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

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

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

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