Nrf2 Deficiency Exaggerates Doxorubicin-Induced Cardiotoxicity and Cardiac Dysfunction

The anticancer therapy of doxorubicin (Dox) has been limited by its acute and chronic cardiotoxicity. In addition to a causative role of oxidative stress, autophagy appears to play an important role in the regulation of Dox-induced cardiotoxicity. However, the underlying mechanisms remain unclear. Accordingly, we explored a role of nuclear factor erythroid-2 related factor 2 (Nrf2) in Dox-induced cardiomyopathy with a focus on myocardial oxidative stress and autophagic activity. In wild type (WT) mice, a single intraperitoneal injection of 25 mg/kg Dox rapidly induced cardiomyocyte necrosis and cardiac dysfunction, which were associated with oxidative stress, impaired autophagy, and accumulated polyubiquitinated protein aggregates. However, these Dox-induced adverse effects were exaggerated in Nrf2 knockout (Nrf2−/−) mice. In cultured cardiomyocytes, overexpression of Nrf2 increased the steady levels of LC3-II, ameliorated Dox-induced impairment of autophagic flux and accumulation of ubiquitinated protein aggregates, and suppressed Dox-induced cytotoxicity, whereas knockdown of Nrf2 exerted opposite effects. Moreover, the exaggerated adverse effects in Dox-intoxicated Nrf2 depleted cardiomyocytes were dramatically attenuated by forced activation of autophagy via overexpression of autophagy related gene 5 (Atg5). Thus, these results suggest that Nrf2 is likely an endogenous suppressor of Dox-induced cardiotoxicity by controlling both oxidative stress and autophagy in the heart

Irisin Controls Growth, Intracellular Ca²⁺ Signals, and Mitochondrial Thermogenesis in Cardiomyoblasts

<div><p>Exercise offers short-term and long-term health benefits, including an increased metabolic rate and energy expenditure in myocardium. The newly-discovered exercise-induced myokine, irisin, stimulates conversion of white into brown adipocytes as well as increased mitochondrial biogenesis and energy expenditure. Remarkably, irisin is highly expressed in myocardium, but its physiological effects in the heart are unknown. The objective of this work is to investigate irisin’s potential multifaceted effects on cardiomyoblasts and myocardium. For this purpose, H9C2 cells were treated with recombinant irisin produced in yeast cells (r-irisin) and in HEK293 cells (hr-irisin) for examining its effects on cell proliferation by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and on gene transcription profiles by qRT-PCR. R-irisin and hr-irisin both inhibited cell proliferation and activated genes related to cardiomyocyte metabolic function and differentiation, including myocardin, follistatin, smooth muscle actin, and nuclear respiratory factor-1. Signal transduction pathways affected by r-irisin in H9C2 cells and C57BL/6 mice were examined by detecting phosphorylation of PI3K-AKT, p38, ERK or STAT3. We also measured intracellular Ca²⁺ signaling and mitochondrial thermogenesis and energy expenditure in r-irisin-treated H9C2 cells. The results showed that r-irisin, in a certain concentration rage, could activate PI3K-AKT and intracellular Ca²⁺ signaling and increase cellular oxygen consumption in H9C2 cells. Our study also suggests the existence of irisin-specific receptor on the membrane of H9C2 cells. In conclusion, irisin in a certain concentration rage increased myocardial cell metabolism, inhibited cell proliferation and promoted cell differentiation. These effects might be mediated through PI3K-AKT and Ca²⁺ signaling, which are known to activate expression of exercise-related genes such as follistatin and myocardin. This work supports the value of exercise, which promotes irisin release.</p></div

Activation of different signaling pathways in H9C2 cells after r-irisin treatment.

<p>H9C2 cells were treated with r-irisin (50 nM). After 0, 5, 20, and 30 min, protein was collected for western blotting. Total β-actin or AKT served as protein-loading control. Bottom is typical blots; the top bar graphs represent densitometry analyses for p-AKT/AKT (A), p-ERK/β-actin (B) and p-p38/β-actin (C). All data were normalized against the control group. Results represent the means from three independent experiments. (* indicating <i>p</i> < 0.05 statistical differences compared to control, respectively.)</p

Detection of r-irisin-his binding to cell membrane of H9C2 cells.

<p>A&B: Detection of irisin binding to cell surface by flow cytometry. Flow cytometry using anti-his-PE antibody detected r-irisin-his that bound to H9C2 cells. Cells were pre-incubated with 5 uM r-irisin-his for 1h. A: Intensity of fluorescence in histogram of flow cytometry. Red is auto-fluorescence of H9C2 cells (red), blue for isotype control, and orange line for irisin-bound cells (r-irisin-his:anti-His-PE-positive). B: R-irisin binding to H9C2 cells was dose dependent. C: R-irisin competed with r-irisin-his in binding to H9C2 cells. H9C2 cells were incubated with r-irisin-his (50nM) or both r-irisin (50μM) and r-irisin-his (50nM) for 1h. Green line, isotype control; Orange line, r-irisin and r-irisin-his competition; Blue line, r-irisin-his only; Red line, auto fluorescence. The same results were expressed in bar graph (right panel).</p

Stimulation of H9C2 cells with r-irisin increased intracellular Ca²⁺ concentration.

<p>Real-time Ca²⁺ imaging analysis was used to measure intracellular Ca²⁺ levels in H9C2 cells after stimulation with low (10 nM, A), intermediate (50 nM, B), and high (150 nM, C) r-irisin concentrations, followed by 20 mM KCl treatment. Gray lines = single cell traces; Black line = average of all cells. D: The average Ca²⁺ peak by each r-irisin concentration compared to basal levels prior to stimulation. Values represent the mean ± SD from 95–113 cells per concentration. (*, **and *** indicating <i>p</i><0.05, <i>p</i><0.01, and <i>p</i><0.001 statistical differences compared to control, respectively.)</p

H9C2 growth responses to r-irisin treatment.

<p>A: SDS-PAGE and western blotting analysis of human irisin expressed in <i>Pichia pastoris</i>. <i>P</i>. <i>pastoris</i> culture supernatant was harvested and analyzed by Coomassie blue stained SDS-PAGE (Lane1) and western blotting (Lane 2). The negative control showed no irisin in western blotting analysis (Lane 3). B: The time response of H9C2 cells to r-irisin (50 nM) treatment. Cell proliferation was measured by MTT assay. C: The dose response of H9C2 cells to r-irisin 3 days treatment. D: Expression profile of selected genes in H9C2 cells treated with r-irisin (50 nM) at different time points. The gene transcription was measured by quantitative RT-PCR, and compared to the expression level of β-actin. The data was shown as mean ±SD of three independent experiments. (*, **, and *** indicating <i>p</i> < 0.05, <i>p</i> <0.01, and <i>p</i> < 0.001 statistical differences compared to control, respectively.)</p

Effects of hr-irisin on cell growth.

<p>A: SDS-PAGE and western blotting analysis of human irisin expressed in HEK 293 cells (hr-irisin). The culture medium of irisin transfected cells was harvested for analysis. In Coomassie blue stained SDS—PAGE, Lane 1: medium supernatant containing hr-irisin; Lane 2: medium supernatant containing hr-irisin incubated with PNGase F; and M: Marker. In western blotting analysis, Lane 3: medium supernatant containing hr-irisin; lane 4: medium supernatant containing hr-irisin incubated with PNGase F; and Lane 5: medium supernatant of GFP transfected HEK 293 cells. Anti-irisin rabbit antibody was used in western blotting analysis. B: The time response of H9C2 cells to hr-irisin treatment. Cell proliferation was measured by MTT assay. C: Expression profile of selected genes in H9C2 cells treated with hr-irisin at different time points. Gene transcription was measured by quantitative RT-PCR, and compared to the expression level of β-actin. D: Effects of hr-irisin on H9C2 baseline OCR. H9C2 cells were incubated with control (GFP-containing medium) or same volume hr-irisin for 72 h and then measured with the XF96 Extracellular Flux Analyzer. In all studies, the data were shown as mean ±SD of three independent experiments. (*, **, and *** indicating p<0.05, p<0.01, and p<0.001 statistical differences compared to control, respectively.)</p