Kalyan C. Chapalamadugu

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.

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.

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.

Smad7 interrupts TGF-β 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)-β-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-β) 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-β 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-β, which, further increased Smad7 expression in a feed-forward loop. We show that Smad7 induced IKK-β expression through direct binding to the IKK-β promoter and its transcriptional activation.Conclusion:Smad7 expression in NEC macrophages interrupts TGF-β signaling and promotes NF-κB-mediated inflammatory signaling in these cells through increased expression of IKK-β.

Deletion of Kvβ1.1 subunit leads to electrical and haemodynamic changes causing cardiac hypertrophy in female murine hearts

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 β1 (Kvβ1) subunit in the heart. What is the main finding and its importance? Genetic ablation of Kvβ1.1 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. Kvβ1.1 binds the MHCα isoform at the protein level, and small interfering RNA targeted knockdown of Kvβ1.1 upregulated MHCα.

Kvβ1.1 (AKR6A8) senses pyridine nucleotide changes in the mouse heart and modulates cardiac electrical activity

The present study investigates the physiological role of Kvβ1 subunit for sensing pyridine nucleotide (NADH/NAD+) changes in the heart. We used Kvβ1.1 knockout (KO) or wild-type (WT) mice and established that Kvβ1.1 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 Kvβ1.1 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 Kvβ1.1 KO mouse hearts did not show APD changes upon lactate perfusion. COS-7 cells coexpressing Kv4.2 and Kvβ1.1 were used for whole cell patch-clamp recordings to evaluate changes caused by NADH (lactate). These data reveal that Kvβ1.1 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 Kvβ1.1 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 Kvβ1.1 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 Kvβ1.1 subunit. In the present study, we identify Kvβ1.1 as a sensor of pyridine nucleotide changes and as a modulator of Kv4.2 gating, action potential duration, and ECG in the mouse heart.

Interactions of muscle regulatory factors and isoforms of insulin growth factor‐1 in the muscle regenerative process

Skeletal muscle regeneration following damage is mainly attributed to activation, proliferation and myogenic differentiation of muscle stem cells known as satellite cells. Satellite cells lie beneath the basal lamina that surrounds muscle fibres. These pluripotent cells respond to muscle injury to initiate the process of satellite cell myogenesis, regulated by several transcription factors known as myogenic regulatory factors (MRFs): MyoD, Myf5, myogenin and MRF4. Pax-7-expressing quiescent satellite cells, upon activation, up-regulate MyoD and Myf5 expression to generate myogenic lineage cells, or myoblasts. Myoblasts proliferate and subsequently up-regulate myogenin and MRF4 levels leading to terminal differentiation into muscle fibres (Charge & Rudnicki, 2004). On the other hand, insulin-like growth factor−1 (IGF-1) signalling was implicated in the process of myogenesis. In vitro studies demonstrated that IGF-1 positively regulated myogenesis in a two-step process, first by cell proliferation of stem cells and later by enhancing differentiation process (Rosenthal & Cheng, 1995; Engert et al. 1996). Transgenic mice with muscle-specific IGF-1 isoform supplementation have muscle regenerative capacity in advanced ages, suggesting muscle-specific IGF-1 in the regenerative process (Musaro et al. 2001). To date, three isoforms of IGF-1 have been detected in skeletal muscle: IGF-1Ea, IGF-1Eb and IGF-1Ec (IGF-1Ec is referred to as mechano-growth factor, MGF) (Rotwein, 1986; Yang et al. 1996). The roles of these isoforms in muscle repair, particularly their regulation of MRFs, was not elucidated in detail. A recent report by McKay et al. (2008) in The Journal of Physiology investigated the temporal gene expression of MRFs and IGF-1 isoforms in human quadriceps femoris upon exercise-induced muscle damage. Eight male subjects were recruited to perform intensive muscle-lengthening exercise. Muscle biopsies were collected before (0 h), and after exercise at 4, 24, 72 and 120 h. Immunofluorescence assays were done to determine localization of IGF-1 and Pax-7 proteins after the exercise-induced damage. The authors speculate about the interaction of IGF-1 signalling and transcription of MRFs based on gene expression and immunoflourescence data. In summary, MyoD and Myf5 gene expressions were significantly up-regulated at 4 h and 24 h post-exercise, respectively. Up-regulation of myogenin gene expression was observed from 4 h onwards, and MRF4 expression was significantly up-regulated at 72 and 120 h. These observations are partly inconsistent with accepted cascade of myogenic differentiation, as the authors observed up-regulation of myogenin prior to Myf5. This clearly suggests limitations of interpretations based on crude muscle samples and gene expression analyses to acutely delineate a cascade in response to damage. The authors also measured transcript levels of IGF-1Ea, IGF-1Eb and MGF. MGF and IGF-1Ea were significantly up-regulated at 24 h and 72 h, respectively, whereas IGF-1Eb was up-regulated at 72 and 120 h. These data were then used to determine correlations between IGF-1 isoforms and MRF gene expressions. The authors report a correlation of MGF with Myf5, while IGF-1Ea and IGF-1Eb correlated with MRF4 expression. The observed correlations are limited in their capacity to provide biological information, because IGF-1Ea and IGF-1Eb correlated with only one of the differentiation markers (MRF4), and MGF was correlated with only one of the myogenic precursor markers (Myf5). Adding to this conundrum as noted above is the paradoxical up-regulation of myogenin before Myf5. In essence, the gene expression analyses along with statistical analyses are limited to provide insight into signalling pathways connecting IGF-1 isoforms and MRFs. The observations from similar studies were contradictory to the current report; for example, Psilander et al. (2003) reported that MyoD was up-regulated immediately after exercise and reached baseline after 1 h onwards, whereas MRF4 and myogenin were significantly up-regulated only at 2 h and 6 h, respectively. It has to be noted that the changes in gene expression reported by Psilander et al. were rapid i.e. within 6 h of exercise. At later time-points the levels were not different from baseline. They report data suggesting a decline in IGF-1a levels at 1 h and 6 h post-exercise. These gene expression patterns are difficult to interpret and the apparent decline of IGF-1a in the muscle regeneration process is unclear. These apparent discrepancies in the timeline of gene expression changes and relationship of IGF-1 and MRFs may be attributed to limitations of interpretations solely based upon gene expression changes, different muscle injury protocols and biological variation in subjects chosen for the two studies. Interpretations based on gene expression analyses are highly influenced by temporal effects depending on kinetics of promoter activation (pulsatile association of transcription factors on promoter), pre-mRNA to mature mRNA formation and half-life of mature mRNA. Due to intrinsic differences in these regulatory processes among transcripts, several minutes to days may be necessary to reliably quantify gene expression changes in response to injury. Thus, interpretations supported by associated protein changes will provide strength to delineate muscle regenerative process. On the other hand, McKay et al. claim that IGF-1 protein was localized only in satellite cells at 24 h, but present both in satellite cells and myofibres at 72 h and 120 h post-exercise. Immunofluorescence data were difficult to interpret due to colocalization of DAPI with IGF-1 but, nonetheless, suggest that IGF-1 is localized to the nucleus of satellite cells. This is unexpected of IGF-1, a secreted protein that is localized in secretory vesicles. Thus, it is difficult to decipher biological information that links different isoforms of IGF and MRFs based on the immunoflourescence data. Although in vitro/rodent models laid foundations for fundamental understanding of muscle regeneration process, understanding details of myogenic regulatory mechanisms in human beings is needed for realization of potential for therapeutic interventions. In this regard, use of human subjects by authors is highly appreciated, as it has the potential for discovering physiologically relevant molecular mechanisms. Future studies should include an examination as to whether the regenerative process is influenced by the extent of muscle damage and associated inflammatory response upon injury. This can be accomplished by determining the levels of serum creatine kinase, an indicator of muscle damage, and also by quantifying the inflammatory response by histological measurement of invading neutrophils and macrophages in injured muscle. This could then be followed by further analyses to determine the relationship between serum creatine kinase, inflammatory response and expression profiles of MRFs/IGF-1 isoforms. The extent of the regenerative process may also depend on basal satellite cell population, their activation and proliferation. In this context, measuring transcript levels of satellite cell marker Pax-7 will provide in indirect measure of relative satellite cell population/activity at different stages of the repair process. The presence of three isoforms of IGF-1 in muscle and their putative roles in repair underlines the importance of understanding their complicated regulatory and signalling mechanisms. Toward understanding IGF-1 biology in the muscle regenerative process, it is important to understand if one or more of the MRFs directly/indirectly regulate transcription or splicing of IGF-1 genes. There is a greater need for distinguishing the three isoforms of IGF-1 at the protein level and quantifying each of these isoforms, and also to determine their signalling. This becomes complex, as IGF proteins upon secretion bind to IGF-binding proteins (IGFBPs). There are several IGFBPs, and specific interactions of the IGF isoforms and IGFBPs may lead to different results, such as inhibitition or stimulation of IGF-1 signalling. This complexity can be partially addressed by conducting studies using transgenic rodent models with over-expression of each of the splice variants in muscle and determine IGF signalling and the myogenic process after injury. Further research in vitro may also be used to determine isoform-specific IGF signalling to gene expression of different MRFs, which involves determining the activated transcription factors and their responsive cis-elements on different MRFs. In addition, recent genetic models such as zebrafish can also be used to better understand the fundamentals of molecular regulation of the myogenic process, as the mechanisms of embryonic muscle development are similar to muscle regeneration. In conclusion, there is a clear need for complementary approaches using different models to understand regulatory and signalling mechanisms of each of the IGFs and MRFs. In the long term, studies using these approaches will help us to better understand and treat muscle degeneration.

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.

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 rtpax7β. 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 rtpax7β that results in putative serine elimination at the N-terminus and a single nucleotide polymorphism (SNP) in majority of the rtpax7β 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.