The Physiological And Biochemical Mechanisms Of Adaptation To Hypoxia In Phrynocephalus Erythrurus

The red tail toad-headed lizard Phrynocephalus erythrurus, which inhabits at elevations from 4500-5300 m on the northeastern Qinghai-Tibetan Plateau, is considered to be the highest living reptile in the world. The Qinghai-Tibetan Plateau is the highest plateau in the world with an average altitude of more than 4000 m above sea level. Hypoxia and cold are its main environmental features. In such extremely harsh environments, natives have evolved a series of unique adaptations to high altitude over a long-term natural selection. To investigate the possible mechanisms of adaptation to high-altitude hypoxia, Phrynocephalus erythrurus and another low altitude living lizard Phrynocephalus przewalskii (Lacertilia:Agamidae) were selected in the present study. We used physiological, biochemical and molecular biological methods to analyse some hypoxia-related characteristics including cardiopulmonary structure, cardiac and skeletal muscle capillary density, myoglobin (Mb) concentration, nitric oxide synthase (NOSs) activity, nitric oxide (NO) level and the differences in the expression of hypoxia-inducible factor (HIFs), vascular endothelial growth factor (VEGF-A), Mb and NOSs between two species. The results are presented as follows:Compared with P. przewalskii, the lung of P. erythrurus has more capillary branches and smaller capillary mesh which showed "regular polygon" arrangement. At the same time, the interior of lung in P. erythrurus contains numerous unique "honeycomb-like" structure, which is an effective form to increase the gas-exchange area between capillaries and faveolar. Thus, the P. erythrurus has a more complex lung structure and greater lung surface area, this may be a strategy to improve gas exchange efficiency. In addition, we also found the capillary density of cardiac and skeletal muscle in P. erythrurus was also significantly higher than low-altitude species. Increased capillary is one of adaptive characters to hypoxic environment in P. erythrurus, which is beneficial to the oxygen transport between the blood and tissues.HIF-la and HIF-2α(Hypoxia-inducible factor, HIFs) are key transcription factors that regulate a variety of cellular and systemic adaptations to hypoxia. Vascular endothelial growth factor (VEGF-A) has been proved to be a key regulator of angiogenesis, which is mainly regulated by HIFs. Studies showed that hypoxia may induce the expression of these genes. In this study, we found that HIF-la and HIF-2a mRNA expression in lung of P. erythrurus were significantly lower than those of P. przewalskii, which decreased by 50.3% and 65.1% (P<0.001). However, there was no difference in cardiac and skeletal muscle between two species. In P. erythrurus, VEGF-A mRNA expression in cardiac and skeletal muscle were significantly lower than P. przewalskii while no significant difference was observed in lung. These results indicated that the expressions of these genes in P. erythrurus exhibit not only obvious tissue specificity but also special passivation of response to hypoxia. Compared with P. przewalskii, the capillary density of lung, cardiac and skeletal muscle in P. erythrurus were increased, but the levels of VEGF-A mRNA expression did not increase. We suppose VEGF-A be regulated by post-transcriptional mechanism in P. erythrurus. NO consistent positive correlation was found between HIF-la, HIF-2a and VEGF-A mRNA expression in different tissues of two species, which may be associated with the existence of multiple regulation of these genes. In addition, we found some amino acid substitutions from the coding sequence of HIF-1α, HIF-2α and VEGF-A between two species. It remains uncertain whether these substitutions are related to high-altitude adaptations, and further research is needed.Myoglobin (Mb) is a cytoplasmic hemoprotein which plays an important role in oxygen storage and transport in cardiac and skeletal muscle under hypoxia. Using the RACE-PCR, we obtained the full-length of Mb from two species. The amino acid substitutions between Mb sequences of two species were found at Thr13Ile, Lys87Thr and His118Asn. Homology modeling results indicated that P. erythrurus myoglobin has a greater heme pocket, which may be in favour of oxygen binding and unloading with less steric hindrance. On the other hand, the levels of Mb mRNA in both cardiac and skeletal muscle in P. erythrurus were significantly larger than those in P. przewalskii. At protein level, Mb concentration in skeletal muscle in P. erythrurus was notably increased, but no significant difference was observed in cardiac muscle. Meanwhile, we also found that both Mb mRNA expression and protein concentration in cardiac muscle were approximately 2-fold higher than those in skeletal muscle in both species. Our study is consistent with the other researches that the Mb was increased in the long-term adaptive response to hypoxia. These results suggested that increased Mb is involved in the high-altitude adaptation to improve oxygen supply efficiency in P. erythrurus.Nitric oxide (NO), produced from L-arginine by nitric oxide synthases (NOS) including eNOS, nNOS and iNOS isoform, plays important roles in systemic and pulmonary vasodilation. Compared with P. przewalskii, there was no significant difference in eNOS mRNA level in tested tissues between two species. The nNOS mRNA levels in both cardiac and skeletal muscle in P. erythrurus were significantly increased by 248.9% and 119.2%. But nNOS expression in lung was not significantly different in two species. In P. erythrurus, iNOS expression was also increased significantly (up to 4.1-fold) in cardiac muscle. No significant difference was observed in lung and skeletal muscle between two species. The total NOS activity in skeletal muscle of P. erythrurus was significantly less than that of P. przewalskii while no difference was found in other tissues. Similarly, lower iNOS activity was found in cardiac and skeletal muscle in P. erythrurus compared to P. przewalskii, decreased by 66.9% and 46.7%. The iNOS activity in serum and lung between two species also showed no significant changes. In addition, the NO levels were significantly lower in P. erythrurus in all tested tissues. Our results indicate that nNOS and iNOS mRNA expression are selectively upregulated in cardiac and skeletal muscle of high altitude lizard but the long-term high altitude environment inhibits iNOS activity in cardiac and skeletal muscle and there are lower NO levels in different tissues in P. erythrurus. We speculate that the feature may be associated with the long-term exposure to hypoxic environment of high altitude, and it should be one of important adaptations in P. erythrurus.In conclusion, we found that P. erythrurus showed a series of special physiological and biochemical adaptations and molecular regulation mechanisms in cardiopulmonary and vascular systems and hypoxia-associated gene expression. This study will enrich and improve the knowledge and theory about adaptations to altitude hypoxia, help the conservation of biodiversity of the plateau, and may also provide a reference for the research and development of the high altitude medicine.