Xiaochen Wang1,2, Ziwei Pan1,2, Jue Wang1,2, Hongkun Wang1,2, Hangping Fan1,2, Tingyu Gong1,2, Qiming Sun3, Ye Feng2,4, Ping Liang1,2. 1. Key Laboratory of combined Multi-organ Transplantation, Ministry of Public Health, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310003, China. 2. Institute of Translational Medicine, Zhejiang University, Hangzhou, 310029, China. 3. Department of Biochemistry, Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China. 4. Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310016, China.
Dear Editor,We performed a comprehensive study to characterize the molecular mechanisms underlying azithromycin (AZM)‐induced cardiotoxicity (AIC) using human‐induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs). Electrophysiologically, high‐concentration AZM causes accelerated beating rate and dramatically shortened QT in the absence of proarrhythmic risk. Morphologically, high‐concentration AZM interferes with lysosomal activity to impair autophagy flux and autophagosome maturation. The futile and excessive autophagosome formation and accumulation confers vacuole formation, sarcomeric damage, and cardiomyocyte death. Our study uncovered a novel molecular mechanism underlying AIC, and reducing the accumulation of autophagosomes may offer a novel therapeutic strategy for potential AZM‐induced cardiovascular risk.AZM is one of the most frequently used antibiotics linked to an increased risk of fatal ventricular arrhythmias and cardiovascular death. Results of AZM‐associated cardiovascular risk in existing retrospective studies were discordant, and underlying mechanisms remain unclear., , Three healthy iPSC‐CM lines were utilized to investigate AIC, such as arrhythmias, reduced cell viability and morphological damage (Figure S1‐S2). Field potentials (FPs) and action potentials were recorded from iPSC‐CMs by multi‐electrode array and patch clamp, and baseline electrophysiological parameters were comparable between three iPSC‐CM lines (Figure S3‐S4). Dimethyl sulfoxide (DMSO) and moxifloxacin (MXF) were firstly tested as negative and positive drugs. As expected, DMSO showed negligible effect on FPs, whereas MXF caused prominent prolongation of FP duration (FPD) (Figure 1A–D, Table S1). Having established a stable drug testing platform, we next assessed the acute effects of AZM on electrophysiology (Figure S5). Strikingly, AZM resulted in strong effects on FPs in a concentration‐dependent manner (Figure 1E). Starting from 3 μM, acute AZM treatment caused significantly increased beating rate and shortened FPD (Figure 1F, Table S1). We observed no arrhythmic activity even at the maximal tested concentration of AZM. Consistent with previous studies,, we observed that 100 μM AZM caused approximately 20% inhibition of human ether‐à‐go‐go‐related gene (hERG) currents, indicating a minimal effect on hERG (Figure S6). Ca2+ currents isolated from 10 and 30 μM AZM‐treated iPSC‐CMs were significantly reduced, and steady‐state activation (SSA) curve in 30 μM AZM‐treated iPSC‐CMs was significantly right‐shifted (Figure 1G‐L, Table S2). Moreover, acutely treated with 30 μM AZM, Na+ current density was significantly decreased, and steady‐state inactivation (SSI) was significantly left‐shifted (Figure 1M‐R, Table S2). No significant change was observed in K+ current recordings (Figure S7).
FIGURE 1
Electrophysiological effects of acute azithromycin (AZM) in induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs). (A) Representative field potential (FP) tracings recorded from iPSC‐CMs with acute treatment of 0, 0.1%, 0.3%, and 0.5% dimethyl sulfoxide (DMSO), respectively. (B) Acute effects of DMSO on beating period (BP), field potential duration (FPD), corrected field potential duration (FPDc), and field potential amplitude (FPA). (C) Representative FP tracings recorded from iPSC‐CMs with acute treatment of 0, 3, 10, 30, and 100 μM moxifloxacin (MXF), respectively. (D) Acute effects of MXF on BP, FPD, FPDc, and FPA. Prominent prolongation of FPD and FPDc was observed in response to acute treatment of MXF in a concentration‐dependent manner. BP was significantly increased at 30 and 100 μM, suggesting a slower effect on beating rate induced by MXF. Note that 30 and 100 μM MXF had largely decreased effect on FPA. Mycoytes were derived from three different iPSC lines. *
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p < 0.0001. (E) Representative FP tracings recorded from iPSC‐CMs with acute treatment of 0, 1, 3, 10, and 30 μM AZM, respectively. (F) Acute effects of AZM on BP, FPD, FPDc, and FPA. Beating rate, reflected by BP, was significantly increased started at the concentration of 3 μM AZM. Starting from 3 μM, acute treatment of AZM gave rise to markedly shortened FPD and FPDc. The FPA was significantly decreased by 10 and 30 μM AZM treatment. Mycoytes were derived from three different iPSC lines. *
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p < 0.0001. (G) Representative Ca2+ current tracings isolated from control iPSC‐CMs and iPSC‐CMs with acute treatment of 30 μM AZM. Myocytes were derived from iPSC #3. (H) Comparison of Ca2+ current‐voltage relationship curve (IV curve) between control iPSC‐CMs and iPSC‐CMs with acute treatment of 1, 10, and 30 μM AZM. (I) Bar graph to compare peak Ca2+ current density at 0 mV between different groups in H. n = 8–13. We observed concentration‐dependent inhibition of Ca2+ currents in AZM‐treated iPSC‐CMs, and significant changes were achieved at 10 and 30 μM. ***
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p < 0.0001. (J) Comparison of steady‐state activation (SSA) and steady‐state inactivation (SSI) of Ca2+ current between control iPSC‐CMs and iPSC‐CMs with acute treatment of 1, 10, and 30 μM AZM. (K and L) Bar graphs to compare V1/2 of SSA and SSI of Ca2+ current between different groups in J. SSA, n = 7–12; SSI, n = 8–9. SSA curve of Ca2+ currents in 30 μM AZM‐treated iPSC‐CMs was significantly right‐shifted as compared to controls, whereas SSI curves of Ca2+ currents stayed unchanged between control and AZM‐treated iPSC‐CMs. *
p < 0.05. (M) Representative Na+ current tracings isolated from control iPSC‐CMs and iPSC‐CMs with acute treatment of 30 μM AZM. (N) Comparison of Na+ IV curve between control iPSC‐CMs and iPSC‐CMs with acute treatment of 10 and 30 μM AZM. Myocytes were derived from iPSC #3. (O) Bar graph to compare peak Na+ current density at −30 mV between different groups in N. n = 5‐11. Slight reduction of Na+ currents was noted by acute treatment of 10 μM AZM, and this change turned to be statistically significant upon 30 μM AZM treatment. ***
p < 0.001. (P) Comparison of SSA and SSI of Na+ current between control iPSC‐CMs and iPSC‐CMs with acute treatment of 10 and 30 μM AZM. (Q and R) Bar graphs to compare V1/2 of SSA and SSI of Na+ current between different groups in P. SSA, n = 5–10; SSI, n = 5–11. SSA curves of Na+ currents were comparable between control and AZM‐treated iPSC‐CMs. However, SSI curve of Na+ currents in 30 μM AZM‐treated iPSC‐CMs was significantly left‐shifted. *
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Electrophysiological effects of acute azithromycin (AZM) in induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs). (A) Representative field potential (FP) tracings recorded from iPSC‐CMs with acute treatment of 0, 0.1%, 0.3%, and 0.5% dimethyl sulfoxide (DMSO), respectively. (B) Acute effects of DMSO on beating period (BP), field potential duration (FPD), corrected field potential duration (FPDc), and field potential amplitude (FPA). (C) Representative FP tracings recorded from iPSC‐CMs with acute treatment of 0, 3, 10, 30, and 100 μM moxifloxacin (MXF), respectively. (D) Acute effects of MXF on BP, FPD, FPDc, and FPA. Prominent prolongation of FPD and FPDc was observed in response to acute treatment of MXF in a concentration‐dependent manner. BP was significantly increased at 30 and 100 μM, suggesting a slower effect on beating rate induced by MXF. Note that 30 and 100 μM MXF had largely decreased effect on FPA. Mycoytes were derived from three different iPSC lines. *
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p < 0.0001. (E) Representative FP tracings recorded from iPSC‐CMs with acute treatment of 0, 1, 3, 10, and 30 μM AZM, respectively. (F) Acute effects of AZM on BP, FPD, FPDc, and FPA. Beating rate, reflected by BP, was significantly increased started at the concentration of 3 μM AZM. Starting from 3 μM, acute treatment of AZM gave rise to markedly shortened FPD and FPDc. The FPA was significantly decreased by 10 and 30 μM AZM treatment. Mycoytes were derived from three different iPSC lines. *
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p < 0.0001. (G) Representative Ca2+ current tracings isolated from control iPSC‐CMs and iPSC‐CMs with acute treatment of 30 μM AZM. Myocytes were derived from iPSC #3. (H) Comparison of Ca2+ current‐voltage relationship curve (IV curve) between control iPSC‐CMs and iPSC‐CMs with acute treatment of 1, 10, and 30 μM AZM. (I) Bar graph to compare peak Ca2+ current density at 0 mV between different groups in H. n = 8–13. We observed concentration‐dependent inhibition of Ca2+ currents in AZM‐treated iPSC‐CMs, and significant changes were achieved at 10 and 30 μM. ***
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p < 0.0001. (J) Comparison of steady‐state activation (SSA) and steady‐state inactivation (SSI) of Ca2+ current between control iPSC‐CMs and iPSC‐CMs with acute treatment of 1, 10, and 30 μM AZM. (K and L) Bar graphs to compare V1/2 of SSA and SSI of Ca2+ current between different groups in J. SSA, n = 7–12; SSI, n = 8–9. SSA curve of Ca2+ currents in 30 μM AZM‐treated iPSC‐CMs was significantly right‐shifted as compared to controls, whereas SSI curves of Ca2+ currents stayed unchanged between control and AZM‐treated iPSC‐CMs. *
p < 0.05. (M) Representative Na+ current tracings isolated from control iPSC‐CMs and iPSC‐CMs with acute treatment of 30 μM AZM. (N) Comparison of Na+ IV curve between control iPSC‐CMs and iPSC‐CMs with acute treatment of 10 and 30 μM AZM. Myocytes were derived from iPSC #3. (O) Bar graph to compare peak Na+ current density at −30 mV between different groups in N. n = 5‐11. Slight reduction of Na+ currents was noted by acute treatment of 10 μM AZM, and this change turned to be statistically significant upon 30 μM AZM treatment. ***
p < 0.001. (P) Comparison of SSA and SSI of Na+ current between control iPSC‐CMs and iPSC‐CMs with acute treatment of 10 and 30 μM AZM. (Q and R) Bar graphs to compare V1/2 of SSA and SSI of Na+ current between different groups in P. SSA, n = 5–10; SSI, n = 5–11. SSA curves of Na+ currents were comparable between control and AZM‐treated iPSC‐CMs. However, SSI curve of Na+ currents in 30 μM AZM‐treated iPSC‐CMs was significantly left‐shifted. *
p < 0.05Electrophysiological effects of chronic AZM in iPSC‐CMs were also assessed. DMSO caused slight changes of FPs on day 1, which were diminished along the treatment and recovered to baseline on day 5 (Figure 2A,B, Table S3). Consistent with the observations of acute AZM treatment, dramatic changes of FPs were seen starting on day 1, resulting in accelerated beating rate and FPD shortening, which stayed constant from day 2 to day 5 postinduction (Figure 2C,D, Table S3). No arrhythmic activities were detected throughout 5‐day recordings. Ca2+ currents were largely reduced in AZM‐treated iPSC‐CMs while Na+ and K+ currents remained unchanged (Figure 2E‐P, Figure S8, Table S4).
FIGURE 2
Electrophysiological effects of chronic AZM in iPSC‐CMs. (A) Representative FP tracings recorded from iPSC‐CMs with chronic treatment of 0.1% DMSO on day 0–5. (B) Chronic effects of 0.1% DMSO on BP, FPD, FPDc, and FPA. *
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p < 0.01. (C) Representative FP tracings recorded from iPSC‐CMs with chronic treatment of 30 μM AZM on day 0–5. (D) Chronic effects of 30 μM AZM on BP, FPD, FPDc, and FPA. In line with the observations of acute effects by AZM, dramatic changes of FPs were seen starting on day 1, resulting in accelerated beating rate and shortened FPD and FPDc, which stayed constant from day 2 to day 5 postinduction. Conversely, we observed increased effect on FPA by chronic treatment of AZM, which was significantly decreased in acute AZM test. Mycoytes were derived from three different iPSC lines. *
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p < 0.0001. (E) Representative Ca2+ current tracings isolated from control and AZM (30 μM, 5 days)‐treated iPSC‐CMs. Myocytes were derived from iPSC #3. (F) Comparison of Ca2+ IV curve between control and AZM (30 μM, 5 days)‐treated iPSC‐CMs. (G) Bar graph to compare peak Ca2+ current density at 0 mV between the two groups in F. n = 9–16. ****
p < 0.0001. (H) Comparison of SSA and SSI of Ca2+ current between control and AZM (30 μM, 5 days)‐treated iPSC‐CMs. (I and J) Bar graphs to compare V1/2 of SSA and SSI of Na+ current between the two groups in H. SSA, n = 9–10; SSI, n = 7–10. (K) Representative Na+ current tracings isolated from control and AZM (30 μM, 5 days)‐treated iPSC‐CMs. Myocytes were derived from iPSC #3. (L) Comparison of Na+ IV curve between control and AZM (30 μM, 5 days)‐treated iPSC‐CMs. (M) Bar graph to compare peak Na+ current density at −30 mV between the two groups in L. n = 11–22. (N) Comparison of SSA and SSI of Na+ current between control and AZM (30 μM, 5 days)‐treated iPSC‐CMs. (O and P) Bar graphs to compare V1/2 of SSA and SSI of Na+ current between the two groups in N. SSA, n = 11–17; SSI, n = 11–12
Electrophysiological effects of chronic AZM in iPSC‐CMs. (A) Representative FP tracings recorded from iPSC‐CMs with chronic treatment of 0.1% DMSO on day 0–5. (B) Chronic effects of 0.1% DMSO on BP, FPD, FPDc, and FPA. *
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p < 0.01. (C) Representative FP tracings recorded from iPSC‐CMs with chronic treatment of 30 μM AZM on day 0–5. (D) Chronic effects of 30 μM AZM on BP, FPD, FPDc, and FPA. In line with the observations of acute effects by AZM, dramatic changes of FPs were seen starting on day 1, resulting in accelerated beating rate and shortened FPD and FPDc, which stayed constant from day 2 to day 5 postinduction. Conversely, we observed increased effect on FPA by chronic treatment of AZM, which was significantly decreased in acute AZM test. Mycoytes were derived from three different iPSC lines. *
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p < 0.0001. (E) Representative Ca2+ current tracings isolated from control and AZM (30 μM, 5 days)‐treated iPSC‐CMs. Myocytes were derived from iPSC #3. (F) Comparison of Ca2+ IV curve between control and AZM (30 μM, 5 days)‐treated iPSC‐CMs. (G) Bar graph to compare peak Ca2+ current density at 0 mV between the two groups in F. n = 9–16. ****
p < 0.0001. (H) Comparison of SSA and SSI of Ca2+ current between control and AZM (30 μM, 5 days)‐treated iPSC‐CMs. (I and J) Bar graphs to compare V1/2 of SSA and SSI of Na+ current between the two groups in H. SSA, n = 9–10; SSI, n = 7–10. (K) Representative Na+ current tracings isolated from control and AZM (30 μM, 5 days)‐treated iPSC‐CMs. Myocytes were derived from iPSC #3. (L) Comparison of Na+ IV curve between control and AZM (30 μM, 5 days)‐treated iPSC‐CMs. (M) Bar graph to compare peak Na+ current density at −30 mV between the two groups in L. n = 11–22. (N) Comparison of SSA and SSI of Na+ current between control and AZM (30 μM, 5 days)‐treated iPSC‐CMs. (O and P) Bar graphs to compare V1/2 of SSA and SSI of Na+ current between the two groups in N. SSA, n = 11–17; SSI, n = 11–12We next sought to determine whether AZM can induce cell death and morphological changes. Starting from 10 μM, we observed cytotoxic effects in AZM‐treated iPSC‐CMs in a concentration‐ and time‐dependent manner (Figure 3A, FigureS9). Significantly reduced cell viability was noted in three iPSC‐CM lines starting from 30 μM AZM (Figure 3B). We thus selected the 5‐day 30 μM AZM treatment plan for downstream assays. Importantly, we observed severe morphological phenotypes in AZM‐treated iPSC‐CMs, including formation of intracellular vacuoles and sarcomeric damage (Figure 3C,D, Figures S10‐S12). Reactive oxygen species (ROS) was significantly increased in AZM‐treated iPSC‐CMs, whereas anti‐oxidant markedly reversed the ROS amount elevation and ameliorated AZM‐induced cell viability reduction (Figure S13).
FIGURE 3
AZM causes cell death and morphological changes by interfering with lysosomes to impair autophagy flux and autophagosome maturation in iPSC‐CMs. (A) Line graph to compare the cell viability by CCK8 assay between control and AZM‐treated myocytes derived from three different iPSC lines at different concentrations. n = 4. *
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p < 0.0001. (B) Bar graph to compare the cell viability between control and 30 μM AZM‐treated myocytes derived from three different iPSC lines. n = 4. *
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p < 0.001. (C) Representative morphology of control and 30 μM AZM‐treated myocytes derived from iPSC #1. Red arrows indicate the vacuole formation. Scale bar = 100 μm. (D) Immunofluorescent staining of control and 30 μM AZM‐treated myocytes derived from iPSC #1 using TNNT2 (green) and α‐actinin (red). DAPI indicates nuclear staining (blue). Enlarged views showing cardiac sarcomeres in the two groups. Scale bar = 20 μm. (E) Representative confocal images of brightfield and LysoTracker staining in control iPSC‐CMs, AZM‐treated iPSC‐CMs, iPSC‐CMs treated with Bafilomycin A1 (BafA1) (10 nM, 12 h), and iPSC‐CMs treated with chloroquine (CQ) (30 μM, 12 h), respectively. Myocytes were derived from iPSC #3. Scale bar = 20 μm. As positive controls, iPSC‐CMs were treated with CQ or BafA1, which are both lysosomal inhibitors. Treatment of BafA1 expectedly decreased the acidity of lysosomes by inhibiting H+‐ATPase and led to a decrease of LysoTracker puncta staining. However, such effect was not seen in CQ‐treated iPSC‐CMs, which exhibited enhanced LysoTracker puncta staining as comapared to controls. Similar with CQ, AZM treatment resulted in markedly enlarged, dilatate and accumulated lysosomes. (F) Western blot analysis of the LAMP2 expression in control and AZM‐treated myocytes from three different iPSC lines. (G) Bar graph to compare the lysosomal associated membrane protein 2 (LAMP2) expression between the two groups. n = 3. **
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p < 0.001. (H) Representative transmission electron microscope (TEM) images of control and AZM‐treated myocytes derived from iPSC #3. Scale bar = 2 μm. Red and blue arrows indicate lysosomes and autophagosomes, respectively. (I) Representative confocal images of mCherry‐GFP‐LC3 expressed in control, AZM‐treated and CQ‐treated iPSC‐CMs. Red fluorescence (mCherry+GFP‐) indicates autolysosomes whereas yellow fluorescence (mCherry+GFP+) indicates autophagosomes. Myocytes were derived from iPSC #3. Scale bar = 10 μm. (J) Bar graph to compare the percentage of mCherry+GFP‐ puncta in control, AZM‐treated or CQ‐treated iPSC‐CMs. n = 15. ****
p < 0.0001. (K‐M) Western blot analysis of the microtubule associated protein 1 light chain (LC3)‐II/LC3‐I expression in control and AZM‐treated myocytes from three different iPSC lines. (N) Bar graph to compare the LC3‐II/LC3‐I expression between control and AZM‐treated iPSC‐CMs. n = 3. **
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p < 0.001. (O) Western blot analysis of the p62 expression in control and AZM‐treated myocytes from three different iPSC lines. (P) Bar graph to compare the p62 expression between control and AZM‐treated myocytes from three different iPSC lines. n = 3. *
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p < 0.001. (Q) Western blot analysis of the p62 and LC3‐II/LC3‐I expression in control iPSC‐CMs, BafA1‐treated iPSC‐CMs, AZM‐treated iPSC‐CMs, and iPSC‐CMs treated with AZM and BafA1. Myocytes were derived from iPSC #3. (R and S) Bar graphs to compare the p62 and LC3‐II/LC3‐I expression between different groups in Q. n = 3. **
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AZM causes cell death and morphological changes by interfering with lysosomes to impair autophagy flux and autophagosome maturation in iPSC‐CMs. (A) Line graph to compare the cell viability by CCK8 assay between control and AZM‐treated myocytes derived from three different iPSC lines at different concentrations. n = 4. *
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p < 0.0001. (B) Bar graph to compare the cell viability between control and 30 μM AZM‐treated myocytes derived from three different iPSC lines. n = 4. *
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p < 0.001. (C) Representative morphology of control and 30 μM AZM‐treated myocytes derived from iPSC #1. Red arrows indicate the vacuole formation. Scale bar = 100 μm. (D) Immunofluorescent staining of control and 30 μM AZM‐treated myocytes derived from iPSC #1 using TNNT2 (green) and α‐actinin (red). DAPI indicates nuclear staining (blue). Enlarged views showing cardiac sarcomeres in the two groups. Scale bar = 20 μm. (E) Representative confocal images of brightfield and LysoTracker staining in control iPSC‐CMs, AZM‐treated iPSC‐CMs, iPSC‐CMs treated with Bafilomycin A1 (BafA1) (10 nM, 12 h), and iPSC‐CMs treated with chloroquine (CQ) (30 μM, 12 h), respectively. Myocytes were derived from iPSC #3. Scale bar = 20 μm. As positive controls, iPSC‐CMs were treated with CQ or BafA1, which are both lysosomal inhibitors. Treatment of BafA1 expectedly decreased the acidity of lysosomes by inhibiting H+‐ATPase and led to a decrease of LysoTracker puncta staining. However, such effect was not seen in CQ‐treated iPSC‐CMs, which exhibited enhanced LysoTracker puncta staining as comapared to controls. Similar with CQ, AZM treatment resulted in markedly enlarged, dilatate and accumulated lysosomes. (F) Western blot analysis of the LAMP2 expression in control and AZM‐treated myocytes from three different iPSC lines. (G) Bar graph to compare the lysosomal associated membrane protein 2 (LAMP2) expression between the two groups. n = 3. **
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p < 0.001. (H) Representative transmission electron microscope (TEM) images of control and AZM‐treated myocytes derived from iPSC #3. Scale bar = 2 μm. Red and blue arrows indicate lysosomes and autophagosomes, respectively. (I) Representative confocal images of mCherry‐GFP‐LC3 expressed in control, AZM‐treated and CQ‐treated iPSC‐CMs. Red fluorescence (mCherry+GFP‐) indicates autolysosomes whereas yellow fluorescence (mCherry+GFP+) indicates autophagosomes. Myocytes were derived from iPSC #3. Scale bar = 10 μm. (J) Bar graph to compare the percentage of mCherry+GFP‐ puncta in control, AZM‐treated or CQ‐treated iPSC‐CMs. n = 15. ****
p < 0.0001. (K‐M) Western blot analysis of the microtubule associated protein 1 light chain (LC3)‐II/LC3‐I expression in control and AZM‐treated myocytes from three different iPSC lines. (N) Bar graph to compare the LC3‐II/LC3‐I expression between control and AZM‐treated iPSC‐CMs. n = 3. **
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p < 0.001. (O) Western blot analysis of the p62 expression in control and AZM‐treated myocytes from three different iPSC lines. (P) Bar graph to compare the p62 expression between control and AZM‐treated myocytes from three different iPSC lines. n = 3. *
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p < 0.001. (Q) Western blot analysis of the p62 and LC3‐II/LC3‐I expression in control iPSC‐CMs, BafA1‐treated iPSC‐CMs, AZM‐treated iPSC‐CMs, and iPSC‐CMs treated with AZM and BafA1. Myocytes were derived from iPSC #3. (R and S) Bar graphs to compare the p62 and LC3‐II/LC3‐I expression between different groups in Q. n = 3. **
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p < 0.001To further elucidate the underlying mechanisms, we performed RNA sequencing of control and AZM‐treated iPSC‐CMs, which demonstrated that differentially expressing genes (DEGs) were enriched in lysosome pathway (Figures S14‐S15). To test if AZM‐induced cytotoxicity correlates with lysosomes, we performed live‐cell confocal microscopy using pH‐sensitive LysoTracker as a specific dye for lysosomes., Similar with chloroquine (CQ), AZM caused markedly enlarged, dilatate, and accumulated lysosomes (Figure 3E). Expression of lysosomal associated membrane protein 2 (LAMP2), a specific marker for lysosomes, was significantly enhanced in AZM‐treated iPSC‐CMs (Figure 3F,G), whereas cathespin D expression was unchanged (Figure S16). Transmission electron microscope exhibited greatly increased number of lysosomes and autophagosomes in AZM‐treated iPSC‐CMs (Figure 3H). Given that the lysosome is a key factor in autophagy, we next investigated if AZM‐induced lysosomal changes may affect autophagy flux. We observed a high proportion of red puncta in untreated cells, indicating the basal state of the autolysosomes. In contrast, the proportion of red puncta was significantly lower in AZM‐ or CQ‐treated iPSC‐CMs (Figure 3I,J).Moving forward, we sought to determine how AZM affects autophagy. AZM‐treated iPSC‐CMs demonstrated a significantly increased protein expression of microtubule associated protein 1 light chain (LC3)‐II/LC3‐I and p62, pointing to suppressed late‐stage autophagy (Figure 3K‐P). Moreover, iPSC‐CMs were treated with bafilomycin A1 (BafA1) to block fusion of autophagosomes with lysosomes. BafA1 did not affect AZM‐induced p62 and LC3‐II/LC3‐I upregulation, further indicating that AZM causes impaired autophagy flux and autophagosome maturation (Figure 3Q‐S, Figure S17). However, expression of beclin 1 remained unchanged (Figure S18).We next assessed whether intervention of autophagy may rescue AZM‐induced cytotoxic phenotypes in iPSC‐CMs. Induction of autophagy by specific mTOR inhibitor Torin showed no rescuing effects (Figure 4A‐C, G, Figure S19‐S20). In contrast, inhibition of early‐stage autophagy by ULK1 inhibitor MRT68921 significantly restored AZM‐induced p62 and LC3‐II/LC3‐I upregulation, and effectively rescued the reduced cell viability phenotype, although failed to rescue vacuole formation and enhanced LAMP2 expression (Figure 4D‐F, H‐M, Figure S21‐S22). Interestingly, we found that removal of AZM‐containing medium significantly rescued AZM‐induced cytotoxic phenotypes, exhibiting not only restored cell viability, but also suppressed vacuole formation and rescued lysosomes (Figure 4N,O).
FIGURE 4
Rescuing AZM‐induced cytotoxicity by inhibition of early‐stage autophagy and medium removal in iPSC‐CMs. (A) Western blot analysis of p62 and LC3‐II/LC3‐I expression in control and AZM‐treated myocytes derived from iPSC #3 with or without Torin (2.5 nM, 5 days). (B and C) Bar graphs to compare p62 and LC3‐II/LC3‐I expression between different groups in A. n = 3. Torin did not alleviate AZM‐induced upregulation of p62 and even exacerbated the LC3‐II/LC3‐I upregulation phenotype caused by AZM. *
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p < 0.0001. (D) Western blot analysis of the p62 and LC3‐II/LC3‐I expression in control and AZM‐treated myocytes derived from iPSC #3 with or without MRT68921 (1 μM, 5 days), an ULK1 inhibitor (ULK1i). (E and F) Bar graphs to compare p62 and LC3‐II/LC3‐I expression between different groups in D. n = 3. *
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p < 0.0001. (G) Bar graph to compare the cell viability between control and AZM‐treated myocytes derived from iPSC #1 with or without Torin. n = 4. *
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p < 0.0001. (H) Bar graph to compare the cell viability between control and AZM‐treated myocytes derived from iPSC #1 with or without ULK1i. n = 4. 5‐day treatment of ULK1i effectively rescued the reduced cell viability phenotype in AZM‐treated iPSC‐CMs, whereas Torin did not show any rescuing effect and even exacerbated the cytotoxic phenotypes. *
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p < 0.0001. (I) Representative confocal images of brightfield and LysoTracker staining in control iPSC‐CMs, AZM‐treated iPSC‐CMs, ULK1i‐treated iPSC‐CMs, or iPSC‐CMs treated with AZM and ULK1i. Myocytes were derived from iPSC #1. Scale bar = 20 μm. (J) Western blot analysis of LAMP2, p62 and LC3‐II/LC3‐I expression in control and AZM‐treated myocytes derived from iPSC #1 with or without ULK1i. (K‐M) Bar graphs to compare LAMP2, p62 and LC3‐II/LC3‐I expression between different groups in J. n = 3. *
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p < 0.0001. (N) Bar graph to compare the cell viability between control iPSC‐CMs, AZM‐treated iPSC‐CMs, and AZM‐treated iPSC‐CMs with 1‐day, 2‐day or 3‐day AZM withdrawal. Myocytes were derived from iPSC #1. n = 12. ***
p < 0.001 and ****
p < 0.0001. (O) Representative confocal images of brightfield and LysoTracker staining in control iPSC‐CMs, AZM‐treated iPSC‐CMs, AZM‐treated iPSC‐CMs with 2‐day AZM withdrawal. Myocytes were derived from iPSC #1. Scale bar = 20 μm. Removal of AZM‐containing medium for 2 days significantly rescued AZM‐induced cytotoxic phenotypes in iPSC‐CMs. (P) Proposed work model of AZM‐induced cardiotoxicity. Electrophysiologically, high‐concentration AZM causes accelerated beating rate and dramatically shortened QTc in the absence of proarrhythmic risk in healthy control populations by acutely suppressing Na+ and Ca2+ channels, or chronically suppressing Ca2+ channel. Morphologically, high‐concentration AZM interferes with lysosomal activity to impair autophagy flux and autophagosome maturation. The futile and excessive autophagosome formation and accumulation confers vacuole formation, sarcomeric damage, and cardiomyocyte death. Inhibition of early‐stage autophagy to alleviate the burden of autophagosome accumulation by ULK1i may partially rescue the deleterious phenotypes
Rescuing AZM‐induced cytotoxicity by inhibition of early‐stage autophagy and medium removal in iPSC‐CMs. (A) Western blot analysis of p62 and LC3‐II/LC3‐I expression in control and AZM‐treated myocytes derived from iPSC #3 with or without Torin (2.5 nM, 5 days). (B and C) Bar graphs to compare p62 and LC3‐II/LC3‐I expression between different groups in A. n = 3. Torin did not alleviate AZM‐induced upregulation of p62 and even exacerbated the LC3‐II/LC3‐I upregulation phenotype caused by AZM. *
p < 0.05, **
p < 0.01, ***
p < 0.001, and ****
p < 0.0001. (D) Western blot analysis of the p62 and LC3‐II/LC3‐I expression in control and AZM‐treated myocytes derived from iPSC #3 with or without MRT68921 (1 μM, 5 days), an ULK1 inhibitor (ULK1i). (E and F) Bar graphs to compare p62 and LC3‐II/LC3‐I expression between different groups in D. n = 3. *
p < 0.05, ***
p < 0.001, and ****
p < 0.0001. (G) Bar graph to compare the cell viability between control and AZM‐treated myocytes derived from iPSC #1 with or without Torin. n = 4. *
p < 0.05, **
p < 0.01, and ****
p < 0.0001. (H) Bar graph to compare the cell viability between control and AZM‐treated myocytes derived from iPSC #1 with or without ULK1i. n = 4. 5‐day treatment of ULK1i effectively rescued the reduced cell viability phenotype in AZM‐treated iPSC‐CMs, whereas Torin did not show any rescuing effect and even exacerbated the cytotoxic phenotypes. *
p < 0.05, ***
p < 0.001, and ****
p < 0.0001. (I) Representative confocal images of brightfield and LysoTracker staining in control iPSC‐CMs, AZM‐treated iPSC‐CMs, ULK1i‐treated iPSC‐CMs, or iPSC‐CMs treated with AZM and ULK1i. Myocytes were derived from iPSC #1. Scale bar = 20 μm. (J) Western blot analysis of LAMP2, p62 and LC3‐II/LC3‐I expression in control and AZM‐treated myocytes derived from iPSC #1 with or without ULK1i. (K‐M) Bar graphs to compare LAMP2, p62 and LC3‐II/LC3‐I expression between different groups in J. n = 3. *
p < 0.05, **
p < 0.01, ***
p < 0.001, and ****
p < 0.0001. (N) Bar graph to compare the cell viability between control iPSC‐CMs, AZM‐treated iPSC‐CMs, and AZM‐treated iPSC‐CMs with 1‐day, 2‐day or 3‐day AZM withdrawal. Myocytes were derived from iPSC #1. n = 12. ***
p < 0.001 and ****
p < 0.0001. (O) Representative confocal images of brightfield and LysoTracker staining in control iPSC‐CMs, AZM‐treated iPSC‐CMs, AZM‐treated iPSC‐CMs with 2‐day AZM withdrawal. Myocytes were derived from iPSC #1. Scale bar = 20 μm. Removal of AZM‐containing medium for 2 days significantly rescued AZM‐induced cytotoxic phenotypes in iPSC‐CMs. (P) Proposed work model of AZM‐induced cardiotoxicity. Electrophysiologically, high‐concentration AZM causes accelerated beating rate and dramatically shortened QTc in the absence of proarrhythmic risk in healthy control populations by acutely suppressing Na+ and Ca2+ channels, or chronically suppressing Ca2+ channel. Morphologically, high‐concentration AZM interferes with lysosomal activity to impair autophagy flux and autophagosome maturation. The futile and excessive autophagosome formation and accumulation confers vacuole formation, sarcomeric damage, and cardiomyocyte death. Inhibition of early‐stage autophagy to alleviate the burden of autophagosome accumulation by ULK1i may partially rescue the deleterious phenotypesIn conclusion, low‐concentration AZM is electrophysiologically and morphologically noncardiotoxic, whereas high‐concentration AZM may cause dramatic QT shortening, cardiomyocyte death, and structural damage but has no proarrhythmic risk in healthy control populations. Our findings suggest that AZM can be prescribed when warranted, but attention should be paid to high‐risk patients with preexisting comorbidities (Figure 4P).
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
AUTHOR CONTRIBUTIONS
P.L., Y.F. and Q.S. designed and supervised the study. X.W., Z.P., J.W., H.W., H.F., T.G. and Y.F. performed the experiments and analyzed data. Z.P. and P.L. wrote the manuscript.
DATA AVAILABILITY STATEMENT
The data that support the findings of the study are available from the corresponding author upon reasonable request.Supporting InformationClick here for additional data file.
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Anand Krishnan V Iyer; José M Izquierdo; Yotaro Izumi; Valentina Izzo; Marja Jäättelä; Nadia Jaber; Daniel John Jackson; William T Jackson; Tony George Jacob; Thomas S Jacques; Chinnaswamy Jagannath; Ashish Jain; Nihar Ranjan Jana; Byoung Kuk Jang; Alkesh Jani; Bassam Janji; Paulo Roberto Jannig; Patric J Jansson; Steve Jean; Marina Jendrach; Ju-Hong Jeon; Niels Jessen; Eui-Bae Jeung; Kailiang Jia; Lijun Jia; Hong Jiang; Hongchi Jiang; Liwen Jiang; Teng Jiang; Xiaoyan Jiang; Xuejun Jiang; Xuejun Jiang; Ying Jiang; Yongjun Jiang; Alberto Jiménez; Cheng Jin; Hongchuan Jin; Lei Jin; Meiyan Jin; Shengkan Jin; Umesh Kumar Jinwal; Eun-Kyeong Jo; Terje Johansen; Daniel E Johnson; Gail Vw Johnson; James D Johnson; Eric Jonasch; Chris Jones; Leo Ab Joosten; Joaquin Jordan; Anna-Maria Joseph; Bertrand Joseph; Annie M Joubert; Dianwen Ju; Jingfang Ju; Hsueh-Fen Juan; Katrin Juenemann; Gábor Juhász; Hye Seung Jung; Jae U Jung; Yong-Keun Jung; Heinz Jungbluth; Matthew J Justice; Barry Jutten; Nadeem O Kaakoush; Kai Kaarniranta; Allen Kaasik; Tomohiro Kabuta; Bertrand Kaeffer; Katarina Kågedal; Alon Kahana; Shingo Kajimura; Or Kakhlon; Manjula Kalia; Dhan V Kalvakolanu; Yoshiaki Kamada; Konstantinos Kambas; Vitaliy O Kaminskyy; Harm H Kampinga; Mustapha Kandouz; Chanhee Kang; Rui Kang; Tae-Cheon Kang; Tomotake Kanki; Thirumala-Devi Kanneganti; Haruo Kanno; Anumantha G Kanthasamy; Marc Kantorow; Maria Kaparakis-Liaskos; Orsolya Kapuy; Vassiliki Karantza; Md Razaul Karim; Parimal Karmakar; Arthur Kaser; Susmita Kaushik; Thomas Kawula; A Murat Kaynar; Po-Yuan Ke; Zun-Ji Ke; John H Kehrl; Kate E Keller; Jongsook Kim Kemper; Anne K Kenworthy; Oliver Kepp; Andreas Kern; Santosh Kesari; David Kessel; Robin Ketteler; Isis do Carmo Kettelhut; Bilon Khambu; Muzamil Majid Khan; Vinoth Km Khandelwal; Sangeeta Khare; Juliann G Kiang; Amy A Kiger; Akio Kihara; Arianna L Kim; Cheol Hyeon Kim; Deok Ryong Kim; Do-Hyung Kim; Eung Kweon Kim; Hye Young Kim; Hyung-Ryong Kim; Jae-Sung Kim; Jeong Hun Kim; Jin Cheon Kim; Jin Hyoung Kim; Kwang Woon Kim; Michael D Kim; Moon-Moo Kim; Peter K Kim; Seong Who Kim; Soo-Youl Kim; Yong-Sun Kim; Yonghyun Kim; Adi Kimchi; Alec C Kimmelman; Tomonori Kimura; Jason S King; Karla Kirkegaard; Vladimir Kirkin; Lorrie A Kirshenbaum; Shuji Kishi; Yasuo Kitajima; Katsuhiko Kitamoto; Yasushi Kitaoka; Kaio Kitazato; Rudolf A Kley; Walter T Klimecki; Michael Klinkenberg; Jochen Klucken; Helene Knævelsrud; Erwin Knecht; Laura Knuppertz; Jiunn-Liang Ko; Satoru Kobayashi; Jan C Koch; Christelle Koechlin-Ramonatxo; Ulrich Koenig; Young Ho Koh; Katja Köhler; Sepp D Kohlwein; Masato Koike; Masaaki Komatsu; Eiki Kominami; Dexin Kong; Hee Jeong Kong; Eumorphia G Konstantakou; Benjamin T Kopp; Tamas Korcsmaros; Laura Korhonen; Viktor I Korolchuk; Nadya V Koshkina; Yanjun Kou; Michael I Koukourakis; Constantinos Koumenis; Attila L Kovács; Tibor Kovács; Werner J Kovacs; Daisuke Koya; Claudine Kraft; Dimitri Krainc; Helmut Kramer; Tamara Kravic-Stevovic; Wilhelm Krek; Carole Kretz-Remy; Roswitha Krick; Malathi Krishnamurthy; Janos Kriston-Vizi; Guido Kroemer; Michael C Kruer; Rejko Kruger; Nicholas T Ktistakis; Kazuyuki Kuchitsu; Christian Kuhn; Addanki Pratap Kumar; Anuj Kumar; Ashok Kumar; Deepak Kumar; Dhiraj Kumar; Rakesh Kumar; Sharad Kumar; Mondira Kundu; Hsing-Jien Kung; Atsushi Kuno; Sheng-Han Kuo; Jeff Kuret; Tino Kurz; Terry Kwok; Taeg Kyu Kwon; Yong Tae Kwon; Irene Kyrmizi; Albert R La Spada; Frank Lafont; Tim Lahm; Aparna Lakkaraju; Truong Lam; Trond Lamark; Steve Lancel; Terry H Landowski; Darius J R Lane; Jon D Lane; Cinzia Lanzi; Pierre Lapaquette; Louis R Lapierre; Jocelyn Laporte; Johanna Laukkarinen; Gordon W Laurie; Sergio Lavandero; Lena Lavie; Matthew J LaVoie; Betty Yuen Kwan Law; Helen Ka-Wai Law; Kelsey B Law; Robert Layfield; Pedro A Lazo; Laurent Le Cam; Karine G Le Roch; Hervé Le Stunff; Vijittra Leardkamolkarn; Marc Lecuit; Byung-Hoon Lee; Che-Hsin Lee; Erinna F Lee; Gyun Min Lee; He-Jin Lee; Hsinyu Lee; Jae Keun Lee; Jongdae Lee; Ju-Hyun Lee; Jun Hee Lee; Michael Lee; Myung-Shik Lee; Patty J Lee; Sam W Lee; Seung-Jae Lee; Shiow-Ju Lee; Stella Y Lee; Sug Hyung Lee; Sung Sik Lee; Sung-Joon Lee; Sunhee Lee; Ying-Ray Lee; Yong J Lee; Young H Lee; Christiaan Leeuwenburgh; Sylvain Lefort; Renaud Legouis; Jinzhi Lei; Qun-Ying Lei; David A Leib; Gil Leibowitz; Istvan Lekli; Stéphane D Lemaire; John J Lemasters; Marius K Lemberg; Antoinette Lemoine; Shuilong Leng; Guido Lenz; Paola Lenzi; Lilach O Lerman; Daniele Lettieri Barbato; Julia I-Ju Leu; Hing Y Leung; Beth Levine; Patrick A Lewis; Frank Lezoualc'h; Chi Li; Faqiang Li; Feng-Jun Li; Jun Li; Ke Li; Lian Li; Min Li; Min Li; Qiang Li; Rui Li; Sheng Li; Wei Li; Wei Li; Xiaotao Li; Yumin Li; Jiqin Lian; Chengyu Liang; Qiangrong Liang; Yulin Liao; Joana Liberal; Pawel P Liberski; Pearl Lie; Andrew P Lieberman; Hyunjung Jade Lim; Kah-Leong Lim; Kyu Lim; Raquel T Lima; Chang-Shen Lin; Chiou-Feng Lin; Fang Lin; Fangming Lin; Fu-Cheng Lin; Kui Lin; Kwang-Huei Lin; Pei-Hui Lin; Tianwei Lin; Wan-Wan Lin; Yee-Shin Lin; Yong Lin; Rafael Linden; Dan Lindholm; Lisa M Lindqvist; Paul Lingor; Andreas Linkermann; Lance A Liotta; Marta M Lipinski; Vitor A Lira; Michael P Lisanti; Paloma B Liton; Bo Liu; Chong Liu; Chun-Feng Liu; Fei Liu; Hung-Jen Liu; Jianxun Liu; Jing-Jing Liu; Jing-Lan Liu; Ke Liu; Leyuan Liu; Liang Liu; Quentin Liu; Rong-Yu Liu; Shiming Liu; Shuwen Liu; Wei Liu; Xian-De Liu; Xiangguo Liu; Xiao-Hong Liu; Xinfeng Liu; Xu Liu; Xueqin Liu; Yang Liu; Yule Liu; Zexian Liu; Zhe Liu; Juan P Liuzzi; Gérard Lizard; Mila Ljujic; Irfan J Lodhi; Susan E Logue; Bal L Lokeshwar; Yun Chau Long; Sagar Lonial; Benjamin Loos; Carlos López-Otín; Cristina López-Vicario; Mar Lorente; Philip L Lorenzi; Péter Lõrincz; Marek Los; Michael T Lotze; Penny E Lovat; Binfeng Lu; Bo Lu; Jiahong Lu; Qing Lu; She-Min Lu; Shuyan Lu; Yingying Lu; Frédéric Luciano; Shirley Luckhart; John Milton Lucocq; Paula Ludovico; Aurelia Lugea; Nicholas W Lukacs; Julian J Lum; Anders H Lund; Honglin Luo; Jia Luo; Shouqing Luo; Claudio Luparello; Timothy Lyons; Jianjie Ma; Yi Ma; Yong Ma; Zhenyi Ma; Juliano Machado; Glaucia M Machado-Santelli; Fernando Macian; Gustavo C MacIntosh; Jeffrey P MacKeigan; Kay F Macleod; John D MacMicking; Lee Ann MacMillan-Crow; Frank Madeo; Muniswamy Madesh; Julio Madrigal-Matute; Akiko Maeda; Tatsuya Maeda; Gustavo Maegawa; Emilia Maellaro; Hannelore Maes; Marta Magariños; Kenneth Maiese; Tapas K Maiti; Luigi Maiuri; Maria Chiara Maiuri; Carl G Maki; Roland Malli; Walter Malorni; Alina Maloyan; Fathia Mami-Chouaib; Na Man; Joseph D Mancias; Eva-Maria Mandelkow; Michael A Mandell; Angelo A Manfredi; Serge N Manié; Claudia Manzoni; Kai Mao; Zixu Mao; Zong-Wan Mao; Philippe Marambaud; Anna Maria Marconi; Zvonimir Marelja; Gabriella Marfe; Marta Margeta; Eva Margittai; Muriel Mari; Francesca V Mariani; Concepcio Marin; Sara Marinelli; Guillermo Mariño; Ivanka Markovic; Rebecca Marquez; Alberto M Martelli; Sascha Martens; Katie R Martin; Seamus J Martin; Shaun Martin; Miguel A Martin-Acebes; Paloma Martín-Sanz; Camille Martinand-Mari; Wim Martinet; Jennifer Martinez; Nuria Martinez-Lopez; Ubaldo Martinez-Outschoorn; Moisés Martínez-Velázquez; Marta Martinez-Vicente; Waleska Kerllen Martins; Hirosato Mashima; James A Mastrianni; Giuseppe Matarese; Paola Matarrese; Roberto Mateo; Satoaki Matoba; Naomichi Matsumoto; Takehiko Matsushita; Akira Matsuura; Takeshi Matsuzawa; Mark P Mattson; Soledad Matus; Norma Maugeri; Caroline Mauvezin; Andreas Mayer; Dusica Maysinger; Guillermo D Mazzolini; Mary Kate McBrayer; Kimberly McCall; Craig McCormick; Gerald M McInerney; Skye C McIver; Sharon McKenna; John J McMahon; Iain A McNeish; Fatima Mechta-Grigoriou; Jan Paul Medema; Diego L Medina; Klara Megyeri; Maryam Mehrpour; Jawahar L Mehta; Yide Mei; Ute-Christiane Meier; Alfred J Meijer; Alicia Meléndez; Gerry Melino; Sonia Melino; Edesio Jose Tenorio de Melo; Maria A Mena; Marc D Meneghini; Javier A Menendez; Regina Menezes; Liesu Meng; Ling-Hua Meng; Songshu Meng; 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Authors: Robert W Button; Sheridan L Roberts; Thea L Willis; C Oliver Hanemann; Shouqing Luo Journal: J Biol Chem Date: 2017-07-03 Impact factor: 5.157