Response by Dunham-Snary and Archer, Regarding Letter by Hüttemann et al. and Article, “Ndufs2, a Core Subunit of Mitochondrial Complex I, Is Essential for Acute Oxygen-Sensing and Hypoxic Pulmonary Vasoconstriction”

To the Editor:

This Response Letter addresses discussion initiated by Hüttemann et al. in their Letter to the Editor, in response to our manuscript “Ndufs2, a Core Subunit of Mitochondrial Complex I, Is Essential for Acute Oxygen-Sensing and Hypoxic Pulmonary Vasoconstriction”¹. We stand by the results presented in the original publication and wish to expand our rationale regarding the screening of COX4I2 in vitro, and further the discussion of pre-clinical models of the complex pulmonary vascular oxygen-sensing mechanism.

Ndufs2 was identified as a candidate oxygen-sensor as a result of over 3 decades of research demonstrating that the Complex I inhibitor rotenone, mimics hypoxic pulmonary vasoconstriction (HPV) and recapitulates the effects of hypoxia in other oxygen-sensing tissues, including type 1 glomus cells of the carotid body² and adrenomedullary chromaffin cells³. As the quinone binding site within Complex I, Ndufs2 is a primary driver of Complex I activity. It is also the rotenone binding site⁴ and is both redox-sensitive⁵ and a target of posttranslationalmodification⁶. Our finding that Ndufs2 is essential for acute oxygen-sensing in the pulmonary vasculature reinforces previously published work by Fernández-Agüera et al. identifying Ndufs2 as the oxygen-sensor of the carotid body⁷, as well as the hypothesis that oxygen-sensing tissues have common sensor(s)⁸. We also made every effort to examine the theories of other groups by screening multiple subunits implicated in pulmonary vascular oxygen-sensing beyond our own hypothesis, including the Riekse Fe-S subunit⁹ and COX4i2¹⁰.

We do not believe the COX4i2 data in our original manuscript was misinterpreted, given the information available at the time of publication. While we cannot comment on unpublished data from other groups, we found that an ~50% knockdown of COX4i2 (verified at the gene and protein level with appropriate statistical replicates) did not result in any reduction of HPV, dose-dependent or otherwise.

It is also pertinent to address the importance of modelling robust HPV as well as significant differences in experimental design utilized by various groups. While Sommer et al. presents a thorough examination of COX4i2 in oxygen-sensing, it cannot be ignored that the range for ΔPAP in their acute hypoxia studies spans only 0–1 mmHg¹⁰. ΔPAP of 1.0 ± 0.11 mmHg for wild-type mice is far below the previously published hypoxia-induced ΔPAP of ~10 mmHg reported by Schwenke et al.¹¹, and thus it remains difficult to interpret the physiological relevance of this model with such small hypoxic changes in mPAP. Further, since in vitro and in vivo models as well as experimental conditions, including degree of hypoxia, duration of hypoxic exposure, and numerous measurement techniques/assays are not identical, somewhat divergent results with respect to the exact identity of the oxygen-sensor, are not entirely surprising. In the fullness of time, it is likely to be confirmed that all proposed subunits play different roles in pulmonary vascular oxygen-sensing, providing a degree of redundancy similar to that found in other biological systems¹².

The evolution of the vascular oxygen-sensing field has resulted in a large degree of consensus: it is widely accepted that the mitochondrion is the oxygen-sensor, that at least one (and likely multiple) electron transport chain protein(s) sense changes in PO₂ in a redoxdependent manner, and that mitochondrially-derived reactive oxygen species are the secondary messenger, altering the cellular milieu to stimulate the closing of voltage-gated potassium (Kv) channels, opening of voltage-gated L-type calcium channels, and subsequent vasoconstriction.

In conclusion, we appreciate the concerns raised by Hüttemann et al. and encourage other groups to consider testing the molecular targets identified by their colleagues. Discussing divergent findings in the field of oxygen sensing with curiosity and openness is consistent with the best spirit of scientific debate.