardiac function in an infarcted pig heart failure model. These studies assess the ability of BB-R12 to acutely treat diseased hearts and act as a preventative agent in progressive heart failure.

Abstract 197: Determining the Potential Role of cTnT Isoform Switching In the Development of Early Childhood Tropomyosin-Linked DCM

An oft-noted component of sarcomeric HCM and DCM is the observation that patients within families, carrying the same primary mutation, often exhibit significant phenotypic variability. This lack of a distinct link between genotype and phenotype has greatly complicated clinical management. In a recent study of two large unrelated multigenerational families carrying the tropomyosin (Tm) mutation Asp230Asn (D230N), a striking “bimodal” distribution of severity was observed. In these families, many children (&lt;1 year) with the mutation presented with a severe form of DCM that led to sudden, often fatal congestive heart failure, while adults developed a mild to moderate DCM in mid-life. Of note, children who survived the initial presentation often recovered significant systolic function in adolescence and young adulthood. Therefore, to better understand the mechanism of this “bimodal” phenotype, we began to investigate the potential modulating role of isoform switching by other sarcomeric components. We hypothesize that the age-dependent remodeling seen in children with D230N Tm is a result of temporal isoform switches involving a closely linked Tm binding partner cardiac Troponin T (cTnT). Initial biophysical studies (circular dichroism and regulated in vitro motility, R-IVM) show that while D230N does not alter Tm’s thermal stability it does have a profound impact on myofilament activation. Both maximal velocity of filament sliding and calcium sensitivity were decreased. Furthermore, an additive decrease was observed in these parameters for R-IVM solutions containing cTnT 1 (fetal)+D230N Tm filaments as compared to cTnT 3 (adult)+D230N. Preliminary in vivo studies utilizing our novel double transgenic Tm-D230N x cTnT 1 mice show profound changes in wall thickness and chamber dilation, as compared to age-matched non-transgenic mice and D230N Tm mice. Further studies aim to model the “bimodal” clinical phenotype seen in families with D230N Tm and assess the potential for disease reversibility using a cardiac specific inducible cTnT 1 transgenic mouse model. Our goal is to use a translational approach to better understand the mechanism by which primary mutations lead to distinct clinical phenotypes in order to improve clinical management.

Deletion of CD24 impairs development of heat shock protein gp96-driven autoimmune disease through expansion of myeloid-derived suppressor cells.

CD24 binds to and suppresses inflammation triggered by danger-associated molecular patterns such as heat shock proteins (HSPs) and high-mobility group box 1. Paradoxically, CD24 has been shown to enhance autoimmune disease. In this study, we attempt to reconcile this paradox by deletion of CD24 (24KO) in a lupus-like disease model driven by forced expression of HSP gp96 at the cell surface (transgenic mice [tm]). As expected, tm24KO mice showed increased CD11c(+) dendritic cell activation coupled to a significant increase in dendritic cell-specific IL-12 production compared with tm mice. However, tm24KO mice showed less CD4 T cell activation and peripheral inflammatory cytokine production in comparison with tm mice. We characterized an enhanced immune suppressive milieu in tm24KO mice distinguished by increased TGF-β and greater regulatory T cell-suppressive capacity. We found greater absolute numbers of myeloid-derived suppressor cells (MDSCs) in tm24KO mice and showed that the Ly6C(+) MDSC subset had greater suppressive capacity from tm24KO mice. Deletion of CD24 in tm mice led to diminished lupus-like pathology as evidenced by anti-nuclear Ab deposition and glomerulonephritis. Finally, we show that expanded MDSC populations were mediated by increased free high-mobility group box 1 in tm24KO mice. Thus, the deletion of CD24 in an HSP-driven model of autoimmunity led to the unexpected development of regulatory T cell and MDSC populations that augmented immune tolerance. Further study of these populations as possible negative regulators of inflammation in the context of autoimmunity is warranted.

Contributions of thrombin targets to tissue factor‐dependent metastasis in hyperthrombotic mice

Tumor cell tissue factor (TF)-initiated coagulation supports hematogenous metastasis by fibrin formation, platelet activation and monocyte/macrophage recruitment. Recent studies identified host anticoagulant mechanisms as a major impediment to successful hematogenous tumor cell metastasis. Here we address mechanisms that contribute to enhanced metastasis in hyperthrombotic mice with functional thrombomodulin deficiency (TM(Pro) mice). Pharmacological and genetic approaches were combined to characterize relevant thrombin targets in a mouse model of experimental hematogenous metastasis. TF-dependent, but contact pathway-independent, syngeneic breast cancer metastasis was associated with marked platelet hyperreactivity and formation of leukocyte-platelet aggregates in immune-competent TM(Pro) mice. Blockade of CD11b or genetic deletion of platelet glycoprotein Ibα excluded contributions of these receptors to enhanced platelet-dependent metastasis in hyperthrombotic mice. Mice with very low levels of the endothelial protein C receptor (EPCR) did not phenocopy the enhanced metastasis seen in TM(Pro) mice. Genetic deletion of the thrombin receptor PAR1 or endothelial thrombin signaling targets alone did not diminish enhanced metastasis in TM(Pro) mice. Combined deficiency of PAR1 on tumor cells and the host reduced metastasis in TM(Pro) mice. Metastasis in the hyperthrombotic TM(Pro) mouse model is mediated by platelet hyperreactivity and contributions of PAR1 signaling on tumor and host cells.

Decreasing Tropomyosin Phosphorylation Rescues Tropomyosin-induced Familial Hypertrophic Cardiomyopathy

Studies indicate that tropomyosin (Tm) phosphorylation status varies in different mouse models of cardiac disease. Investigation of basal and acute cardiac function utilizing a mouse model expressing an α-Tm protein that cannot be phosphorylated (S283A) shows a compensated hypertrophic phenotype with significant increases in SERCA2a expression and phosphorylation of phospholamban Ser-16 (Schulz, E. M., Correll, R. N., Sheikh, H. N., Lofrano-Alves, M. S., Engel, P. L., Newman, G., Schultz Jel, J., Molkentin, J. D., Wolska, B. M., Solaro, R. J., and Wieczorek, D. F. (2012) J. Biol. Chem. 287, 44478-44489). With these results, we hypothesized that decreasing α-Tm phosphorylation may be beneficial in the context of a chronic, intrinsic stressor. To test this hypothesis, we utilized the familial hypertrophic cardiomyopathy (FHC) α-Tm E180G model (Prabhakar, R., Boivin, G. P., Grupp, I. L., Hoit, B., Arteaga, G., Solaro, R. J., and Wieczorek, D. F. (2001) J. Mol. Cell. Cardiol. 33, 1815-1828). These FHC hearts are characterized by increased heart:body weight ratios, fibrosis, increased myofilament Ca(2+) sensitivity, and contractile defects. The FHC mice die by 6-8 months of age. We generated mice expressing both the E180G and S283A mutations and found that the hypertrophic phenotype was rescued in the α-Tm E180G/S283A double mutant transgenic animals; these mice exhibited no signs of cardiac hypertrophy and displayed improved cardiac function. These double mutant transgenic hearts showed increased phosphorylation of phospholamban Ser-16 and Thr-17 compared with the α-Tm E180G mice. This is the first study to demonstrate that decreasing phosphorylation of tropomyosin can rescue a hypertrophic cardiomyopathic phenotype.

Use of Jak2 Inhibitors to Limit Ineffective Erythropoiesis and Iron Absorption in Mice Affected by β-Thalassemia and Other Disorders of Red Cell Production.

Abstract 2020Poster Board I-1042β-thalassemia intermedia (TI) and major (TM) are characterized by Ineffective Erythropoiesis (IE). We hypothesized that the kinase Jak2 plays a major role in IE and splenomegaly. To test this hypothesis we administered a Jak2 inhibitor (TG101209) to mice affected by TI, showing that this treatment was associated with a marked decrease in IE, and a moderate decrease in hemoglobin (Hb) levels (∼1 g/dL). This last observation indicates that the use of a Jak2 inhibitor might exacerbate anemia in thalassemia. However, we hypothesized that using standard transfusion to treat TM mice would also be adequate to prevent any further anemia caused by Jak2 inhibition while still allowing for decreased splenomegaly. Therefore, we analyzed the erythropoiesis and iron metabolism in TM animals treated with a Jak2 inhibitor and transfused. Use of TG101209 in TM mice not only reduced the spleen size dramatically (0.42±0.15 g and 0.19±0.10 g respectively in transfused+placebo (N=4) vs transfused+TG101209 (N=8), P= 0.007), but also allowed the mice to maintain higher Hb levels (respectively 7.3±1.1 g/dl vs 9.3±1.2 g/dl, P=0.019). This was likely due to reduced spleen size and limited red cell sequestration. Contrary to TM mice treated with transfusion+placebo, no foci of extra-medullary hematopoiesis were detectable in the parenchema of mice treated with TG101209. Hamp1 expression inversely correlated with the spleen weight, possibly indicating that suppression of IE (due both to blood transfusion and TG101209 administration) had a positive effect on Hamp1 expression. Hb levels also directly correlated with Hamp1 expression in the same animals. In this case, however, only transfusion played a role in increasing Hamp1 expression, although TG101209 undoubtedly had a positive effect by reducing the spleen size and thereby indirectly increasing the Hb levels.The suppression of erythropoiesis by blood transfusion limits the extent of our interpretations as it may mask the effect of the Jak2 inhibitor. Therefore we hypothesized that the administration of a tailored and reduced dose of the drug could be effective in reducing the splenomegaly in non-transfused TI mice, without affecting the Hb levels. We also hypothesized that the suppression of erythropoiesis would also lead to increased Hamp1 expression in the presence of iron overload. Compared to mice treated with placebo (N=5), analysis of TI mice treated with a tailored dose of 100mg/kg/day of the drug (N=11) showed a significant decrease in spleen size (0.18±0.05 g and 0.27±0.05 g, P=0.006 for drug treated mice and placebo treated mice respectively). Of note no significant difference of Hb levels was detectable between the 2 groups. In the drug treated mice we observed a significant decrease of the immature erythroid cell population (P=0.012) and amelioration of the architecture of the spleen, with the reappearance of white pulp foci and a significant restoration of the splenic lymphocitic populations. Drug treated mice showed increased levels of Hamp1 mRNA that inversely correlated with the spleen weight, suggesting a direct feedback between erythropoietic rate and expression of Hamp1.To determine if the use of Jak2 inhibitors could be beneficial in a mouse model mimicking a human form of hereditary ellyptocytosis, we treated mice KO for the 4.1R protein isoforms with TG101209, in presence or absence of blood transfusions. These mice exhibit moderate splenomegaly and anemia and the drug treatment was effective in reducing the spleen weight and the associated IE. We also plan to analyze Sickle Cell mice that we are treating with a Jak2 inhibitor. In conclusion our data show that the administration of Jak2 inhibitors is efficient in decreasing the spleen size and ameliorating the pathologic iron metabolism in thalassemia, both in the presence or absence of blood transfusions. Moreover we show that Jak2 inhibitors could transform the therapeutic approach for other forms of anemias. Disclosures:No relevant conflicts of interest to declare.

Deregulation of Transcription Factors GATA-1, GATA-2 and C/EBPa in Acute Promyelocytic Leukemia.

Acute promyelocytic leukemia (APL) accounts for ~10% of all acute myelogenous leukemia (AML) cases, and is characterized by accumulation of abnormal promyelocytes in the bone marrow. APL is uniquely associated with balanced chromosomal translocations involving the retinoic acid receptor alpha (RARA) locus. A functional chimeric protein, X-RARA, is created, whose N-terminus is derived from the partner (“X”) gene, and retains its oligomerization domain. The C-terminus is derived from RARA. X-RARA contributes to the APL phenotype by interfering with granulocyte differentiation, through acquisition of novel transactivating activity and/or interference with the normal functions of RARA and X. Several studies have indicated that defective retinoid signaling, though sufficient to block myeloid differentiation, cannot induce a leukemia. Thus, pathways external to retinoid signaling must be deregulated by X-RARA in order to give rise to APL. We previously reported whole genome gene expression analysis of the hCG-NuMA-RARA transgenic mouse model (Sukhai et al, Blood, Blood , 2006: a2247), and that the NuMA-RARA fusion protein deregulated a wide array of hematopoietic and myeloid transcription factors in leukemic cells derived from transgenic mice. Here, we report that the deregulated expression of this transcription factor set (specifically, Gata-1, Gata-2, C/ebpa and Pu.1) accurately distinguished leukemic TM mice from WT animals, and was dependent upon both the presence of functional RXRA (Sukhai et al, Oncogene, 2008) and transgene dosage. We thus formulated the hypothesis that NuMA-RARA initiated the deregulation of a range of transcription factors, which in turn were responsible for deregulating pathways within the cell. In order to determine whether this transcription factor signature was unique to APL, we focused on GATA-1, GATA-2, PU.1 and C/EBPA, and examined their expression in a range of AML cell lines. Strikingly, the over-expression of GATA-1 was unique to APL cell lines and the OCI/AML4 cell line. GATA-2 was over-expressed in most cell systems tested, suggesting that its up-regulation may play a general role in leukemogenesis. C/EBPA was under-expressed in NB4 cells specifically, while PU.1 was not significantly deregulated in any cell system tested. Having identified that APL cell lines recapitulated our observation of deregulated myeloid transcription factor expression in transgenic mice, we sought to extend these studies to human patients. We analyzed the expression of GATA- 1, GATA-2, PU.1 and C/EBPA in a series of 12 APL patients, 10 AML patients with a range of diagnoses, and 12 normal BM samples. We observed specific up-regulation of GATA-1 (2.0–20.0-fold change, 7/12 patients) and GATA-2 (2.0–25.0-fold change, 9/12 patients) and under-expression of C/EBPA (0.05–0.5-fold change, 11/12 patients), in APL, but not AML, patient samples, in comparison to normal BM. The deregulated expression of these three transcription factors could accurately distinguish APL patient samples from AML and normal BM in both principle component analysis and hierarchical clustering analysis. We therefore report herein that NuMA-RARA deregulated a wide array of myeloid transcription factors, suggesting that a state of globally deregulated myeloid transcription exists in APL cells. Furthermore, a specific subset of transcription factors, GATA-1, GATA-2 and C/EBPA, can be used to specifically classify APL patient samples. These deregulated transcription factors may therefore serve as potential therapeutic targets and a means of distinguishing APL from other forms of acute myelogenous leukemia.

Autoimmune Th2-Mediated Dacryoadenitis in MRL/MpJ Mice Becomes Th1-Mediated in IL-4 Deficient MRL/MpJ Mice

MRL/MpJ mice of substrains MRL/MpJ-fas(+)/fas(+) (MRL/+) and MRL/MpJ-fas(lpr)/fas(lpr) (MRL/lpr) spontaneously develop autoimmune dacryoadenitis and sialadenitis and are a model for the human disorder Sjögren syndrome. The dacryoadenitis in both substrains appears to be Th2 in nature, with little IFN-gamma and substantial IL-4 at the site of lacrimal gland inflammation. MRL/MpJ mice with a defective IL-4 gene-both MRL/+-IL-4(tm)/IL-4(tm) (MRL/+/IL-4(tm)) and MRL/lpr-IL-4(tm)/IL-4(tm) (MRL/lpr-IL-4(tm))-that resulted in a loss of IL-4 production were bred and evaluated for dacryoadenitis. MRL/+/IL-4(tm) and MRL/lpr/IL-4(tm) mice developed dacryoadenitis of similar onset, appearance, and severity as found in MRL/MpJ mice with an intact IL-4 gene. Immunohistochemistry examination revealed a substantially greater number of inflammatory cells staining for IFN-gamma than for IL-13 in the dacryoadenitis of IL-4-deficient MRL/MpJ mice (MRL/+/IL-4(tm), 66% vs. 0.8%, P = 0.001; MRL/lpr/IL-4(tm), 67% vs. 1.2%, P = 0.002). Real-time PCR demonstrated greater amounts of IFN-gamma than IL-13 mRNA relative transcripts in lacrimal glands of MRL/lpr/IL-4(tm) mice (mean difference, 28.6; P = 0.035). Greater CD86 (B7-2) than CD80 (B7-1) expression was present in MRL/+/IL-4(tm) mice (11% vs. 3%, P = 0.003) and MRL/lpr/IL-4(tm) mice (10% vs. 3%, P = 0.002). These results suggest that a Th2 autoimmune process can be converted to a Th1 process in the absence of IL-4.

Cardiac dysfunction in hypertrophic cardiomyopathy mutant tropomyosin mice is transgene-dependent, hypertrophy-independent, and improved by beta-blockade.

Familial hypertrophic cardiomyopathy (FHC) has been linked to mutations in proteins of the cardiac contractile apparatus, including alpha-tropomyosin (Tm). Mice expressing alphaTm in the heart were developed to determine the effects of FHC mutant Tm on cardiac structure and function from single cardiac myocytes to whole organ function in vivo. Expression of E180G mutant Tm did not produce cardiac hypertrophy or detectable changes in cardiac muscle morphology. However, E180G mutant Tm expression increased the Ca2+ sensitivity of force production in single cardiac myocytes in a transgene expression-dependent manner. Contractile dysfunction in single myocytes manifested organ level dysfunction, as conductance-micromanometry showed E180G Tm mice had significantly slowed relaxation (diastolic dysfunction) under physiological conditions. The diastolic dysfunction in E180G Tm mice was no longer evident during beta-blockade because propranolol eliminated the effect of E180G Tm to slow myocardial relaxation. Cellular and organ level dysfunction were evident in E180G Tm mice in the absence of significant cardiac structural abnormalities normally associated with FHC. These findings therefore suggest that diastolic dysfunction in FHC may be a direct consequence of FHC mutant protein expression. In addition, because diastolic dysfunction in E180G Tm mice is dependent on inotropic status, cardiovascular stress may play an important role in FHC pathogenesis.