Changes in cardiac function and structure during the aging process in Drosophila

(A-E) Heart rate (HR), heart period (HP), diastolic interval (DI), systolic interval (SI), and arrhythmia index (AI) in w1118 10-day-old, 30-day-old, and 50-day-old Drosophila. (F) Representative M-mode traces of heartbeats in 10d, 30d, and 50d Drosophila. The blue horizontal line represents a complete cardiac cycle, with red indicating the DI and green indicating the SI. Red vertical lines represent diastolic diameter (DD), and green vertical lines represent systolic diameter (SD). “*” indicates bradycardia, and “#” indicates incomplete relaxations. Scale bar: 100 μm. (G) Percentage of Drosophila with a diastolic interval (DI) greater than 1 second (indicating bradycardia) and percentage with a DI less than 0.06 seconds (indicating incomplete relaxations), calculated based on M-mode traces. (H-K) DD, SD, fractional shortening (FS), and cardiac output (CO) in 10d, 30d, and 50d Drosophila. Cardiac function measurements: n=25-35. (L-N) Phalloidin-stained images of Drosophila hearts acquired using the Leica somatic fluorescence microscopy. The arrow points in the direction of the head of the fruit fly. Magnified images of the heart tube at the 2nd-3rd abdominal segments are shown in L’-N’, corresponding to the regions imaged by M-mode. Prominent myofibrillar gaps are indicated by white arrows. n=5-8. (O) Left: Instantaneous climbing height at the 5th second for 10d, 30d, and 50d Drosophila. The climbing tube is divided into nine equal sections, with scores ranging from 1 to 9 from bottom to top. The average climbing score for each group is calculated as the climbing index (right). n=100-150. One-way ANOVA followed by Tukey multiple comparisons test. *** p < 0.001, ** p < 0.01, * p < 0.05.

Effects of miR-283 knockout and overexpression on heart function and climbing ability

(A) RT-qPCR analysis of miR-283-5p expression levels in the whole body, heart muscle, and brain of control (w1118), miR-283 heterozygous knockout (mir-283KO/+), homozygous knockout (mir-283KO), as well as control (Tub> w1118) and miR-283 overexpression (Tub>mir-283OE) Drosophila. Whole body: n=3-5; heart muscle and brain: n=20-30. (B-H) Heart rhythm-related indicators. Scale bar: 100 μm. (I-L) Heart pumping capacity-related indicators. n=25-35. (M) Left: Representative climbing heights at the 5th second for each group of Drosophila. Right: Corresponding climbing index. n=100-150. Exceptionally, Tub>mir-283OE group had a high lethality rate, and thus the number of flies available for testing was n=6. One-way ANOVA followed by Tukey multiple comparisons test was used for comparisons between w1118, mir-283KO/+, and mir-283KO; Student t-test was used for comparisons between Tub> w1118 and Tub>mir-283OE. *** p < 0.001, ** p < 0.01, * p < 0.05.

Effects of miR-283-specific knockdown in myocardium or LNvs on cardiac function, climbing ability, and lifespan

(A) Virgin flies expressing UAS-mCherry.mir-283.sponge were crossed with w1118, Hand-Gal4, and Pdf-Gal4 male flies. Spontaneous fluorescence images of the myocardium in the F1 generation were captured using a Leica stereomicroscope. White arrows points in the direction of the head of the flies. (B) Virgin flies expressing UAS-mCherry.mir-283.sponge were crossed with w1118 and Pdf-Gal4 male flies. Brightfield (left), spontaneous fluorescence (middle), and merged (right) images of the Drosophila brain in the F1 generation were captured using a Leica stereomicroscope. Scale bar: 100 μm. (C-I) and (J-M) Represent heart rhythm-related indicators and heart pumping capacity-related indicators, respectively. n=20-35. One-way ANOVA followed by Tukey multiple comparisons test. *** p < 0.001, ** p < 0.01, * p < 0.05. Scale bar: 100 μm. (N) RT-qPCR analysis of the relative expression levels of miR-283-5p in the myocardium of miR-283 knockdown flies (Hand>mir-283SP) compared to the control group (w1118>mir-283SP) using U6 as the reference gene. n=20-30. Student t-test. * p < 0.05. (O) Climbing index of control, myocardial miR-283 knockdown, and LNvs miR-283 knockdown flies. n=110-200. One-way ANOVA followed by Tukey multiple comparisons test. *** p < 0.001, ** p < 0.01. (P) Survival curves (left) and mean lifespan (right) of control, myocardial miR-283 knockdown, and LNvs miR-283 knockdown flies. n=115-135. One-way ANOVA followed by Tukey multiple comparisons test. * p < 0.05.

miR-283 Knockdown in LNvs triggers brain and heart aging

(A) RT-qPCR analysis of miR-283 expression levels in the myocardium and whole brain of 10d and 30d control (w1118>mir-283SP), myocardial miR-283 knockdown (Hand>mir-283SP), and LNvs miR-283 knockdown (Pdf>mir-283SP) Drosophila. n=20-30. (B-D) Representative images of SA-β-gal staining in the brain of w1118>mir-283SP flies at 10d and 30d, and(Pdf>mir-283SP flies at 10d. n=5-8. (E) Percentage of SA-β-gal-positive staining in the brain analyzed using Image J. (F-H) Representative images of SA-β-gal staining in the heart of w1118>mir-283SP flies at 10d and 30d, and Pdf>mir-283SP flies at 10d. The outline of the heart tube is outlined with a red dashed line. Black arrows points in the direction of the head of the flies. Red arrows point to significant SA-β-gal precipitate areas. n=5-8. (I) SA-β-gal-positive staining was analyzed as a percentage of the total myocardium using Image J. One-way ANOVA, * p < 0.05. (J) Complete myocardium Phalloidin staining and imaging. The red dashed box corresponds to the M-mode capture area, which was imaged at 600× magnification to obtain (K-M). Transverse circular fibers represent the muscles responsible for myocardial contractile function, while longitudinal fibers represent the ventral longitudinal muscles. Significant gaps between myofibrils are indicated with“*”. n=5-8. One-way ANOVA followed by Tukey multiple comparisons test. *** p < 0.001, ** p < 0.01.

Prediction and validation of potential target genes of miR-283

(A) Homology alignment of the Drosophila miR-283-5p sequence with other species. (B) Prediction and enrichment analysis of target genes of miR-283-5p using the DIANA microT CDS online tool. (C-J) qPCR analysis of the effects of aging (10d and 30d w1118>mir-283SP) and miR-283 knockdown in LNvs (Pdf>mir-283SP) on the expression levels of miR-283-5p and its predicted target genes in the brain and heart. n=20-30. (K) Prediction of the targeted binding site between miR-283-5p and the 3’UTR of cwo using the RNAHybrid online tool, including thermodynamic values and secondary structure. (L) Dual-luciferase activity assay to determine the relationship between miR-283 and the 3’UTR of cwo. One-way ANOVA followed by Tukey multiple comparisons test.*** p < 0.001, ** p < 0.01, * p < 0.05.

Exercise regulates miR-283 expression to improve heart health

(A-E) Heart rhythm-related indices in age-matched sedentary and exercised groups of w1118 and miR-283KO/+ Drosophila. n=20-32. Student t-test. (F-J) Heart rhythm -related indices in age-matched sedentary and exercised groups of w1118>mir-283SP and Pdf>mir-283SP Drosophila. n=15-25. Student t-test. (K-N) Myocardial Phalloidin staining and imaging in age-matched sedentary and exercised groups of w1118>mir-283SP and Pdf>mir-283SP Drosophila. Significant gaps between myofibrils are indicated with “*”. n=5-8. (O-P) qPCR analysis of the expression levels of miR-283-5p and its target gene cwo in the brain and heart. n=20-30. Student t-test was used to analyze differences between aging (10d vs. 30d) and exercise (30d_C vs. 30d_E). (Q) Effects of exercise on the survival curves and average lifespan of w1118>mir-283SP and Pdf>mir-283SP Drosophila. n=120-150. Student t-test. *** p < 0.001, ** p < 0.01, * p < 0.05.

Schematic illustration of the findings obtained from this study

Based on this study, we propose that exercise ameliorates aging-associated bradyarrhythmias by modulating miR-283. First, we determined by systemic overexpression and knockout of miR-283 that abnormal expression of miR-283 can induce age-related HR reduction, prolonged DI, and increased incidence of bradycardia, which are features similar to bradyarrhythmias. Second, we specifically knocked down miR-283 in myocardium and LNvs, respectively, and found that miR-283 knockdown in LNvs leads to accumulation of miR-283 expression throughout the brain, inducing bradyarrhythmic features consistent with aging, a process that may be mediated by targeting regulation of cwo. Furthermore, exercise ameliorates the bradyarrhythmic features of aging or miR-283 knockdown in LNvs by modulating brain miR-283/cwo expression.