Experiment
Decompensated heart failure is characterized by impaired cardiac performance despite cardiac hypertrophy. The authors previously showed that Balb/cJ and C57BL/6J mice develop cardiac hypertrophy to the same degree when treated with a combination of angiotensin II and high-salt diet (ANG II + Salt), but only Balb/cJ show impaired cardiac function associated with edema development and substantial mortality. The authors hypothesized that the different response to ANG II + Salt is due to the different genetic backgrounds of Balb/cJ and C57BL/6J.
To address their hypothesis, the authors performed QTL mapping of second filial generation (F2) of mice derived from a backcross between Balb/cJ and first filial generation (F1) of mice. Eleven 20 week-old male F1 mice derived from a cross between female Balb/cJ and male C57BL/6J mice (Janvier Labs) and 103 male F2 mice derived from a backcross between female Balb/cJ and male F1 mice (Taconic) 710 weeksA of age were used in the experiments.
Cardiac function was measured with echocardiography, glomerular filtration rate using FITC-inulin clearance, fluid and electrolyte balance in metabolic cages, and blood pressure with tail-cuff at baseline and on the fourth day of treatment with ANG II + Salt.
QTL analysis was performed on 88 individuals using data from 58,996 SNP markers (genome coordinates relative to GRCm38/mm10). The analysis was performed using R/qtl package. Standard interval mapping was used for normally distributed phenotypes and binary trait mapping for binary traits such as ascites. Genotype probabilities were calculated at the genetic markers assuming a genotyping error rate of 0.1% and genetic distances converted to recombination fraction with Carter-Falconer map function.
A total of nine QTL were found to be linked to different phenotypes in ANG II + Salt-treated F2 mice:
Dcardo1 (delta cardiac output 1) is significant for delta cardiac output and maps to Chr 3. The marker with the highest LOD score (3.3, approximated from Figure 2E) had a strong effect on delta cardiac output and the gene Sertm1 was closest to this marker.
Ivrt1 (isovolumic relaxation time 1) is significant for isovolumic relaxation time and maps to Chr 12. Mice heterozygous for the marker with highest LOD score (3.05, approximated from Figure 4B) had larger increase in isovolumic relaxation time compared with mice homozygous for the marker. Mice heterozygous for the marker with highest LOD score (3.05) had larger increase in isovolumic relaxation time compared with mice homozygous for the marker and Gphn was closest to this marker. Gene Ahr in the region had a previously known coding nonsynonymous SNP that differed between Balb/cJ and C57BL/6J.
QTL on chromosome 2 and 3 were linked to urine excretion and sodium excretion. Balb/cJ mice treated with ANG II + Salt excreted less sodium and water compared with C57BL/6J.
Urex1 (urine excretion 1) is significant for urine excretion and maps to Chr 3. F2 mice that were homozygous for the marker with the highest LOD score (3.1, approximated from Figure 5B) had higher increases in urine excretion than mice that were heterozygous for the marker. Negr1 was closest to the peak. ANG II + Salt also increased sodium excretion in F2 mice as is expected during high-salt intake.
QTL on chromosomes 2 and 3 were associated with differences in sodium excretion:
Sodex1 (sodium excretion 1) is significant for sodium excretion and maps to Chr 2, with a peak LOD score of 3.1 (approximated from Figure 6B). Ccm2 was closest to the peak of Sodex1.
Sodex2 (sodium excretion 2) is significant for sodium excretion and maps to Chr 3, with a peak LOD score of 3.4 (approximated from Figure 6B). Negr1 was closest to the peak of Sodex2.
The marker with highest LOD score on chromosomes 2 and 3 had different effects on delta sodium excretion in homozygous and heterozygous animals. There was an additive effect of Sodex1 and Sodex2, where animals that were homozygous for the top marker on Sodex1 and heterozygous for the top marker on Sodex2 excreted the least sodium. On the other hand, animals that were heterozygous for the top marker on Sodex1 and homozygous for the top marker on Sodex2, excreted the most sodium.
Sodex3 (sodium excretion 3, fractional) is significant for fractional sodium excretion and maps to Chr 2, with a peak LOD score of 3.9 (approximated from Figure 6F).
Ascdev1 (ascites development 1) is significant for ascites development and maps to Chr 7. Stk32c was closest to the QTL peak (peak LOD score of 4.25, approximated from Figure 7B). Balb/cJ mice treated with ANG II + Salt develop massive edema and ascites after 4 6 days on the treatment, while C57BL/6J mice do not. In F2 mice, 25% developed ascites during ANG II + Salt treatment. F2 mice that were homozygous for the marker with the highest LOD score had a much greater risk of developing ascites than animals that were heterozygous.
Bldph1 (blood pH 1) is significant for blood pH and maps to Chr 18, with a peak LOD score of 3.6 (approximated from Figure 8B). During heart failure, acid-base disturbances can be present due to shifts in renal electrolyte handling, lactatemia, and renal failure. In this study, the authors investigated if acid-base and blood gas parameters are genetically linked. A large variance in pH between F2 animals was linked to a QTL on chromosome 18. F2 mice homozygous for the marker with the highest LOD score (3.6) had lower pH than mice heterozygous for the marker. Tcf4 was closest to the peak, and Mc5r and 4930503L19Rik contained a previously known coding nonsynonymous SNP that differed between Balb/cJ and C57BL/6J.
Diadys1 (diastolic dysfunction 1, peak E velocity) is significant for peak E velocity (diastolic dysfunction) during ANG II + Salt treatment and maps to Chr 8, with a peak LOD score of 3.3 (approximated from Figure 3B). F2 mice heterozygous for the marker with the highest LOD score (3.3) had lower peak E velocity compared with F2 mice homozygous for the marker. Lrrc8e was closest to the peak, and Plat contained a previously known coding nonsynonymous SNP that differed between Balb/cJ and C57BL/6J.
Foxo1 was the only gene containing a previously known coding nonsynonymous SNP within the QTL linked to difference in cardiac output. The authors performed immunohistochemical staining of heart sections to validate the difference in Foxo1 activation between Balb/cJ and C57BL/6J. A higher percentage of nuclei were stained
in C57BL/6J after ANG II + Salt than in Balb/cJ, indicating a translocation of Foxo1 from the cytoplasm to the nuclei after ANG II + Salt treatment in C57BL/6J but not in Balb/cJ.
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