Hydrogen Attenuates Chronic Intermittent Hypoxia-Induced Cardiac Hypertrophy by Regulating Iron Metabolism

Abstract

The present study aimed to investigate the impact of hydrogen (H₂) on chronic intermittent hypoxia (CIH)-induced cardiac hypertrophy in mice by modulating iron metabolism. C57BL/6N mice were randomly allocated into four groups: control (Con), CIH, CIH + H₂, and H₂. The mice were exposed to CIH (21-5% FiO₂, 3 min/cycle, 8 h/d), and received inhalation of a hydrogen-oxygen mixture (2 h/d) for 5 weeks. Cardiac and mitochondrial function, levels of reactive oxygen species (ROS), and iron levels were evaluated. The H9C2 cell line was subjected to intermittent hypoxia (IH) and treated with H₂. Firstly, we found H₂ had a notable impact on cardiac hypertrophy, ameliorated pathological alterations and mitochondrial morphology induced by CIH (p < 0.05). Secondly, H₂ exhibited a suppressive effect on oxidative injury by decreasing levels of inducible nitric oxide synthase (i-NOS) (p < 0.05) and 4-hydroxynonenal (4-HNE) (p < 0.01). Thirdly, H₂ demonstrated a significant reduction in iron levels within myocardial cells through the upregulation of ferroportin 1 (FPN1) proteins (p < 0.01) and the downregulation of transferrin receptor 1 (TfR1), divalent metal transporter 1 with iron-responsive element (DMT1(+ire)), and ferritin light chain (FTL) mRNA or proteins (p < 0.05). Simultaneously, H₂ exhibited the ability to decrease the levels of Fe²⁺ and ROS in H9C2 cells exposed to IH (p < 0.05). Moreover, H₂ mediated the expression of hepcidin, hypoxia-inducible factor-1α (HIF-1α) (p < 0.01), and iron regulatory proteins (IRPs), which might be involved in the regulation of iron-related transporter proteins. These results suggested that H₂ may be beneficial in preventing cardiac hypertrophy, a condition associated with reduced iron toxicity.

Keywords: cardiac hypertrophy; chronic intermittent hypoxia; ferroportin 1; hepcidin; hydrogen; mitochondrial dysfunction; obstructive sleep apnea.

Figure 1

The cardiac hypertrophy and dysfunction induced by CIH exposure in mice. (A) M-model echocardiography in mice (n = 6). (B) The ejection fraction of the left ventricle (n = 6). (C) The fractional shortening (n = 6). (D) The left ventricular end-diastolic diameter (n = 6). (E) The left ventricular end-systolic diameter (n = 6). (F) The left ventricular posterior wall depth (n = 6). (G) The WGA immunofluorescence stain from Con, CIH, CIH+H₂, and H₂ groups (scale bar = 50 μm, n = 3). (H) The MYH7, Nppa, and Nppb mRNA levels in heart tissue. The data are presented as the means ± SEM. * p < 0.05, ** p < 0.01 vs. Con group. ^# p < 0.05 vs. CIH group.

Figure 2

The mitochondrial damage in the heart of CIH mice. (A) The TEM images of mitochondria in the heart (scale bar = 2 or 1 μm, n = 3). (B) The mitochondrial membrane potential (n = 6). (C,D) The expression and statistics of Fis-1, Drp-1, and Opa-1 protein levels (n = 6). (E) The Fis-1, Drp-1, and Opa-1 mRNA levels in heart tissue (n = 3). The data are presented as the means ± SEM. * p < 0.05, ** p < 0.01 vs. Con group. ^# p < 0.05 vs. CIH group.

Figure 3

The oxidative stress level in the cardiac tissue subjected to CIH. (A,B) The expression and statistics of 4-HNE and i-NOS protein levels (n = 4–6). (C,D) The expression and statistics of Nrf2, Keap-1, and HO-1 protein levels (n = 6). (E,F) The immunohistochemical staining and statistics of Nrf2 protein (scale bar = 100 μm, n = 3). The results are presented as the mean ± SEM. * p < 0.05, ** p < 0.01 vs. Con group. ^# p < 0.05, ^## p < 0.01 vs. CIH group.

Figure 4

The iron content and iron-related transport proteins in the cardiac tissue during CIH. (A) The Perls’ staining of heart tissue (scale bar = 100 μm, n = 3). (B) The mean density of Fe content as shown in panel A. (C) The total iron content in the cardiac tissue (n = 5). (D,E) The immunohistochemical staining of FPN1 protein (scale bar = 100 μm, n = 3). (F) The expression and statistics of FPN1 protein levels were measured by Western blot (n = 6). (G) The FPN1, DMT1(+ire), DMT1(-ire) mRNA levels in heart tissue (n = 6). (H,I) The expression and statistics of TfR1, FTL, FTH, and MtFt protein levels (n = 6). The results are presented as the mean ± SEM. ** p < 0.01 vs. Con group. ^# p < 0.05, ^## p< 0.01 vs. CIH group.

Figure 5

The Hepcidin level in the cardiac tissue during CIH and the effects on H9C2 cells after IH exposure. (A) The Hepcidin mRNA levels in heart tissue (n = 3). (B,C) The immunohistochemical staining of Hepcidin protein (scale bar = 100 μm, n = 3). (D) The cell viability of H9C2 cells treated with hydrogen for 0, 30, 60, 90, and 120 min (n = 6). (E) The cell viability of H9C2 cells treated with IH and hydrogen for 0, 30, 60, 90, and 120 min (n = 5). (F) The cell viability of H9C2 cells treated with 60 min (n = 6). (G) The ROS level induced by IH (n = 6). (H,I) The fluorescence intensity of Fe²⁺ (scale bar = 100 μm, n = 3). (J) The immunofluorescence double-label staining of FPN1 and Hepcidin (scale bar = 100 μm, n = 3). The results are presented as the mean ± SEM. * p < 0.05, ** p < 0.01 vs. Con group. ^# p < 0.05, ^## p < 0.01 vs. IH group.

Figure 6

The HIF-1α and related proteins in the cardiac tissue during CIH. (A,B) The immunohistochemical staining of HIF-1α protein (scale bar = 100 μm, n = 3). (C) The expression and statistics of HIF-1α protein levels (n = 6). (D,E) The expression and statistics of FBXL5, IRP-1, and IRP-2 protein levels (n = 6). The results are presented as the mean ± SEM. * p < 0.05, ** p < 0.01 vs. Con group. ^## p < 0.01 vs. CIH group.

Figure 7

A schematic representation of the proposed cardioprotective mechanism of hydrogen after CIH exposure. On the one hand, hydrogen attenuated ROS levels through the Keap-1/Nrf2 signaling pathway. On the other hand, hydrogen could inhibit iron overload-induced ROS generation and oxidative damage by adjusting hepcidin-FPN1, and HIF-1α-related IRPs’ signaling pathways.