Differential involvement of various sources of reactive oxygen species in thyroxin-induced hemodynamic changes and contractile dysfunction of the heart and diaphragm muscles

Abstract

Thyroid hormones are key regulators of basal metabolic state and oxidative metabolism. Hyperthyroidism has been reported to cause significant alterations in hemodynamics, and in cardiac and diaphragm muscle functions, all of which have been linked to increased oxidative stress. However, the definite source of increased reactive oxygen species (ROS) in each of these phenotypes is still unknown. The goal of the current study was to test the hypothesis that thyroxin (T4) may produce distinct hemodynamic, cardiac, and diaphragm muscle abnormalities by differentially affecting various sources of ROS. Wild-type and T4 mice with and without 2-week treatments with allopurinol (xanthine oxidase inhibitor), apocynin (NADPH oxidase inhibitor), L-NIO (nitric oxide synthase inhibitor), or MitoTEMPO (mitochondria-targeted antioxidant) were studied. Blood pressure and echocardiography were noninvasively evaluated, followed by ex vivo assessments of isolated heart and diaphragm muscle functions. Treatment with L-NIO attenuated the T4-induced hypertension in mice. However, apocynin improved the left-ventricular (LV) dysfunction without preventing the cardiac hypertrophy in these mice. Both allopurinol and MitoTEMPO reduced the T4-induced fatigability of the diaphragm muscles. In conclusion, we show here for the first time that T4 exerts differential effects on various sources of ROS to induce distinct cardiovascular and skeletal muscle phenotypes. Additionally, we find that T4-induced LV dysfunction is independent of cardiac hypertrophy and NADPH oxidase is a key player in this process. Furthermore, we prove the significance of both xanthine oxidase and mitochondrial ROS pathways in T4-induced fatigability of diaphragm muscles. Finally, we confirm the importance of the nitric oxide pathway in T4-induced hypertension.

Keywords: Allopurinol; Apocynin; Cardiac muscle; Diaphragm; Free radicals; L-NIO; MitoTEMPO; Thyroxin.

Fig 1

Blood pressure measurement showed that the NOS pathway is a major determinant of T4-induced hemodynamic changes in mice. Representative bar graphs show (A) systolic, (B) diastolic, and (C) mean arterial blood pressure and (D) heart rate in mice. T4, thyroxin; L-NIO, N⁵-(1-iminoethyl)-l-ornithine dihydrochloride; BPM, beats per minute. Control, n=13; T4, n=12; allopurinol, n=11; apocynin, n=12; L-NIO, n=10; MitoTEMPO, n=12. *Significant change compared with T4 group (one-way ANOVA followed by Dunnett multiple comparisons post hoc test, comparing all groups to T4).

Fig 2

Echocardiography analysis of the mouse hearts showed that the NADPH oxidase pathway is a key player in the T4-induced LV dysfunction regardless of the increased LV mass. Representative bar graphs show (A) left-ventricular (LV) mass, (B) stroke volume, (C) ejection fraction (EF), and (D) fractional shortening (FS) in mice. T4, thyroxin; L-NIO, N⁵-(1-iminoethyl)-l-ornithine dihydrochloride. Control, n=12; T4, n=13; allopurinol, n=11; apocynin, n=13; L-NIO, n=10; MitoTEMPO, n=12. *Significant change compared with T4 group (one-way ANOVA followed by Dunnett multiple comparisons post hoc test, comparing all groups to T4).

Fig 3

Contractile profile of isolated right-ventricular papillary muscles indicates rapid contraction and relaxation in T4-treated mice. Representative bar graphs show (A) mean isometric developed force (F_dev), (B) their corresponding time to peak (TTP), and (C) 50% relaxation time (RT₅₀) at 4 Hz stimulation frequency and 2 mmol/L Ca²⁺. Control, n=12; T4, n=11; allopurinol, n=10; apocynin, n=10; L-NIO, n=9; MitoTEMPO, n=9. *Significant change compared with T4 group (one-way ANOVA followed by Dunnett multiple comparisons post hoc test, comparing all groups to T4).

Fig 4

Isolated right-ventricular papillary muscles from T4-treated mice preserved their length-dependent activation, but lost their ability to respond to increased frequency or β-adrenergic stimulation. (A) Length-dependent activation. Peak isometric developed force (F_dev) value is expressed as a fraction of its corresponding F_dev at optimal length (L_opt) and presented as the mean ± SEM. (B) Force–frequency relationship. F_dev value is expressed as a fraction of its corresponding F_dev at the basal frequency of 4 Hz and presented as the mean ± SEM. (C) β-Adrenergic stimulation. F_dev value is expressed as a fraction of its corresponding F_dev at the basal frequency of 4 Hz before isoproterenol addition and presented as the mean ± SEM. Control, n=12; T4, n=11; allopurinol, n=10; apocynin, n=10; L-NIO, n=9; MitoTEMPO, n=9. ⁺Significant change as revealed by two-way ANOVA. *Significant change as revealed by one-way ANOVA followed by Dunnett multiple comparisons post hoc test, comparing all groups to T4.

Fig 5

T4-induced fatigability in diaphragm can be repressed by xanthine oxidase inhibitors and mitochondrial-targeted antioxidants at early and late stages, respectively. (A) Representative bar graphs show mean tetanic forces at different frequencies in the range of 20–180 Hz. (B) Fatigue/injury induced by repetitive contractions, once per second (frequency 100 Hz, duration 500 ms) for 66 s, followed by 15 min of rest and a repeat in diaphragm muscles. (C) Representative bar graphs show mean % change in relative tension before and after rest (rest recovery). Control, n=12; T4, n=11; allopurinol, n=13; apocynin, n=11; L-NIO, n=12; MitoTEMPO, n=13; n represents the number of muscles isolated from 8 mice/group. ⁺ Significant change as revealed by two-way ANOVA; *only control exhibited a significant change compared to T4, as revealed by one-way ANOVA; ^#both control and allopurinol exhibited significant changes compared to T4, as revealed by one-way ANOVA; ^×only MitoTEMPO exhibited a significant change compared to T4, as revealed by one-way ANOVA. One-way ANOVA was followed by Dunnett multiple comparisons post hoc test, comparing all groups to T4.