1. Introduction

Normal aging hearts experience left ventricular hypertrophy, fibroblast proliferation, altered diastolic function, and decreased systolic reversal capacity, ultimately leading to decreased cardiac output and increased myocardial fibrosis [1]. This age-dependent change in cardiac structure and function is the main cause of the high prevalence of cardiovascular disease in the elderly population [1, 2], and is also the combined result of aging cardiovascular system dysfunction and autonomic nervous system imbalance [3–5]. Studies have shown that the imbalance of the renin-angiotensin system and sympathetic-vagal system homeostasis with age is an important trigger of cardiovascular aging [3, 5, 6], with the ultimate result being a decrease in the heart rate and myocardial contractility, increasing the risk of heart failure [3, 7]. Furthermore, research further reveals that changes at the cellular level are the main determinants of structural remodeling and functional changes in the aging heart [8–10]. MicroRNAs (miRNAs), a class of small non-coding RNAs, have been implicated at the post-transcriptional level by targeting hundreds of transcripts to regulate a variety of important cellular functions including stem cell self-renewal, cell proliferation, apoptosis, metabolism, and oxidative stress, and are considered to be regulators of cellular senescence [11, 12]. Several studies have shown that miRNAs play important roles in cardiovascular tissue homeostasis and cardiac aging processes. For example, age-dependent alterations of miR-34a [13], miR-195 [14, 15], and miR-217 [16] induce myocardial fibrosis, promote apoptosis, and cardiovascular dysfunction, and have been identified as circulating markers of de novo cardiac aging and cardiovascular risk, providing new perspectives for studying the biological processes of cardiac aging and therapeutic targets for delaying aging. We noted that circulating miR-216a is significantly upregulated in patients with heart failure [17] and induces endothelial senescence [18]. However, whether age-related decline in cardiac function is associated with altered miR-216a expression requires further investigation.

Aging and its consequences cannot be completely stopped; however, cardiac aging can be slowed down by appropriate means. Previous research has shown that appropriate physical exercise can reduce the incidence of age-related heart disease [19]. Rodent studies have further revealed the widespread benefits of exercise training in preventing apoptosis in the aging heart [20], reversing or “re-sensitizing” aged hearts to adrenergic stimulation [19, 21], improving Ca²⁺ cycling[22], and activating the IGF-1/PI3K/Akt signaling pathway [19]. Therefore, regular physical exercise has been proposed as a healthy lifestyle to delay aging and prevent and treat diseases [23–25]. In recent years, an increasing number of studies have found that exercise protects heart health and prevents cardiac dysfunction by regulating miRNAs with apoptotic potential [26–30]. However, the role of miRNAs in exercise-improving function in aging hearts remains largely unknown.

The Drosophila heart, also referred to as the dorsal vessel, has some conserved regulatory mechanisms related to vertebrate heart development and aging [31], and has been widely used to study the physiological, genetic, and epigenetic basis of cardiac aging [32]. Furthermore, the detection of mature heart function in Drosophila allows the accurate assessment of the functional characteristics of aging hearts [33, 34]. Exercise based on the innate anti-gravity climbing trait of Drosophila has been shown to be prominently effective in preventing age-related cardiac dysfunction [35–38]. In this study, we used a Drosophila melanogaster model to reveal the regulatory role of dme-miR-283 (miR-283, a homolog of miR-216a in Drosophila) in age-dependent cardiac function decline, and further explored whether the ameliorative effects of exercise on the aging heart are associated with exercise resistance to age-dependent changes in miR-283 expression.

2. Results

2.1 Functional and structural deterioration of the myocardium in aging Drosophila

Analysis of cardiac function in aging w¹¹¹⁸ flies (10 days of age corresponding to the young stage, 30 days of age for middle age, and 50 days of age for old age) [39] using SOHA revealed that a significant reduction in HR and an increase in HP were already present at midlife and persisted into old age (Figure 1A and 1B), mainly caused by the prolonged DI and SI (Figure 1C and 1D). M-mode traces from the SOHA showed a more prevalent incidence of bradycardia in middle-aged and older hearts (Figure 1F and 1G). The occurrence of diastolic insufficiency was also significantly increased compared to that in younger flies (Figure 1I), suggesting a more abnormal cardiac rhythm in middle-aged and older Drosophila. In addition, another change with aging was manifested by narrowing of the heart lumen canal diameter, with significant shortening of SD and DD at 30d and 50d (Figure 1F and 1G), showing an increase in FS due to a greater narrowing of the SD (Figure 1J) but a significant decrease in cardiac output (Figure 1K). Moreover, the incidence of incomplete relaxations was also significantly increased in Drosophila at 30d and 50d (Figure 1F and 1G). Myocardial SA-β-gal staining also showed a significant increase in a blue precipitated area at 30d that persisted until 50d (Figure S 1A-1D). Therefore, our results show that Drosophila has already experienced comprehensive age-related deterioration of cardiac rhythm and pumping capacity at 30d, and this deterioration continues until 50d.

Figure 1.

Visualization of cardiac filamentous actin (F-actin) by Phalloidin staining similarly showed an increase in myofibrillar gaps in middle-aged Drosophila hearts and a larger staining deficit in aged myocardium (Figure 1L-1N). These results suggest a progressive deterioration of Drosophila myocardial structure with age, which may contribute to the decline in myocardial pumping function and the increase in arrhythmias.

Meanwhile, we observed a significant enhancement of SA-β-gal activity in the brain at 30d that persisted until 50d (Figure S 1E-1H), a change similar to that of aging myocardium. And the climbing ability was continuously decreased with age at 30d and 50d (Figure 1O), and this age-related decline in behavior also suggests that Drosophila undergoes neurodegeneration during aging [40, 41]. Our study reveals that age-dependent deterioration of cardiac function with neurologic deficits is already occurring in Drosophila at the age of 30d.

2.2 Dysregulation of miR-283 expression triggers aging-related cardiac functional alterations in young Drosophila

To determine whether miR-283 regulates cardiac function, we examined cardiac performance in young miR-283 heterozygous knockout (mir-283^KO/+), homozygous knockout (mir-283^KO) and overexpressing (Tub>mir-283^OE) flies. And whole-body miR-283 expression levels were examined (Figure 2A, whole body), while the myocardium and brain also showed consistent partial knockdown, complete knockout, and overexpression (Figure 2A, heart and brain). Analysis of heart rhythm indices found that both miR-283 knockout and overexpression resulted in decreased HR (Figure 1B), prolonged HP and DI (Figure 1C and 1D), and a general increase in bradycardia (Figure 1G and 1H), but did not induce an aging-associated increase in SI (Figure 1E). These results suggest that miR-283 knockout and overexpression lead to an increased incidence of long DI-associated bradycardia with reduced heart rate, which is phenotypically similar to bradyarrhythmia defined clinically on the basis of heartbeat rate[42, 43]. Moreover, differing from miR-283 knockout, miR-283 overexpression also resulted in an overarching reduction in pumping capacity (Figure 1I-1L), as well as a decrease in the climbing index (Figure 1M), suggesting that systemic overexpression of miR-283 produced more widespread negative effects. Additionally, it is worth stating that we drove miR-283 systemic overexpression with Tub-Gal4, which generated a particularly high lethality rate, further suggesting that systemic increase in miR-283 expression triggers severe consequences.

Figure 2.

We next explored whether the slowing of heart beat caused by altered miR-283 expression was consistent with an age-induced decrease in HR. We performed a two-way ANOVA for gene*age on HR, HP and DI in mir-283^KO/+ and mir-283^KO senescent Drosophila and found a significant interaction between miR-283 knockout and aging (Table S2). In addition, there was a significant rise or trend towards an increase in HP and DI in 10d and 30d miR-283 knockout flies (Figure S2B-S2C), which resulted in a decrease in HR (Figure S2A). Collectively, these findings suggest that miR-283 regulates age-related HR reduction and DI prolongation, and that dysregulation of its expression may contribute to the development of bradycardic arrhythmias.

2.3 miR-283 knockdown in LN_vs, but not heart, leads to bradyarrhythmia

We examined changes in miR-283 expression in senescent myocardium, including wild-type w¹¹¹⁸ and hybridized controls (w¹¹¹⁸>mir-283^SP). The results showed that the expression of miR-283 in the myocardium of w¹¹¹⁸ flies exhibited a dynamic change of decrease at 30 d and increase at 50 d (Figure S3A). In contrast, w¹¹¹⁸>mir-283^SP Drosophila showed a dynamic change of increase at 30d and decrease at 50d (Figure S3B). This difference may be related to the fact that these two types of Drosophila are in different life states. Typically, wild-type w¹¹¹⁸ “aged” faster than the hybridized control. Overall, however, our results suggest that myocardial miR-283 expression changes during aging.

To determine whether age-related decline in cardiac function is associated with downregulation of myocardial miR-283 expression, we used the GAL4/UAS system to express mir-283 sponge in the heart, resulting in a specific knockdown of miR-283. We crossed UAS-mCherry.mir-283.sponge virgin strains with heart-specific tissue drivers (Hand-Gal4) and observed that the cardiomyocytes and paracardiocytes of the offspring generation were labeled with mCherry fluorescence (Figure 3A), and the expression of miR-283 in the myocardial tissue was significantly reduced (Figure 3N), indicating that myocardial successful knockdown of miR-283.

Figure 3.

Next, we examined cardiac function in young Hand>mir-283^SP Drosophila. Compared to age-matched controls (w¹¹¹⁸>mir-283^SP), myocardial tissue miR-283 knockdown did not have a significant effect on heart beating and contractility (Figure 3C-3M). This indicates that partial decrease of miR-283 in the heart does not affect cardiac function or that there are other compensatory regulatory mechanisms to maintain normal performance of the heart. mir-283^KO/+ Drosophila exhibited features of bradyarrhythmia along with a significant reduction in miR-283 expression in the myocardium and brain (Figure 2A-2L). However, specific knockdown of myocardial miR-283 did not affect cardiac function (Figure 3C-3M). Therefore, we hypothesized that the cardiac phenotype of mir-283^KO/+ Drosophila may be regulated by neuronal miR-283.

In addition to its own pacing regulation, the heartbeat is also regulated by the “brain-heart” axis from the vagus and sympathetic nerves of the brain [44]. The mechanism of brain-heart regulation is based on the hierarchical clock system, where the main biological clock that coordinates the circadian activity of the whole body is located in the suprachiasmatic nucleus (SCN) of the hypothalamus, which transmits neurotransmitters to the vagus and sympathetic nerves through an anatomical connection with the paraventricular nucleus of the hypothalamus. This enforces endogenous rhythm enforcement to the heart and other organs, and regulates HR and rhythm via the sinus node [44]. The Drosophila circadian pacemaker is located in LN_vs of the brain. We used LN_vs-expressing Gal4 (Pdf-Gal4) to drive the mir-283 sponge to investigate whether miR-283 regulates cardiac rhythm through the brain-heart axis. We observed mCherry fluorescence in the LN_vs of Pdf>mir-283^SP Drosophila (Figure 3B), indicating successful driving. The results of cardiac function in young Drosophila with Pdf>mir-283^SP showed that 10d flies exhibited a significantly reduced HR (Figure 3C) and prolonged HP and DI (Figure 3D - 3E) heartbeat pattern, especially a significantly increased incidence of bradycardia (Figure 3H - 3I), which is consistent with the senescent heartbeat phenotype induced by mir-283 knockdout or overexpression. Similarly, we did not find a function of miR-283 in LN_vs to regulate cardiac contraction (Figure 3J - 3L), but resulted in a significant reduction in cardiac output due to decreased HR (Figure 3M).

Furthermore, we observed a decline in climbing ability in Hand>mir-283^SP and Pdf>mir-283^SP 10d flies (Figure 3O), suggesting that imbalances in miR-283 homeostasis in the heart or brain accelerate neurosenescence. A leftward shift in the survival curve of Hand>mir-283^SP and Pdf>mir-283^SP Drosophila was also observed (Figure 3P), especially a significant decrease in the median survival time (Figure 3P) and mean lifespan (Figure 3P) in Pdf>mir-283^SP flies, further suggesting that stable expression of miR-283 in LN_vs is important for healthy aging.

We further analyzed the effect of aging combined with miR-283 knockdown in LN_vs on heartbeat rhythm in Drosophila. Results of a two-way ANOVA on aging and miR-283 knockdown showed an interaction between aging and knockdown in HR, HP, and DI (Table S3). Senescence changes with reduced HR, prolonged HP and DI consistent with wild type were observed in control group (w¹¹¹⁸>mir-283^SP, Figure S3). Meanwhile, Pdf>mir-283^SP exhibited cardiac senescence characteristics (significantly reduced HR and prolonged HP and DI) in youth and persisted into middle age (Figure S3). In conclusion, our findings suggest that specific knockdown of miR-283 in LN_vs, but not in the myocardium, leads to an age-associated decrease in HR, along with an increased incidence of DI prolongation-associated bradycardia, and ultimately an increase in bradyarrhythmia.

2.4 Knockdown of miR-283 in LN_vs induces heart and brain aging and impairs cardiac morphological

We next examined the effect of miR-283-specific knockdown in LN_vs on miR-283 expression in the myocardium and brain. qPCR results showed a significant increase in miR-283 expression in the brain and myocardium of 10d Pdf>mir-283^SP Drosophila, which compared with age-matched controls (w¹¹¹⁸>mir-283^SP) (Figure 4A). To determine whether this increased expression was consistent with changes in aging, we further examined miR-283 expression levels in 10d and 30d Drosophila. The results revealed an elevation of miR-283 expression in both myocardium and brain of Drosophila in the 30d w¹¹¹⁸>mir-283^SP and Hand>mir-283^SP groups, as compared to their 10d counterparts of the same genotype (Figure 4A). Brain miR-283 expression was also significantly increased in the senescent Pdf>mir-283^SP Drosophila, whereas a high level of expression consistent with 10d was maintained in the myocardium (Figure 4A). These results suggest that miR-283 knockdown in LN_vs leads to an accumulation of miR-283 expression in the myocardium and brain consistent with senescence.

Figure 4.

Senescence-associated β-galactosidase (SA-β-gal) is a marker of aging cells in vitro, vertebrates, and Drosophila, and histochemical assays are considered the most reliable and easily detectable age-dependent markers [45, 46]. To further characterize that miR-283 knockdown in LN_vs induces senescence, we stained brain and heart tissues of w¹¹¹⁸>mir-283^SP (10d and 30d) and Pdf>mir-283^SP (10d) Drosophila using SA-β-gal. The results showed a significant increase in blue precipitated areas in 30d brains of w¹¹¹⁸>mir-283^SP as well as in the 10d brains of Pdf>mir-283^SP (Figure 4B-4E), indicating that both aging and miR-283 knockdown in LN_vs resulted in enhanced SA-β-gal activity throughout the brain. In senescent hearts, conical chambers and within the walls of the heart tube showed sporadic granular staining (Figure 4G), unlike in LN_vs knockdown of miR-283-induced senescent hearts, where stained regions covered most of the conical chambers and heart tube (Figure 4F-4I), further suggesting that knockdown of miR-283 in LN_vs simultaneously induces heart and brain aging.

The adult Drosophila heart consists of circumferential muscles, longitudinal muscles, and alary muscles, of which the circumferential muscles are the working cells of the heart [47]. To investigate whether aging and miR-283 knockdown in LN_vs result in structural changes in filamentous actin (F-actin) in myogenic fibers, we stained semi-exposed hearts with phalloidin and obtained 600-fold magnified images of cardiac structures at the third ventral segment (Figure 4J). In control young hearts, myogenic fibers were circular and tightly aligned (Figure 4K) to promote consistent cardiac contraction. While in aged w¹¹¹⁸>mir-283^SP Drosophila (Figure 4L) as well as in young Pdf>mir-283^SP Drosophila (Figure 4M), consistent disorganization of myogenic fiber arrangement and increased gaps were observed. This suggests that both aging and miR-283 knockdown in LN_vs lead to structural degradation of myogenic fibers, which may contribute to the development of bradyarrhythmias.

2.5 Bioinformatics prediction and experimental validation of potential targets of miR-283

Drosophila miR-283 is located on the intron of the X-chromosome-encoded gene Gmap, and the pre-mir-283 is processed to generate the mature miR-283-5p, which is conserved with miR-216a in species such as humans, rats, and mice (Figure 5A). Previous studies have shown that miR-216a may play a pivotal role in aging cardiovascular diseases (atherosclerosis and heart failure) [18] and has been proposed as a new biomarker for the diagnosis of heart failure and related diseases [17], but its regulatory mechanisms remain unclear. We predicted by DIANA microT-CDS online tool that miR-283-5p target genes are mainly enriched in Notch signaling pathway (Nct, mam, Ser, Delta, Hairless) (Figure 5B). In our study, the expression level of miR-283 in the brain and heart of 10d Pdf>mir-283^SP flies exhibited a consistent increase with that of the control aging Drosophila (Figure 5C). Examination of the predicted target genes of the Notch signaling pathway showed that Nct, Ser and mam were significantly downregulated in both senescent and Pdf>mir-283^SP brains. However, the predicted target genes were significantly upregulated or tended to increase in both aging and Pdf>mir-283^SP hearts (Figure 5D-H) and were not consistent with the expected effect of miRNA sponge inhibition.

Figure 5.

Considering that it is miR-283 in circadian pacemaker neurons LN_vs that plays a major regulatory role in bradyarrhythmias, we sought to investigate whether miR-283 affects heart beating by regulating the expression of biological clock genes. One of our previous studies predicted that the core rhythm genes PAR-domain protein 1 (Pdp1) and clockwork orange (cwo) are potential target genes of miR-283-5p[48] and that Pdp1 and cwo can be rhythmically expressed in multiple tissues[49]. We therefore further examined the expression of Pdp1 in brain and heart, and found that Pdp1 showed a consistent increase in transcription in senescent heart and brain, as well as increased expression in the heart of Drosophila with Pdf>mir-283^SP (Figure 5I), which is inconsistent with the target-binding degradation of miR-283-5p to function as a degradation mRNA.

Examination of cwo transcription levels revealed a significant downregulation of cwo in the brain and heart of aging flies and those with miR-283 knockdown in LN_vs flies (Figure 5J), consistent with the miRNA-mRNA targeting relationship. We further predicted the potential binding targets of miR-283-5p to the 3′-untranslated region (3’ UTR) of cwo by the RNAHybrid online tool (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid) and found a potential binding site at the 489th position of the cwo 3’ UTR (Figure 5K). We next determined whether miR-283 could regulate cwo by a Dual-Luciferase Reporter Assay. The results showed that the expression of miR-283 inhibited the luciferin reporter activity of the wild-type 3’ UTR of cwo, but not the cwo mutant 3’ UTR reporter activity (Figure 5L), suggesting that miR-283 can be targeted to regulate the expression of cwo.

2.6 Exercise ameliorates bradyarrhythmias due to aging or miR-283-specific knockdown in LN_vs

Exercise enhances cardiac health and may even partially reverse pathological cardiac remodeling in the elderly [19]. However, the underlying miRNA mechanisms by which exercise provides cardioprotection against aging are not well understood. Our study identified a role for miR-283 in LN_vs in regulating aging-associated bradyarrhythmia and that miR-283 targeting of the core rhythm gene cwo may mediate this process. We next determined whether exercise improves aging heart health by modulating miR-283 expression.

First, we performed endurance exercise on w¹¹¹⁸ and miR-283 ^KO/+ Drosophila for 3 weeks. Cardiac function analysis showed that exercise had a significant ameliorative effect on bradyarrhythmia due to aging in w¹¹¹⁸ flies. Specifically, exercise enhanced HR and reduced the incidence of bradycardia, and this improvement was mainly attributed to the effective shortening of DI and SI (Figure 6A-D). In addition, exercise also significantly reduced the incidence of incomplete relaxation in w¹¹¹⁸ flies (Figure 6E), suggesting that exercise exerts multifaceted benefits on cardiac function in aging Drosophila. However, in the miR-283^KO/+ flies, exercise did not improve the incidence of bradycardia, although it increased HR and decreased HP by decreasing DI (Figure 6E). This result suggests that the ameliorative effect of exercise on cardiac rhythm may depend on the intact expression of miR-283.

Figure 6.

We next analyzed the effects of exercise on cardiac rhythms in w¹¹¹⁸>mir-283^SP and Pdf>mir-283^SP Drosophila. The results showed that 3 weeks of endurance exercise was also effective in reducing bradyarrhythmia caused by aging or miR-283 knockdown in LN_vs. The main manifestations were synchronized improvement of reduced HR, prolonged DI, and high-frequency onset bradycardia (Figure 6F-J). These improvements may be related to the fact that endurance exercise improves the morphology of contractile circumferential muscles. Drosophila experiencing exercise had more closely aligned ring myogenic fibers and fewer gaps than the age-matched non-exercising group (Figure 6K-N). In addition, we also observed denser non-contractile longitudinal fibers (Figure 6K-N).

Improvements in cardiac structure and arrhythmia may be due in part to the regulation of miR-283 by exercise. Exercise significantly reduced brain and myocardial miR-283 expression accumulation due to aging and miR-283 knockdown in LN_vs (Figure 6O), and its target gene cwo was significantly upregulated in the brain (Figure 6P). This result suggests that the ameliorating effect of exercise on cardiac rhythm may act through nervous system miR-283-targeted regulation of cwo. In addition, endurance exercise significantly prolonged the mean and median lifespans of w¹¹¹⁸>mir-283^SP flies (Figure 6Q). Although exercise improved lifespan in Pdf>mir-283^SP Drosophila, it did not reach a statistically significant difference (Figure 6Q). This provides further evidence that the overall beneficial regulatory effects of exercise on the organism may depend on the stable expression of miR-283.

In conclusion, our study suggests that 3 weeks of endurance exercise exerts a cardioprotective effect by modulating the stable expression of miR-283/cwo in the brain, thereby counteracting bradyarrhythmia caused by aging or miR-283 knockdown in LN_vs.

3. Discussion

miRNAs play an important role in regulating deleterious changes in cardiac aging and related diseases [50], however, a limited number of role-playing miRNAs have been identified. Circulating miR-216a was found to be a circulating marker for early heart failure screening [17], and its overexpression promotes the proliferation of human cardiac fibroblasts and enhances fibrosis [51], however whether it is involved in the regulation of cardiac aging remains unknown. In this study, we systemically overexpressed or knocked out miR-283, the homolog of miR-216a in Drosophila, both causing age-related HR reduction and rhythm abnormalities, suggesting that the stable expression of miR-283 plays an important role in aging heart health. Further studies revealed that specific knockdown of miR-283 in LN_vs resulted in upregulation of miR-283 throughout the brain, which in turn induced bradyarrhythmias consistent with aging. This process may be mediated by the target gene cwo of miR-283. In addition, inducing endurance exercise of anti gravity climbing in flies with miR-283 knockdown in LN_vs can improve bradyarrhythmic features by decreasing brain miR-283 expression (mechanism diagram shown in Figure 7). Notably, we detected dynamic expression of miR-283, either decreasing or increasing, during different stages of cardiac aging. However, the specific knockdown of miR-283 in the heart did not affect cardiac function. This suggests that miR-283 in the heart may not be the main miRNA regulating cardiac aging or that there is compensatory regulation from the brain or other parts to maintain stable cardiac function after specific knockdown of miR-283 in the heart.

Figure 7.

Age-related cardiovascular risks are not only closely related to the dysfunction of peripheral organ systems such as the heart and vascular system, but also closely associated with the imbalance of the autonomic nervous system[3]. Previous studies have confirmed that, neurohumoral regulation plays a critical role in cardiac remodeling to minimize changes in cardiac function [52, 53]. Thus, the autonomic nervous system plays an important role in the regulation of cardiac electrophysiology and arrhythmogenesis [54]. An in situ hybridization experiment on mir-283 showed that miR-283 is centrally expressed in the peripheral nervous system of Drosophila embryos, and silencing of miR-283 using the 2′Ome oligos technique induced neurological defects [55], further defining the autonomic modulatory role of miR-283. As revealed by the results of miR-283 overexpression or knockdown, the effects of miR-283 on cardiac function mainly focused on reduced HR and abnormal rhythm without affecting cardiac contractility, which predicts that the regulation of the aging heart by miR-283 may act through the autonomic nervous system. In particular, the specific knockdown of miR-283 in LN_vs resulted in an increased range of SA-β-gal staining in the brain and heart, showing strong aging-specific changes. In addition, knockdown of miR-283 in LN_vs resulted in disorganized myocardial myogenic fiber arrangement, and which may have contributed to more generalized bradycardia.

miRNAs regulate gene expression by inhibiting the translation of target transcripts or by inducing mRNA degradation mainly at the post-transcriptional level through sequence-specific binding [12]. In this study, we further explored the potential mechanisms by which miR-283 regulates the heart rate and rhythm. Using bioinformatics, we predicted that miR-283-5p target genes are mainly enriched in the Notch signaling pathway (Nct, mam, Ser, Delta, and Hairless) and could potentially regulate biological rhythm by targeting multi-tissue expression of cwo and Pdp1. Of the potential target genes tested, only cwo was downregulated in the heart and brain of middle-aged w¹¹¹⁸>mir-283^SP and young Pdf>mir-283^SP flies, consistent with the trend for miR-283 to accumulate in senescent and LN_vs knockdown hearts and brains and thus degrade target genes. As a transcriptional repressor, CWO can competitively bind to E-box to inhibit sustained transcriptional activation of PER-TIM by CLK-CYC [56]. Where the function of cwo is absent, the direct target genes of Clk (per, tim, vri, and Pdp1) manifest high trough values and low amplitude oscillations [57], which accounts for the significant downregulation of cwo and significant increase in Pdp1 following aging and miR-283 knockdown in our results. The orthologs of the CWO in mammals are DEC1 and DEC2. In a mouse model of transverse aortic constriction-induced cardiac hypertrophy, Dec1 deficiency protected the heart from fibrosis [58, 59]. In addition, it has been shown that DEC1 and DEC2 are involved in cardiogenesis and central nervous system development in zebrafish [60], further revealing the potential role of cwo in regulating cardiac function.

Exercise plays an important role in preventing cardiovascular diseases and delaying cardiovascular aging [61]. Therefore, exploring the mechanisms underlying the benign regulation of cardiac aging by exercise is necessary to reduce or reverse age-related cardiac decline. More recently, a growing number of studies have shown that miRNAs are important mediators of exercise-induced cardiac remodeling and myocardial regeneration, exerting cardioprotective effects via multiple pathways [26, 62, 63]. In our study, both aged w¹¹¹⁸ flies and w¹¹¹⁸ >mir-283^SP flies, as well as those with miR-283 specifically knocked down in LN_vs, showed effective improvements in heart rate reduction and bradycardia after undergoing a three-week exercise training program. Additionally, compared to age-matched non-exercise group, the myocardial fibers of the exercised Drosophila were more neatly and densely arranged. Those benign improvement in cardiac function may be attributed to the lowering of accumulated miR-283 in the heart and brain by exercise, thereby relieving the inhibitory effect on cwo. Furthermore, we also observed a significant increase in the mean lifespan and a significant rightward shift in the survival curve in w¹¹¹⁸>mir-283^SP group after exercise, suggesting that exercise produces a broad benefit in Drosophila.

In conclusion, our study shows that miR-283 regulates aging-associated bradyarrhythmias. In particular, miR-283 knockdown in LN_vs, but not the myocardium, promotes the development of bradyarrhythmias and induces brain and myocardial senescence, as well as cardiac structural decline. These changes are associated with accumulation of miR-283 in the brain and heart due to miR-283 knockdown in LN_vs, and the target gene cwo may mediate the regulatory effects of miR-283 on cardiac rhythm. Endurance exercise from youth effectively downregulated miR-283 accumulation in the heart and brain caused by aging or miR-283 knockdown in LN_vs, thereby rescuing reduced heart rate and frequent bradycardia and improving myocardial structure. However, the regulatory role of miR-283 in LN_vs on the whole brain as well as heart and even other peripheral tissues needs to be investigated in depth. In addition, we speculate that miR-283 regulation in the aging heart may act through the autonomic nervous system, but electrophysiological indicators still need to be further investigated.

4. Materials and Methods

4.1 Drosophila stocks and rearing conditions

White¹¹¹⁸ (w¹¹¹⁸) was obtained from the Bloomington Drosophila Stock Center (BDSC#3605) as a wild-type line. MiR-283 expression regulatory strains included knockout (mir-283^KO, BDSC#58912), overexpression (mir-283^OE, BCF132#, y M{vas-int.Dm}ZH-2A w[*]; P{CaryP}attP40, obtained from the Core Facility of Drosophila Resource and Technology, CEMCS, CAS), and knockdown (mir-283^SP,BDSC#61415, UAS-mCherry. mir-283.sponge). Tub-Gal4/TM3 (a gift from the Heart Development Center of Hunan Normal University) was used as a systemic driver. Pdf-Gal4 (BDSC#41286)[64] and Hand-Gal4 (BDSC#48396) driver lines are specifically expressed in ventral-lateral neurons (LN_vs) and heart, respectively. The flies were reared on standard sugar/yeast/agar medium at a density of 30-35 flies per tube in a controlled environment at 25°C, 50% humidity, and 12 h light:12 h dark cycles (12:12h LD). The study was approved by the Ethics Committee of Hunan Normal University (No. 2024-375) and the experimental procedures were conducted in strict compliance with the ethical guidelines set by the committee. All procedures conformed to the guidelines from the Directive 2010/63/EU of the European Parliament.

4.2 Exercise training devices and protocols

We developed a climbing locomotion device that exploits the natural negative geotaxis of Drosophila. By placing the test tube (230 mm×900 mm) containing the fruit flies vertically into the card slot, the card slot rotates along its horizontal axis under the drive of the motor, and the rotation speed and angle can be set. In this study, the card slot is set for 180° vertical flip, the motor speed is 0.45 rev/s, and the reserved climbing time is 5s. Under this setting, most flies performed anti-gravity climbing movements throughout the exercise period, and a few fruit flies that failed to climb in the later period of the exercise could also actively walk up the inner wall of the test tube.

Exercise-trained Drosophila were subjected to a continuous 3-week exercise intervention starting from 10 days of age. Following the exercise protocol established in our previous studies[65], the flies were exercised for 2.5 hours per day for five consecutive days, followed by a two-day rest period. Data were collected at 30 days of age. The exercise sessions were conducted between ZT5 and ZT8.

4.3 Semi-intact Drosophila preparation and cardiac function

Prepare Drosophila semi-intact hearts using previously described methods[33]. Briefly, after anesthetizing flies with CO₂, the head, ventral thorax, ventral abdominal cuticle, and organs were quickly removed to expose the heart. In the oxygenated artificial Drosophila hemolymph (ADH, 108 mM NaCl₂, 5 mM KCl, 2 mM CaCl₂, 8 mM MgCl₂, 1 mM NaH₂PO₄, 4 mM NaHCO₃, 15 mM 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid, 10 mM sucrose, and 5 mM trehalose, pH 7.1), the fat around the second and third abdominal segments of the heart tube is carefully aspirated with a finely drawn glass capillarie to expose the heart edge. Then, high-speed digital movies of beating hearts were taken with a Hamamatsu EMCCD 9300 camera (Hamamatsu, Japan) at ∼124 fps for 30 s and were recorded using HCImage software (Hamamatsu, Japan). The functional cardiac parameters were assessed using the semi-automatic optical heartbeat analysis software (SOHA, kindly gifted by Ocorr and Bodmer), which can accurately detect and quantify heart rate (HR), heart period (HP), arrhythmicity index (AI, StdDev/Median of HP), systolic and diastolic intervals (SI and DI), diastolic and systolic diameters (SD and DD), and percent fractional shortening (%FS=(DD-SD/DD) ×100). Cardiac output (CO) was calculated as [π r(d)² - π r(s)²] × HR, where r(d) is the diastolic cardiac tube radius and r(s) is the systolic cardiac tube radius[66]. In addition, a DI longer than 1 s was considered bradycardia, and a DI shorter than 0.06 s indicative of incomplete relaxations were used to estimate cardiac rhythmicity and abnormal contractions[34].

4.4 Fluorescence imaging

Pdf-Gal4 and Hand-Gal4 were used to drive the UAS-mCherry.mir-283.sponge Drosophila express mCherry fluorescence in PDF neurons, and in cardiac and paracardial cells. Drosophila brains [67] and semi-exposed hearts [33] were prepared according to a previously described method, and hearts were treated with hemolymph containing 10 mM EGTA after dissection to maintain a diastolic state. Drosophila LN_vs and heart fluorescence expression were photographed from 3 to 5 days using a stereomicroscope (Leica DVM6, Leica Microsystems, Germany).

For F-actin staining in the heart, semi-exposed hearts were fixed with 4% paraformaldehyde at room temperature for 20 min in a relaxed state, washed with PBS three times, and then incubated with AbFluor™ 488-Phalloidin (1:500, Abbkine, BMD0082) at room temperature for 1 h, then washed 2-3 times with PBS, and mounted in an anti-fluorescence quenching agent. Confocal images were taken using a NIKON ECLIPSE TI microscope in conjunction with a NIKON C2 imaging system.

4.5 SA-β-gal staining and quantification

According to the manufacturer’s instructions (Senescence β-Galactosidase Staining Kit, C0602, Beyotime Bio., Shanghai, China), the dissected Drosophila brain and half-exposed heart were fixed in fixative solution for 30 min and washed 3 times with PBS, 5 min each. Transfer the tissue to a 1.5 ml Ep tube, add 1 ml staining working solution, and incubate overnight at 37°C. Carefully clip the brain or clip out the heart tube individually, and place it on a concave glass slide for mounting. The stained brain and heart tubes was photographed using a Leica stereomicroscope (Leica, DVM6) under the same conditions. Image J software were used to calculate the percentage of positive staining area in the whole brain or whole heart tube area by adjusting the threshold for quantitative analysis. For the analysis of the heart tube, since the accessory cardiomyocytes showed a strong β blue color, the LASSO tool in Adobe Photoshop was used to select the myocardial tissue before quantitative analysis, and the background outside the heart tube was removed.

4.6 Gene expression analysis

The brain, heart, and whole body of age-matched flies were collected. Total RNA was extracted using the RNA fast 200 kit (Fastagen Biotech, Shanghai, China) and cDNA was synthesized using PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Japan) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed on Bio-Rad CFX Connect Real-Time System with TB Green (TaKaRa, Japan) and the corresponding primer sets. The primer sequences are shown in Table S1.

4.7 Rapid iterative negative geotaxis (RING)

To assess the locomotion and neuromuscular coordination of Drosophila, negative geotaxis was performed according to the method described by Julia et al. [68, 69]. Briefly, age-appropriate flies were transferred into five cylindrical glass tubes (18 cm × 2.8 cm side by side) with sponge plugs at both ends to prevent escape and injury. During the RING process, nine shake-downs were performed, each time the fruit flies were allowed to climb for 10 s, and the complete RING was videotaped. Abandon the first three adaptive climbing times, and obtained the climbing height of the 5th seconds of 4-9 climbing videos for analysis. Since the autonomous activities of Drosophila show a clear rhythm with peaks of activity before and after light is turned on and off [70], we uniformly acquired RING videos during ZT11 to ZT12.

4.8 Survival statistics and analysis

Randomly selected 100-150 flies from each group were used for lifespan analysis. The number of dead flies was recorded at ZT14∼ZT15 each day. The average lifespan of Drosophila was recorded and calculated in Excel, and survival curves were plotted using the Hiplot online tool (https://hiplot.com.cn/home/index.html).

4.9 Bioinformatics analysis

The DIANA microT-CDS online tool (http://diana.imis.athena-innovation.gr/DianaTools/) was used to predict target genes for dme-miR-283-5p. The DAVID Bioinformatics Resources (https://david.ncifcrf.gov/) was utilized to perform pathway enrichment analysis of the predicted target genes. Use the online resource RNAhybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid) to predict the binding targets of dme-miR-283-5p and corresponding mRNA 3’UTR, and generate a secondary structure.

4.10 Cell culture, transfection, and luciferase assay

293T cells (human embryonic kidney cells) were cultured in DMEM (BasalMedia, Shanghai, China) containing 10% FBS and 1% P/S under conditions of 37°C and 5% CO₂. The reporter genes for the wild-type 3’UTR of cwo and the mutant 3’UTR with predicted binding sites were cloned into the multiple cloning sites of the psiCheck-2 vector (1643-1674). Both the plasmids and miR-283 mimics were synthesized by Tsingke Biotech Co. (Beijing, China). The transfection process of plasmids and miRNA mimics was mediated using Lipo8000^TM transfection reagent (Beyotime Biotechnology Co., Shanghai, China) in a 96-well plate. The transfection amount of plasmids was 100 ng / well, and the transfection amount of miRNA was 5 pmol / well. Luciferase activity was measured according to the instructions of the Dual-Luciferase^®Reporter (DLR™) Assay System (Promega, UAS) and normalized to the Firefly luciferase data.

4.11 Statistical analysis

Data were processed and statistical analysis using SPSS 22.0. In the SOHA analysis, data were normalized using the Z-score normalization method prior to data analysis, and abnormal data with absolute values ≥ 3 were removed. Subsequently, data that conformed to a normal distribution and variance homogeneity were subjected to an independent samples t-test or one-way ANOVA with Tukey’s multiple comparison test. Two-way ANOVA was used to detect the interaction effects of miR-283 knockout and aging, as well as the specific knockdown of miR-283 in LN_vs and aging, multiple comparisons were performed using Tukey’s test. GraphPad Prism was used for graph generatio and is displayed as mean ± SEM. Statistical significance was set at at p < 0.05.

Acknowledgements

We are grateful to the Center for Heart Development of Hunan Normal University for providing Tub-Gal4/TM3 flies.

Additional information

Funding

This work was supported by grants from the National Natural Science Foundation of China (grant number 32371182).

Author Contribution

Qiufang Li: Conceptualization; methodology; data curation; formal analysis; visualization; writing—original draft. Lan Zheng: Conceptualization; resources; supervision; funding acquisition; project administration; writing—review and editing. Xu Ping: Data curation; formal analysis; Zhengwen Yu: Data curation; formal analysis. Qin Yi: Data curation; formal analysis. Chao Tang: Data curation. Xiaoya Wang: Data curation.