A modified mouse model of Friedreich's ataxia with conditional Fxn allele homozygosity delays onset of cardiomyopathy.

Friedreich's ataxia (FA) is an autosomal recessive disorder caused by a deficiency in frataxin (FXN), a mitochondrial protein that plays a critical role in the synthesis of iron-sulfur clusters (Fe-S), vital inorganic cofactors necessary for numerous cellular processes. FA is characterized by progressive ataxia and hypertrophic cardiomyopathy, with cardiac dysfunction as the most common cause of mortality in patients. Commonly used cardiac-specific mouse models of FA use the muscle creatine kinase (MCK) promoter to express Cre recombinase in cardiomyocytes and striated muscle cells in mice with one conditional Fxn allele and one floxed-out/null allele. These mice quickly develop cardiomyopathy that becomes fatal by 9-11 wk of age. Here, we generated a cardiac-specific model with floxed Fxn allele homozygosity (MCK-Fxnflox/flox). MCK-Fxnflox/flox mice were phenotypically normal at 9 wk of age, despite no detectable FXN protein expression. Between 13 and 15 wk of age, these mice began to display progressive cardiomyopathy, including decreased ejection fraction and fractional shortening and increased left ventricular mass. MCK-Fxnflox/flox mice began to lose weight around 16 wk of age, characteristically associated with heart failure in other cardiac-specific FA models. By 18 wk of age, MCK-Fxnflox/flox mice displayed elevated markers of Fe-S deficiency, cardiac stress and injury, and cardiac fibrosis. This modified model reproduced important pathophysiological and biochemical features of FA over a longer timescale than previous cardiac-specific mouse models, offering a larger window for studying potential therapeutics.NEW & NOTEWORTHY Previous cardiac-specific frataxin knockout models exhibit rapid and fatal cardiomyopathy by 9 wk of age. This severe phenotype poses challenges for the design and execution of intervention studies. We introduce an alternative cardiac-specific model, MCK-Fxnflox/flox, with increased longevity and delayed onset of all major phenotypes. These phenotypes develop to the same severity as previous models. Thus, this new model provides the same cardiomyopathy-associated mortality with a larger window for potential studies.

Fully automated mouse echocardiography analysis using deep convolutional neural networks.

Echocardiography (echo) is a translationally relevant ultrasound imaging modality widely used to assess cardiac structure and function in preclinical models of heart failure (HF) during research and drug development. Although echo is a very valuable tool, the image analysis is a time-consuming, resource-demanding process, and is susceptible to interreader variability. Recent advancements in deep learning have enabled researchers to automate image processing and reduce analysis time and interreader variability in the field of medical imaging. In the present study, we developed a fully automated tool, mouse-echocardiography neural net (MENN), for the analysis of both long-axis brightness (B)-mode and short-axis motion (M)-mode images of left ventricle. MENN is a series of fully convolutional neural networks that were trained and validated using manually segmented B-mode and M-mode echo images of the left ventricle. The segmented images were then used to compute cardiac structural and functional metrics. The performance of MENN was further validated in two preclinical models of HF. MENN achieved excellent correlations (Pearson's r = 0.85-0.99) and good-to-excellent agreement between automated and manual analyses. Further interreader variability analysis showed that MENN has better agreements with an expert analyst than both a trained analyst and a novice. Notably, the use of MENN reduced manual analysis time by >92%. In conclusion, we developed an automated echocardiography analysis tool that allows for fast and accurate analysis of B-mode and M-mode mouse echo data and mitigates the issue of interreader variability in manual analysis.NEW & NOTEWORTHY Echocardiography is commonly used in preclinical research to evaluate cardiac structure and function. Despite the broad applications across therapeutic areas, the analysis of echo data is laborious and susceptible to interreader variability. In this study, we developed a fully automated mouse-echocardiography neural net (MENN). Cardiac measurements from MENN showed excellent correlations with manual analysis. Furthermore, the use of MENN leads to >92% reduction in analysis time and potentially eliminates the interobserver variability issue.

An assessment of thermoneutral housing conditions on murine cardiometabolic function.

Mouse models are used to model human diseases and perform pharmacological efficacy testing to advance therapies to humans; most of these studies are conducted in room temperature conditions. At room temperature (22°C), mice are cold-stressed and must use brown adipose tissue (BAT) to maintain body temperature. This cold stress increases catecholamine tone to maintain adipocyte lipid release via lipolysis, which will fuel adaptive thermogenesis. Maintaining rodents at thermoneutral temperatures (28°C) ameliorates the need for adaptive thermogenesis, thus reducing catecholamine tone and BAT activity. Cardiovascular tone is also determined by catecholamine levels in rodents, as ß-adrenergic stimuli are primary drivers of not only lipolytic but also ionotropic and chronotropic responses. As mice have increased catecholamine tone at room temperature, we investigated how thermoneutral housing conditions would impact cardiometabolic function. Here, we show a rapid and reversible effect of thermoneutrality on both heart rate and blood pressure in chow-fed animals, which was blunted in animals fed a high-fat diet. Animals subjected to transverse aortic constriction displayed compensated hypertrophy at room temperature, whereas animals displayed less hypertrophy and a trend toward worse systolic function at thermoneutrality. Despite these dramatic changes in blood pressure and heart rate at thermoneutral housing conditions, enalapril effectively improved cardiac hypertrophy and gene expression alterations. There were surprisingly few differences in cardiac parameters in high-fat-fed animals at thermoneutrality. Overall, these data suggest that thermoneutral housing may alter some aspects of cardiac remodeling in preclinical mouse models of heart failure.NEW & NOTEWORTHY Thermoneutral housing conditions cause rapid and reversible changes in mouse heart rate and blood pressure. Despite dramatic reductions in heart rate and blood pressure, thermoneutrality reduced the compensatory hypertrophic response in a pressure overload heart failure model compared with room temperature housing, and ACE inhibitors were still efficacious to prevent pressure overload-induced cardiac remodeling. The effects of thermoneutrality on heart rate and blood pressure are abrogated in the context of diet-induced obesity.

Loss of Roundabout Guidance Receptor 2 (Robo2) in Podocytes Protects Adult Mice from Glomerular Injury by Maintaining Podocyte Foot Process Structure.

Roundabout guidance receptor 2 (ROBO2) plays an important role during early kidney development. ROBO2 is expressed in podocytes, inhibits nephrin-induced actin polymerization, down-regulates nonmuscle myosin IIA activity, and destabilizes kidney podocyte adhesion. However, the role of ROBO2 during kidney injury, particularly in mature podocytes, is not known. Herein, we report that loss of ROBO2 in podocytes [Robo2 conditional knockout (cKO) mouse] is protective from glomerular injuries. Ultrastructural analysis reveals that Robo2 cKO mice display less foot process effacement and better-preserved slit-diaphragm density compared with wild-type littermates injured by either protamine sulfate or nephrotoxic serum (NTS). The Robo2 cKO mice also develop less proteinuria after NTS injury. Further studies reveal that ROBO2 expression in podocytes is up-regulated after glomerular injury because its expression levels are higher in the glomeruli of NTS injured mice and passive Heymann membranous nephropathy rats. Moreover, the amount of ROBO2 in the glomeruli is also elevated in patients with membranous nephropathy. Finally, overexpression of ROBO2 in cultured mouse podocytes compromises cell adhesion. Taken together, these findings suggest that kidney injury increases glomerular ROBO2 expression that might compromise podocyte adhesion and, thus, loss of Robo2 in podocytes could protect from glomerular injury by enhancing podocyte adhesion that helps maintain foot process structure. Our findings also suggest that ROBO2 is a therapeutic target for podocyte injury and podocytopathy.

GPAT1 Deficiency in Mice Modulates NASH Progression in a Model-Dependent Manner.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD), and its more severe form, nonalcoholic steatohepatitis (NASH), is the leading cause for liver failure and liver cancer. Although the etiology is likely multifactorial, genes involved in regulating lipid metabolism are enriched in human NAFLD genome-wide association studies (GWAS), pointing to dysregulated lipid metabolism as a major pathogenic factor. Glycerol-3-phosphate acyltransferase 1 (GPAT1), encoded by GPAM, converts acyl-CoAs and glycerol-3-phosphate into lysophosphatidic acid and has been shown to regulate lipid accumulation in the liver. However, its role in mediating the progression from NAFLD to NASH has not been explored. METHODS: GPAT1-deficient mice were generated and challenged with diets inducing hepatic steatosis and NASH. Effects of GPAT1 deficiency on lipid and systemic metabolic end points were evaluated. RESULTS: Ablating GPAT1 globally or specifically in mouse hepatocytes reduced hepatic steatosis in the context of diet-induced or genetic obesity. Interestingly, blunting of progression from NAFLD to NASH in global GPAT1 knockout (KO) mice was model dependent. GPAT1 KO mice were protected from choline deficient, amino acid defined high-fat diet-induced NASH development, but not from the high fat, high carbohydrate, and high cholesterol diet-induced NASH. CONCLUSIONS: Our preclinical data support the notion that lipid metabolism pathways regulated by GPAT1 in hepatocytes play an essential role in NASH progression, albeit in a model-dependent manner.

Increased circulating progranulin is not sufficient to induce cardiac dysfunction or supraventricular arrhythmia.

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia, and the incidence of new-onset AF has been increasing over the past two decades. Several factors contribute to the risk of developing AF including age, preexisting cardiovascular disease, chronic kidney disease, and obesity. Concurrent with the rise in AF, obesity has followed the same two-decade trend. The contribution of circulating proteins to obesity-related AF is of particular interest in the field. In this study, we investigated the effects of increased circulating levels of the glycoprotein progranulin on the development of supraventricular arrhythmias and changes to cardiac function. AAV8-mediated overexpression of full-length mouse progranulin was used to increase plasma protein levels and determine susceptibility to supraventricular arrhythmias and changes in cardiac structure and function. C57Bl/6N mice were subjected to increased circulating levels of progranulin for 20 weeks. Cardiac conduction was evaluated by surface ECG with and without isoproterenol challenge, and cardiac structure and function were measured by echocardiography after 20 weeks of circulating progranulin overexpression. Increased circulating levels of progranulin were maintained throughout the 20-week study. The cardiac structure and function remained unchanged in mice with increased circulating progranulin. ECG indices (P wave duration, P amplitude, QRS interval) were unaffected by increased progranulin levels and no arrhythmogenic events were observed following the isoproterenol challenge. In our model, increased levels of circulating progranulin were not sufficient to induce changes in cardiac structure and function or elicit ECG abnormalities suggestive of susceptibility to supraventricular arrhythmias.

Neutralization of GDF15 Prevents Anorexia and Weight Loss in the Monocrotaline-Induced Cardiac Cachexia Rat Model.

Growth and differentiation factor 15 (GDF15) is a cytokine reported to cause anorexia and weight loss in animal models. Neutralization of GDF15 was efficacious in mitigating cachexia and improving survival in cachectic tumor models. Interestingly, elevated circulating GDF15 was reported in patients with pulmonary arterial hypertension and heart failure, but it is unclear whether GDF15 contributes to cachexia in these disease conditions. In this study, rats treated with monocrotaline (MCT) manifested a progressive decrease in body weight, food intake, and lean and fat mass concomitant with elevated circulating GDF15, as well as development of right-ventricular dysfunction. Cotreatment of GDF15 antibody mAb2 with MCT prevented MCT-induced anorexia and weight loss, as well as preserved lean and fat mass. These results indicate that elevated GDF15 by MCT is causal to anorexia and weight loss. GDF15 mAb2 is efficacious in mitigating MCT-induced cachexia in vivo. Furthermore, the results suggest GDF15 inhibition is a potential therapeutic approach to alleviate cardiac cachexia in patients.

In vivo overexpression of frataxin causes toxicity mediated by iron-sulfur cluster deficiency.

Friedreich's ataxia is a rare disorder resulting from deficiency of frataxin, a mitochondrial protein implicated in the synthesis of iron-sulfur clusters. Preclinical studies in mice have shown that gene therapy is a promising approach to treat individuals with Friedreich's ataxia. However, a recent report provided evidence that AAVrh10-mediated overexpression of frataxin could lead to cardiotoxicity associated with mitochondrial dysfunction. While evaluating an AAV9-based frataxin gene therapy using a chicken ß-actin promoter, we showed that toxic overexpression of frataxin could be reached in mouse liver and heart with doses between 1 × 1013 and 1 × 1014 vg/kg. In a mouse model of cardiac disease, these doses only corrected cardiac dysfunction partially and transiently and led to adverse findings associated with iron-sulfur cluster deficiency in liver. We demonstrated that toxicity required frataxin's primary function by using a frataxin construct bearing the N146K mutation, which impairs binding to the iron-sulfur cluster core complex. At the lowest tested dose, we observed moderate liver toxicity that was accompanied by progressive loss of transgene expression and liver regeneration. Together, our data provide insights into the toxicity of frataxin overexpression that should be considered in the development of a gene therapy approach for Friedreich's ataxia.

Application of Ultrasound and Shear Wave Elastography Imaging in a Rat Model of NAFLD/NASH.

Nonalcoholic Steatohepatitis (NASH) is a condition within the spectrum of Non-Alcoholic Fatty Liver Disease (NAFLD), which is characterized by liver fat accumulation (steatosis) and inflammation leading to fibrosis. Preclinical models closely recapitulating human NASH/NAFLD are essential in drug development. While liver biopsy is currently the gold standard for measuring NAFLD/NASH progression and diagnosis in the clinic, in the preclinical space, either collection of whole liver samples at multiple timepoints during a study or biopsy of liver is needed for histological analysis to assess the disease stage. Conducting a liver biopsy mid-study is an invasive and labor-intensive procedure, and collecting liver samples to assess disease level increases the number of research animals needed for a study. Thus, there is a need for a reliable, translatable, non-invasive imaging biomarker to detect NASH/NAFLD in these preclinical models. Non-invasive ultrasound-based B-mode images and Shear Wave Elastography (SWE) can be used to measure steatosis as well as liver fibrosis. To assess the utility of SWE in preclinical rodent models of NASH, animals were placed on a pro-NASH diet and underwent non-invasive ultrasound B-mode and shear wave elastography imaging to measure hepatorenal (HR) index and liver elasticity, measuring progression of both liver fat accumulation and tissue stiffness, respectively, at multiple time points over the course of a given NAFLD/NASH study. The HR index and elasticity numbers were compared to histological markers of steatosis and fibrosis. The results showed strong correlation between the HR index and percentage of Oil Red O (ORO) staining, as well as between elasticity and Picro-Sirius Red (PSR) staining of livers. The strong correlation between classic ex vivo methods and in vivo imaging results provides evidence that shear wave elastography/ultrasound-based imaging can be used to assess disease phenotype and progression in a preclinical model of NAFLD/NASH.

Acetyl-CoA Carboxylase Inhibition Improves Multiple Dimensions of NASH Pathogenesis in Model Systems.

BACKGROUND & AIMS: Disordered metabolism, steatosis, hepatic inflammation, and fibrosis contribute to the pathogenesis of nonalcoholic steatohepatitis (NASH). Acetyl-CoA carboxylase (ACC) catalyzes the first committed step in de novo lipogenesis (DNL) and modulates mitochondrial fatty acid oxidation. Increased hepatic DNL flux and reduced fatty acid oxidation are hypothesized to contribute to steatosis. Some proinflammatory cells also show increased dependency on DNL, suggesting that ACC may regulate aspects of the inflammatory response in NASH. PF-05221304 is an orally bioavailable, liver-directed ACC1/2 inhibitor. The present studies sought to evaluate the effects of PF-05221304 on NASH pathogenic factors in experimental model systems. METHODS: The effects of PF-05221304 on lipid metabolism, steatosis, inflammation, and fibrogenesis were investigated in both primary human-derived in vitro systems and in vivo rodent models. RESULTS: PF-05221304 inhibited DNL, stimulated fatty acid oxidation, and reduced triglyceride accumulation in primary human hepatocytes, and reduced DNL and steatosis in Western diet-fed rats in vivo, showing the potential to reduce hepatic lipid accumulation and potentially lipotoxicity. PF-05221304 blocked polarization of human T cells to proinflammatory but not anti-inflammatory T cells, and suppressed activation of primary human stellate cells to myofibroblasts in vitro, showing direct effects on inflammation and fibrogenesis. Consistent with these observations, PF-05221304 also reduced markers of inflammation and fibrosis in the diethylnitrosamine chemical-induced liver injury model and the choline-deficient, high-fat-fed rat model. CONCLUSIONS: The liver-directed dual ACC1/ACC2 inhibitor directly improved multiple nonalcoholic fatty liver disease/NASH pathogenic factors including steatosis, inflammation, and fibrosis in both human-derived in vitro systems and rat models.

AKAP150 participates in calcineurin/NFAT activation during the down-regulation of voltage-gated K(+) currents in ventricular myocytes following myocardial infarction.

The Ca(2+)-responsive phosphatase calcineurin/protein phosphatase 2B dephosphorylates the transcription factor NFATc3. In the myocardium activation of NFATc3 down-regulates the expression of voltage-gated K(+) (Kv) channels after myocardial infarction (MI). This prolongs action potential duration and increases the probability of arrhythmias. Although recent studies infer that calcineurin is activated by local and transient Ca(2+) signals the molecular mechanism that underlies the process is unclear in ventricular myocytes. Here we test the hypothesis that sequestering of calcineurin to the sarcolemma of ventricular myocytes by the anchoring protein AKAP150 is required for acute activation of NFATc3 and the concomitant down-regulation of Kv channels following MI. Biochemical and cell based measurements resolve that approximately 0.2% of the total calcineurin activity in cardiomyocytes is associated with AKAP150. Electrophysiological analyses establish that formation of this AKAP150-calcineurin signaling dyad is essential for the activation of the phosphatase and the subsequent down-regulation of Kv channel currents following MI. Thus AKAP150-mediated targeting of calcineurin to sarcolemmal micro-domains in ventricular myocytes contributes to the local and acute gene remodeling events that lead to the down-regulation of Kv currents.