Knockout of Hsp70 genes modulates age-related transcriptomic changes in leg muscles, reduces locomotion speed and lifespan of Drosophila melanogaster

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Abstract

The study investigated the effect of knockout of six Hsp70 genes (orthologues of the mammalian genes Hspa1a, Hspa1b, Hspa2 and Hspa8) on age-related changes in gene expression in the legs of Drosophila melanogaster, which contain predominantly skeletal muscle bundles. For this, the leg transcriptomic profile was examined in males of the w1118 control line and the Hsp70– line on the 7th, 23rd and 47th days of life. In w1118 flies, an age-related decrease in the locomotion (climbing) speed (a marker of functional state and endurance) was accompanied by a pronounced change in the transcriptomic profile of the leg skeletal muscles, which is conservative in nature. In Hsp70– flies, the median lifespan was shorter and the locomotion speed was significantly lower compare to the control; at the same time, complex changes in the age-related dynamics of the skeletal muscle transcriptome were observed. Mass spectrometry-based quantitative proteomic showed that 47-day-old Hsp70– flies, compared with w1118, demonstrated multidirectional changes in the content of key enzymes of glucose metabolism and fat oxidation (glycolysis, pentose phosphate pathway, Krebs cycle, beta-oxidation and oxidative phosphorylation). Such dysregulation may be associated with a compensatory increase in the expression of other genes encoding chaperones (small Hsp, Hsp40, 60, and 70), which regulate specific sets of target proteins. Taken together, our data show that knockout of six Hsp70 genes slightly reduces the median lifespan of flies, but significantly reduces the locomotion speed, which may be associated with complex changes in the transcriptome of the leg skeletal muscles and with multidirectional changes in the content of key enzymes of energy metabolism.

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About the authors

I. V. Kukushkina

Institute of Biomedical Problems, Russian Academy of Sciences; Lomonosov Moscow State University

Email: danil-popov@yandex.ru

Биологический факультет, Московский государственный университет им. М.В. Ломоносова

Russian Federation, Moscow, 123007; Moscow, 119234

P. A. Makhnovskii

Institute of Biomedical Problems, Russian Academy of Sciences

Email: danil-popov@yandex.ru
Russian Federation, Moscow, 123007

V. G. Zgoda

Orekhovich Research Institute of Biomedical Chemistry

Email: danil-popov@yandex.ru
Russian Federation, Moscow, 119121

N. S. Kurochkina

Institute of Biomedical Problems, Russian Academy of Sciences

Email: danil-popov@yandex.ru
Russian Federation, Moscow, 123007

D. V. Popov

Institute of Biomedical Problems, Russian Academy of Sciences

Author for correspondence.
Email: danil-popov@yandex.ru
Russian Federation, Moscow, 123007

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Supplementary files

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2. Fig. 1. Knockout of six Hsp70 family genes reduces the median lifespan of D. melanogaster and has a pronounced negative effect on the speed of locomotion in the negative geotaxis test. a – Normalized number of reads (RNA sequencing) per open reading frame of each gene. n = 12 pools (legs of 15 flies) of each line. b and c – Proportion of surviving flies with normal and chronically elevated levels of motor (locomotor) activity. The p value is shown for comparison of curves, as well as for the median and maximum lifespan (proportion of survivors 50 and 10%); n > 150 in each line. TP – training. d – Age-related changes in the speed of locomotion in the negative geotaxis test. One and three asterisks represent p values ​​< 0.05 and < 0.001, respectively. n = 5–6 pools (10–30 flies per pool) of each line.

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3. Fig. 2. Aging induces pronounced and progressive changes in the transcriptome profile of the legs (composed predominantly of muscle fiber bundles) of w1118 flies. Knockout of six Hsp70 family genes modulates age-related changes in the transcriptome of leg skeletal muscles. (a) Number of unique and common genes that are expressed differently in 23- and 47-day-old flies compared to 7-day-old flies. n = 4 pools (legs of 15 flies) of each strain. (b and c) Functional enrichment analysis of genes that are up-regulated (b) and down-regulated (c). The number of genes in each functional category is shown; the heat map shows the p-value.

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4. Fig. 3. Knockout of six genes of the Hsp70 family disrupts the age-related dynamics of gene expression. The line represents the normalized expression values ​​(z-scale) of each gene of some enriched functional groups from Fig. 2b and c. The names and numbers of functional categories are given; n = 4 pools (legs of 15 flies) of each line.

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5. Fig. 4. Knockout of six Hsp70 family genes causes opposite changes in the levels of key enzymes of glucose and lipid metabolism (glycolysis, pentose phosphate pathway, Krebs cycle, beta-oxidation, and oxidative phosphorylation) in the leg muscles of 47-day-old flies. a – Changes in mRNA and protein levels in skeletal leg muscles of Hsp70– flies relative to w1118 flies. mRNA n = 4 pools (legs of 15 flies) for each line; protein n = 7–8 pools (legs of 10 flies) for each line. b – Functional enrichment analysis showed that proteins with different patterns of regulation at the mRNA level were enriched in different functional categories. The number of genes in each category is shown; the heat map shows the p-value. c – Opposite changes in the content of oxidative phosphorylation proteins in the legs of 47-day-old Hsp70– flies and w1118 flies (see Fig. S1, Supplementary Materials). N.S. – no change in protein content detected; N.D. – no protein detected.

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6. Supplementary Figure S1
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7. Supplementary Table S1
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