Ntg Littermates Research Articles

Chromosome Y‐haplotype mediates cognitive resilience and differential expression of transposable elements in 5xFAD‐carrying AD‐BXD males

AbstractBackgroundIn Alzheimer’s disease (AD), risk factors and clinical progression differ between sexes. Men have lower post‐diagnosis life‐expectancies 1,2, while women display worsened cognitive decline than men with similar neuropathology3‐5 or shared genetic risk6,7. Investigation of sex‐specific genetic contributions to AD has largely focused on the X‐chromosome, but emerging evidence has also linked somatic Y‐chromosomal instability to age‐linked disease outcomes, including AD8‐10. Notably, the Y‐chromosome contains few coding genes, but is enriched with transposable elements (TEs)11, enigmatic DNA sequences capable of introducing variation into the genome12. Differential expression of TEs has recently been reported in AD13‐15. We hypothesize that TEs could contribute to sex‐specific variation observed in AD onset and progression.MethodHere, we utilized bulk hippocampal RNAseq and contextual fear memory (CFM) data from a 28‐strain AD‐BXD dataset comprised of male and female 5xFAD‐carrying animals and non‐transgenic (Ntg) littermates to investigate whether expression of coding genes and/or TEs contribute to AD‐relevant sex differences and cognitive outcomes. Data were stratified by sex and Y‐chromosome haplotype (C57BL/6J‐type vs. DBA/2J‐type). TEs were quantified in RNAseq data using TETranscripts16. For each TE, we predicted genomic localization using DFAM17, and calculated sex‐specific heritability.ResultIn 5xFAD animals, C57BL/6J‐type males significantly out‐performed both DBA/2J‐type males and females on a CFM task (Fig.1). TEs identified in this dataset were differentially expressed due to both sex and Y‐chromosome haplotype in 5xFAD animals (Fig.2). Interestingly, Y‐haplotype did not affect Y‐chromosome coding gene expression. We calculated that a large portion of TEs identified in our dataset are highly heritable, with differential heritability associated with sex and 5xFAD‐transgene status (Fig.3)ConclusionWe report that the C57BL/6J Y‐chromosome confers cognitive resilience in 5xFAD males, which may undermine the utility of models maintained on a homogenous C57BL/6J background as tools for investigating age‐related cognitive decline. We speculate that differential TE expression/activity between Y‐haplotypes and between sexes may account for differential resilience, but further investigation is needed to establish this link directly. Finally, our data suggest that not only are TEs likely influencers of AD risk and disease progression, but that TE expression in disease states may be heritable, rather than stochastically induced.

Abstract P1028: Elucidating Differential Mechanisms That Underlie Phenotypic Variability In Two Independent Dcm Models

Mutations in components of the cardiac thin filament cause disease via complex, multi-faceted myocellular mechanisms, ultimately presenting as heterogeneous, symptomatic cardiomyopathies. The heterogeneity of both the myocellular mechanism(s) involved and the clinical outcome(s) represents a challenge in ongoing efforts to link genotype to phenotype. Two DCM-associated thin filament mutations (D230N-Tm and R173W-cTnT) with similar clinical phenotypes, were selected explore the molecular basis of disease trajectory. In patient populations, D230N-Tm exhibits an earlier time of onset compared to R173W-cTnT, with children (<1yr) presenting with an often-fatal DCM. We hypothesize that the difference in time of onset can be explained by distinct myocellular mechanisms. We utilized transgenic mice expressing D230N-Tm and R173W-cTnT and defined the progression of pathogenic remodeling across 3 independent timepoints via 2D-echo. At 2 months the %FS of D230N-Tm was significantly reduced (21.79 ± 1.19) compared to NTg littermates (33.45±1.45). R173W-cTnT, however, exhibited no reduction in %FS until 4 months (22.53±0.78), thus phenocopying the patient population. Using these timepoints to guide our myocellular mechanics, contractility measurements were then utilized to assess the myocellular mechanics associated with the mutations. At 2 months D230N-Tm exhibited a decrease in the rate of contraction (4.53±0.24μm/sec) and rate of relaxation (2.88±0.23) compared to NTg. R173W-cTnT did not diverge from NTg until 4 months, exhibiting a later onset of impaired contractility. By 4 months, both mutations displayed the opposite, an increase in rate of contraction and relaxation (rate of contraction, D230N-Tm 4.49±0.18 and R173W-cTnT 5.83±0.27; and rate of relaxation, D230N-Tm 3.14±0.19 and R173W-cTnT 3.70±0.24). Our results suggest that the accelerated time course for the development of D230N-Tm – linked DCM is caused by differential myocellular mechanism(s) that induce the early reduction in contractility and relaxation observed. Ongoing studies aim to further identify possible myocellular mechanism(s) by which these mutations exhibit different myocellular mechanics.

Early alterations in brain glucose metabolism and vascular function in a transgenic rat model of Alzheimer's disease.

Alteration in brain metabolism predates clinical onset of Alzheimer's Disease (AD). Realizing its potential as an early diagnostic marker, however, requires understanding how early AD metabolic dysregulation manifests on non-invasive brain imaging. We presently utilized magnetic resonance imaging and spectroscopy to map glucose and ketone metabolic profiles and image cerebrovascular function in a rat model of early stage AD - 9-month-old TgF344-AD (TgAD) rats - and their age-matched non-transgenic (nTg) littermates. Compared to the nTg rats, TgAD rats displayed attenuation in global cerebral and hippocampal vasoreactivity to hypercapnia, by 49±17% and 58±19%, respectively, while their functional hyperemia to somatosensory stimulation diminished by 69±5%. To assess brain glucose uptake, rats were fasted overnight and then challenged with an intravenous infusion of 2-deoxy-D-glucose (2DG). Compared to their non-transgenic littermates, TgAD rats exhibited 99±10% and 52±5% smaller glucose uptake in the entorhinal cortex and the hippocampus, respectively. Moreover, hippocampal glucose uptake reduction in male TgAD rats compared to the nTg was 54±36% greater than the reduction seen in female TgAD rats. TgAD rats also showed a 59±42% increase in total choline level in the hippocampus, suggesting increased membrane turnover. In combination with our earlier findings of impaired electrophysiological metrics at this early stage of AD pathology progression, our findings suggest that subtle neuronal function alterations that would be difficult to assess in a clinical population may be accompanied by MRI-detectable changes in brain glucose metabolism and cerebrovascular function.

Prostaglandin EP2 receptor antagonist ameliorates neuroinflammation in a two-hit mouse model of Alzheimer\u2019s disease

BackgroundAlzheimer’s disease (AD) causes substantial medical and societal burden with no therapies ameliorating cognitive deficits. Centralized pathologies involving amyloids, neurofibrillary tangles, and neuroinflammatory pathways are being investigated to identify disease-modifying targets for AD. Cyclooxygenase-2 (COX-2) is one of the potential neuroinflammatory agents involved in AD progression. However, chronic use of COX-2 inhibitors in patients produced adverse cardiovascular effects. We asked whether inhibition of EP2 receptors, downstream of the COX-2 signaling pathway, can ameliorate neuroinflammation in AD brains in presence or absence of a secondary inflammatory stimuli.MethodsWe treated 5xFAD mice and their non-transgenic (nTg) littermates in presence or absence of lipopolysaccharide (LPS) with an EP2 antagonist (TG11-77.HCl). In cohort 1, nTg (no-hit) or 5xFAD (single-hit—genetic) mice were treated with vehicle or TG11-77.HCl for 12 weeks. In cohort 2, nTg (single-hit—environmental) and 5xFAD mice (two-hit) were administered LPS (0.5 mg/kg/week) and treated with vehicle or TG11-77.HCl for 8 weeks.ResultsComplete blood count analysis showed that LPS induced anemia of inflammation in both groups in cohort 2. There was no adverse effect of LPS or EP2 antagonist on body weight throughout the treatment. In the neocortex isolated from the two-hit cohort of females, but not males, the elevated mRNA levels of proinflammatory mediators (IL-1β, TNF, IL-6, CCL2, EP2), glial markers (IBA1, GFAP, CD11b, S110B), and glial proteins were significantly reduced by EP2 antagonist treatment. Intriguingly, the EP2 antagonist had no effect on either of the single-hit cohorts. There was a modest increase in amyloid–plaque deposition upon EP2 antagonist treatment in the two-hit female brains, but not in the single-hit genetic female cohort.ConclusionThese results reveal a potential neuroinflammatory role for EP2 in the two-hit 5xFAD mouse model. A selective EP2 antagonist reduces inflammation only in female AD mice subjected to a second inflammatory insult.

Characterization of an APP/tau rat model of Alzheimer\u2019s disease by positron emission tomography and immunofluorescent labeling

BackgroundTo better understand the etiology and pathomechanisms of Alzheimer’s disease, several transgenic animal models that overexpress human tau or human amyloid-beta (Aβ) have been developed. In the present study, we generated a novel transgenic rat model by cross-breeding amyloid precursor protein (APP) rats with tau rats. We characterized this model by performing positron emission tomography scans combined with immunofluorescent labeling and cerebrospinal fluid analyses.MethodsAPP/Tau rats were generated by cross-breeding male McGill-R-Thy1-APP transgenic rats with female hTau-40/P301L transgenic rats. APP/Tau double transgenic rats and non-transgenic (ntg) littermates aged 7, 13, and 21 months were subjected to dynamic [11C] PiB scan and dynamic [18F]THK-5317 scans. For regional brain analysis, a template was generated from anatomical MR images of selected animals, which was co-registered with the PET images. Regional analysis was performed by application of the simplified reference tissue model ([11C]PiB data), whereas [18F]THK-5317 data were analyzed using a 2-tissue compartment model and Logan graphical analysis. In addition, immunofluorescent labeling (tau, amyloid) and cerebrospinal fluid analyses were performed.Results[11C]PiB binding potential (BPND) and [18F]THK-5317 volume of distribution (VT) showed an increase with age in several brain regions in the APP/Tau group but not in the ntg control group. Immunohistochemical analysis of brain slices of PET-scanned animals revealed a positive correlation between Aβ labeling and [11C]PiB regional BPND. Tau staining yielded a trend towards higher levels in the cortex and hippocampus of APP/Tau rats compared with ntg littermates, but without reaching statistical significance. No correlation was found between tau immunofluorescence labeling results and the respective [18F]THK-5317 VT values.ConclusionsWe thoroughly characterized a novel APP/Tau rat model using combined PET imaging and immunofluorescence analysis. We observed an age-related increase in [11C]PiB and [18F]THK-5317 binding in several brain regions in the APP/Tau group but not in the ntg group. Although we were able to reveal a positive correlation between amyloid labeling and [11C]PiB regional brain uptake, we observed relatively low human tau and amyloid fibril expression levels and a somewhat unstable brain pathology which questions the utility of this animal model for further studies.

Evaluation of Neuropathological Features in the SOD1-G93A Low Copy Number Transgenic Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) still depicts an incurable and devastating disease. Drug development efforts are mostly based on superoxide dismutase 1 gene (SOD1)-G93A mice that present a very strong and early phenotype, allowing only a short time window for intervention. An alternative mouse model is available, that is based on the same founder line but has a reduced SOD1-G93A copy number, resulting in a weaker and delayed phenotype. To be able to use these SOD1-G93A/low mice for drug testing, we performed a characterization of ALS-typical pathologies. All analyses were performed compared to non-transgenic (ntg) littermates of the same sex and age. In vivo analysis of SOD1-G93A/low mice was performed by weekly body weight measurements, analysis of the survival rate, and measurement of the muscle strength of 24–30 weeks old female and male SOD1-G93A/low mice. Immunofluorescent labeling of SOD1, glial fibrillary acidic protein (GFAP), and ionized calcium-binding adaptor molecule 1 (Iba1) protein was performed in the cervical, thoracic, and lumbar ventral horn of the spinal cord of 24–30 weeks old male and female SOD1-G93A/low mice. The musculus gastrocnemius of male SOD1-G93A/low mice was labeled with fluorophore-conjugated α-bungarotoxin and antibodies against phosphorylated neurofilaments. Fluorescent labeling was detected and quantified by macro-based image analysis. Although SOD1 protein levels were highly increased in both sexes and all age groups, levels strongly peaked in 30 weeks old male SOD1-G93A/low mice. Astrocytosis and activated microglia in the spinal cord ventral horn and phosphorylated neurofilaments in the motor unit of the musculus gastrocnemius progressively increased, while muscle strength progressively decreased in male SOD1-G93A/low mice. In female SOD1-G93A/low mice, only activated microglia increased progressively, while muscle strength was constantly reduced starting at 26 weeks. These differences result in a shorter survival time of male SOD1-G93A/low mice of about 3 weeks compared to female animals. The results suggest that male SOD1-G93A/low mice present a stronger pathology and are, therefore, better suitable to evaluate the efficacy of new drugs against ALS as most pathological features are developing progressively paralleled by a survival time that allows treatment to start before symptom onset.

Peripherally misfolded proteins exacerbate ischemic stroke-induced neuroinflammation and brain injury

BackgroundProtein aggregates can be found in peripheral organs, such as the heart, kidney, and pancreas, but little is known about the impact of peripherally misfolded proteins on neuroinflammation and brain functional recovery following ischemic stroke.MethodsHere, we studied the ischemia/reperfusion (I/R) induced brain injury in mice with cardiomyocyte-restricted overexpression of a missense (R120G) mutant small heat shock protein, αB-crystallin (CryABR120G), by examining neuroinflammation and brain functional recovery following I/R in comparison to their non-transgenic (Ntg) littermates. To understand how peripherally misfolded proteins influence brain functionality, exosomes were isolated from CryABR120G and Ntg mouse blood and were used to treat wild-type (WT) mice and primary cortical neuron-glia mix cultures. Additionally, isolated protein aggregates from the brain following I/R were isolated and subjected to mass-spectrometric analysis to assess whether the aggregates contained the mutant protein, CryABR120G. To determine whether the CryABR120G misfolding can self-propagate, a misfolded protein seeding assay was performed in cell cultures.ResultsOur results showed that CryABR120G mice exhibited dramatically increased infarct volume, delayed brain functional recovery, and enhanced neuroinflammation and protein aggregation in the brain following I/R when compared to the Ntg mice. Intriguingly, mass-spectrometric analysis of the protein aggregates isolated from CryABR120G mouse brains confirmed presence of the mutant CryABR120G protein in the brain. Importantly, intravenous administration of WT mice with the exosomes isolated from CryABR120G mouse blood exacerbated I/R-induced cerebral injury in WT mice. Moreover, incubation of the CryABR120G mouse exosomes with primary neuronal cultures induced pronounced protein aggregation. Transduction of CryABR120G aggregate seeds into cell cultures caused normal CryAB proteins to undergo dramatic aggregation and form large aggregates, suggesting self-propagation of CryABR120G misfolding in cells.ConclusionsThese results suggest that peripherally misfolded proteins in the heart remotely enhance neuroinflammation and exacerbate brain injury following I/R likely through exosomes, which may represent an underappreciated mechanism underlying heart-brain crosstalk.

Abstract 539: Buthionine Sulfoximine (BSO) Prevents Reductive Stress-induced Pathological Hypertrophic Cardiomyopathy

Background: Chronic reductive stress (RS) induces pathological cardiac remodeling and diastolic dysfunction. Here, we hypothesized that preventing RS via glutathione (GSH) depletion, through selective inhibition of γ-glutamyl cysteine ligase (γGCL), mitigates cardiac pathology in cRS mice. Methods: Cardiac-specific constitutively active Nrf2 TG-mice (α-MHc-caNrf2-TG), at 6 weeks of age, were administered with buthionine sulfoximine (BSO; 5.0 mM/Kg; daily for 16-weeks). At the end of 22 weeks, cardiac structure and function (systole & diastole using echocardiography), myocardial redox state, levels of ROS (using dihydroethidium (DHE) fluorescence), and antioxidant proteome were assessed in TG mice treated with PBS or BSO and compared with the age matched NTG littermates (n=6/group). Results: While the TG mice experiencing RS (GSH; 426.3±22.55 vs. 141.8±3.9 & GSH/GSSG; 61.04±5.4 vs. 23.27±1.3 in TG vs. NTG), this was significantly curtailed in BSO-treated TG mice (GSH 161.8±12.6 & GSH/GSSG; 25.1±4.3). This was coupled with the normal cardiac functions (EF; ~53% & MV E/A; 1.57) in the BSO-treated TG when compared to TG mice experiencing a hyper systolic function (>80% ejection fraction) with decreased cardiac volume and diastolic dysfunction with restricted filling (MV E/A ratio; >3.0), Of note, BSO treatment did not alter the protein levels of antioxidants (i.e. GCLC, GCLM, NQO1 and CAT), but only depleted the GSH pool (via inhibiting GCL activity) and restored the basal ROS signaling in the myocardium. Moreover, the BSO-treated TG mice did not develop cardiac hypertrophy, which was assessed by heart weight/body weight ratio, qPCR-based gene expression for hypertrophy ( Anf, Bnf, α-MHc and β-Mhc ). Conclusion: Our results suggest that pharmacological manipulation of myocardial redox and repletion of basal ROS signaling prevented RS-mediated pathological processes and rescued the cardiac structure and function.

Abstract 303: Fast Skeletal Myosin Binding Protein-c Expression in Heart Failure

Background: Heart failure is a devastating disease affecting nearly 5 million Americans. We recently identified the upregulation of fast skeletal myosin binding protein-C (fsMyBP-C) in models of heart failure. fsMyBP-C is a homologue of cardiac MyBP-C, which is normally expressed in the heart. This family of proteins regulate contractility by modulating thick and thin filament interactions. However, the functional role(s) of fsMyBP-C expression in the heart is unknown. Methods: To understand the role of fsMyBP-C expression in the heart, we generated cardiac specific transgenic mice expressing fsMyBP-C under the control of the alpha myosin heavy chain promoter. Mice underwent serial echocardiography to monitor cardiac function. Western blotting and qRT-PCR were performed to determine transgene expression levels. Additionally, fsMyBP-C localization in cardiomyocytes was established by immunofluorescence. Furthermore, to understand the temporal expression profiles of the MyBP-C homologues, we performed qRT-PCR on 9 muscle groups at different developmental stages in healthy mice. Results: qRT-PCR demonstrated an upregulation of the slow MyBP-C transcript in the developing heart, while fsMyBP-C transcript levels increased steadily following birth in WT mice. At 3 months of age, transgenic mice displayed a significant increase in ventricular dimensions compared to NTG littermates by echocardiography (LVID d : 3.72 ± 0.07 vs. 4.17 ± 0.09 mm, p<0.01; LVID s : 2.44 ± 0.09 vs. 2.81 ± 0.09, p<0.05), with no changes in wall thickness. This was accompanied by a 25% increase in stroke volume (p<0.01) and a 30% increase in cardiac output (p<0.05). fsMyBP-C correctly localized to the C-zone of the sarcomere. Furthermore, protein levels showed mild expression of fsMyBP-C, in line with the small upregulation seen in diseased hearts, while qRT-PCR confirmed overexpression of fsMyBP-C in the ventricles. Conclusion: Mice expressing fsMyBP-C in the heart display a mild hypertrophic phenotype at 3 months, with preserved function, possibly indicating compensated hypertrophy. Ongoing studies investigate cardiac function at later stages and cardiac specific conditional knockout of fsMyBP-C to determine the long-term effect of fsMyBP-C regulation in the heart.

Cyclin D2 is a critical mediator of exercise-induced cardiac hypertrophy.

A number of signaling pathways underlying pathological cardiac hypertrophy have been identified. However, few studies have probed the functional significance of these signaling pathways in the context of exercise or physiological pathways. Exercise studies were performed on females from six different genetic mouse models that have been shown to exhibit alterations in pathological cardiac adaptation and hypertrophy. These include mice expressing constitutively active glycogen synthase kinase-3β (GSK-3βS9A), an inhibitor of CaMK II (AC3-I), both GSK-3βS9A and AC3-I (GSK-3βS9A/AC3-I), constitutively active Akt (myrAkt), mice deficient in MAPK/ERK kinase kinase-1 (MEKK1-/-), and mice deficient in cyclin D2 (cyclin D2-/-). Voluntary wheel running performance was similar to NTG littermates for five of the mouse lines. Exercise induced significant cardiac growth in all mouse models except the cyclin D2-/- mice. Cardiac function was not impacted in the cyclin D2-/- mice and studies using a phospho-antibody array identified six proteins with increased phosphorylation (greater than 150%) and nine proteins with decreased phosphorylation (greater than 33% decrease) in the hearts of exercised cyclin D2-/- mice compared to exercised NTG littermate controls. Our results demonstrate that unlike the other hypertrophic signaling molecules tested here, cyclin D2 is an important regulator of both pathologic and physiological hypertrophy. Impact statement This research is relevant as the hypertrophic signaling pathways tested here have only been characterized for their role in pathological hypertrophy, and not in the context of exercise or physiological hypertrophy. By using the same transgenic mouse lines utilized in previous studies, our findings provide a novel and important understanding for the role of these signaling pathways in physiological hypertrophy. We found that alterations in the signaling pathways tested here had no impact on exercise performance. Exercise induced cardiac growth in all of the transgenic mice except for the mice deficient in cyclin D2. In the cyclin D2 null mice, cardiac function was not impacted even though the hypertrophic response was blunted and a number of signaling pathways are differentially regulated by exercise. These data provide the field with an understanding that cyclin D2 is a key mediator of physiological hypertrophy.

Identification of transcriptome signature for myocardial reductive stress

The nuclear factor erythroid 2 like 2 (Nfe2l2/Nrf2) is a master regulator of antioxidant gene transcription. We recently identified that constitutive activation of Nrf2 (CaNrf2) caused reductive stress (RS) in the myocardium. Here we investigate how chronic Nrf2 activation alters myocardial mRNA transcriptome in the hearts of CaNrf2 transgenic (TG-low and TG-high) mice using an unbiased integrated systems approach and next generation RNA sequencing followed by qRT-PCR methods. A total of 246 and 1031 differentially expressed genes (DEGs) were identified in the heart of TGL and TGH in relation to NTG littermates at ~ 6 months of age. Notably, the expression and validation of the transcripts were gene-dosage dependent and statistically significant. Ingenuity Pathway Analysis identified enriched biological processes and canonical pathways associated with myocardial RS in the CaNrf2-TG mice. In addition, an overrepresentation of xenobiotic metabolic signaling, glutathione-mediated detoxification, unfolded protein response, and protein ubiquitination was observed. Other, non-canonical signaling pathways identified include: eNOS, integrin-linked kinase, glucocorticoid receptor, PI3/AKT, actin cytoskeleton, cardiac hypertrophy, and the endoplasmic reticulum stress response. In conclusion, this mRNA profiling identified a "biosignature" for pro-reductive (TGL) and reductive stress (TGH) that can predict the onset, rate of progression, and clinical outcome of Nrf2-dependent myocardial complications. We anticipate that this global sequencing analysis will illuminate the undesirable effect of chronic Nrf2 signaling leading to RS-mediated pathogenesis besides providing important guidance for the application of Nrf2 activation-based cytoprotective strategies.

Abstract 69: Cardiac Fibrosis Induced by Mkk6-p38 Activation Alters Myocardial Stiffness and Myofilament Function

Heart failure with preserved ejection fraction (HFpEF) accounts for at least half of the patients with heart failure, but to date there are no proven therapies for these patients. Cardiac fibrosis and increased collagen content plays an important role in the pathophysiology of HFpEF. Studies in human HFpEF patients have indicated increased cardiac fibrosis, myocardial stiffness and myofilament calcium sensitivity. However, it is unclear how and if cardiac fibrosis directly alters myofilament function. In this study we used a new model of cardiac fibrosis genetically induced by the constitutive activation of MKK6-p38 (MKK6-Tg) signaling specifically in resident cardiac fibroblasts to determine the effect of fibrosis on myofilament stiffness and function. The MKK6-Tg mice develop interstitial and perivascular fibrosis in the heart accompanied with severe diastolic dysfunction, although systolic function remains normal. We measured the passive stiffness and isometric contractile properties of demembranated papillary muscle preparations. Our results show an increase in passive stiffness in demembranated papillary muscles from MKK6-Tg mice compared to NTG littermates when stretched to 24% from sarcomere length of 2.0 μM. Passive tension at SL=2.3 was significantly increased in MKK6-Tg mice (28±4 vs. 13±3 mN/mm 2 , P<0.05) compared to NTG mice, which was ascribed to changes in Titin isoform as well as extracellular matrix composition. The MKK6-Tg mice also show an increase in Ca 2+ sensitivity (pCa 50 =5.72±0.05 vs. 5.56±0.02, P<0.05). This was associated with an increased rate of force redevelopment in MKK6-Tg mice compared to NTG littermates (k TR =35±8 vs. 22±3 s -1 ). Towards understanding the mechanism of these myofilament changes we performed phosphate affinity electrophoresis and saw decreased phosphorylation of both Troponin I and Troponin T in MKK6-Tg hearts. There was no change in troponin I phosphorylation level at Ser23/24, indicating role of none-PKA mediated phosphorylation in mechanical changes seen in this model.

Liver-specific NG37 overexpression leads to diet-dependent fatty liver disease accompanied by cardiac dysfunction.

BackgroundEnvironmental factors are well-known causes of diseases. However, aside from a handful of risk indicators, genes’ encoding susceptibility to chronic illnesses and their associated environmental triggers are largely unknown. In this era of increasingly rich diets, such genetic predispositions would be immensely helpful from a public health perspective. The novel transgenic mouse model with liver-specific NG37 overexpression characterized in this article identifies the diet-dependent function of NG37 in the pathogenesis of fatty liver disease and cardiac arrhythmia.ResultsThe liver-specific NG37 overexpression transgenic mouse model described here was generated using the Alb-SV40 polyA expression plasmid backbone. NG37 cDNA under control of the albumin promoter for liver-specific expression was fused with a 5′ terminal M2 FLAG sequence and a SV40 early region transcription terminator/polyadenylation site attached at the 3′-UTR. These NG37 transgenic mice developed normally and were physiologically normal on a standard diet. However, in comparison to non-transgenic (nTG) litter mates, these mice develop dramatic phenotypes within 12–18 days of starting a high-fat diet: (i) increased body weight (28.5 ± 12.3 g), (ii) increased liver weight (87.4 ± 35.7 mg), (iii) increased heart weight (140 ± 38.4 mg), and (iv) cardiac arrhythmia. The enlarged livers of high-fat diet NG37 transgenic mice was histologically similar to human fatty liver disease and contained Maltese cross birefringent active depositions in hepatocytes that are indicative of fatty liver disease. We also confirmed via X-ray diffraction the steatotic vesicles in the diseased hepatocytes of our high-fat diet NG37 mice was composed of cholesteryl derivatives also found in human fatty liver disease. In addition to cardiac enlargement, NG37 transgenic mice on high-fat diet also exhibited highly irregular bradycardia not present in either high-fat diet nTG littermates or normal-diet transgenic litter mates.ConclusionsThe dramatic high-fat diet-dependent symptoms (increased body weight, cardiac enlargement, fatty liver, and cardiac arrhythmias) characterized in our liver-specific NG37 overexpression mouse model identifies NG37 as a gene encoding latent lipid metabolism pathology induced only in the presence of an environmental factor relevant to human health: high-fat diet.

Abstract 423: Proteasome Priming by Protein Kinase G Protects Against Myocardial Ischemia-reperfusion Injury

Cardiac proteasome functional insufficiency is implicated in a large subset of heart disease and has been experimentally demonstrated to play an essential role in cardiac proteotoxicity, including desmin-related cardiomyopathy and myocardial ischemia-reperfusion (I-R) injury. Pharmacological inhibition of phosphodiesterase 5 (PDE5) via sildenafil for example, which can stabilize cGMP and thereby increase cGMP-dependent protein kinase (PKG) activity, is consistently reported to protect against I-R injury; however, the underlying mechanism is not fully understood. We have recently discovered that PKG activation enhances proteasomal degradation of misfolded proteins (Ranek, et al. Circulation 2013), prompting us to hypothesize that proteasome-priming may contribute to cardioprotection-induced by PDE5 inhibition. Here we used a cardiomyocyte-restricted proteasome inhibition transgenic mouse line (Tg) and non-Tg (Ntg) littermates to interrogate the action of sildenafil on I-R injury created by left anterior descending artery (LAD) ligation (30 min) and release (24 hr). Sildenafil was administered 30 min before LAD ligation. Results showed that (1) the 26S proteasome activity of the Ntg I-R hearts was significantly elevated by sildenafil but this elevation was blocked in the Tg line; (2) the infarct size reduction by sildenafil treatment in Ntg mice was completely abolished in the Tg mice with the same treatment; and (3) systolic and diastolic function impairment after I/R was markedly attenuated in sildenafil-treated Ntg mice, but not in the sildenafil-treated Tg mice. Additionally, immunoprecipitation assays show that PKG interacted with the proteasome in cultured cardiomyocytes, and this interaction appeared to be augmented by sildenafil treatment. Moreover, in vitro incubation of active PKG with purified human 26S proteasomes increased proteasome peptidase activities and the phosphorylation at specific serine residues of a 19S proteasome subunit as revealed by “gel-free” nano-LC-MS/MS. We conclude that active PKG directly interacts with, phosphorylates, and increases the activities of, the proteasome and that proteasome priming mediates to cardioprotection of PDE5 inhibition against I-R injury.

Su1885 Insulin Resistance Is a Key Factor for Metabolic and Inflammatory Biomarkers in Children Obesity

protein integrity. Hypothesis: Epithelial cell Hsp70 overexpression maintains tight junction protein levels in spite of high fat diet. Materials & Methods: Five week old male mice, [non-transgenic (NTG; n=19) and Hsp70 transgenic littermates (TG; n=14; villin promoter driven epithelial cell Hsp70 expression)] were randomly divided into low fat (LF; 10 kcal% fat) or high fat (HF; 60 kcal% fat) diet groups (NTG/LF, NTG/HF, TG/LF, TG/HF). All mice continued on the protocol for 12 weeks. At week 13, colonic scrapings were collected and homogenized for determining Hsp70 levels (ELISA, R&D Systems) and tight junction protein expression (Western blot). Western blots probed for occludin (Life Technologies) and constitutive heat shock protein 70 (HSC70, Santa Cruz Biotech) were run using pooled samples of each group (n= 6-10 per group). Densitometry readings were completed using a ChemiDoc MP Imager (Bio-Rad) and data normalized using HSC70 as a loading control with NTG/LF as the reference sample. Hsp70 data was analyzed by 2-way ANOVA and presented as a mean ± SEM. Results: Measurement of colonic Hsp70 levels showed a significant (p < 0.05, Fig. 1), 100 fold, increase in the TG group compared to its NTG littermates. Western blot for occludin using pooled samples representative of each group suggested TG mice expressed an increased amount of this protein compared to their NTG littermates regardless of diet (Fig. 2). Discussion: Hsp70 TG mice demonstrated increased levels of occludin, suggesting that it may enhance gut barrier function by increasing tight junction proteins independent of diet. Further examination of Hsp70's affect on colonic tight junction proteins may provide a possible target for the prevention and treatment of obesity

Abstract 159: Cardiac Hypertrophy and Sudden Death in a Mouse Model of STIM1 Overexpression

Background: STIM1, an ER/SR resident Ca 2+ sensing protein regulates Ca 2+ entry following internal Ca 2+ store depletion in a broad range of tissues and cell types. However their putative roles in excitable tissue such as cardiac myocytes is uncertain. Results: Here we generated a mouse model of STIM1 overexpression in cardiac and skeletal muscle. Western blot analysis suggested approximately 4-6 fold STIM1 overexpression in Tg mouse hearts compared to Ntg littermates. Immunocytochemistry carried out in ventricular myocytes revealed that STIM1 and the cardiac ryanodine receptor (RyR2) co-localize. Functionally, the amplitude of Ca 2+ entry following SR Ca 2+ depletion was 2-fold greater in myocytes isolated from STIM1 Tg mice compared to NTg littermates. Echocardiographic analysis in STIM1 Tg mice showed age dependent remodeling of the myocardium with a significant decrease in fractional shortening at 16 weeks of age (14.4.5±3.8 in STIM1 Tg vs. 36.9±1.5 in Ntg). These changes were accompanied by a significant increase in heart weight to tibia length (13.6 +/- 1.4 vs 6.5 +/- 0.24) and increased lung weight to tibia length ratio (11.6+/- 2.1 vs 8.1 +/- 0.38) in STIM1 Tg mice compared to Ntg littermates. Photometry experiments in isolated ventricular myocytes demonstrated significantly increased Ca 2+ transient amplitude with an unexpected decrease in the SR Ca 2+ load associated with STIM1 overexpression. In addition transgenic mice showed increased calcineurin-nuclear factor of activated T cells (NFAT) activation in vivo, increased CaMKII activity, interstitial fibrosis and exaggerated hypertrophy following two weeks of neuroendocrine agonist or pressure overload stimulation. Conclusion: Our observations suggest that STIM1 overexpression by itself can lead to cardiac hypertrophy and contribute to pathological cardiac remodeling and possibly sudden cardiac death. The molecular mechanisms underlying these phenomena are currently under investigation.

Abstract TMP82: Increased Intracerebral Hemorrhage During Acute and Chronic Hypertension in Alzheimer's Transgenic Mice

Hypertension is a major risk factor for intracerebral hemorrhage (ICH), and the accumulation of amyloid-beta (Aβ) in the cerebrovascular system, cerebral amyloid angiopathy (CAA), is also a significant risk factor for intracerebral hemorrhage ICH. Currently, there are no animal studies demonstrating a direct involvement of hypertension in the accumulation of Alzheimer’s disease-like pathology. To address this issue we have developed several mouse models that combine hypertension protocols with amyloid precursor protein (APP) transgenic mice (Tg2576), which accumulate significant CAA in the large cerebral vessels and the meninges by 18 months of age. The goal of this study was to determine the effect of acute and chronic hypertension on ICH in wildtype and a transgenic mouse model overexpressing a mutant human amyloid precursor protein (Tg2576 mice) associated with early onset AD and CAA. Fifteen-month-old Tg2576 mice and non-transgenic (nTg) littermates were treated with an angiotensin II (AngII) infusion (1000 ng/kg/min) and L-NAME (100 mg/kg/day) in drinking water to produce chronic hypertension. One week later, transient acute hypertension was induced by daily AngII injections (0.5 μg/g, s.c., twice daily) to produce ICH. A similar increase in mean blood pressure was observed in Tg2576 and nTg mice when evaluated 2 weeks after initiation of treatment. However Tg2576 mice had a higher incidence of signs of stroke compared with nTg littermates (P &gt; 0.05). These data suggest that the accumulation of Aβ in the brain has an important role in development of ICH. Moreover, there was robust glial activation and increase in CAA in the gray matter of Tg2576 mice showing that hypertension may affect gray as well as white matter in the brain. Further studies may provide insights into the hypertension-induced changes in the cerebral vascular system that initiated the increase in CAA. The accumulation of Aβ in the cerebrovascular system is a significant risk factor for intracerebral hemorrhage (ICH), and has been linked to endothelial transport failure and blockage of perivascular drainage. While management of hypertension and atherosclerosis can reduce the incidence of ICH, there are currently no approved therapies for attenuating CAA.

Microarray analysis of CA1 pyramidal neurons in a mouse model of tauopathy reveals progressive synaptic dysfunction

The hTau mouse model of tauopathy was utilized to assess gene expression changes in vulnerable hippocampal CA1 neurons. CA1 pyramidal neurons were microaspirated via laser capture microdissection followed by RNA amplification in combination with custom-designed microarray analysis and qPCR validation in hTau mice and nontransgenic (ntg) littermates aged 11–14 months. Statistical analysis revealed ~ 8% of all the genes on the array platform were dysregulated, with notable downregulation of several synaptic-related markers including synaptophysin ( Syp), synaptojanin, and synaptobrevin, among others. Downregulation was also observed for select glutamate receptors (GluRs), Psd-95, TrkB, and several protein phosphatase subunits. In contrast, upregulation of tau isoforms and a calpain subunit were found. Microarray assessment of synaptic-related markers in a separate cohort of hTau mice at 7–8 months of age indicated only a few alterations compared to the 11–14 month cohort, suggesting progressive synaptic dysfunction occurs as tau accumulates in CA1 pyramidal neurons. An assessment of SYP and PSD-95 expression was performed in the hippocampal CA1 sector of hTau and ntg mice via confocal laser scanning microscopy along with hippocampal immunoblot analysis for protein-based validation of selected microarray observations. Results indicate significant decreases in SYP-immunoreactive and PSD-95-immunoreactive puncta as well as downregulation of SYP-immunoreactive and PSD-95-immunoreactive band intensity in hTau mice compared to age-matched ntg littermates. In summary, the high prevalence of downregulation of synaptic-related genes indicates that the moderately aged hTau mouse may be a model of tau-induced synaptodegeneration, and has profound effects on how we perceive progressive tau pathology affecting synaptic transmission in AD.

At the Source: Treating Heart Failure by Altering Muscle Motor Function

### Cardiac Myosin Activation: A Potential Therapeutic Approach for Systolic Heart Failure Malik et al Science . 2011;331:1439–1443. A new study in Science provides proof of the principle for directly modulating the cardiac muscle's motor, myosin, as a therapeutic target in heart failure. The human and economic tolls from heart failure (HF) are significant in both developed and undeveloped countries.1–3 Although HF can take many forms,4,5 it is most commonly associated with decreased cardiac contractility and is, therefore, referred to as systolic HF (sHF).6 Current therapeutic strategies rely on blocking neurohormonal activation by inhibiting associated pathways or by receptor blockade using either β-adrenergic or aldosterone blockers.7,8 Alternatively, some therapies are directed at increasing cardiac contractility in an effort to restore sufficient blood flow, usually by activating second messenger signaling pathways that increase cardiomyocyte calcium levels and augment sarcomere contraction.9 Both strategies suffer from a lack of specificity in that they target proteins upstream of the actual contractile events that underlie force production. Affecting important signaling pathways or receptor-ligand interactions can impact cellular function and perturb homeostasis in ways unrelated to the contractile apparatus, leading to undesirable side effects, such as hypotension, tachycardia, and even arrhythmias that can lead to sudden death.10 Recognizing the significant limitations of current therapeutic modalities, Malik and co-workers have directed their efforts at directly modulating cardiac contractility by tuning motor function at the source, the myosin heavy chain. The cardiac myosin heavy chains are encoded by two genes, MYH6 , which encodes α myosin heavy chain, and MYH7 , which encodes β myosin heavy chain, the latter being the predominant isoform in human ventricle. Although having greater than 90% amino acid homology, the two proteins differ significantly at the biochemical level. In comparison with α myosin, β myosin is relatively more efficient in force production as a function of energy consumption, with …

Mouse HCM Model Expressing E99K ACTC Mutation Reproduces Phenotypes As Found In Human Patients

The mutation Gly99lys (E99K) in the cardiac actin (ACTC) gene was reported to cause Hypertrophic Cardiomyopathy in extensive clinical studies. Transgenic (TG) mice expressing 50% E99K mutant cardiac actin in their hearts were generated and studied. The mice show high mortality between 28 and 45 days old (70% females, 34% males).Thin filaments reconstituted with purified mouse f-actin from the survivors and human heart tropomyosin and troponin were studied by in vitro motility assay. The E99K thin filaments were 2.5 ± 0.6 times more Ca2+ sensitive than NTG thin filaments (p = 0.05). E99K actin thin filaments also exhibited a reduced response to troponin dephosphorylation (EC50 E99K/E99KdP = 1.1 ± 0.1 compared with 3.0 ± 0.3 for NTG /NTGdP).7 month-old E99K TG mice (n=9) and their NTG littermates (n=7) were studied using in vivo cine MRI. Abnormal cardiac morphology and significantly lower ejection fractions (56.5 vs. 65.2%) and reduced stroke volumes (26.0 vs. 42.3 μl) were observed in TG mice. Peak LV ejection rates were also reduced (188 ± 41 vs. 252 ± 49 μl/min). LV mass was similar between groups, but septal wall thickness was increased (1.5 vs. 1.0 mm).Left ventricular function of 9 month-old female E99K NTG (n=4) and TG (n=5) mice were studied with an in vivo conductance catheter. In TG mice ejection fraction was 20.2% less, end-diastolic pressure was 39.6% higher and relaxation rate was 50.0% slower.We conclude that the basic effect of E99K mutation is increased Ca2+-sensitivity and blunted response to troponin dephosphorylation and this leads to the high rate of sudden death at early ages, alterations to cardiac function and hypertrophy as observed in patients with hypertrophic cardiomyopathy.Supported by a grant from the British Heart Foundation