Cardioprotective Effects of 1-(3,6-Dibromo-carbazol-9-yl)-3-Phenylamino-Propan-2-Ol in Diabetic Hearts via Nicotinamide Phosphoribosyltransferase Activation.

Diabetes is associated with increased cardiac injury and sudden death. Nicotinamide phosphoribosyltransferase (Nampt) is an essential enzyme for the NAD+ salvage pathway and is dysregulated in diabetes. Nampt activation results in rescued NADH/NAD+ ratios and provides pharmacological changes necessary for diabetic cardioprotection. Computer docking shows that 1-(3,6-Dibromo-carbazol-9-yl)-3-phenylamino-propan-2-ol (P7C3) allows for enhanced Nampt dimerization and association. To test the pharmacological application, we used male leptin receptor-deficient (db/db) mice and treated them with Nampt activator P7C3. The effects of 4-week P7C3 treatment on cardiac function were evaluated along with molecular signaling changes for phosphorylated protein kinase B (p-AKT), phosphorylated endothelial nitric oxide synthase (p-eNOS), and sirtuin 1 (SIRT1). The cardiac function evaluated by ECG and echocardiography were significantly improved after 4 weeks of P7C3 treatment. Biochemically, higher NADH/NAD+ ratios in diabetic hearts were rescued by P7C3 treatment. Moreover, activities of Nampt and SIRT1 were significantly increased in P7C3-treated diabetic hearts. P7C3 treatment significantly decreased the blood glucose in diabetic mice with 4-week treatment as noted by glucose tolerance test and fasting blood glucose measurements compared with vehicle-treated mice. P7C3 activated Nampt enzymatic activity both in vitro and in the 4-week diabetic mouse hearts, demonstrating the specificity of the small molecule. P7C3 treatment significantly enhanced the expression of cardioprotective signaling of p-AKT, p-eNOS, and Beclin 1 in diabetic hearts. Nampt activator P7C3 allows for decreased infarct size with decreased Troponin I and lactose dehydrogenase (LDH) release, which is beneficial to the heart. Overall, the present study shows that P7C3 activates Nampt and SIRT1 activity and decreases NADH/NAD+ ratio, resulting in improved biochemical signaling providing cardioprotection. SIGNIFICANCE STATEMENT: This study shows that 1-(3,6-Dibromo-carbazol-9-yl)-3-phenylamino-propan-2-ol (P7C3) is effective in treating diabetes and cardiovascular diseases. The novel small molecule is antiarrhythmic and improves the ejection fraction in diabetic hearts. The study successfully demonstrated that P7C3 decreases the infarct size in hearts during myocardial infarction and ischemia-reperfusion injury. Biochemical and cellular signaling show increased NAD+ levels, along with Nampt activity involved in upregulating protective signaling in the diabetic heart. P7C3 has high therapeutic potential for rescuing heart disease.

Nampt activator P7C3 ameliorates diabetes and improves skeletal muscle function modulating cell metabolism and lipid mediators.

BACKGROUND: Nicotinamide phosphoribosyltransferase (Nampt), a key enzyme in NAD salvage pathway is decreased in metabolic diseases, and its precise role in skeletal muscle function is not known. We tested the hypothesis, Nampt activation by P7C3 (3,6-dibromo-α-[(phenylamino)methyl]-9H-carbazol-9-ethanol) ameliorates diabetes and muscle function. METHODS: We assessed the functional, morphometric, biochemical, and molecular effects of P7C3 treatment in skeletal muscle of type 2 diabetic (db/db) mice. Nampt+/- mice were utilized to test the specificity of P7C3. RESULTS: Insulin resistance increased 1.6-fold in diabetic mice compared with wild-type mice and after 4 weeks treatment with P7C3 rescued diabetes (P < 0.05). In the db-P7C3 mice fasting blood glucose levels decreased to 0.96-fold compared with C57Bl/6J wild-type naïve control mice. The insulin and glucose tolerance tests blood glucose levels were decreased to 0.6-fold and 0.54-folds, respectively, at 120 min along with an increase in insulin secretion (1.76-fold) and pancreatic ß-cells (3.92-fold) in db-P7C3 mice. The fore-limb and hind-limb grip strengths were increased to 1.13-fold and 1.17-fold, respectively, together with a 14.2-fold increase in voluntary running wheel distance in db-P7C3 mice. P7C3 treatment resulted in a 1.4-fold and 7.1-fold increase in medium-sized and larger-sized myofibres cross-sectional area, with a concomitant 0.5-fold decrease in smaller-sized myofibres of tibialis anterior (TA) muscle. The transmission electron microscopy images also displayed a 1.67-fold increase in myofibre diameter of extensor digitorum longus muscle along with 2.9-fold decrease in mitochondrial area in db-P7C3 mice compared with db-Veh mice. The number of SDH positive myofibres were increased to 1.74-fold in db-P7C3 TA muscles. The gastrocnemius and TA muscles displayed a decrease in slow oxidative myosin heavy chain type1 (MyHC1) myofibres expression (0.46-fold) and immunostaining (6.4-fold), respectively. qPCR analysis displayed a 2.9-fold and 1.3-fold increase in Pdk4 and Cpt1, and 0.55-fold and 0.59-fold decrease in Fgf21 and 16S in db-P7C3 mice. There was also a 3.3-fold and 1.9-fold increase in Fabp1 and CD36 in db-Veh mice. RNA-seq differential gene expression volcano plot displayed 1415 genes to be up-regulated and 1726 genes down-regulated (P < 0.05) in db-P7C3 mice. There was 1.02-fold increase in serum HDL, and 0.9-fold decrease in low-density lipoprotein/very low-density lipoprotein ratio in db-P7C3 mice. Lipid profiling of gastrocnemius muscle displayed a decrease in inflammatory lipid mediators n-6; AA (0.83-fold), and n-3; DHA (0.69-fold) and EPA (0.81-fold), and a 0.66-fold decrease in endocannabinoid 2-AG and 2.0-fold increase in AEA in db-P7C3 mice. CONCLUSIONS: Overall, we demonstrate that P7C3 activates Nampt, improves type 2 diabetes and skeletal muscle function in db/db mice.

Metabolic regulation of Kv channels and cardiac repolarization by Kvß2 subunits.

Voltage-gated potassium (Kv) channels control myocardial repolarization. Pore-forming Kvα proteins associate with intracellular Kvß subunits, which bind pyridine nucleotides with high affinity and differentially regulate channel trafficking, plasmalemmal localization and gating properties. Nevertheless, it is unclear how Kvß subunits regulate myocardial K+ currents and repolarization. Here, we tested the hypothesis that Kvß2 subunits regulate the expression of myocardial Kv channels and confer redox sensitivity to Kv current and cardiac repolarization. Co-immunoprecipitation and in situ proximity ligation showed that in cardiac myocytes, Kvß2 interacts with Kv1.4, Kv1.5, Kv4.2, and Kv4.3. Cardiac myocytes from mice lacking Kcnab2 (Kvß2-/-) had smaller cross sectional areas, reduced sarcolemmal abundance of Kvα binding partners, reduced Ito, IK,slow1, and IK,slow2 densities, and prolonged action potential duration compared with myocytes from wild type mice. These differences in Kvß2-/- mice were associated with greater P wave duration and QT interval in electrocardiograms, and lower ejection fraction, fractional shortening, and left ventricular mass in echocardiographic and morphological assessments. Direct intracellular dialysis with a high NAD(P)H:NAD(P)+ accelerated Kv inactivation in wild type, but not Kvß2-/- myocytes. Furthermore, elevated extracellular levels of lactate increased [NADH]i and prolonged action potential duration in wild type cardiac myocytes and perfused wild type, but not Kvß2-/-, hearts. Taken together, these results suggest that Kvß2 regulates myocardial electrical activity by supporting the functional expression of proteins that generate Ito and IK,slow, and imparting redox and metabolic sensitivity to Kv channels, thereby coupling cardiac repolarization to myocyte metabolism.

Nanodrug delivery platform for glucocorticoid use in skeletal muscle injury.

Glucocorticoids are utilized for their anti-inflammatory properties in the skeletal muscle and arthritis. However, the major drawback of use of glucocorticoids is that it leads to senescence and toxicity. Therefore, based on the idea that decreasing particle size allows for increased surface area and bioavailability of the drug, in the present study, we hypothesized that nanodelivery of dexamethasone will offer increased efficacy and decreased toxicity. The dexamethasone-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles were prepared using nanoprecipitation method. The morphological characteristics of the nanoparticles were studied under scanning electron microscope. The particle size of nanoparticles was 217.5 ± 19.99 nm with polydispersity index of 0.14 ± 0.07. The nanoparticles encapsulation efficiency was 34.57% ± 1.99% with in vitro drug release profile exhibiting a sustained release pattern over 10 days. We identified improved skeletal muscle myoblast performance with improved closure of the wound along with increased cell viability at 10 nmol/L nano-dexamethasone-PLGA. However, dexamethasone solution (1 µmol/L) was injurious to cells because the migration efficiency was decreased. In addition, the use of dexamethasone nanoparticles decreased lipopolysaccharide-induced lactate dehydrogenase release compared with dexamethasone solution. Taken together, the present study clearly demonstrates that delivery of PLGA-dexamethasone nanoparticles to the skeletal muscle cells is beneficial for treating inflammation and skeletal muscle function.

Pleiotropic effects of metformin to rescue statin-induced muscle injury and insulin resistance: A proposed mechanism and potential clinical implications.

The 2013 American Heart Association Blood Cholesterol Guidelines increased the number of patients recommended for statin therapy in the United States to 56million. Two common statin side effects are muscle pain, referred to as "statin-associated muscle symptoms", and increased risk for new onset type-2-diabetes mellitus. Up to 25% of statin users report muscle symptoms resulting in many patients being switched to lower dose or lower potency statins, or refusing statins altogether. The most likely signaling mechanisms for statin-associated muscle symptoms overlaps with the proposed mechanism of statin-induced insulin resistance. Metformin has outstanding utility in reducing insulin resistance and preventing type-2-diabetes mellitus, but has not been studied for statin-associated muscle symptom rescue or prevention. The overlapping mechanisms of statin-associated muscle symptoms, statin-induced insulin resistance, and metformin intervention offers the potential to address two common and detrimental side effects of statins. As statins are the single best medication class for preventing cardiovascular events the potential for clinical benefit is large given metabolic syndrome's growing prevalence in the United States. Herein we hypothesize that metformin will rescue and prevent patients from statin-associated muscle symptoms. This hypothesis can benefit two patient groups: 1) patients at risk for diabetes who are taking a statin and experiencing muscle symptoms; and 2) patients with diabetes taking metformin who are to be started on a statin. Method to test Group 1) Symptom Rescue: randomized control trial of metformin versus placebo in patients with prediabetes who are already taking a statin, and are experiencing mild-to-moderate muscle symptoms. Method to test Group 2) Symptom Prevention: meta-analysis, of statin randomized control trials, with patient level data, comparing patients taking metformin at baseline to patients not taking metformin when a statin is started. An efficient method to simulate both symptom rescue and symptom prevention is a skeletal muscle cell culture model of statin-associated muscle symptom markers. These experiments would identify if metformin reverses (rescues) or prevents markers of statin-associated muscle symptoms. As metformin is recommended by the American Diabetes Association for type-2-diabetes mellitus prevention, yet not frequently used, validating this hypothesis will lead towards research and practice change including: a) decreases in the frequency of statin-associated muscle symptoms; leading to subsequent increases in statin therapy compliance; b) increases in metformin use in prediabetes with subsequent decrease in the incidence of type-2-diabetes mellitus; and c) decreases in complications of both cardiovascular disease and diabetes due to improved statin compliance and type-2-diabetes mellitus prevention.

Analysis of Longissimus thoracis Protein Expression Associated with Variation in Carcass Quality Grade and Marbling of Beef Cattle Raised in the Pacific Northwestern United States.

Longissimus thoracis (LD) samples from 500 cattle were screened for protein expression differences relative to carcass quality grade. The LD of the top 5% (low prime and high choice, HQ) and bottom 5% (low select, LQ) carcasses were analyzed using two-dimensional difference gel electrophoresis and Western blot. Following initial screening, 11 candidate proteins were selected for Western blot analyses. Differentially expressed proteins were clustered into four groups: (1) heat shock proteins and oxidative protection, (2) sarcomeric proteins (muscle maturity and fiber type), (3) metabolism and energetics, and (4) miscellaneous proteins. Proteins from groups 1 and 2 were greater in HQ carcasses. Alternatively, increased quantities of proteins from group 3 were observed in LQ carcasses. Proteomic differences provide insights into pathways contributing to carcass quality grade. A deeper understanding of the physiological pathways involved in carcass quality grade development may allow producers to employ production practices that improve quality grade.

Kvß1.1 (AKR6A8) senses pyridine nucleotide changes in the mouse heart and modulates cardiac electrical activity.

The present study investigates the physiological role of Kvß1 subunit for sensing pyridine nucleotide (NADH/NAD+) changes in the heart. We used Kvß1.1 knockout (KO) or wild-type (WT) mice and established that Kvß1.1 preferentially binds with Kv4.2 and senses the pyridine nucleotide changes in the heart. The cellular action potential duration (APD) obtained from WT cardiomyocytes showed longer APDs with lactate perfusion, which increases intracellular NADH levels, while the APDs remained unaltered in the Kvß1.1 KO. Ex vivo monophasic action potentials showed a similar response, in which the APDs were prolonged in WT mouse hearts with lactate perfusion; however, the Kvß1.1 KO mouse hearts did not show APD changes upon lactate perfusion. COS-7 cells coexpressing Kv4.2 and Kvß1.1 were used for whole cell patch-clamp recordings to evaluate changes caused by NADH (lactate). These data reveal that Kvß1.1 is required in the mediated inactivation of Kv4.2 currents, when NADH (lactate) levels are increased. In vivo, isoproterenol infusion led to increased NADH in the heart along with QTc prolongation in wild-type mice; regardless of the approach, our data show that Kvß1.1 recognizes NADH changes and modulates Kv4.2 currents affecting AP and QTc durations. Overall, this study uses multiple levels of investigation, including the heterologous overexpression system, cardiomyocyte, ex vivo, and ECG, and clearly depicts that Kvß1.1 is an obligatory sensor of NADH/NAD changes in vivo, with a physiological role in the heart.NEW & NOTEWORTHY Cardiac electrical activity is mediated by ion channels, and Kv4.2 plays a significant role, along with its binding partner, the Kvß1.1 subunit. In the present study, we identify Kvß1.1 as a sensor of pyridine nucleotide changes and as a modulator of Kv4.2 gating, action potential duration, and ECG in the mouse heart.

Deletion of Kvß1.1 subunit leads to electrical and haemodynamic changes causing cardiac hypertrophy in female murine hearts.

NEW FINDINGS: What is the central question of this study? The goal of this study was to evaluate sex differences and the role of the potassium channel ß1 (Kvß1) subunit in the heart. What is the main finding and its importance? Genetic ablation of Kvß1.1 in females led to cardiac hypertrophy characterized by increased heart size, prolonged monophasic action potentials, elevated blood pressure and increased myosin heavy chain α (MHCα) expression. In contrast, male mice showed only electrical changes. Kvß1.1 binds the MHCα isoform at the protein level, and small interfering RNA targeted knockdown of Kvß1.1 upregulated MHCα. Cardiovascular disease is the leading cause of death and debility in women in the USA, and cardiac arrhythmias are a major concern. Voltage-gated potassium (Kv) channels along with the binding partners; Kvß subunits are major regulators of the action potential (AP) shape and duration (APD). The regulation of Kv channels by the Kvß1 subunit is unknown in female hearts. In the present study, we hypothesized that the Kvß1 subunit is an important regulator of female cardiac physiology. To test this hypothesis, we ablated (knocked out; KO) the KCNAB1 isoform 1 (Kvß1.1) subunit in mice and evaluated cardiac function and electrical activity by using ECG, monophasic action potential recordings and echocardiography. Our results showed that the female Kvß1.1 KO mice developed cardiac hypertrophy, and the hearts were structurally different, with enlargement and increased area. The electrical derangements caused by Kvß1.1 KO in female mice included long QTc and QRS intervals along with increased APD (APD20-90% repolarization). The male Kvß1.1 KO mice did not develop cardiac hypertrophy, but they showed long QTc and prolonged APD. Molecular analysis showed that several genes that support cardiac hypertrophy were significantly altered in Kvß1.1 KO female hearts. In particular, myosin heavy chain α expression was significantly elevated in Kvß1.1 KO mouse heart. Using a small interfering RNA strategy, we identified that knockdown of Kvß1 increases myosin heavy chain α expression in H9C2 cells. Collectively, changes in molecular and cell signalling pathways clearly point towards a distinct electrical and structural remodelling consistent with cardiac hypertrophy in the Kvß1.1 KO female mice.

Smad7 interrupts TGF-ß signaling in intestinal macrophages and promotes inflammatory activation of these cells during necrotizing enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants. Based on our recent findings of increased Smad7 expression in surgically resected bowel affected by NEC, we hypothesized that NEC macrophages undergo inflammatory activation because increased Smad7 expression renders these cells resistant to normal, gut-specific, transforming growth factor (TGF)-ß-mediated suppression of inflammatory pathways. METHODS: We used surgically resected human NEC tissue, murine models of NEC-like injury, bone marrow-derived and intestinal macrophages, and RAW264.7 cells. Smad7 and IκB kinase-beta (IKK-ß) were measured by quantitative PCR, western blots, and immunohistochemistry. Promoter activation was confirmed in luciferase reporter and chromatin immunoprecipitation assays. RESULTS: NEC macrophages showed increased Smad7 expression, particularly in areas with severe tissue damage and high bacterial load. Lipopolysaccharide-induced Smad7 expression suppressed TGF-ß signaling and augmented nuclear factor-kappa B (NF-κB) activation and cytokine production in macrophages. Smad7-mediated NF-κB activation was likely mediated via increased expression of IKK-ß, which, further increased Smad7 expression in a feed-forward loop. We show that Smad7 induced IKK-ß expression through direct binding to the IKK-ß promoter and its transcriptional activation. CONCLUSION: Smad7 expression in NEC macrophages interrupts TGF-ß signaling and promotes NF-κB-mediated inflammatory signaling in these cells through increased expression of IKK-ß.

Oncorhynchus mykiss pax7 sequence variations with comparative analyses against other teleost species.

The paired box-7 (pax7) transcription factor expressed in satellite cells (SCs) is an essential regulator of skeletal muscle growth and regeneration in vertebrates including fish. Characterization of rainbow trout (Oncorhynchus mykiss) pax7 gene/s may offer novel insights into skeletal myogenesis by SCs in this indeterminate growth species. Further, evaluation of promoters for cis-regulatory regions may shed light on the evolutionary fate of the duplicated genes. Employing standard PCR, cloning and computational approach, we identified and report complete coding sequences of two pax7 paralogs of rainbow trout (rt); rtpax7α and rtpax7ß. Both genes show significant identity in the nucleotide (97%) and the predicted amino acid (98%) sequences, and bear the characteristic paired domain (PD), octapeptide (OP) and homeodomain (HD) motifs. We further report several splice variants of each gene and nucleotide differences in coding sequence that predicts six putative amino acid changes between the two genes. Additionally, we noted a trinucleotide deletion in rtpax7ß that results in putative serine elimination at the N-terminus and a single nucleotide polymorphism (SNP) in majority of the rtpax7ß variants (6/10) that predicts an arginine substitution for a lysine. We also deciphered the genomic organization up to the first three exons and the upstream putative promoter regions of both genes. Comparative in silico analysis of both the trout pax7 promoters with that of zebrafish pax7 duplicates; zfpax7a and zfpax7b; predicts several important cis-elements/transcription factor binding sites (TFBS) in these teleost pax7 promoter regions.

High level of oxygen treatment causes cardiotoxicity with arrhythmias and redox modulation.

Hyperoxia exposure in mice leads to cardiac hypertrophy and voltage-gated potassium (Kv) channel remodeling. Because redox balance of pyridine nucleotides affects Kv function and hyperoxia alters cellular redox potential, we hypothesized that hyperoxia exposure leads to cardiac ion channel disturbances and redox changes resulting in arrhythmias. In the present study, we investigated the electrical changes and redox abnormalities caused by 72h hyperoxia treatment in mice. Cardiac repolarization changes were assessed by acquiring electrocardiogram (ECG) and cardiac action potentials (AP). Biochemical assays were employed to identify the pyridine nucleotide changes, Kv1.5 expression and myocardial injury. Hyperoxia treatment caused marked bradycardia, arrhythmia and significantly prolonged (ms) the, RR (186.2 ± 10.7 vs. 146.4 ± 6.2), PR (46.8 ± 3.1 vs. 39.3 ± 1.6), QRS (10.8 ± 0.6 vs. 8.5 ± 0.2), QTc (57.1 ± 3.5 vs. 40 ± 1.4) and JT (13.4 ± 2.1 vs. 7.0 ± 0.5) intervals, when compared with normoxia group. Hyperoxia treatment also induced significant increase in cardiac action potential duration (APD) (ex-APD90; 73.8 ± 9.5 vs. 50.9 ± 3.1 ms) and elevated levels of serum markers of myocardial injury; cardiac troponin I (TnI) and lactate dehydrogenase (LDH). Hyperoxia exposure altered cardiac levels of mRNA/protein expression of; Kv1.5, Kvß subunits and SiRT1, and increased ratios of reduced pyridine nucleotides (NADH/NAD & NADPH/NADP). Inhibition of SiRT1 in H9C2 cells using Splitomicin resulted in decreased SiRT1 and Kv1.5 expression, suggesting that SiRT1 may mediate Kv1.5 downregulation. In conclusion, the cardiotoxic effects of hyperoxia exposure involve ion channel disturbances and redox changes resulting in arrhythmias.

Maternal bisphenol a exposure impacts the fetal heart transcriptome.

Conditions during fetal development influence health and disease in adulthood, especially during critical windows of organogenesis. Fetal exposure to the endocrine disrupting chemical, bisphenol A (BPA) affects the development of multiple organ systems in rodents and monkeys. However, effects of BPA exposure on cardiac development have not been assessed. With evidence that maternal BPA is transplacentally delivered to the developing fetus, it becomes imperative to examine the physiological consequences of gestational exposure during primate development. Herein, we evaluate the effects of daily, oral BPA exposure of pregnant rhesus monkeys (Macaca mulatta) on the fetal heart transcriptome. Pregnant monkeys were given daily oral doses (400 µg/kg body weight) of BPA during early (50-100 ± 2 days post conception, dpc) or late (100 ± 2 dpc--term), gestation. At the end of treatment, fetal heart tissues were collected and chamber specific transcriptome expression was assessed using genome-wide microarray. Quantitative real-time PCR was conducted on select genes and ventricular tissue glycogen content was quantified. Our results show that BPA exposure alters transcription of genes that are recognized for their role in cardiac pathophysiologies. Importantly, myosin heavy chain, cardiac isoform alpha (Myh6) was down-regulated in the left ventricle, and 'A Disintegrin and Metalloprotease 12', long isoform (Adam12-l) was up-regulated in both ventricles, and the right atrium of the heart in BPA exposed fetuses. BPA induced alteration of these genes supports the hypothesis that exposure to BPA during fetal development may impact cardiovascular fitness. Our results intensify concerns about the role of BPA in the genesis of human metabolic and cardiovascular diseases.

Integrated approach for smart implantable cardioverter defibrillator (ICD) device with real time ECG monitoring: use of flexible sensors for localized arrhythmia sensing and stimulation.

Arrhythmias are the most common cause of death associated with sudden death and are common in US and worldwide. Cardiac resynchronization therapy (CRT), evolving from pacemakers and development of implantable cardioverter defibrillator (ICD), has been adopted for therapeutic use and demonstrated benefits in patients over the years due to its design and intricate functionality. Recent research has been focused on significant design improvement and efforts are dedicated toward device size reduction, weight and functionality in commercially available ICD's since its invention in the 1960's. Commercially available CRT-D has shown advancement on both clinical and technical side. However, improved focus is required on the device miniaturization, technologically supported and integrated wireless based system for real time heart monitoring electrocardiogram (ECG). In the present report a concise overview for the state-of-the art technology in ICDs and avenues for future development are presented. A unique perspective is also included for ICD device miniaturization and integration of flexible sensing array. Sensor array integration along with its capabilities for identifying localized arrhythmia detection and targeted stimulation for enhancing ICD device capabilities is reviewed.

MicroRNA-301a mediated regulation of Kv4.2 in diabetes: identification of key modulators.

Diabetes is a metabolic disorder that ultimately results in major pathophysiological complications in the cardiovascular system. Diabetics are predisposed to higher incidences of sudden cardiac deaths (SCD). Several studies have associated diabetes as a major underlying risk for heart diseases and its complications. The diabetic heart undergoes remodeling to cope up with the underlying changes, however ultimately fails. In the present study we investigated the changes associated with a key ion channel and transcriptional factors in a diabetic heart model. In the mouse db/db model, we identified key transcriptional regulators and mediators that play important roles in the regulation of ion channel expression. Voltage-gated potassium channel (Kv4.2) is modulated in diabetes and is down regulated. We hypothesized that Kv4.2 expression is altered by potassium channel interacting protein-2 (KChIP2) which is regulated upstream by NFkB and miR-301a. We utilized qRT-PCR analysis and identified the genes that are affected in diabetes in a regional specific manner in the heart. At protein level we identified and validated differential expression of Kv4.2 and KChIP2 along with NFkB in both ventricles of diabetic hearts. In addition, we identified up-regulation of miR-301a in diabetic ventricles. We utilized loss and gain of function approaches to identify and validate the role of miR-301a in regulating Kv4.2. Based on in vivo and in vitro studies we conclude that miR-301a may be a central regulator for the expression of Kv4.2 in diabetes. This miR-301 mediated regulation of Kv4.2 is independent of NFkB and Irx5 and modulates Kv4.2 by direct binding on Kv4.2 3'untranslated region (3'-UTR). Therefore targeting miR-301a may offer new potential for developing therapeutic approaches.

Adipocyte differentiation-specific gene transcriptional response to C18 unsaturated fatty acids plus insulin.

Adipocyte differentiation (AD) and AD-specific gene expression was studied in 3T3-L1 cells in response to oleic acid (OA) or linoleic acid (LA) alone and in combination with insulin. This system facilitated the study of key regulators of adipogenesis PPARγ and C/EBPα and other AD-specific genes, in the absence of dexamethasone (DEX) and isobutyl-1-methyl xanthine (IBMX) (components of the traditional AD medium, DMI). Lipid accumulation and expression levels of AD-specific genes were enhanced by both OA and LA in the presence of insulin but not by OA or LA alone. Gene expression levels of PPARγ, C/EBPα, FABP4, and SREBP1c induced by OA plus insulin, were comparable to DMI medium, by study day 10. The response to long-chain fatty acids (LCFA) plus insulin in the presence or absence of LY294002 demonstrated that the insulin-induced PI 3-kinase pathway regulates AD and AD-specific gene expression levels. Insulin treatment in the presence or absence of genistein suggested that genistein invoked inhibition of AD and AD-specific gene expression. In contrast when LCFA were also included with insulin, the presence of genistein invoked a pronounced and opposite effect on AD to that in the absence of LCFA. This effect may be modulated via C/EBPα as C/EBPα but not PPARγ expression patterns closely reflected the changes in AD. DMI invoked a rapid expression of all genes studied, and LCFA plus insulin invoke more gradual increases in gene expression, to similar levels to those invoked by DMI. The model system is valuable for study of transactivators and response elements of PPARγ and C/EBPα genes.

Dietary carbohydrate level affects transcription factor expression that regulates skeletal muscle myogenesis in rainbow trout.

Understanding the effects of dietary carbohydrates on transcription factors that regulate myogenesis provides insight into the role of nutrient sensing by satellite cells towards myocyte differentiation. We evaluated the influence of dietary carbohydrate level (0, 15, 25 or 35%) on the temporal mRNA expression patterns (4, 8 or 12 weeks) of transcription factors that regulate satellite cell myocyte addition (MA) in rainbow trout (Oncorhynchus mykiss), a vertebrate with indeterminate growth. Relative to the 0% carbohydrate (NC) diet, 15 (IC-15) and 25% (IC-25) carbohydrate containing diets significantly up-regulate MyoD and Myf5, but not Pax7, after 12 weeks of feeding. Simultaneously, the Pax7/MyoD mRNA expression ratio declined significantly with both the IC diets. Myogenin mRNA expression also increased in rainbow trout (RBT) fed the IC-15 diet. The high carbohydrate (HC) diet (35%) attenuated the increased mRNA expression of these transcription factors. It is of note that the 4 and 8 week samples lacked the promyogenic expression patterns. The myogenic gene expression in fish fed the IC-15 diet for 12 weeks indicate a transcriptional signature that reflects increased satellite cell myogenesis. Our results suggest a potential role for satellite cells in the nutrient sensing ability of a vertebrate with indeterminate skeletal muscle growth.

Taenia taeniaeformis: effectiveness of staining oncospheres is related to both temperature of treatment and molecular weight of dyes utilized.

Methods to determine viability of taeniid oncospheres following treatments with potential lethality have practical application in efforts to control transmission. Here we investigated several methods, in lieu of infectivity studies, to assess oncosphere viability and determine lethal temperature treatment regimens. In the first experiment, a standard treatment to exshell oncospheres with 0.5% hypochlorite was assessed for influence on oncosphere recovery of Taenia taeniaeformis eggs. Recovery of eggs and exshelled oncospheres decreased with increasing time in hypochlorite, which indicated that hypochlorite can damage eggs and oncospheres, translating into potential overestimation of lethality of experimental treatments. Losses in hypochlorite were accentuated when eggs were pretreated at 75 degrees C, but not lower temperatures, including 65 degrees C, indicating a sharp threshhold between 65 degrees C and 75 degrees C where eggs and oncospheres became hypersensitive to subsequent hypochlorite treatment. To further investigate this change in relation to temperature, non-vital (acridine orange, AO) and vital (propidium iodide, PI; trypan blue, TB) dyes were used to assess staining of oncospheres (exshelled or not) under conditions ranging from room temperature up to 95 degrees C. The behaviors of dyes as related to internal staining of oncospheres were described using non-linear regression and a sigmoid four-parametric model to determine the inflection point (T50). Each of the dyes differed significantly in T50 estimates, e.g. AO (69.22+/-0.53), PI (73.89+/-0.52) and TB (79.43+/-0.45). For these dyes, the T50 increased in relation to the increasing molecular weight of the dyes. Collectively, the results suggested that barriers to chemical permeability exist in eggs that breakdown incrementally with increasing temperatures above 65 degrees C. This staining behavior and the likelihood that the temperatures involved are above a lethal threshhold clarify a basic limitation in the use of vital dyes to assess oncosphere viability. The results may be relevant to other Taenia spp.