Cardiac Timeless Trans-Organically Regulated by miR-276 in Adipose Tissue Modulates Cardiac Function

Abstract The interconnection between cardiac function and circadian rhythms is of great importance. While the role of the biological clock gene Timeless (Tim) in circadian rhythm has been extensively studied, its impact on cardiac function remains largely been unexplored. Previous research has provided experimental evidence for the regulation of the heart by adipose tissue and the targeting of miR-276a/b on Timeless. However, the extent to which adipose tissue regulates cardiac Timeless genes trans-organically through miR-276a/b, and subsequently affects cardiac function, remains uncertain. Therefore, the objective of this study was to investigate the potential trans-organ modulation of the Timeless gene in the heart by adipose tissue through miR-276a/b. We found that cardiac-specific Timeless knockdown and overexpression resulted in a significant increase in heart rate (HR) and a significant decrease in Heart period (HP), diastolic intervals (DI), systolic intervals (SI), diastolic diameter (DD), and systolic diameter (SD). miR-276b systemic knockdown resulted in a significant increase in DI, arrhythmia index (AI), and fractional shortening (FS) significantly increased and SI, DD and SD significantly decreased. Adipose tissue-specific miR-276a/b knockdown and miR-276a overexpression resulted in a significant increase in HR and a significant decrease in DI and SI, which were improved by exercise intervention. This study presents a novel finding that highlights the significance of the heart circadian clock gene Timeless in heart function. Additionally, it demonstrates that adipose tissue exerts trans-organ modulation on the expression of the heart Timeless gene via miR-276a/b.


Introduction
Car dio vascular disease (CVD) poses a huge health and economic burden w orldwide .Gi v en its r ole as the pumping organ of the human body, the function of the heart is vital to human health.Heart failure and arrhythmia often occur during acute and scar healing in patients with m y ocardial infar ction, whic h is a significant cause of death.Yet, the pathogenesis of car dio vascular disease remains incompletely understood Circadian rhythms are pervasive life activities in animals that occur in cycles of day and night, 1 and at the molecular level consist of a tr anscription-tr anslation feedbac k loop (TTFL) composed of 4 biological clock cogs, P eriod (P er), Timeless (Tim), CLOCK, and CYCLE. 2 More than 13% of genes in the heart exhibit rhythmic expression. 3The circadian clock of heart muscle cells and all cell types associated with cardiov ascular disease ar e r ecognized, 4 and be found to r egulate intrinsic properties of the heart, including its growth and remodeling, transcription, and contractile function. 5Circadian rhythms and sleep disorders are strongly associated with increased incidences of heart and car dio vascular disease. 6Epidemiologic observations also show an increase in car dio vascular disease among night shift workers. 7All of this suggests that changes in circadian rhythms are closely linked to heart function.
Timeless is the second biological clock gene discov er ed to regulate circadian rhythms through photo-regulation or by forming a heterodimer with Per that regulates circadian rhythms thr ough positi v e and negati v e r e gulation. 8Resear c h on other biological clock genes Per, Bmall has shown that altered expression of these genes leads to impaired cardiac contractile function, 9 decreased heart rate (HR), and increased risk of arrhythmias, 10 brad ycar dia, and a generalized hypotensi v e phenotype . 11How ev er, the r elationship between Tim and heart function has been largely unexplored, and here we innovatively explore the effect of the cir cadian cloc k gene Tim on heart function.
In recent years, adipose tissue has been identified to play an important role in regulating car dio vascular, 12 , 13 affecting normal/patient cardiac function, 14 , 15 It is also closely associated with m y ocardial fibrosis and ather oscler osis. 16Bioacti v e pr oducts secreted by adipose tissue include adipokine, micr ov esicles, and microRNAs (miRNAs). 17Concurr entl y, studies have demonstrated that adipose tissue miRNAs regulate the function of heart organs, 18 and circulating miRNAs originate from adipose tissue. 19While recent studies have found that neuronal miR276a/b in the brain target Tim and thus have an important effect on sleep, 20 , 21 with a corr esponding degr ee of circadian d ysrhythmia, 22 ho wev er, ther e ar e few r e ports on adipose tissue cr oss-organ r egulation of car diac Tim and thus affecting car diac function.
Fruit flies serve as pioneering models to uncover the effects of heart development, function, 23 , 24 and exercise on heart health, [25][26][27] and their cardiac function can be measured by M-mode. 28Adipose bodies in fruit flies are analogous mammalian equi v alent of adipose tissue , 29 whic h is important in cross-organ regulatory studies. 303][34] Furthermore, exercise can also affect the release of miRNAs from adipose tissue, 35 , 36 and the role of exercise is initially explored in this paper.

Exercise-Tr aining De vice and Protocols
We developed a Drosophila locomotion device that takes advantage of the negative anisotropy of Drosophila to scale up by flipping the glass tube and adjusting the distance between the glass tube and sponge plug the locomotion distance at a constant rate of 8 cm.Referring to various exercise protocols and modifying them appr opriatel y, 26 , 49 Drosophila flies of the long-term regular exercise gr oup wer e allowed to exercise from the second day after fledging, and then rested for 2 d after exercising for 5 d, and exercised for 5 d again.Exercise was ended by 13 d.Exercise time was the same time period every day for 2.5 h.The exercise speed of the exercise device was 24 s/r.Each group of fruit flies was tested for a variety of indicators the day after the exercise intervention.

Real-Time Quantitati v e PCR (qPCR)
Ten whole bodies or 40 hearts or 20 adipose tissue tissues were collected from each group of Drosophila, and total RNA was extracted from the lysates using Trizol (Invitrogen, CA, USA) r ea gents, according to the kit instructions. 1 μl of RT product was mixed with 10 μl of SYBR qPCR Mastermix (TaKaRa) containing the appropriate PCR primers.qPCR amplification reactions were replicated, thermocycled, and fluoresced three times using an ABI7300 real-time PCR instrument (Applied Biosystems, USA): (30 s at 95 • C, 5 s at 95 • C, and 30 s at 60 • C) ×40.rp49/u6 standard SYBR Green (Takara) for real-time PCR.The relative abundance of the measured genes was calculated by the 2 − Ct method and normalized with GAPDH.The primer used were as follows (5 -3 ): U6 F: TGGCCCCTGCGCAAGGATG miR-276a F: ctC AGTC AGACCCC A TGT AGGAACTTC miR-276b F : ctC AGTC AGACCCC A TGT AGGAACTT A miRNA R: Reverse primer from a miRcute miRNA quantitati v e RT-PCR detection kit (Takara) Rp49 F: CTAA GCTGTCGCA CAAATGG Rp49 R: AACTTCTTGAATCCGGTGGG T imeless (T im) F: CGGA GTGGTCTCAA GGTTCC T imeless (T im) R: TGGATTGCTCGTTGTTCCAC

Semi-intact Drosophila Heart Prepar a tion and Cardiac Function Analysis
F ollowing pre vious resear c h methods, 50 artificial hemolymph fluid w as pr e par ed and placed at r oom temperatur e to pump oxygen for 30 min.Drosophila are anesthetized with carbon dioxide and fixed to a Petri dish coated with medical petroleum jelly.Add artificial lymph.Under a ster eo-micr oscope, the cuticles of the head, chest, and abdomen were removed with disemboweling scissors and pr ecision force ps, the viscera was scraped a wa y, and the accumulated fat was sucked into a microstraw to expose the heart.The HR of fruit flies (124 fps in 30 ss of video frame rate) w as r ecorded using a scientifically developed high-sensitivity camera, 51 HR data were collected using HC Ima ge softw ar e, and the ima ge data wer e anal yzed using semiautomated optical HR analysis to obtain quantitative indicators: heart rate (HR), Heart Period(HP), diastolic intervals(DI), systolic intervals(SI), arrhythmia index (AI), diastolic diameter (DD), systolic diameter (SD) and fractional shortening (FS).The sample of each group was 20 ± 5.

Negati v e Geotaxis Assay
In order to test the climbing ability of Drosophila, a Drosophila climbing ability test device was designed using the negati v e fast iterati v e negati v e appr oach of fruit flies. 52Drosophila was transferred to an 18-cm glass tube with an inner diameter of 2.8 cm, and Drosophila crawled along the wall of the glass tube out of instinct, and the climbing height of Drosophila was recorded when Dr osophila w as crawling for 8 s.The glass tube was shaken ev er y 30 s so that the Drosophila returned to the bottom of the tube, and the pr ocess w as r e peated 8 times.The entire climbing process was filmed with a speed camera and we had the fruit flies acclimatized in a glass tube for 10 min before filming.The fourth, fifth, and sixth images of the climb were intercepted at the end of the tenths and the number of fruit flies reaching the top area was counted.Climbing index = number of fruit flies/total number of fruit flies in the highest area, N = 100.

293T Cell Culture and Luciferase Reporter Assay
Luciferase assays (Dual-Luciferase Reporter Assay System, Prome ga) w ere performed for 48 h after transfection.The full length 3'UTR of Tim was cloned into a psiCheck-2 vector (Tsingke), m utations wer e constructed by altering the binding site of miR-276a on the Tim 3'UTR in vitro, media used DMEM (BasalMedia).

Sta tistical Anal ysis
The data were mapped using GraphPad Prism 9 softw ar e and Statistical Analysis using Statistical Package for (SPSS) version 25.W 1118 and W 1118 + E gr oups wer e compar ed using inde pendent t-assays.Another inter-gr oup anal ysis w as used by onew ay anal ysis of v ariance (ANOVA) and minimal significant difference (LSD).The significance level was set as P < 0.05.

Modifying Cardiac Timeless Expression Results in Low Climbing Ability and Impaired Cardiac Function
Using the Gal4/UAS transgenic system (HandGal4 > UAS-GFP) with W 1118 > UAS-GFP as a control, we observed Hand-Gal4 dri v es the m y ocardium gene expression ( Figure 1 A).The effect of Tim on cardiac function was explored using Hand-Gal4-driven knockdown and ov er-expr ession in the heart, which showed a 41% ov er-expr ession efficiency of Hand-Gal4 > UAS-tim and a 31% knockdo wn efficienc y of Hand-Gal4 > tim RNAi compared with the Hand-Gal4 > W 1118 group ( Figure 1 B).Cardiac protein Western b lot anal ysis showed a significant incr ease in Tim pr otein content in the Hand-Gal4 > UAS-tim group ( P < 0.05) and a significant decrease in the Hand-Gal4 > tim RNAi group ( P < 0.01) ( Figure 1 C and D).Climbing ability was significantly reduced in the Hand-Gal4 > UAS-tim gr oup compar ed to the control group ( P < 0.01) ( Figure 1 E).M-mode was utilized to quantify HR, heart period (HP), SI, and DI, arrhythmia index (AI), systolic diameter (SD), diastolic diameter (DD), and F r antional Shortening (FS), and the results showed that compared with the contr ol gr oup, the Hand-Gal4 > UAS-tim versus Hand-Gal4 > tim RNAi gr oup HR wer e significantl y incr eased ( P < 0.01) ( Figur e 1 F), and HP, DI, SI, DD, and SD were significantly decreased ( P < 0.01 or P < 0.05) ( Figure 1 G, H, I, K, and L).These results suggest that elevated Tim expression in the heart significantly reduces the ability of fruit flies to exer cise , while elevated or decreased Tim expression in the heart leads to impaired heart function.

Reduced Expression of miR-276a and miR-276b Correlates With Increased Timeless Expression in the Heart and Impaired Cardiac Function
Gene expr ession lev els in whole body, fat, heart, and cardiac function were measured using miR-276aKO and miR-276bKO Drosophila lines as a control group using W 1118 .The knockdown efficiency of miR-276aKO line miR-276a was 74%, 78%, and 63% for the whole body, fat, and heart, r especti v el y ( Figur e 2 A).miR-276bKO line had knockdown efficiency of 54%, 78%, and 99% of miR-276b, r especti v el y ( Figur e 2 B).In miR-276aKO and miR-276bK O , mRNA levels of Tim in the heart respectively elevated by 84% and 124% ( Figure 2 C).miR-276aKO showed a significant increase in HR, FS ( P < 0.01) ( Figure 2 D, K) and a significant decrease in HP, DI, SI, DD, and SD ( P < 0.01 or P < 0.05) ( Figure 2 E, F, G, I, and J).miR-276bKO increased significantly in DI, AI, FS ( Figure 2 G, H, and K) ( P < 0.05 or P < 0.001) and decreased significantly in SI, DD, SD ( P < 0.01 or P < 0.001) ( Figure 2 G, I, and J).These results indicated a significant decrease in miR-276a and miR-276b expression in miR276aKO and miR276bKO and a significant increase in Tim transcript levels in fruit flies.In terms of heartbeat rhythm, the knockdown of miR-276a caused the sim ultaneous r eduction of cardiac inter-systolic and DIs, resulting in accelerated heartbeat.miR-276b knockdown caused significant arrhythmia.In terms of cardiac pumping function, both knockdowns caused shorter diastolic and systolic diameters, which led to compensatory enhancement of m y ocardial pumping function.The results showed that reduction/increase adipose tissue miR-276a/b in adipose tissue led to decr ease/incr ease of miR-276a/b in adipose tissue and heart, and incr ease/decr ease in Tim expression in the heart, which is another indication of adipose tissue-heart cross-organ regulation in Drosophila.In wild-type Drosophila, exercise reduced miR-276a/b in adipose tissue and heart, ho wever, car diac Tim lev els strangel y did not increase, but significantly decreased.In addition, To test whether Tim can be actually regulated by miR-276a, 293T cells w ere tr ansfected with plasmids expressing Tim 3'UTR connected downstream from a Renilla luciferase reporter.The results showed that miR-276a increased fluorescent expression by 65.7%, which could be rescued by mutation of the Tim 3'UTR binding site ( Figure 3 H).

Effects of Adipose Tissue miR-276a/B and Exercise Intervention on Athletic Abilities and Cardiac Function
The climbing index was significantly lower in the Cg-Gal4 > UAS-276a/b-Sponge gr oup compar ed with Cg-Gal4 > W 1118 in each group within the group ( P < 0.001), and there was no signif- icant difference in the over-expression group ( P > 0.05), the climbing index was significantly higher in each of the longterm re gular exer cise groups betw een the gr oups compar ed with the control group, respectively ( P < 0.01 or P < 0.001) ( Figure 4

Discussion
The identification of the circadian rhythm gene has significantly enhanced the comprehension of circadian rhythms acr oss v arious organisms, encompassing insects, mammals, and humans.In humans, the discov er y of ov er a dozen circadian clock genes has shed light on the intricate mechanisms underlying circadian rhythms.Notably, a tr anscription-tr anslation feedback loop (TTFL) comprising four circadian gears (Per, Tim, CLOCK, and CYCLE) assumes a crucial role in the regulation of circadian rhythms in fruit flies. 2 Prior investigations on additional cir cadian cloc k genes hav e r ev ealed their significant impact on cardiac function.Our findings indicate that the presence of PER2S662G and PER2S662D tr ansgenic mice , whic h alter circadian rhythms, resulted in the inhibition of cardiac function and a reduction in FS. 9 Additionally, Bmal1 knockout mice displayed dilated heart disease, 37 while car diomyoc ytes with Bmal1-specific knockout experienced cardiac systolic dysfunction, 38 , 39 a decrease in HR, and an increased risk of arrhythmia. 10Furthermore, Yu's study demonstrated that the specific knockout of Bmal1 in the left stellate ganglion effecti v el y pr ev ented v entricular arrhythmias following m y ocardial ischemia. 40Another study r ev ealed that Bmal1 −/ − mice exhibited brad ycar dia and a generalized hypotensi v e phenotype. 11egr etta b l y, ther e exists a dearth of literatur e pertaining to the correlation between the circadian clock gene Tim and its impact on cardiac function.This may be related to the fact that Tim transgenic mice are technically difficult to realize.Based on the aforementioned analyses, we have formulated a scientific hypothesis positing that the circadian clock gene Tim plays a r egulator y r ole in cardiac function.To test this hypothesis, our resear c h team employ ed the Hand-Gal4 tec hnique to induce knockdown and ov er expr ession of cardiac Tim.The findings depicted in Figure 1 demonstrate that both heightened and diminished expression of cardiac Tim result in an increased HR in Drosophila, accompanied by a decrease in diastolic duration (DD) and impaired cardiac function to a certain de gree .These results indicate that deviations from the normal state of Tim gene expression can exert an influence on cardiac function.Ther e w as no statisticall y significant disparity observ ed in the impact of various circadian clock genes on AI or FS in comparison to their impact on circadian clock genes on heart function, whereas Per and Bmal1 exhibited a mor e pr onounced influence on both parameters.These findings imply that Tim and other circadian clock genes exhibit compara b le yet not identical effects on cardiac function.
Following the identification of Tim' influence on cardiac function, our resear c h focused on elucidating the mechanisms of miRN A re gulation upstream of this gene followed by an explor ation of mec hanisms inv olv ed in subsequent studies regarding up-down regulation.In the initial investigation, a cohort of resear c hers successfully identified miRNAs that specifically target the RNA endonuclease Drosha, thereby providing confirmation of the r egulator y functions exhibited by miR-276. 41ubsequent studies have documented the r emov al miR-276a/b binding sites from Tim binding sites, as well as alterations in Tim levels resulting from modified miR-276a expression in the brain.1][22] Our resear c h team conducted a comprehensi v e bioinformatics analysis, which revealed the presence of a distinct miR-276a/b seed sequence within the 3'UTR of Tim (Supplementary Figure S1 ).Collectively, these reports and anal yses str ongl y suggest a potential association between miR-276a/b and Tim, underscoring their r elev ance in the context of slee p r e gulation and cir cadian rhythm disturbances.
Secondly, adipose tissue, being a crucial organ in the organism, adipose tissue releases hormones and miRs either fr eel y or through vesicle pathways.It has been widely acknowledged for its inter-organ role in the regulation of other tissue functions. 42 , 43Furthermor e, r ecent r esear c h has highlighted the significance of certain miRNAs in adipose tissue in the regulation of cardiac function.For instance, Fang et al.'s investigation r ev ealed that miR-200a in adipose tissue exosomes targets cardiom y ocytes, resulting in cardiac h ypertroph y . 44Similarly , miR-320d in adipose tissue inhibits cardiom y ocyte apoptosis associated with atrial fibrillation. 45Additionally, Hao's resear c h r ev ealed a corr elation between adipose tissue-deri v ed miR-134-5p and remodeling following m y ocardial infar ction. 46Building upon this information, our team aims to further investigate the scientific hypothesis that adipose tissue miR276a/b transorganically modulates the expression of the Tim gene in the heart, consequently impacting cardiac function.
To validate this hypothesis, we employed a systematic knoc kout approac h for miR-276a/b and conducted specific knockout and ov er-expr ession experiments for miR-276a or miR-276b in adipose tissue, assessing their effects on Tim gene expression and cardiac function.Figure 2 illustrates that the elevation of cardiac Tim levels resulting from systemic miR-276a and miR-276b decreases leads to a reduction in both DD and SD.
The diminished DD indicates a certain degree of impaired cardiac function, while the shortened SD may be associated with the compensatory augmentation of cardiac pumping function.Specifically, the impairment of cardiac function induced by the knockdown of miR-276a or miR-276b is primarily manifested as an increase in HR or the occurrence of cardiac arrhythmia, r especti v el y.Figur e 3 and 4 demonstr ate that the alter ation of adipose tissue miR-276a/b levels leads to corresponding changes in adipose tissue and cardiac miR-276a/b expression, as well as cardiac Tim expression.These findings strongly suggest a partial origin of that cardiac miR-276a/b from adipose.We re vie wed the literature and guessed that miR-276 is transported from adipose tissue to the heart via exosomes as carriers, but it has not been pr ov ed mor e dir ectl y, and we will further inv estigate this part in the future.Manipulation of adipose tissue miR-276a/b, such as knockout or ov er-expr ession, r esults in incr eased HR.Furthermore , knoc kdown of adipose tissue miR-276a/b leads to shorter diastolic duration and systolic duration, thereby impairing cardiac pumping function.Conv ersel y, ov er-expr ession of miR-276b in adipose tissue leads to a reduction in DD and FS.
Based on the aforementioned findings, it is evident that adipose tissue miR-276a/b exerts a substantial direct influence on the levels of cardiac miR-276a/b and Tim.This observation underscores the regulatory role of adipose tissue in modulating the expression of the Tim gene in the heart, thereby impacting cardiac function through miR-276a/b.Furthermore, the alterations observed in the systemic knockout of miR-276a align closely with those observed in the specific knockout of miR-276a/b in adipose tissue, both of which result in changes in cardiac Tim levels and cardiac function.These findings suggest that adipose tissue exerts trans-organ regulation on cardiac Tim in the heart, thereby influencing cardiac function, mainly through miR-276a, not miR276b.
Pr evious r esear c h has demonstr ated that physical activity enhances car dio vascular function, m y ocardial structure , and behavioral aspects (such as sleep patterns and negati v e emotions) in Drosophila melanogaster. 47 , 48Furthermore, exercise exerts a nota b le influence on the release of miR within adipose tissue. 35 , 36For instance, a study conducted by Kristensen et al. r ev ealed that a combination of exercise and dietary modifications over a 15-wk period resulted in the upregulation of miR-29a-3p and miR-29a-5p, while downregulating of miR-20b-5p in subcutaneous adipose tissue during weight loss. 36Ther efor e, our study aimed to examine the potential impact of exercise on the inter-organ regulation of miR276a/b in adipose tissue, specifically focusing on its influence on Tim expression and cardiac function in the heart.Nota b l y, our findings, as depicted in Figure 3 , demonstrated a significant reduction in the expression levels of both miR-276a/b and Tim gene in adipose tissue and the heart following exer cise .We attribute this to the complexity of the way in which exercise regulates gene expression in the body under normal physiological conditions, and Tim is a time-clockcritical gene that is influenced by multiple factors.We believe that this phenomenon r equir es mor e in-de pth studies in the future to elucidate its mechanisms.Furthermore, the findings de picted in Figur e 4 indicate that a decline in miR-276a/b levels resulted in a decrease in athletic performance.However, engaging in regular exercise over an extended period of time has the potential to ameliorate the diminished athletic ability caused by reduced miR-276a/b levels.The enduring impacts of consistent physical activity on cardiac function in fruit flies primarily inv olv e a reduction in HR, while the pumping function remains unaffected.Pr eliminar y evidence suggests that exercise might regulate miR-276a/b levels in the adipose tissue of fruit flies, thereby enhancing athletic performance and reducing HR during exer cise .Despite the a bsence of elev ated Tim gene expression in the heart as a result of diminished miR-276a/b levels induced by exer cise , this intriguing discov er y w arrants further investigation in subsequent resear c h endeavors.

Figure 2 .
Figure 2. Effects of miR-276a/b on Timeless expression and cardiac function.[Note: (A) Relati v e expr ession of miR-276a in whole body, fat, and heart under miR-276a systemic knockdown condition (B) Relati v e expr ession of miR-276b in whole body, fat, and heart under miR-276b systemic knockdown condition (C) Relati v e expr ession of TIM in the heart under miR-276a, miR-276b systemic knockdown condition (2D-2K).Effect of miR-276a, miR-276b systemic knockdown on cardiac function with M-mode indexes of HR, HP, DI, SI, AI, DD , SD , and FS.n = 20.(L) Model M. electrocardiogram with a 10-s interception time for each group horizontal green line: DI horizontal blue line: SI, horizontal red line: HP; vertical dotted line: DD.Vertical solid lines: All P -values of SD were derived from one-way ANOVA, * P < 0.05, * * P < 0.01, * * * P < 0.001].
A), indicating that decreased miR-276a/b levels led to decreased exercise capacity, however, long-term regular exercise impr ov ed the decr eased exercise capacity caused by decreased miR-276a/b levels.In terms of cardiac function, the HR in the control group Cg-Gal4 > UAS-276a/b-Sponge and Cg-Gal4 > UAS-miR-276a was significantly increased compared to Cg-Gal4 > W 1118 ( P < 0.01 or P < 0.001), and the HR was significantl y r educed in the long-term r outine exercise gr oup U AS-miR-276a > U AS-miR-276b compared to the contr ol gr oup ( Figure4 B).The cardiac cycle, as the r ecipr ocal of HR, showed a trend opposite to HR ( Figure4 C).The car diac c ycle included SI and DI, and in the Cg-Gal4 > UAS-276a/b-Sponge and Cg-Gal4 > UAS-miR-276a groups, the HR increased due to DI and SI, r especti v el y ( P < 0.01).The ameliorati v e effect on HR from exercise was mainly due to longer DI ( Figure4 D, E).There was no significant difference in the arrhythmia index between the two groups ( P > 0.05) ( Figure4F).Compared to Cg-Gal4 > W 1118 , the DD decreased in the Cg-Gal4 > UAS-miR-276b group ( P < 0.05) ( Figure 4 G) and FS decreased ( P < 0.05) ( Figure 4 I), while the DD and SD decreased in the Cg-Gal4 > UAS-276a/b-Sponge group ( P < 0.01 or P < 0.05) ( Figure 4 G).

Figure 3 .
Figure 3. Effects of adipose tissue miR-276a/b and exercise intervention on cardiac Timeless.[Note: (A) UAS-GFP > Cg-Gal4 Fruit Fly Fluorescence, W 1118 > Cg-Gal4 Contr ol, arr ow dir ection fr om tail to head, white line for Drosophila dorsal plate outline, red line for Drosophila heart outline.Scale = 1 mm.(B) miR-276a in adipose tissue and heart of Cg-Gal4 > UAS-276a/b-sponge, Cg-Gal4 > UAS-miR-276a compared to Cg-Gal4 > W 1118 .(C) Levels of miR-276b in Cg-Gal4 > UAS-276a/b-Sponge, Cg-Gal4 > UAS-miR-276b adipose tissue, heart, with Cg-Gal4 > W 1118 as contr ol.(D) Expr ession lev els of Timeless in cardiac Cg-Gal4 > Cg-Gal4 > UAS-276a/b-Sponge, Cg-Gal4 > UAS-miR-276a, UAS-miR-276b adipose tissues.(E) Levels of miR-276a in cardiac adipose tissue from W 1118 exercise group, as compared to W 1118 quiet group.(F) Levels of miR-276b in adipose tissue, the heart of W 1118 exercise gr oup with W 1118 quiet gr oup as the contr ol gr oup.(G) Expr ession lev els of Timeless in the heart of W 1118 exercise gr oup wer e contr olled by W 1118 quiet gr oup.(H) Identification of miR-276a targets.Renilla luciferase is used as a primar y r e porter gene, and the tim 3'UTR can be cloned into the multiple cloning region located downstream of the Renilla lucifer ase tr anslational stop codon.P -value in the B-D gr oup w as deri v ed fr om one-way ANOVA, and P -value in the E-H group was derived from t -testing of independent samples, * P < 0.05, * * P < 0.01, * * * P < 0.001.]

Figure 4 .
Figure 4. Effects of adipose tissue miR-276a/b and exercise intervention on athletic abilities and heart function.[Note: (A) Effects of knockdown, ov er-expr ession of miR-276a/b in adipose tissue and exercise intervention on Drosophila athletic ability, N = 100.(B-I) Effects of adipose tissue miR-276a/b knockout, ov er-expr ession, and exercise intervention on cardiac function, with M-mode indicators being HR, HP, DI, SI, AI, DD, SD, and FS.N = 20.All P -values are from one-way ANOVA.Values ar e fr om one-w ay ANOVA.* P < 0.05, * * P < 0.01, * * * P < 0.001]