Vidarabine, an anti-herpes agent, prevents occlusal-disharmony-induced cardiac dysfunction in mice

Abstract

We recently reported a positive relationship between occlusal disharmony and cardiovascular disease via activation of β-adrenergic signaling in mice. Furthermore, inhibition of type 5 adenylyl cyclase (AC5), a major cardiac subtype in adults, protects the heart against oxidative stress. Here, we examined the role of AC5 in the development of occlusal-disharmony-induced cardiovascular disease in bite-opening (BO) mice, prepared by cementing a suitable appliance onto the mandibular incisor. We first examined the effects of BO treatment on cardiac function in mice treated or not treated for 2 weeks with vidarabine, which we previously identified as an inhibitor of cardiac AC. Cardiac function was significantly decreased in the BO group compared to the control group, but vidarabine ameliorated the dysfunction. Cardiac fibrosis, myocyte apoptosis and myocyte oxidative DNA damage were significantly increased in the BO group, but vidarabine blocked these changes. The BO-induced cardiac dysfunction was associated with increased phospholamban phosphorylation at threonine-17 and serine-16, as well as increased activation of the Ca²⁺-calmodulin-dependent protein kinase II/receptor-interacting protein 3 signaling pathway. These data suggest that AC5 inhibition with vidarabine might be a new therapeutic approach for the treatment of cardiovascular disease associated with occlusal disharmony.

Keywords: Adenylyl cyclase; Apoptosis; Fibrosis; Occlusal disharmony; Signal transduction; β-Adrenergic signaling.

Fig. 1

Schematic illustrations of experimental procedure and bite-opening treatment, and comparison of body weight, cardiac muscle weight and lung weight among the groups. a Male 16-week-old C57BL/6 mice were divided into four groups: a normal control group (CTRL), a bite-opening (BO)-treated group, a vidarabine-treated group (V), and a BO plus vidarabine-treated (BO + V) group. Long-term infusion of vidarabine was performed for 14 days at a dose of 15 mg/kg/day with the osmotic mini-pumps, and the indicated measurements were made. b Schematic representation of a bite-opening (BO) in the form of a 0.7 mm increase in the vertical height of occlusion, obtained by cementing a composite resin onto the mandibular incisors to cause occlusal disharmony in mice. c Body weight was measured daily for all animals throughout the 2-week experimental period. ****P < 0.0001 (Control (n = 5) vs. BO (n = 5), ^####P < 0.0001 (Control vs. BO + V (n = 5), ^‡‡‡‡P < 0.0001 (BO vs. V (n = 5)), ^※※※※P < 0.0001 (V vs. BO + V) by two-way repeated-measures ANOVA followed by the Bonferroni post hoc test. d, e No significant difference in heart (d) or lung (e) size in terms of weight per tibial length ratio (mg/mm) at 2 weeks after BO treatment (Control (n = 5), BO (n = 5), vidarabine (n = 5) and BO + vidarabine (n = 5) groups; P = NS, not significantly different, vs. Control by one-way ANOVA) followed by the Tukey–Kramer post hoc test. Data are presented as mean ± SD

Fig. 2

Effect of vidarabine on BO-induced fibrosis in the heart. a Representative images of Masson-trichrome-stained sections of cardiac muscle in the Control (CTRL) (upper left), BO (upper right), vidarabine (V) (lower left) and BO + V; lower right) groups. b The area of fibrosis was significantly increased in the BO group (n = 5), but this increase was blocked in the BO + V group (n = 4). ^**P < 0.01, ^***P < 0.001 by one-way repeated-measures ANOVA followed by the Tukey–Kramer post hoc test. c Expression of α-SMA, a fibrosis-related gene, was significantly increased in the BO group (n = 4), but this increase was blocked in the BO + V group (n = 4). ^*P < 0.05, ^**P < 0.01 by one-way ANOVA followed by the Tukey–Kramer post hoc test. Data are presented as mean ± SD. Full-size images of immunoblots are presented in Additional file 1: Fig. S3

Fig. 3

Effect of vidarabine on BO-induced cardiac myocyte apoptosis. a TUNEL-positive nuclei (black arrows) in representative TUNEL-stained sections were counted in cardiac muscle in the Control (CTRL; upper left), BO (upper right), Vidarabine (V; lower left) and BO + V (lower right) groups. b The number of TUNEL-positive nuclei was significantly increased in the BO group (n = 5), but this increase was blocked in the BO + V group (n = 4). ^*P < 0.05 by one-way ANOVA followed by the Tukey–Kramer post hoc test. c The Bax/BCL-2 ratio was significantly increased in the BO group (n = 4), but this increase was blocked in the BO + V group (n = 5). ^*P < 0.05 by one-way ANOVA followed by the Tukey–Kramer post hoc test. Data are presented as mean ± SD. Full-size images of immunoblots are presented in Additional file 1: Fig. S4

Fig. 4

Effect of vidarabine on BO-induced oxidative stress in cardiac muscle. a Representative immunohistochemical images of oxidative DNA damage (8-OHdG) in cardiac muscle in the Control (CTRL; upper left), BO (upper right), vidarabine (V; lower left) and BO + V (lower right) groups. b 8-OHdG-positive nuclei were significantly increased in the BO group (n = 5), but this increase was blocked in the BO + V group (n = 4). ^*P < 0.05 by one-way ANOVA followed by the Tukey–Kramer post hoc test. c Representative SDS-PAGE of oxidized proteins in cardiac muscle homogenate prepared from Control (CTRL; lane 1), BO (lane 2), V (lane 3) and BO + V (lane 4) groups using the OxiSelect^TM Protein Carbonyl Immunoblot Kit. Full-size images of immunoblots are presented in Additional file 1: Fig. S6. d Oxidized proteins were significantly increased in the BO group (n = 5), but this increase was blocked in the BO + V group (n = 4). ^*P < 0.05, ^**P < 0.01 by one-way ANOVA followed by the Tukey–Kramer post hoc test. Data are presented as mean ± SD

Fig. 5

Effect of vidarabine on AC5, NOX4/2, XO, phospho-p38 and phospho-ASK1 in the heart of BO mice. a AC5 expression was similar in all four groups. NS, not significantly different, by one-way ANOVA followed by the Tukey–Kramer post hoc test. Full-size images of immunoblots are presented in Additional file 1: Fig. S7. b NOX4 expression was significantly increased in the BO group (n = 8), and this increase was significantly blocked in the BO + V group (n = 8). ^**P < 0.01, ^***P < 0.001 by one-way ANOVA followed by the Tukey–Kramer post hoc test. Full-size images of immunoblots are presented in Additional file 1: Fig. S8. c NOX2 expression was similar among the four groups (n = 5 each). NS, not significantly different, by one-way ANOVA followed by the Tukey–Kramer post hoc test. Full-size images of immunoblots are presented in Additional file 1: Fig. S9. d Expression of XO was significantly increased in the BO group (n = 5), and this increase was significantly blocked in the BO + V group (n = 7). ^**P < 0.01, ^***P < 0.001, ^***P < 0.001 by one-way ANOVA followed by the Tukey–Kramer post hoc test. Full-size images of immunoblots are presented in Additional file 1: Fig. S10. e Expression of phospho-p38 was significantly increased in the BO group (n = 4) and this was significantly blocked in the BO + V group (n = 5). ^*P < 0.05 by one-way ANOVA followed by the Tukey–Kramer post hoc test. Full-size images of immunoblots are presented in Additional file 1: Fig. S11. f Expression of phospho-ASK1 (Thr-845) was significantly increased in the BO group (n = 4) and this increase was significantly blocked in the BO + V group (n = 5). ^*P < 0.05 by one-way ANOVA followed by the Tukey–Kramer post hoc test. Full-size images of immunoblots are presented in Additional file 1: Fig. S12. Data are presented as mean ± SD

Fig. 6

Effect of vidarabine on BO-induced RIP3, phospho-CaMKII and phospho-PLN in the heart of BO mice. a Expression of RIP3, a key mediator of necroptosis, was significantly increased in the BO group (n = 4), but this increase was significantly blocked in the BO + V group (n = 4). ^**P < 0.01, ^***P < 0.001 by one-way ANOVA followed by the Tukey–Kramer post hoc test. Full-size images of immunoblots are presented in Additional file 1: Fig. S13. b Expression of phospho-CaMKII (Thr-286) was significantly increased in the BO group (n = 5), but this increase was significantly blocked in the BO + V group (n = 4). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 by one-way ANOVA followed by the Tukey–Kramer post hoc test. Full-size images of immunoblots are presented in Additional file 1: Fig. S14. c Expression of phospho-PLN (Thr-17) was significantly increased in the BO group (n = 5), but this increase was blocked in the BO + V group (n = 4). ^*P < 0.05, ^**P < 0.01 by one-way ANOVA followed by the Tukey–Kramer post hoc test. Full-size images of immunoblots are presented in Additional file 1: Fig. S15. d Expression of phospho-PLN (Ser-16) was significantly increased in the BO group (n = 5), but this increase was significantly blocked in the BO + V group (n = 5). ^*P < 0.05, ^**P < 0.01 by one-way ANOVA followed by the Tukey–Kramer post hoc test. Full-size images of immunoblots are presented in Additional file 1: Fig. S16. Data are presented as expressed as mean ± SD

Fig. 7

This scheme illustrates the proposed role of β-AR/Gsα/AC5 signaling in the heart of BO mice. β-AR/Gsα/AC5 signaling is activated by the BO treatment, leading to oxidative stress via activation of NOX4/ASK1/p38 and phosphorylation of CaMKII (Thr-286), which mediates PLN phosphorylation at Thr-17. In addition, cAMP derived from AC5 mediates oxidative stress and PLN phosphorylation at Ser-16. These changes might cause fibrosis, myocyte apoptosis and oxidative stress in the heart of BO mice, leading to cardiac dysfunction