Experiment
The current study was designed to characterize the cardiopulmonary response to hyperoxia, and to identify candidate susceptibility genes in mice. Electrocardiogram and ventilatory data were recorded continuously from 4 inbred (A/J, C3H/HeJ (C3), C57BL/6J (B6) and DBA/2J (D2)) and 29 recombinant inbred strains (AXB/BXA) during 96 hours of hyperoxia (100% oxygen).
AXB and BXA RI mice are inbred F2 lines derived from A/J and B6 parental strains. Cardiopulmonary function during hyperoxia (100% oxygen to 96 h until moribund or when HR reached z200 bpm) was assessed after baseline ECG and pulmonary function recording. ECG data were then used to calculate HR and heat rate variability (HRV). HRV calculations were made in two frequency ranges. Low-frequency (LF) HRV indicated the level of variation in sympathetic regulation of HR. High-frequency (HF) HRV indicated the level of variation in parasympathetic regulation of HR. Changes in HRV suggested a disturbance in the autonomic nervous system control of the heart. Total power (TP) was also calculated, which was a summation of LF and HF as a measure of total HR variation in the frequencies measured.
Genome-wide scans for QTL were performed for basal HR, HRV, ventilatory phenotypes and hyperoxia-induced changes in HR and HRV phenotypes in RI strains using RI phenotypes from 27 RI lines and the WebQTL resource (http://www.genenetwork.org). For linkage analyses with the RI strains, minute ventilation was quantified (VE), HR, and HRV responses by calculating the area under the curve for each parameter from 40 hours of exposure to the time at which HRs reached 220 bpm. The calculations, or time indices (TIs), were referred to as VE TI, HRTI, HFTI, LFTI, and TPTI. Higher TIs indicated greater resistance to hyperoxia.
Table 1: Linkage analysis identified the following QTL:
A significant QTL, Hrtiq1 (heart rate time index, QTL 1), mapped to Chromosome 9 between flanking markers rs3723670 and rs3655098, with a LOD score of 4.298, p<0.0134. The C57BL/6J allele increased the trait value at this locus.
A significant QTL, Lfhrvq1 (low frequency time index heart rate varaiblity, QTL 1), mapped to Chromosome 9 between markers rs4227700 and rs3655098, with a LOD score of 3.346, p<0.0340. The C57BL/6J allele increased the trait value at this locus.
Both significant QTL overlap on Chr 9 between 58.3229 and 89.4965 Mbp. A nonsynonymous coding SNP (G/T) was identified that causes an amino acid substitution in exon 8 of timeless interacting protein (Tipin) and the HRTI and LFTI responses in RI strains homozygous for the G allele were significantly greater than in the RI strains with the T allele. Similarly, a nonsynonymous coding SNP (C/T) in thrombospondin type I, domain containing 4 (Thsd4) was significantly associated with greater HRTI and LFTI responses in RI strains homozygous for the C allele compared with those with the T allele.
Suggestive QTL for heart rate time index were identified on Chr 3 mapping between rs3691363 and rs13477106, with a LOD score of 2.579, p<0.0705 and on Chr 5 mapping between rs365477 and rs13478527, with a LOD score of 2.403, p<0.0829.
A suggestive QTL for low frequency time index heart rate variablity was identified mapped to Chr 5 between rs3671202 and rs13478527 with a LOD score of 2.632, p<0.0671.
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