Knockout of Hsp70 genes significantly affects locomotion speed and gene expression in leg skeletal muscles of Drosophila melanogaster
The functions of the Hsp70 genes were studied using a line of D. melanogaster with knockout of six these genes out of thirteen. Namely, effect of knockout of Hsp70 genes on negative geotaxis climbing (locomotor) speed and the ability to adapt to climbing training (0.5-1.5 h/day, 7 days/week, 19 days) were examined. Seven- and 23-day-old Hsp70– flies demonstrated a comparable reduction (2-fold) in locomotor speed and widespread changes in leg skeletal muscle transcriptome (RNA-seq), compared to w1118 flies. To identify the functions of genes related to decreased locomotor speed the overlapped differentially expressed genes at both time points were analyzed: the up-regulated genes encoded extracellular proteins, regulators of drug metabolism and antioxidant response, while down-regulated genes encoded regulators of carbohydrate metabolism and transmembrane proteins. Additionally, in Hsp70– flies, activation of transcription factors related to disruption of the fibril structure and heat shock response (Hsf) were predicted, using the position weight matrix approach. In the control flies, adaptation to chronic exercise training was associated mainly with gene response to a single exercise bout, while the predicted transcription factors were related to stress/immune (Hsf, NF-kB, etc.) and early gene response. In contrast, Hsp70– flies demonstrated no adaptation to training, as well as significantly impaired gene response to a single exercise bout. In conclusion, the knockout of Hsp70 genes not only reduced physical performance, but also disrupted adaptation to chronic physical training, which is associated with changes in leg skeletal muscle transcriptome and impaired gene response to a single exercise bout. Overall design: The w1118 males (Vienna Drosophila Resource Center, ID 60000) and Hsp70– males (genotype w1118; Df(3R)Hsp70A, Df(3R)Hsp70B, Bloomington Drosophila Stock Center, ID 8841) with a knockout of 6 genes (Hsp70Aa, Hsp70Ab, Hsp70Ba, Hsp70Bb, Hsp70Bbb, Hsp70Bc) were used in this study. 1) Flies (both strains) were anesthetized with diethyl ether on the 7th, 23rd, and 47th days of life (after eclosion), legs were removed in phosphate-buffered saline and then placed in RNA extraction buffer for RNA isolation and RNA-sequencing (n = 4 pools (15 flies per pool) for each strain). 2) Flies (both strains) were trained from the 4th to the 22nd day after eclosion (19 days) using a homemade device, which rotated the tubes around the transverse axis every 15 s. Each training session began at 12:00; the training duration was 30 min on the first 2 days, 60 min on the second 2 days, and 90 min on the remaining days. Flies were anesthetized 24 h after the last training session; legs were removed for RNA isolation and RNA-sequencing (n = 4 pools (15 flies per pool) for each strain). 3) Untrained flies (7 days old, both strains) exercised for 2 h. Immediately thereafter, exercised and non-exercised flies were anesthetized and legs were removed for RNA isolation and RNA-sequencing (n = 4 pools (15 flies per pool) for each strain). https://link.springer.com/article/10.1134/S0026893324020109