Inhibition of hydrogen sulfide restores normal breathing stability and improves autonomic control during experimental heart failure

Abstract

Cardiovascular autonomic imbalance and breathing instability are major contributors to the progression of heart failure (CHF). Potentiation of the carotid body (CB) chemoreflex has been shown to contribute to these effects. Hydrogen sulfide (H2S) recently has been proposed to mediate CB hypoxic chemoreception. We hypothesized that H2S synthesis inhibition should decrease CB chemoreflex activation and improve breathing stability and autonomic function in CHF rats. Using the irreversible inhibitor of cystathione γ-lyase dl-propargylglycine (PAG), we tested the effects of H2S inhibition on resting breathing patterns, the hypoxic and hypercapnic ventilatory responses, and the hypoxic sensitivity of CB chemoreceptor afferents in rats with CHF. In addition, heart rate variability (HRV) and systolic blood pressure variability (SBPV) were calculated as an index of autonomic function. CHF rats, compared with sham rats, exhibited increased breath interval variability and number of apneas, enhanced CB afferent discharge and ventilatory responses to hypoxia, decreased HRV, and increased low-frequency SBPV. Remarkably, PAG treatment reduced the apnea index by 90%, reduced breath interval variability by 40-60%, and reversed the enhanced hypoxic CB afferent and chemoreflex responses observed in CHF rats. Furthermore, PAG treatment partially reversed the alterations in HRV and SBPV in CHF rats. Our results show that PAG treatment restores breathing stability and cardiac autonomic function and reduces the enhanced ventilatory and CB chemosensory responses to hypoxia in CHF rats. These results support the idea that PAG treatment could potentially represent a novel pathway to control sympathetic outflow and breathing instability in CHF.

Keywords: autonomic function.; breathing; chemoreflex; heart failure; hydrogen sulfide.

Fig. 1.

Chronic heart failure (CHF) induced hypoxic and hypercapnic ventilatory potentiation in rats. A: ventilatory responses to 10% O₂ in awake CHF and sham rats. Note that 6 wk after coronary artery ligation, rats developed increased resting ventilation (21% O₂) as well as enhanced acute ventilatory responses to 10% O₂ compared with the sham-operated group. Minute ventilation (V̇e). B: hypercapnic ventilatory responses in CHF and sham rats. Heart failure rats exhibited enhanced 7% CO₂ ventilatory responses (balanced with 100% O₂ as well as with 21% O₂). *P < 0.05; n = 8 rats per group.

Fig. 2.

H₂S inhibition by dl-propargylglycine (PAG) similarly reduced the hypoxic ventilatory response in sham-operated and CHF rats. A: representative tracings displaying Vt response to a hypoxic insult (Fi_O2 ∼ 10%) before and after PAG treatment in one sham and one CHF rat. B: PAG effects on the ventilatory response to acute hypoxia (Fi_O2 ∼10%) in CHF and sham rats expressed as the difference from the baseline values. ▲, CHF (n = 8); ▼, CHF + PAG (n = 8); ○, sham (n = 8); ●, sham + PAG (n = 8). *P < 0.05 CHF vs. sham; +P < 0.05 CHF vs. CHF + PAG; ‡P < 0.05 sham vs. sham + PAG. C: no difference was found in the percent inhibition of the hypoxic ventilatory responses (HVR = 10% Fi_O2) induced by PAG between sham and CHF rats. P > 0.05; n = 8 rats per group.

Fig. 3.

PAG treatment enhanced the ventilatory responses to hypercapnia in CHF and sham-operated rats. A: effect of PAG treatment on the ventilatory responses to 7% Fi_CO2 in sham rats. PAG potentiated the hyperoxic-hypercapnic (7% Fi_CO2 balanced with 100% O₂) response in sham rats without affecting the ventilatory response to normoxic-hypercapnia (7% Fi_CO2 balanced with 21% O₂). B: effect of PAG treatment on the ventilatory responses to hypercapnia in CHF rats. Note that PAG treatment significantly increased both hypercapnic responses in CHF rats. *P < 0.05; n = 8 rats per group.

Fig. 4.

Effects of PAG on the hypoxic ventilatory response normalized for V̇o₂. Summary of the data showing the response to acute hypoxia (Fi_O2 ∼10%) in CHF and sham rats expressed as the difference from the baseline values and corrected by V̇o₂. ▲, CHF; ▼, CHF + PAG; ○, sham; ●, sham + PAG. *P < 0.05 CHF vs. sham; +P < 0.05 CHF vs. CHF + PAG; ‡P < 0.05 sham vs. sham + PAG. Inset: percent inhibition of the HVR (=10% Fi_O2) induced by PAG between sham and CHF rats. P > 0.05; n = 8 rats per group.

Fig. 5.

H₂S synthesis inhibition by PAG treatment restored breathing stability in CHF rats. A: representative tracings displaying tidal volume (Vt) and respiratory frequency (RR) in one sham-operated rat, and one CHF rat before and after PAG. Breathing instability was evident in rats with myocardial infarction-induced CHF. Note the presence of spontaneous cessation of breathing and changes in Vt and RR at rest. B: representative Poincare plots showing the breath-to-breath interval variability in the same rats illustrated in A. Cystathionine γ-lyase (CSE) inhibition by PAG improved breathing stability in CHF. C: summary of the data showing the effects of PAG on SD1 (short-term variability) and SD2 (long-term variability) in breathing interval. Both SD1 and SD2 were increased in CHF rats. PAG treatment significantly reduced both indices in rats with CHF. *P < 0.05; ***P < 0.001; n = 8 rats per group.

Fig. 6.

Effects of CSE inhibition with PAG on the incidence of apneas and hypopneas in rats with CHF. A: rats with CHF exhibited an increased apnea/hypoapnea index (AHI) compared with sham-operated rats. PAG treatment suppressed the augmented incidence of apneas in CHF rats below levels seen even in sham animals. B: effect of PAG on the frequency of spontaneous sighs. No significant effects of PAG were found in the number of sighs. *P < 0.05; **P < 0.01; n = 8 rats per group.

Fig. 7.

Effects of PAG on HRV and systolic blood pressure variability (SBPV) in CHF rats. A: rats with CHF displayed a marked decrease in HRV total power compared with sham rats (A, left). PAG treatment improved HRV in CHF rats (A, right). Note that PAG did not restore the HRV values of CHF rats back to normal levels. B: effect of PAG treatment on the low-frequency (LF) SBPV. Rats with CHF showed a significant increase in the LF domain compared with sham rats. PAG decrease the LF_SBPV in CHF rats. *P < 0.05; **P < 0.01. Sham, n = 4; sham + PAG, n = 2; CHF, n = 4; CHF + PAG, n = 4.

Fig. 8.

PAG treatment reduced carotid body (CB) chemoreceptor activity in CHF rats in response to hypoxia. A: representative recordings from the CB from a sham rat and the CB from a CHF rat showing the chemosensory response to acute hypoxia (Fi_O2 ∼10%, bar) before and after PAG treatment. B: summary data of the effect of PAG on the CB chemosensory function in CHF. PAG significantly reduced the overall CB chemosensory discharge curve in response to several O₂ levels in both CHF and sham rats (P < 0.05, two-way ANOVA). ▲, CHF (n = 4); ▼, CHF + PAG (n = 4); ○, sham (n = 4); ●, sham + PAG (n = 4).

Fig. 9.

Rats with CHF displayed normal CSE expression levels in the CB. Top: representative immunoblots showing no difference in the CSE expression levels in the CBs from CHF rats compared to those from sham rats. Bottom: summary data from 6 CBs from sham rats and 8 CBs from CHF rats run in triplicate. No significant difference was found between the 2 conditions.