The ages of the HDAC6 KO mice were slightly lower (-3.0%) than those of WT mice whereas the body weights of the HDAC6 KO mice were slightly higher (+ 13.2%) than the WT mice (see Table 1). As such the body weight/age ratio for the HDAC6 KO mice (0.34 ± 0.01) was higher than that of the WT mice (0.29 ± 0.1). The heavier body weights of the HDAC6 KO mice could certainly influence the findings related to flow parameters, namely, tidal volume, minute ventilation (tidal volume x frequency of breathing), peak inspiratory and expiratory flows, and EF50. Accordingly, (1) resting tidal volumes were lower in HDAC6 KO mice than in WT mice but were similar to one another when corrected for body weight, (2) corrections for body weights did not alter the lack of differences between the two groups with respect to minute ventilation, peak inspiratory and expiratory flows, or EF50. As also summarized in Table 1, resting frequency of breathing was lower in the HDAC6 KO mice than in the WT mice and inspiratory and expiratory times were longer in the HDAC6 KO mice. In addition, resting expiratory delay (Te-RT) was longer in HDAC6 KO mice than in WT mice. All other baseline parameters were similar between the two groups. It was also evident that the moment-to moment variability of many parameters was higher in the HDAC6 KO mice than in the WT mice. As shown in Table 2, values of standard deviation/corrected for mean (STDEV/mean) for frequency of breathing, minute ventilation, inspiratory time, expiratory time, end inspiratory pause and inspiratory drive were higher in the HDAC6 KO mice than in the WT mice. The finding that neither NEBI or NEBI/Freq were different between the groups suggests that the variability is due to simple changes in the breath-to-breath levels of frequency of breathing, for example, rather than enhanced expression of non-eupneic breathing including irregular breaths and apneas.
Ventilatory responses to hypoxic gas challenges and upon return to room-air
Frequency of breathing, tidal volume, minute ventilation
The frequency of breathing, tidal volume and minute ventilation values recorded before (Pre-HX), during a 5 min hypoxic gas (HX; 10% O2, 90% N2) challenge and upon return to room-air (Post-HX) in WT mice and in HDAC6 KO mice are shown in the left-hand panels of Fig. 1. As seen in Panel A, resting frequency of breathing prior to the hypoxic gas challenge was similar in WT and HDAC6 KO. The hypoxic challenge in WT mice elicited a typical increase in frequency of breathing associated with expected roll-off. The increases in Freq were somewhat higher in HDAC6 KO mice. The return to room-air elicited a typical increase in frequency of breathing in WT mice that gradually declined over the first 5 min of the recording period. The corresponding frequency of breathing values were higher in HDAC6 KO mice especially over the 5–15 min time-period when values had returned to baseline values in WT mice. The total responses summarized in Panel B show that the total frequency of breathing responses elicited by the hypoxic challenge (HX) and upon return to room-air (RA5 and RA15) were higher in HDAC6 KO mice than in WT mice.
As seen in Panel C, resting tidal volume prior to the hypoxic challenge was consistently higher in the HDAC6 KO mice than WT mice, perhaps due to the slightly larger body weights of the KO mice (see Table 1). The hypoxic challenge in the WT mice elicited a sustained increase in tidal volume that did not display roll-off. The increases in tidal volume were similarly robust in HDAC6 KO mice and reached higher values, mostly because of the higher resting values. Upon return to room-air, tidal volume values returned to pre-hypoxic levels within 5 min in WT mice but remained elevated in the HDAC6 KO mice over the 15 min recording period. The total responses summarized in Panel D shows that the total tidal volume responses elicited by hypoxia (HX) were similar in HDAC6 KO mice than in WT mice. The total responses upon return to room-air over the first 5 min (RA5) were similar in the HDAC6 KO and WT mice. The total tidal volume response over the entire 15 min recording period was significant in the HDAC6 KO mice but not the WT mice.
As seen in Panel E, resting minute ventilation prior to the hypoxic challenge was similar in WT and HDAC6 KO mice. The hypoxic challenge in the WT mice elicited a typical increase in minute ventilation that was associated with the expected roll-off. The increases in minute ventilation were consistently higher in HDAC6 KO mice. The return to room-air elicited a typical increase in minute ventilation in WT mice that subsided within 5 min. The corresponding MV values were higher in HDAC6 KO mice and remained elevated over the 15 min recording period. As seen in Panel F, the total minute ventilation responses elicited by hypoxia (HX) and return to room-air (RA 5 and RA15) were higher in the HDAC6 KO mice than in WT mice.
Inspiratory time, expiratory time, expiratory time/inspiratory time
The Ti, Te and Te/Ti values recorded before (Pre-HX), during a 5 min hypoxic gas (HX; 10% O2, 90% N2) challenge and upon return to room-air (Post-HX) in WT mice and in HDAC6 KO mice are shown in the left-hand panels of Fig. 2. As seen in Panel A, resting Ti prior to the hypoxic gas challenge tended to be higher in HDAC6 KO mice than WT mice during the 5 min period immediately before the hypoxic challenge. The hypoxic challenge in WT mice elicited a decrease in Ti that was associated with the expected roll-off. The decreases in Ti were somewhat greater in HDAC6 KO mice. The return to room-air elicited an initial further decrease in Ti in the WT mice that was followed by a gradual return toward pre-hypoxia values. Corresponding Ti values in HDAC6 KO mice showed the same pattern of changes as in WT mice but reached lower values most probably in part perhaps because of the lower values reached at the end of the hypoxic challenge. The total responses summarized in Panel B shows that the total Ti responses elicited by hypoxia (HX) and upon return to room-air (RA5 and RA15) were greater in HDAC6 KO mice than in WT mice.
As seen in Panel C, resting expiratory time prior to the hypoxic challenge tended to be higher in HDAC6 KO mice than WT mice during the 5 min period before the hypoxic challenge. The hypoxic challenge in WT mice elicited a decrease in Te that was associated with the expected roll-off. The decreases in Te were somewhat greater in HDAC6 KO mice. The return to room-air elicited an initial further decrease in Te in the WT mice that was followed by a rapid return to pre-hypoxia values. The corresponding Te values in HDAC6 KO mice followed the same pattern of changes as in WT mice but stayed at lower values for longer before returning to pre-hypoxia values. The total responses summarized in the Panel D shows that the total Ti responses elicited by hypoxia (HX) and upon return to room-air (RA5 and RA15) were greater in HDAC6 KO mice than in WT mice. As seen in Panel E, prior to the hypoxic challenge, resting Te/Ti was similar in HDAC6 KO and WT mice. The hypoxic challenge elicited minimal increases in Te/Ti in both groups. Upon return to room-air, Te/Ti rose further (with a spike evident in WT mice) before gradually declining to pre-hypoxia levels. As seen in Panel F, the the total Te/Ti responses that occurred during the hypoxic challenge (HX) and upon return to room-air (RA5 and RA15) were similar in HDAC6 KO and WT mice.
End Inspiratory Pause, End Expiratory Pause
EIP and EEP values recorded before (Pre-HX), during a 5 min hypoxic gas (HX; 10% O2, 90% N2) challenge and upon return to room-air (Post-HX) in WT mice and in HDAC6 KO mice are shown in the left-hand panels of Fig. 3. As seen in Panel A, resting EIP and EEP values prior to the hypoxic gas challenge were similar between HDAC6 KO mice and WT mice. The hypoxic challenge elicited similar sustained decreases in EIP in WT and HDAC6 KO mice. Upon return to room-air, the EIP values gradually returned to pre-HX levels in both groups. As seen in Panel B, the total EIP responses elicited by hypoxia and upon return to room-air (RA5 and RA15) were similar in HDAC6 KO and WT mice. As seen in Panel C, resting EEP values tended to be higher in the HDAC6 KO mice. The hypoxic challenge elicited an initial decrease in EEP in both groups of mice that tended to recover toward baseline toward the end of the challenge. Upon return to room-air, EEP rose well above baseline levels in WT mice but stayed at baseline values in the HDAC6 KO mice. As can be seen in Panel D, the total decreases in EEP elicited by the hypoxic challenge (HX) were similar in WT and HDAC6 KO mice. In contrast, the substantial total increases in EEP observed upon return to room-air in the WT mice (RA5 and RA15) were not seen in the HDAC6 KO mice.
Peak Inspiratory and Expiratory Flows
The PIF and PEF values recorded before (Pre-HX), during a 5 min hypoxic gas (HX; 10% O2, 90% N2) challenge and upon return to room-air (Post-HX) in WT mice and in HDAC6 KO mice are shown in the left-hand panels of Fig. 4. As seen in Panels A and C, resting PIF and PEF values were similar between the HDAC6 KO mice and WT mice. The HX challenge elicited sustained increases in PIF and PEF in WT mice. These responses were markedly augmented in HDAC6 KO mice. Upon return to room-air, PIF and PEF spiked upward before gradually returning to baseline levels in WT and HDAC6 KO mice with PIF and PEF values remaining substantially higher in HDAC6 KO mice. As seen in Panels B and D, total PIF and PEF responses elicited by hypoxia (HX) and upon return to room-air (RA5 and RA15) were greater in the HDAC6 KO than in the WT mice.
EF50, relaxation time, expiratory delay (Te-RT)
EF50, relaxation time, expiratory delay (Te-RT) values recorded before (Pre-HX), during a 5 min hypoxic gas (HX; 10% O2, 90% N2) challenge and upon return to room-air (Post-HX) in WT mice and in HDAC6 KO mice are shown in the left-hand panels of Fig. 5. As seen in Panel A, resting EF50 values were similar between the HDAC6 KO mice and WT mice. The HX challenge elicited greater increases in EF50 in the HDAC6 KO mice than in the WT mice. These responses were markedly augmented in HDAC6 KO mice. The responses that occurred upon return to room-air were also markedly greater in the HDAC6 KO mice. As seen in Panel B, the total increases EF50 elicited by the hypoxic challenge and upon return to room-air (RA5 and RA15) were greater in the HDAC6 KO than in the WT mice. As seen in Panel C, resting relaxation time values were similar in HDAC6 KO mice and WT mice. The hypoxic challenge elicited minimal changes in relaxation time in the WT mice but substantial initial falls in the HDAC6 KO mice that recovered within 3 min of the hypoxic challenge. Relaxation time dropped substantially upon return to room-air in the WT and HDAC6 KO mice. Relaxation time then rose above baseline values in the WT mice but returned to baseline values in HDAC6 KO mice. As seen in Panel D, total decreases in relaxation elicited by hypoxia and upon return to room-air (RA5 and RA15) were greater in HDAC6 KO than in WT mice. As seen in Panel E, resting expiratory delay (Te-RT) values tended to be higher in HDAC6 KO mice than in the WT mice immediately before the hypoxic challenge. The hypoxic challenge elicited slightly greater decreases in expiratory delay in HDAC6 KO mice. Expiratory delay returned rapidly to baseline levels in the WT mice whereas these values remained below baseline values in HDAC6 KO mice for 5–6 min. As seen in Panel F, the total decreases in expiratory delay elicited during hypoxic challenge (HX) were greater in HDAC6 KO than in WT mice. Decreases in expiratory delay upon return to room air (RA5 and RA15) occurred in the HDAC6 KO mice only.
Inspiratory and Expiratory Drives
Inspiratory and expiratory drive values recorded before (Pre-HX), during a 5 min hypoxic gas (HX; 10% O2, 90% N2) challenge and upon return to room-air (Post-HX) in WT mice and in HDAC6 KO mice are shown in the left-hand panels of Fig. 6. As seen in Panels A and C, resting drives prior to the hypoxic challenge were similar in HDAC6 KO mice and WT mice. The hypoxic challenge elicited sustained increases in inspiratory drive and expiratory drive in WT mice. These responses were markedly augmented in HDAC6 KO mice. Upon return to room-air, the inspiratory drive and expiratory drive values spiked upward in WT mice before gradually returning to baseline levels. These values were considerable higher in the HDAC 6 KO mice. As seen in Panels B and D, the responses elicited during the hypoxic challenge (HX) and upon return to room-air (RA5 and RA15) were greater in HDAC6 KO mice than WT mice.
NEBI, NEBI/Freq
Non-eupneic breathing index (NEBI) and NEBI/Freq values recorded before (Pre-HX), during a 5 min hypoxic gas (HX; 10% O2, 90% N2) challenge and upon return to room-air (Post-HX) in WT mice and in HDAC6 KO mice are shown in the left-hand panels of Fig. 7. As seen in Panel A, resting NEBI values prior to the hypoxic challenge were similar in HDAC6 KO mice and WT mice. The hypoxic challenge elicited similar increases in NEBI in WT and HDAC6 KO mice. The return to room-air caused remarkable increases in NEBI in both groups with NEBI subsiding more rapidly in the WT mice. These values were considerable higher in the HDAC 6 KO mice. As seen in Panel B, the increases in NEBI elicited during the hypoxic challenge (HX) and during the first 5 min upon return to room-air (RA5) were similar in the WT and HDAC6 KO mice whereas the overall increase in NEBY (RA15) was much greater in the HDAC6 mice. and RA15) were greater in HDAC6 KO mice than WT mice. As seen in Panels C and D, normalizing the changes in NEBI for the changes in frequency of breathing (NEBI/Freq) resulted in the changes during hypoxia (HX) and upon return to room-air (RA5 and RA15) that were similar in both groups of mice.
Body weight considerations
The heavier body weights of HDAC 6 KO mice may influence findings related to the effects of hypoxic challenge and return to room-air on flow parameters; tidal volume, minute ventilation, peak inspiratory and expiratory flows, and EF50. Table 3 summarizes the total arithmetic changes in ventilatory parameters during hypoxic gas challenge with flow parameters also shown corrected for body weight. The total arithmetic changes for frequency of breathing, inspiratory and expiratory times, expiratory time/expiratory time, end inspiratory and expiratory pauses, relaxation time, expiratory time-relaxation time, NEBI and NEBI/Freq provide the exact same conclusions given by the %change data provided in Figs. 1–7 in that hypoxia-mediated changes in these parameters were greater in the HDAC6 KO mice. Second, the delta changes in flow variables corrected for body weights (delta/body weight) for tidal volume, minute ventilation, peak inspiratory and expiratory flows, EF50 and inspiratory and expiratory drives were also consistent with %change data provided in Figs. 1–7 in that except for tidal volume, the hypoxia-mediated changes in these parameters were greater in the HDAC6 KO mice than in the WT mice.
Table 3
Total arithmetic responses during hypoxic gas challenge
Parameter | Parameter | WT mice | HDAC6 KO mice |
Flow-independent parameters |
Frequency (breaths/min) | Delta response | + 1570 ± 157* | + 3076 ± 212*,† |
Inspiratory Time (sec) | Delta response | -0.63 ± 0.07* | -1.33 ± 0.09*,† |
Expiratory Time (sec) | Delta response | -0.85 ± 0.16* | -2.12 ± 0.19*,† |
Expiratory Time/Inspiratory Time | Delta response | + 3.72 ± 2.27 | + 5.66 ± 1.82* |
End Inspiratory Pause (msec) | Delta response | -8.84 ± 1.44* | -10.28 ± 1.41* |
End Expiratory Pause (msec) | Delta response | -227 ± 54* | -781 ± 153*,† |
Relaxation Time (sec) | Delta response | -0.22 ± 0.13 | -0.82 ± 0.11*,† |
Expiratory Time - Relaxation Time | Delta response | -0.63 ± 0.07* | -1.26 ± 0.08*,† |
NEBI (%) | Delta response | + 292 ± 55* | + 420 ± 40*,† |
NEBI/Freq (%/(breaths/min)) | Delta response | + 0.46 ± 0.17* | + 0.40 ± 0.24* |
Flow-dependentparameters |
Tidal Volume (ml) | Delta response | + 0.91 ± 0.14* | + 1.34 ± 0.15* |
| Delta/Body Weight | + 0.033 ± 0.005* | + 0.045 ± 0.006 |
Minute Ventilation (ml/min) | Delta response | + 531 ± 74* | + 1024 ± 54*,† |
| Delta/Body Weight | + 19.3 ± 2.5* | + 33.6 ± 2.2*,† |
PIF (ml/sec) | Delta response | + 46.1 ± 6.6* | + 88.5 ± 4.3*,† |
| Delta/Body Weight | + 1.67 ± 0.22* | + 2.90 ± 0.17*,† |
PEF (ml/sec) | Delta response | + 23.6 ± 3.4* | + 47.7 ± 2.7*,† |
| Delta/Body Weight | + 0.863 ± 0.12* | + 1.55 ± 0.09*,† |
PEF/PIF | Delta response | -0.93 ± 0.79 | -2.39 ± 0.51 |
| Delta/Body Weight | -0.032 ± 0.029 | -0.081 ± 0.016 |
EF50 (ml/sec) | Delta response | + 0.98 ± 0.18* | + 2.34 ± 0.16*,† |
| Delta/Body Weight | 0.035 ± 0.006* | + 0.076 ± 0.006*,† |
Inspiratory Drive (ml/sec | Delta response | + 26.9 ± 2.4* | + 57.5 ± 3.0*,† |
| Delta/Body Weight | + 0.99 ± 0.08* | + 1.88 ± 0.11*,† |
Expiratory Drive (ml/sec) | Delta response | + 11.1 ± 1.6* | 24.3 ± 1.7*,† |
| Delta/Body Weight | + 0.40 ± 0.05* | + 0.80 ± 0.06*,† |
WT, wild-type; HDAC6 KO histone deacetylase 6 knockout mice; NEBI, non-eupneic breathing index; Freq, frequency of breathing; PIF, peak inspiratory flow; PEF, peak expiratory flow; EF50, airflow at 50% expired tidal volume. The data are presented as mean ± SEM. *P < 0.05, histone deacetylase 6 knockout (HDAC6 KO) mice versus wild-type (WT). |