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

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

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

<i>Longissimus thoracis</i> (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

Maternal Bisphenol A Exposure Impacts the Fetal Heart Transcriptome

<div><p>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 (<i>Macaca mulatta</i>) 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 (<i>Myh6</i>) was down-regulated in the left ventricle, and ‘A Disintegrin and Metalloprotease 12’, long isoform (<i>Adam12-l</i>) 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.</p></div

Differentially expressed genes observed using microarray on rhesus fetal heart exposed maternally to BPA or vehicle during early gestation, EG (days; 50–100±2).

<p>ND - below differential expression filter criteria.</p

Total number of significantly altered genes observed using microarray global transcriptome expression analysis.

<p>Whole transcriptome analysis was performed on the left ventricle (LV), right ventricle (RV), left atrium (LA) and the right atrium (RA) of rhesus monkey (Macaca mulatta) fetuses that were exposed maternally to a 400 µg/kg body weight, Bisphenol A (BPA), relative to matched control fetuses, either during, (A) early gestation, EG (days; 50–100±2) or, (B) late gestation, LG (days 100±2–term). Genes that changed by greater than ±1 log₂ fold change (1 LFC = 2 fold change) at an unadjusted p≤0.01 were considered significant and differentially expressed. Bars represent the number of upregulated (positive y-axis) or downregulated (negative y-axis) genes in each of the tissues.</p

Taqman MGB primer & probe system.

<p>Sequences of primers; forward (FP), reverse (RP), Taqman probe (TP) specific to gene transcripts analyzed.</p

Sequence of primers used for quantitative real-time PCR.

<p>Sequence of primers used for quantitative real-time PCR.</p

Pyruvate dehydrogenase kinase isozyme 4 (Pdk4) mRNA expression.

<p>Real-time PCR quantification of Pdk4 gene was performed on the left ventricle (LV), right ventricle (RV), left atrium (LA) and the right atrium (RA) samples of rhesus monkey (Macaca mulatta) fetuses at birth. Fetuses were exposed maternally to a 400 µg/Kg. body weight of BPA dose (B) or 150 µL ethanol (C) during late gestation (LG, days 100±2–term). Data were analyzed by tissue and bars represent the mean ± S.E.M. of the log₂ (fold change). A two sample t-test was performed to identify significant BPA effects and a two-tailed p≤0.05 was considered statistically significant.</p

Kv current recording in H9C2 cells treated with miR-301a inhibitor.

<p>H9C2 cells were either transfected with miR-301a inhibitor (50 nM) or scrambled inhibitor and potassium channel currents were measured by whole cell patch clamp technique after 72 h of treatment. (A) Representative whole cell currents recorded from H9C2 cells normalized to I_peak are shown at +50 mV. (B) The tau (τ) calculated from the recordings with scrambled or miR-301a inhibitor is shown as bar graph representing mean ± SEM with p≤0.05, n = 11 for scrambled and n = 22 for miR-301a inhibitor.</p