TNFR1 contributes to activation-induced cell death of pathological CD4+ T lymphocytes during ischemic heart failure

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Nanocurcumin Prevents Hypoxia Induced Stress in Primary Human Ventricular Cardiomyocytes by Maintaining Mitochondrial Homeostasis

<div><p>Hypoxia induced oxidative stress incurs pathophysiological changes in hypertrophied cardiomyocytes by promoting translocation of p53 to mitochondria. Here, we investigate the cardio-protective efficacy of nanocurcumin in protecting primary human ventricular cardiomyocytes (HVCM) from hypoxia induced damages. Hypoxia induced hypertrophy was confirmed by FITC-phenylalanine uptake assay, atrial natriuretic factor (ANF) levels and cell size measurements. Hypoxia induced translocation of p53 was investigated by using mitochondrial membrane permeability transition pore blocker cyclosporin A (blocks entry of p53 to mitochondria) and confirmed by western blot and immunofluorescence. Mitochondrial damage in hypertrophied HVCM cells was evaluated by analysing bio-energetic, anti-oxidant and metabolic function and substrate switching form lipids to glucose. Nanocurcumin prevented translocation of p53 to mitochondria by stabilizing mitochondrial membrane potential and de-stressed hypertrophied HVCM cells by significant restoration in lactate, acetyl-coenzyme A, pyruvate and glucose content along with lactate dehydrogenase (LDH) and 5' adenosine monophosphate-activated protein kinase (AMPKα) activity. Significant restoration in glucose and modulation of GLUT-1 and GLUT-4 levels confirmed that nanocurcumin mediated prevention of substrate switching. Nanocurcumin prevented of mitochondrial stress as confirmed by c-fos/c-jun/p53 signalling. The data indicates decrease in p-300 histone acetyl transferase (HAT) mediated histone acetylation and GATA-4 activation as pharmacological targets of nanocurcumin in preventing hypoxia induced hypertrophy. The study provides an insight into propitious therapeutic effects of nanocurcumin in cardio-protection and usability in clinical applications.</p></div

Hypoxia induced hypertrophy is independent of translocation of p53 to mitochondria:

<p>The dependency of translocation of p53 to mitochondria on induction of hypertrophy in HVCM cells under hypoxia was estimated by blocking entry of p53 to mitochondria by treatment with CsA. Treatment of HVCM cells with CsA prevented p53 translocation to mitochondria but did not affect ANF levels in HVC cells exposed to hypoxia for 24 h. However, treatment HVCM cells with nanocurcumin significantly reduced p53 translocation to mitochondria and down-regulated ANF levels. Values are mean ± SD, significant values represented as ^@@p≤0.01 <i>vs</i> normoxia, ^#p≤0.01 <i>vs</i> hypoxia, *p≤0.05 <i>vs</i> hypoxia.</p

Effect of nanocurcumin on ΔΨ_m in HVCM cells under hypoxia:

<p>(A) Hypoxia insult disrupted ΔΨ_m as early as 1 h of exposure caused in HVCM cells as depicted by FACS. (B) Curcumin treatment did not show any significant improvement in ΔΨ_m till 6 h of hypoxia and maximum protection was observed by 24 h of hypoxia. (C) Nanocurcumin treatment significantly restored the ΔΨ_m by 3 h of hypoxia than curcumin treated cells and maximum restoration was achieved by 24 h of hypoxia.</p

Nanocurcumin imparts cardio-protection in HVCM cells by activation of c-Jun/c-Fos:

<p>Hypoxia insult prevented c-Fos and c-Jun expression in HVCM cells upto 24 h of hypoxia and thus promoted translocation of p53 to mitochondria. Treatment of HVCM cells with nanocurcumin promoted c-Fos accumulation as early as 1 h of hypoxia and prevented p53 mediated cell-death whereas curcumin treated cells showed significant up-regulation of c-Fos and c-Jun by 6–12 h of treatment under hypoxia showing that nanocurcumin is frequently available to the cells compared to curcumin. Nanocurcumin treated cells showed higher expression of negative regulators of p53 accumulation, i.e. c-Jun and c-Fos in HVCM cells under hypoxia, depicting that improved bio-availability and stability of nanocurcumin prevents hypoxia induced damage in cardiomyocytes.</p

Nanocurcumin prevents hypoxia induced hypertrophy in HVCM cells:

<p>(A, B) Fluorescent staining of HVCM cells (60X) with actin filaments (green) and nuclei (blue) under various experimental groups showed increase in cell size in hypoxia exposed cells compared to normoxia controls. (C) Increment in FITC-phenylalanine uptake was observed in cells exposed to hypoxia compared to normoxia control. (D) Increase in ANF levels were observed within 1 h of hypoxia and reached maximum by 24 h of hypoxia. Treatment with nanocurcumin significantly restored the cell size and FITC-phenylalanine uptake under hypoxia when compared to curcumin treated cells and cells exposed to hypoxia only. Nanocurcumin treated cells showed tremendous restoration in ANF levels under hypoxia at all the time points compared to curcumin treated cells. No significant changes in ANF levels, cell size or FITC-phenylalanine uptake was observed in cells exposed to normoxia after treatment with nanocurcumin or curcumin. Values are mean ± SD, significant values represented as ^@@ p≤0.01 <i>vs</i> normoxia, *p≤0.05 <i>vs</i> hypoxia, ^#p≤0.01 <i>vs</i> hypoxia, ^@ p≤0.005 <i>vs</i> hypoxia and ^$$ p≤0.001 <i>vs</i> hypoxia. Bar represents 50 μm.</p

Effect of nanocurcumin on p-300 HAT and HDAC activities in HVCM cells under hypoxia:

<p>Histone acetylation by p-300 was up-regulated with a corresponding decline in HDAC activity in cardiomyocytes under hypoxia depicting that hypoxia induced hypertrophy dependent upon p-300 HAT and HDAC activity (A and C). Nanocurcumin treatment significantly down-regulated p-300 HAT and up-regulated HDAC activity in HVCM cells under hypoxia compared to cells exposed to hypoxia only. This was further confirmed by western blot analysis of GATA-4 and p-GATA-4 levels along with acetylated histone 3 and 4 (B). However, curcumin treatment did not prevent histone acetylation under hypoxia. Also, nanocurcumin or curcumin treated cells did not show change in p-300 HAT activity under normoxia. Values are mean ± SD, significant values represented as ^@@ p≤0.01 <i>vs</i> normoxia, *p≤0.05 <i>vs</i> hypoxia and ^#p≤0.01 <i>vs</i> hypoxia. Non-significant changes are depicted as ns. Scale bar represents 50μm.</p

Nanocurcumin improves cellular viability under hypoxia:

<p>(A) Hypoxia insult caused decline in cell viability (20%) compared to normoxia control cells by 24 h. (B) Nanocurcumin treatment at 500 ng/ml significantly improved cellular viability (89%) by 24 h of hypoxia exposure and confirmed by FACS. Curcumin treatment did not show significant improvement in cellular viability at any time point. No significant change in cell viability was observed in nanocurcumin or curcumin treated cells under normoxia. Nanocurcumin treatment at 500 ng/ml was used for further experiments. Values are mean ± SD, significant values represented as ^@@p≤0.01 <i>vs</i> normoxia and ^#p≤0.01 <i>vs</i> hypoxia. Non-significant changes are designated as ns.</p

Nanocurcumin protects HVCM cells by restoring oxidative balance:

<p>Hypoxia stress disturbed cellular redox balance as depicted by increase in ROS leakage and lipid peroxidation and concomitant decrease in GSH/GSSG content, xanthine oxidase activity and MnSOD activity. Nanocurcumin treatment showed significant improvement in redox status when compared to curcumin treated cells under hypoxia and cells exposed to hypoxia only. No significant change in redox status was observed in cells treated with curcumin or nanocurcumin under normoxia. Values are mean ± SD, significant values represented as ^@p≤0.05 <i>vs</i> normoxia, ^#p≤0.01 <i>vs</i> hypoxia, *p≤0.05 <i>vs</i> hypoxia.</p