Distribution of cardiomyocyte-selective adeno-associated virus serotype 9 vectors in swine following intracoronary and intravenous infusion.

Limited reports exist regarding adeno-associated virus (AAV) biodistribution in swine. This study assessed biodistribution following antegrade intracoronary and intravenous delivery of two self-complementary serotype 9 AAV (AAV9sc) biologics designed to target signaling in the cardiomyocyte considered important for the development of heart failure. Under the control of a cardiomyocyte-specific promoter, AAV9sc.shmAKAP and AAV9sc.RBD express a small hairpin RNA for the perinuclear scaffold protein muscle A-kinase anchoring protein ß (mAKAPß) and an anchoring disruptor peptide for p90 ribosomal S6 kinase type 3 (RSK3), respectively. Quantitative PCR was used to assess viral genome (vg) delivery and transcript expression in Ossabaw and Yorkshire swine tissues. Myocardial viral delivery was 2-5 × 105 vg/µg genomic DNA (gDNA) for both infusion techniques at a dose ∼1013 vg/kg body wt, demonstrating delivery of ∼1-3 viral particles per cardiac diploid genome. Myocardial RNA levels for each expressed transgene were generally proportional to dose and genomic delivery, and comparable with levels for moderately expressed endogenous genes. Despite significant AAV9sc delivery to other tissues, including the liver, neither biologic induced toxic effects as assessed using functional, structural, and circulating cardiac and systemic markers. These results indicate successful targeted delivery of cardiomyocyte-selective viral vectors in swine without negative side effects, an important step in establishing efficacy in a preclinical experimental setting.

Home Dust Mites Promote MUC5AC Hyper-Expression by Modulating the sNASP/TRAF6 Axis in the Airway Epithelium.

House dust mites (HDMs) are a common source of respiratory allergens responsible for allergic asthma and innate immune responses in human diseases. Since HDMs are critical factors in the triggering of allergen-induced airway mucosa from allergic asthma, we aimed to investigate the mechanisms of Toll-like receptors (TLR) in the signaling of the HDM extract that is involved in mucus hypersecretion and airway inflammation through the engagement of innate immunity. Previously, we reported that the somatic nuclear autoantigenic sperm protein (sNASP)/tumor necrosis factor receptor-associated factor 6 (TRAF6) axis controls the initiation of TLRs to maintain the homeostasis of the innate immune response. The present study showed that the HDM extract stimulated the biogenesis of Mucin 5AC (MUC5AC) in bronchial epithelial cells via the TLR2/4 signaling pathway involving MyD88 and TRAF6. Specifically, sNASP binds to TRAF6 in unstimulated bronchial epithelial cells to prevent the activation of TRAF6-depenedent kinases. Upon on HDMs' stimulation, sNASP is phosphorylated, leading to the activation of TRAF6 downstream of the p38 MAPK and NF-κB signaling pathways. Further, NASP-knockdown enhanced TRAF6 signaling and MUC5AC biogenesis. In the HDM-induced mouse asthma model, we found that the HDM extract promoted airway hyperresponsiveness (AHR), MUC5AC, and allergen-specific IgE production as well as IL-5 and IL-13 for recruiting inflammatory cells. Treatment with the PEP-NASP peptide, a selective TRAF6-blocking peptide, ameliorated HDM-induced asthma in mice. In conclusion, this study indicated that the sNASP/TRAF6 axis plays a regulatory role in asthma by modulating mucus overproduction, and the PEP-NASP peptide might be a potential target for asthma treatment.

Tissue-specific small heat shock protein 20 activation is not associated with traditional autophagy markers in Ossabaw swine with cardiometabolic heart failure.

The small heat shock protein 20 (HSPB6) emerges as a potential upstream mediator of autophagy. Although autophagy is linked to several clinical disorders, how HSPB6 and autophagy are regulated in the setting of heart failure (HF) remains unknown. The goal of this study was to assess the activation of the HSPB6 and its association with other well-established autophagy markers in central and peripheral tissues from a preclinical Ossabaw swine model of cardiometabolic HF induced by Western diet and chronic cardiac pressure overload. We hypothesized HSPB6 would be activated in central and peripheral tissues, stimulating autophagy. We found that autophagy in the heart is interrupted at various stages of the process in a chamber-specific manner. Protein levels of HSPB6, Beclin 1, and p62 are increased in the right ventricle, whereas only HSPB6 was increased in the left ventricle. Unlike the heart, samples from the triceps brachii long head showed only an increase in the protein level of p62, highlighting interesting central versus peripheral differences in autophagy regulation. In the right coronary artery, total HSPB6 protein expression was decreased and associated with an increase in LC3B-II/LC3B-I ratio, demonstrating a different mechanism of autophagy dysregulation in the coronary vasculature. Thus, contrary to our hypothesis, activation of HSPB6 was differentially regulated in a tissue-specific manner and observed in parallel with variable states of autophagy markers assessed by protein levels of LC3B, p62, and Beclin 1. Our data provide insight into how the HSPB6/autophagy axis is regulated in a preclinical swine model with potential relevance to heart failure with preserved ejection fraction.NEW & NOTEWORTHY Our study shows that the activation of HSPB6 is tissue specific and associated with variable states of downstream markers of autophagy in a unique preclinical swine model of cardiometabolic HF with potential relevance to HFpEF. These findings suggest that targeted approaches could be an important consideration regarding the development of drugs aimed at this intracellular recycling process.

Inhibition of Stat3 activation suppresses caspase-3 and the ubiquitin-proteasome system, leading to preservation of muscle mass in cancer cachexia.

Cachexia occurs in patients with advanced cancers. Despite the adverse clinical impact of cancer-induced muscle wasting, pathways causing cachexia are controversial, and clinically reliable therapies are not available. A trigger of muscle protein loss is the Jak/Stat pathway, and indeed, we found that conditioned medium from C26 colon carcinoma (C26) or Lewis lung carcinoma cells activates Stat3 (p-Stat3) in C2C12 myotubes. We identified two proteolytic pathways that are activated in muscle by p-Stat3; one is activation of caspase-3, and the other is p-Stat3 to myostatin, MAFbx/Atrogin-1, and MuRF-1 via CAAT/enhancer-binding protein δ (C/EBPδ). Using sequential deletions of the caspase-3 promoter and CHIP assays, we determined that Stat3 activation increases caspase-3 expression in C2C12 cells. Caspase-3 expression and proteolytic activity were stimulated by p-Stat3 in muscles of tumor-bearing mice. In mice with cachexia caused by Lewis lung carcinoma or C26 tumors, knock-out of p-Stat3 in muscle or with a small chemical inhibitor of p-Stat3 suppressed muscle mass losses, improved protein synthesis and degradation in muscle, and increased body weight and grip strength. Activation of p-Stat3 stimulates a pathway from C/EBPδ to myostatin and expression of MAFbx/Atrogin-1 and increases the ubiquitin-proteasome system. Indeed, C/EBPδ KO decreases the expression of MAFbx/Atrogin-1 and myostatin, while increasing muscle mass and grip strength. In conclusion, cancer stimulates p-Stat3 in muscle, activating protein loss by stimulating caspase-3, myostatin, and the ubiquitin-proteasome system. These results could lead to novel strategies for preventing cancer-induced muscle wasting.

Glucose and fatty acid metabolism in infarcted heart from streptozotocin-induced diabetic rats after 2 weeks of tissue remodeling.

BACKGROUND: The effects of streptozotocin (STZ)-induced diabetes on heart metabolism and function after myocardial infarction (MI) remodelling were investigated in rats. METHODS: Fifteen days after STZ (50 mg/kg b.w. i.v.) injection, MI was induced by surgical occlusion of the left coronary artery. Two weeks after MI induction, contents of glycogen, ATP, free fatty acids and triacylglycerols (TG) and enzyme activities of glycolysis and Krebs cycle (hexokinase, glucose-6-phosphate dehydrogenase, phosphofructokinase, citrate synthase) and expression of carnitine palmitoyl-CoA transferase I (a key enzyme of mitochondrial fatty acid oxidation) were measured in the left ventricle (LV). Plasma glucose, free fatty acids and triacylglycerol levels were determined. Ejection fraction (EF) and shortening fraction (SF) were also measured by echocardiography. RESULTS: Glycogen and TG contents were increased (p < 0.05) whereas ATP content was decreased in the LV of the non-infarcted diabetic group when compared to the control group (p < 0.05). When compared to infarcted control rats (MI), the diabetic infarcted rats (DI) showed (p < 0.05): increased plasma glucose and TG levels, elevated free fatty acid levels and increased activity of, citrate synthase and decreased ATP levels in the LV. Infarct size was smaller in the DI group when compared to MI rats (p < 0.05), and this was associated with higher EF and SF (p < 0.05). CONCLUSIONS: Systolic function was preserved or recovered more efficiently in the heart from diabetic rats two weeks after MI, possibly due to the high provision of glucose and free fatty acids from both plasma and heart glycogen and triacylglycerol stores.

Glucocorticoids increase adipocytes in muscle by affecting IL-4 regulated FAP activity.

An increase in intramuscular adipocyte tissue (IMAT) is associated with glucose dysregulation, decreased muscle strength, and increased risk of disability. Unfortunately, the mechanisms stimulating intramuscular adipogenesis remain unclear. We found that dexamethasone (Dex) administration to mice with injured muscles stimulates the accumulation of IMAT. To identify precursors of these adipocytes, we isolated satellite cells and fibro/adipogenic progenitors (FAPs) from muscle; satellite cells did not differentiate into adipocytes even following Dex treatment. In contrast, Dex stimulated FAP differentiation into adipocytes. In vivo, we transplanted purified FAPs from transgenic, EGFP mice into the injured muscles of C57/BL6 mice and found that Dex administration stimulated adipogenesis from FAP-EGFP. The increase in adipogenesis depended on Dex-induced inhibition of interleukin-4 (IL-4). In the injured muscle of IL-4-knockout mice, the levels of adipocytes were increased, while in the injured muscles of Dex-treated mice with IL-4 injections, adipogenesis was suppressed. In cultured FAPs, IL-4 inhibited Dex-induced conversion of FAPs into adipocytes; this did not occur in FAPs expressing knockdown of the IL-4 receptor. Thus, we concluded that glucocorticoids stimulate FAPs to differentiate into adipocytes in injured muscles. This process is blocked by IL-4, suggesting that interfering with IL-4 signaling could prevent adipogenesis in muscle.

Interactions between p-Akt and Smad3 in injured muscles initiate myogenesis or fibrogenesis.

In catabolic conditions such as aging and diabetes, IGF signaling is impaired and fibrosis develops in skeletal muscles. To examine whether impaired IGF signaling initiates muscle fibrosis, we generated IGF-IR(+/-) heterozygous mice by crossing loxP-floxed IGF-IR (exon 3) mice with MyoD-cre mice. IGF-IR(+/-) mice were studied because we were unable to obtain homozygous IGF-IR-KO mice. In IGF-IR(+/-) mice, both growth and expression of myogenic genes (MyoD and myogenin; markers of satellite cell proliferation and differentiation, respectively) were depressed. Likewise, in injured muscles of IGF-IR(+/-) mice, there was impaired regeneration, depressed expression of MyoD and myogenin, and increased expression of TGF-ß1, α-SMA, collagen I, and fibrosis. To uncover mechanisms stimulating fibrosis, we isolated satellite cells from muscles of IGF-IR(+/-) mice and found reduced proliferation and differentiation plus increased TGF-ß1 production. In C2C12 myoblasts (a model of satellite cells), IGF-I treatment inhibited TGF-ß1-stimulated Smad3 phosphorylation, its nuclear translocation, and expression of fibronectin. Using immunoprecipitation assay, we found an interaction between p-Akt or Akt with Smad3 in wild-type mouse muscles and in C2C12 myoblasts; importantly, IGF-I increased p-Akt and Smad3 interaction, whereas TGF-ß1 decreased it. Therefore, in muscles of IGF-IR(+/-) mice, the reduction in IGF-IR reduces p-Akt, allowing for dissociation and nuclear translocation of Smad3 to enhance the TGF-ß1 signaling pathway, leading to fibrosis. Thus, strategies to improve IGF signaling could prevent fibrosis in catabolic conditions with impaired IGF signaling.

Mechanism-Driven Modeling to Aid Non-invasive Monitoring of Cardiac Function via Ballistocardiography.

Left ventricular (LV) catheterization provides LV pressure-volume (P-V) loops and it represents the gold standard for cardiac function monitoring. This technique, however, is invasive and this limits its applicability in clinical and in-home settings. Ballistocardiography (BCG) is a good candidate for non-invasive cardiac monitoring, as it is based on capturing non-invasively the body motion that results from the blood flowing through the cardiovascular system. This work aims at building a mechanistic connection between changes in the BCG signal, changes in the P-V loops and changes in cardiac function. A mechanism-driven model based on cardiovascular physiology has been used as a virtual laboratory to predict how changes in cardiac function will manifest in the BCG waveform. Specifically, model simulations indicate that a decline in LV contractility results in an increase of the relative timing between the ECG and BCG signal and a decrease in BCG amplitude. The predicted changes have subsequently been observed in measurements on three swine serving as pre-clinical models for pre- and post-myocardial infarction conditions. The reproducibility of BCG measurements has been assessed on repeated, consecutive sessions of data acquisitions on three additional swine. Overall, this study provides experimental evidence supporting the utilization of mechanism-driven mathematical modeling as a guide to interpret changes in the BCG signal on the basis of cardiovascular physiology, thereby advancing the BCG technique as an effective method for non-invasive monitoring of cardiac function.

Resistance exercise shifts the balance of renin-angiotensin system toward ACE2/Ang 1-7 axis and reduces inflammation in the kidney of diabetic rats.

AIMS: We aimed to determine whether resistance training (RT) regulates renal renin-angiotensin system (RAS) components and inflammatory mediators in diabetic rats. MAIN METHODS: Male Wistar rats (3 months old) were randomly assigned into four groups: non-trained (NT), trained (T), non-trained + diabetes (NTD) and trained +diabetes (TD). Diabetes was induced by streptozotocin (50 mg/kg, Sigma Chemical Co., St. Louis, MO, USA), before RT protocol. Trained rats performed RT protocol on a 110-cm ladder (8 ladder climbs, once/day, 5 days/week, 8 weeks), carrying a load corresponding to 50-80% of maximum carrying capacity. Blood glucose, albuminuria and urinary volume were measured. Renal levels of angiotensin peptides (angiotensin I, II and 1-7), inflammatory markers, and also the activities of angiotensin-converting enzyme (ACE) and ACE2 were determined. KEY FINDINGS: Blood glucose and urinary volume were elevated in diabetic animals, and RT decreased albuminuria, renal Ang I and Ang II levels in diabetic rats. RT shifted the balance of renal RAS toward ACE2/Ang 1-7 axis in TD group, and mitigated the high levels of interleukin (IL)-10, IL-1ß and cytokine-induced neutrophil chemoattractant 1 (CINC) in the context of diabetes. Strong positive correlations were found between albuminuria and Ang II, IL-10 and IL-1ß. On the other hand, intrarenal Ang 1-7 levels were negatively correlated with IL-10 and IL-1ß levels. SIGNIFICANCE: RT improved kidney function by modulating intrarenal RAS toward ACE2/Ang 1-7 axis and inflammatory cytokines. RT represents a reasonable strategy to improve the renal complications induced by diabetes, counteracting nephropathy-associated maladaptive responses.

Large Animal Models of Heart Failure: A Translational Bridge to Clinical Success.

Preclinical large animal models of heart failure (HF) play a critical and expanding role in translating basic science findings to the development and clinical approval of novel therapeutics and devices. The complex combination of cardiovascular events and risk factors leading to HF has proved challenging for the development of new treatments for these patients. This state-of-the-art review presents historical and recent studies in porcine, ovine, and canine models of HF and outlines existing methodologies and physiological phenotypes. The translational importance of large animal studies to clinical success is also highlighted with an overview of recent devices approved by the Food and Drug Administration, together with preclinical HF animal studies used to aid both development and safety and/or efficacy testing. Increasing the use of large animal models of HF holds significant potential for identifying the novel mechanisms underlying the clinical condition and to improving physiological and economical translation of animal research to successfully treat human HF.

Short-Term Cigarette Smoking in Rats Impairs Physical Capacity and Induces Cardiac Remodeling.

Despite the strong evidence on the cardiac and renal damages after chronic exposure to cigarette smoke, there is a paucity of data on its short-term effects. The study evaluated the short-term effects of cigarette smoking on left ventricular (LV) remodeling, in vitro myocardial and renal function. Female Wistar rats were randomized to control (C) and cigarette smoking rats for eight weeks. Physical capacity was assessed using an adapted model of exhaustive swim; left ventricle (LV) morphology and function were also evaluated. Renal function was assessed by creatinine clearance and urine protein. The in vitro myocardial performance was analyzed in isolated papillary muscles. Rats exhibited reduced physical capacity after short-term cigarette smoking. Although there was no change on LV function, reduced chamber diameter was found in the smoking group associated with an increased LV wall thickness. There was augmented cardiac mass compared to C that was confirmed by increased cardiomyocyte nucleus volume, but in vitro myocardial performance and renal function were unchanged. A short-term cigarette smoking induces cardiac remodeling without abnormalities in function. The smoking group still preserved renal function and in vitro myocardial performance. However, the reduced physical capacity may suggest an impairment of the cardiac reserve.

Angiotensin II suppresses autophagy and disrupts ultrastructural morphology and function of mitochondria in mouse skeletal muscle.

Angiotensin II (ANG II)-induced skeletal muscle wasting is characterized by activation of the ubiquitin-proteasome system. However, the potential involvement of proteolytic system macroautophagy/autophagy in this wasting process remains elusive. Autophagy is precisely regulated to maintain cell survival and homeostasis; thus its dysregulation (i.e., overactivation or persistent suppression) could lead to detrimental outcomes in skeletal muscle. Here we show that infusion of ANG II for 7 days in male FVB mice suppressed autophagy in skeletal muscle. ANG II blunted microtubule-associated protein 1 light chain 3B (LC3B)-I-to-LC3B-II conversion (an autophagosome marker), increased p62/SQSTM1 (an autophagy cargo receptor) protein expression, and decreased the number of autophagic vacuoles. ANG II inhibited UNC-51-like kinase 1 via inhibition of 5'-AMP-activated kinase and activation of mechanistic target of rapamycin complex 1, leading to reduced phosphorylation of beclin-1Ser14 and Autophagy-related protein 14Ser29, suggesting that ANG II impairs autophagosome formation in skeletal muscle. In line with ANG II-mediated suppression of autophagy, ANG II promoted accumulation of abnormal/damaged mitochondria, characterized by swelling and disorganized cristae and matrix dissolution, with associated increase in PTEN-induced kinase 1 protein expression. ANG II also reduced mitochondrial respiration, indicative of mitochondrial dysfunction. Together, these results demonstrate that ANG II reduces autophagic activity and disrupts mitochondrial ultrastructure and function, likely contributing to skeletal muscle wasting. Therefore, strategies that activate autophagy in skeletal muscle have the potential to prevent or blunt ANG II-induced skeletal muscle wasting in chronic diseases. NEW & NOTEWORTHY Our study identified a novel mechanism whereby angiotensin II (ANG II) impairs mitochondrial energy metabolism in skeletal muscle. ANG II suppressed autophagosome formation by inhibiting the UNC-51-like kinase 1(ULK1)-beclin-1 axis, resulting in accumulation of abnormal/damaged and dysfunctional mitochondria and reduced mitochondrial respiratory capacity. Therapeutic strategies that activate the ULK1-beclin-1 axis have the potential to delay or reverse skeletal muscle wasting in chronic diseases characterized by increased systemic ANG II levels.

Preventive role of exercise training in autonomic, hemodynamic, and metabolic parameters in rats under high risk of metabolic syndrome development.

High fructose consumption contributes to metabolic syndrome incidence, whereas exercise training promotes several beneficial adaptations. In this study, we demonstrated the preventive role of exercise training in the metabolic syndrome derangements in a rat model. Wistar rats receiving fructose overload in drinking water (100 g/l) were concomitantly trained on a treadmill (FT) or kept sedentary (F) for 10 wk. Control rats treated with normal water were also submitted to exercise training (CT) or sedentarism (C). Metabolic evaluations consisted of the Lee index and glycemia and insulin tolerance test (kITT). Blood pressure (BP) was directly measured, whereas heart rate (HR) and BP variabilities were evaluated in time and frequency domains. Renal sympathetic nerve activity was also recorded. F rats presented significant alterations compared with all the other groups in insulin resistance (in mg · dl(-1) · min(-1): F: 3.4 ± 0.2; C: 4.7 ± 0.2; CT: 5.0 ± 0.5 FT: 4.6 ± 0.4), mean BP (in mmHG: F: 117 ± 2; C: 100 ± 2; CT: 98 ± 2; FT: 105 ± 2), and Lee index (in g/mm: F = 0.31 ± 0.001; C = 0.29 ± 0.001; CT = 0.27 ± 0.002; FT = 0.28 ± 0.002), confirming the metabolic syndrome diagnosis. Exercise training blunted all these derangements. Additionally, FS group presented autonomic dysfunction in relation to the others, as seen by an ≈ 50% decrease in baroreflex sensitivity and 24% in HR variability, and increases in sympathovagal balance (140%) and in renal sympathetic nerve activity (45%). These impairments were not observed in FT group, as well as in C and CT. Correlation analysis showed that both Lee index and kITT were associated with vagal impairment caused by fructose. Therefore, exercise training plays a preventive role in both autonomic and hemodynamic alterations related to the excessive fructose consumption.

Exercise attenuates renal dysfunction with preservation of myocardial function in chronic kidney disease.

Previous studies have suggested that exercise improves renal and cardiac functions in patients with chronic kidney disease. The aim of this study was to evaluate the effects of long-term aerobic swimming exercise with overload on renal and cardiac function in rats with 5/6 nefrectomy (5/6Nx). Eight Wistar rats were placed into 4 groups: Control (C), Control+Exercise (E), Sedentary 5/6Nx (NxS) and 5/6Nx+Exercise (NxE). The rats were subjected to swimming exercise sessions with overload for 30 min five days per week for five weeks. Exercise reduced the effect of 5/6Nx on creatinine clearance compared to the NxS group. In addition, exercise minimized the increase in mean proteinuria compared to the NxS group (96.9±10.0 vs. 51.4±9.9 mg/24 h; p<0.05). Blood pressure was higher in the NxS and NxE groups compared to the C and E groups (216±4 and 178±3 vs. 123±2 and 124±2 mm Hg, p<0.05). In the 200 glomeruli that were evaluated, the NxS group had a higher sclerosis index than did the NxE group (16% vs. 2%, p<0.05). Echocardiography demonstrated a higher anterior wall of the left ventricle (LV) in diastole in the NxS group compared with the C, E and NxE groups. The NxS group also had a higher LV posterior wall in diastole and systole compared with the E group. The developed isometric tension in Lmax of the heart papillary muscle was lower in the NxS group compared with the C, E and NxE groups. These results suggested that exercise in 5/6Nx animals might reduce the progression of renal disease and lessen the cardiovascular impact of a reduction in renal mass.