ZnO nanoparticles induce acute arrhythmia and heart failure in mice by disturbing cardiac ion channels

Abstract

Background: The widespread use of zinc oxide nanoparticles (ZnO NPs) has raised safety concerns on human health. However, the effects and underlying mechanisms of ZnO NPs exposure on the heart, especially during acute exposure, have yet to be elucidated.

Methods: Two different sizes of ZnO NPs (40 nm and 100 nm) were selected and their in vivo effects on mouse heart were evaluated by echocardiography and electrocardiograms. Action potential, ion channel currents, and calcium recordings were employed to assess the electrical alterations in individual myocytes. The underlying mechanisms were further investigated by transmission electron microscopy (TEM) imaging, mitochondrial staining, LDH and ROS detection. In addition, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were utilized for translational exploration.

Results: Acute exposure to ZnO NPs induces cardiac dysfunction and arrhythmia in mice. Mechanistically, exposure to ZnO NPs did not significantly affect the IK1, but it markedly decreased I_Na and I_Ca-L currents, resulting in a reduced amplitude and shortened duration of the action potential in cardiomyocytes. These changes not only prolonged PR-interval and blocked A-V conduction that triggered cardiac arrhythmia, but also led to a diminished calcium transient, which contributed to heart failure. The downregulation of calcium transient upon ZnO NPs exposure was further confirmed in hiPSC-CMs. Meanwhile, acute exposure to ZnO NPs did not induce endocytosis, impair membrane integrity, or promote ROS production in the mitochondria of cardiomyocytes.

Conclusion: Acute ZnO NPs exposure causes heart failure and arrhythmia in mice by directly impacting ion channel function.

Keywords: calcium homeostasis; cardiac arrhythmia; heart failure; ion channels; zinc oxide nanoparticles.

Figure 1

Effects of ZnO NPs on mice ECG. (A) Representative images showed the effects of ZnO NPs-40 at different doses and exposure duration on ECG in mice. (B) Representative images showed the effects of ZnO NPs-100 at different doses and exposure duration on ECG in mice. (C) Changes of PR-interval after different doses of ZnO NPs-40 for 0-60 min. (D) Changes of PR-interval after different doses of ZnO NPs-100 for 0-60 min. P, P wave; R, R wave; QRS-T, QRS complex and T wave; PVC, premature ventricular contractions; AVB, atrioventricular conduction block. *P < 0.05, **P < 0.01, ***P < 0.001. n = 6 for control group; n = 6 for ZnO NPs-40 at 5 mg/kg group and 8 mg/kg group; n = 11 for ZnO NPs-40 at 10 mg/kg group; n = 7 for ZnO NPs-40 at 20 mg/kg group; n = 6 for ZnO NPs-100 at 5 mg/kg group, 10 mg/kg group and 20 mg/kg group; n = 10 for ZnO NPs-100 at 30 mg/kg group.

Figure 2

Representative image of transmembrane potential (TMP) of NMVMs before and after exposure to ZnO NPs-40 and ZnO NPs-100, respectively. (A) The effect of 10⁻⁶–10⁻⁴ g/ml ZnO NPs-40 on AP. (B) The effect of 10⁻⁶–10⁻⁴ g/ml ZnO NPs-100 on AP. AP. n = 10 for each group.

Figure 3

Rapid inhibitory effect of ZnO NPs on INa channel in NMVMs. (A,B) Typical I_Na currents recorded before and after exposure to ZnO NPs-40 and ZnO NPs-100 at 10⁻⁴ g/ml for 5 min, respectively. (C,D) Corresponding I–V curves of I_Na channels before and after exposure to ZnO NPs-40 and ZnO NPs-100. (E,F) The activation curves of I_Na channels at baseline and after exposure to ZnO NPs-40 and ZnO NPs-100 at 10⁻⁴ g/ml for 5 min. *P < 0.05, *P < 0.01, ***P < 0.001, ****P < 0.0001. n = 5.

Figure 4

Zno NPs suppress I_Ca−L currents and calcium transients in NMVMs and hiPSC-CMs. (A,C) Typical I_Ca−L current traces before and after exposure to ZnO NPs-40 and ZnO NPs-100 for 5 min, respectively. (B,D) I-V curves of I_Ca−L channels before and after exposure to ZnO NPs-40 and ZnO NPs-100 for 5 min, n = 6. (E,F) Representative line scan image of cardiomyocytes loaded with the Ca²⁺ indicator fluo-4 AM (5 μM) and exposed to ZnO NPs-40 and ZnO NPs-100 at a concentration of 10⁻⁴ g/ml. (G,H) Representative line scan image of hiPSC-CMs loaded with the Ca²⁺ indicator fluo-4 AM (5 μM) and exposed to ZnO NPs-40 and ZnO NPs-100 at a concentration of 10⁻⁴ g/ml. ns. not significant, *P < 0.05, **P < 0.01, n = 10.

Figure 5

Zno NPs suppress cardiac contractility. (A) Representative echocardiographic images of mice after normal saline tail vein injection, 20 mg/kg ZnO NPs-40 and ZnO NPs-100 respectively. (B) Changes in left ventricular ejection fraction, short-axis shortening rate, and heart rate of mice after tail vein injection of normal saline and 10–20 mg/kg of ZnO NPs-40. (C) Changes in left ventricular ejection fraction, short-axis shortening rate and heart rate in mice after tail vein injection of normal saline and 10–20 mg/kg ZnO NPs-100. ns. not significant, *P < 0.05 vs baseline, **P < 0.01 vs. baseline, ***P < 0.001 vs. baseline, ****P < 0.0001 vs. baseline. n = 6.

Figure 6

Effects of ZnO NPs on ROS production and mitochondrial density. (A) Fluorescence images of MitoSOXTM Red labeled ROS in NMVMs treated with 10⁻⁶ g/ml–10⁻⁴ g/ml ZnO NPs-40 for 10 min. (B) Fluorescence images of MitoSOXTM Red labeled ROS in the primary ventricular myocytes treated with 10⁻⁶ g/ml–10⁻⁴ g/ml ZnO NPs-100 for 10 min. (C) Fluorescence images of Mito-Tracker Green labeled mitochondrion in NMVMs treated with two kinds of ZnO NPs for 10 min, respectively. (D) Statistical results of average fluorescence intensity of ROS level in NMVMs exposed to two types of ZnO NPs for 5 min or 10 min respectively. (E) Statistical analysis of average fluorescence intensity of mitochondria in NMVMs exposed to two kinds of ZnO NPs for 10 min respectively. Scale bar, 50 μm. ns. no significance, *P < 0.05 vs. control, **P < 0.01 vs. control, ***P < 0.001 vs. control, ****P < 0.0001 vs. control. n = 3.