Analysis of human collagen sequences

The extracellular matrix is fast emerging as important component mediating cell-cell interactions, along with its established role as a scaffold for cell support. Collagen, being the principal component of extracellular matrix, has been implicated in a number of pathological conditions. However, collagens are complex protein structures belonging to a large family consisting of 28 members in humans; hence, there exists a lack of in depth information about their structural features. Annotating and appreciating the functions of these proteins is possible with the help of the numerous biocomputational tools that are currently available. This study reports a comparative analysis and characterization of the alpha-1 chain of human collagen sequences. Physico-chemical, secondary structural, functional and phylogenetic classification was carried out, based on which, collagens 12, 14 and 20, which belong to the FACIT collagen family, have been identified as potential players in diseased conditions, owing to certain atypical properties such as very high aliphatic index, low percentage of glycine and proline residues and their proximity in evolutionary history. These collagen molecules might be important candidates to be investigated further for their role in skeletal disorders

Hydrogen sulfide modulates basal metabolic circuitry ̶ A transcriptome sequencing assisted insight

Hydrogen Sulfide (H2S), the third gasomessenger to be discovered after nitric oxide (NO) and carbon monoxide (CO), is known for its distinctive health promoting effects on various organ systems, including cardiovascular system. This molecule has now qualified as an authentic mediator of specific cellular signal transduction pathways [1, 2]. The mechanistic insight into its cyto-protective role however remains incompletely understood. To understand the molecular circuitry regulated by H2S augmentation, we utilized an exogenous donor of H2S, Sodium hydrogen sulfide (NaHS), and performed unbiased global transcriptome sequencing (Illumina) in cardiac cells (H9c2 cardiomyoblasts). These experiments yielded differential transcriptome of the cells with varying levels of H2S (with or without NaHS treatment for 6 hrs). We subjected this dataset to multiple pathway mining tools, including gene ontology analysis, functional annotation clustering and co-expression network analysis, to infer biological themes hidden as concerted differential gene expression signatures [3]. We, interestingly, observed common biological processes in different analysis strategies, suggesting authentic, conserved nature of cellular response to H2S. Biological networks, largely associated with metabolic/ redox processes were recognized; within three gross themes - steroid/ isoprenoid biosynthesis, oxidoreductase coenzyme metabolism (representing pentose phosphate pathway, PPP) and glutathione metabolism. Glucose-6- phosphate dehydrogenase (G6PD) - rate-limiting enzyme within PPP stood as the highest degree node in majority of these networks. Also, genes related to oxidative stress and redox signaling were enriched. Interestingly, these pathways appear to be centrally linked by nicotinamide nucleotide cofactor (NADPH) homeostasis. We further supported this proposition at functional level by performing various enzyme activities besides recording cellular NADP/NADPH and GSH levels in established cellular as well as rat model system. In summary, our data suggested profound influence of H2S on integrated cellular metabolic circuitry modulating redox homeostasis in cardiac cells

Curcumin Suppresses Gelatinase B Mediated Norepinephrine Induced Stress in H9c2 Cardiomyocytes

<div><p>Background</p><p>Extracellular matrix (ECM) remodeling facilitates biomechanical signals in response to abnormal physiological conditions. This process is witnessed as one of the major effects of the stress imposed by catecholamines, such as epinephrine and norepinephrine (NE), on cardiac muscle cells. Matrix metalloproteinases (MMPs) are the key proteases involved in degradation of the ECM in heart.</p> <p>Objectives</p><p>The present study focuses on studying the effect of curcumin on Gelatinase B (MMP-9), an ECM remodeling regulatory enzyme, in NE-induced cardiac stress. Curcumin, a bioactive polyphenol found in the spice turmeric, has been studied for its multi-fold beneficial properties. This study focuses on investigating the role of curcumin as a cardio-protectant.</p> <p>Methods</p><p>H9c2 cardiomyocytes were subjected to NE and curcumin treatments to study the response in stress conditions. Effect on total collagen content was studied using Picrosirus red staining. Gelatinase B activity was assessed through Gel-Diffusion Assay and Zymographic techniques. RT-PCR, Western Blotting and Immunocytochemistry were performed to study effect on expression of gelatinase B. Further, the effect of curcumin on the localization of NF-κB, known to regulate gelatinase B, was also examined.</p> <p>Results</p><p>Curcumin suppressed the increase in the total collagen content under hypertrophic stress and was found to inhibit the in-gel and <i>in-situ</i> gelatinolytic activity of gelatinase B. Moreover, it was found to suppress the mRNA and protein expression of gelatinase B.</p> <p>Conclusions</p><p>The study provides an evidence for an overall inhibitory effect of curcumin on Gelatinase B in NE-induced hypertrophic stress in H9c2 cardiomyocytes which may contribute in the prevention of ECM remodeling.</p> </div

Expression levels of MMP-9 on curcumin treatment.

<p><b>A</b>) <b>RT-PCR </b><b>for </b><b>MMP-9</b>: mRNA expression seen through semiqunatitative RT-PCR Samples in different lanes starting from the left represented as Lane-1: Control; Lane-2: NE-treated; Lane-3: NE+Curcumin-treated; Lane-4: Curcumin-treated alone. qPCR results obtained were normalized against beta actin and plotted as a histogram (*P<0.05). <b>B</b>) <b>Western </b><b>Blotting </b><b>for </b><b>MMP-9</b>: Samples in different lanes starting from the left represented as Lane-1: Control; Lane-2: NE-treated; Lane-3: NE+Curcumin-treated; Lane-4: Curcumin-treated alone. Protein expression observed through Western blotting was quantitated by NIH ImageJ software. Results obtained were normalized against beta actin and plotted as a histogram (*P<0.01). <b>C</b>) <b>Immunocytochemistry </b><b>for </b><b>MMP-9</b>: Images captured by fluoresencent microscope at 20X magnifications are represented. NE-treated group showed a significant difference compared to control and NE+Curcumin-treated group in all experiments shown in the figure.</p

Effect of curumin on gelatinolytic activity.

<p><b>A</b>) <b>Gel-diffusion </b><b>assay</b>: Upper gel: Various concentrations of trypsin (1-Blank; 2-5 µg/µl; 3-10 µg/µl; 4-15 µg/µl; 5-20 µg/µl; 6-25 µg/µl; 7-30 µg/µl; 8-35 µg/µl) were added to different wells and protease activity was observed as digested zones around it. A standard curve of the enzyme activity in units as a function of diameter of zone was prepared. The enzyme activity for different samples shown in the lower gel (Control; NE-treated; NE+Curcumin-treated; Curcumin-treated alone) was calculated from the standard graph and represented as a histogram (*P<0.01). The difference of NE-treated was significant to control as well as NE+Curcumin-treated group. <b>B</b>) <b>Gelatin </b><b>Zymography</b>: Samples in different lanes of zymograms starting from the left represented as Lane-1: Control; Lane-2: NE-treated; Lane-3: NE+Curcumin-treated; Lane-4: Curcumin-treated alone. The fold change in the activity for bands corresponding to MMP-9 with respect to control was quantified using ImageJ and plotted as histogram (*P<0.01, **P<0.05). <b>C</b>) <b><i>in-situ</i></b><b>Gelatin </b><b>Zymography</b>: The experiment was carried out under different experimental conditions above and images captured by fluoresencent microscope at 20X magnifications are represented.</p

Proposed Model for the study.

<p>An illustration of the proposed mechanism of prevention of NE-induced cardiotoxicity by curcumin through downregulation of gelatinase B. It shows that curcumin targets different signaling players in the pathway.</p

Nuclear localization of NF-κB.

<p>(<b>A</b>) <b>Immunofluorescence </b><b>for </b><b>NF-κB</b>: Nuclear localization of NF-κB was compared between H9c2 Control cells, NE-treated cells NE+Curcumin-treated and Curcumin-treated alone cells. Blue color represents DAPI staining of nucleus. NF- κB was stained green. (<b>B</b>) <b>Western </b><b>Blotting</b>: Nuclear and cytosolic proteins from different Samples in different lanes for both nuclear and cytosolic extract panels starting from the left represented as Lane-1: Control; Lane-2: NE-treated; Lane-3: NE+Curcumin-treated; Lane-4: Curcumin-treated alone. Protein expression observed through Western blotting was quantitated by NIH ImageJ software. Results obtained were normalized against beta actin for cytoplasmic extracts and Lamin A/C for nuclear extracts and plotted as a histogram (*P<0.01, **P<0.05). NE-treated group showed a significant difference compared to control and NE+Curcumin-treated group.</p

Curcumin prevents Norepinephrine induced cardiac stress in H9c2 cells.

<p><b>A</b>) <b>Analysis </b><b>of </b><b>FACS </b><b>Forward </b><b>Scatter</b>: Histogram represents comparison of cell size in Control (Uninduced cells), NE-treated (Hypertrophic), NE+Curcumin-treated and CurcuminNE-treated alone experimental groups (*P<0.01). <b>B</b>) <b>Analysis </b><b>of </b><b>protein </b><b>content</b>: The statistical representation of total protein concentration (mg/ml) for all four groups (*P<0.05). <b>C</b>) <b>RT-PCR </b><b>analysis </b><b>for </b><b>ANF</b>: Starting from left, samples in different lanes represent, Lane-1: Control; Lane-2: NE-treated; Lane-3: NE+Curcumin-treated; Lane-4: Curcumin-treated. The bands were quantitated by NIH ImageJ software and fold intensity with respect to control after normalization was plotted as a histogram (*P<0.05). The difference in NE-treated group was statistically significant in comparison to control and NE+Curcumin-treated groups in all experiments shown in the figure.</p