Shaker potassium channel mediates an age-sensitive neurocardiac axis regulating sleep and cardiac function in Drosophila

For the cardiac and neuronal specific function of shaker gene the fly stocks were ordered from the Bloomington Drosophila Stock Center (BDSC) and the Vienna Drosophila Resource Center (VDRC). The stocks include UAS-RNAi lines for the Shaker gene (BDSC: 53347; BDSC: 31680 and VDRC:104474), controls (attP2, BDSC: 36303 and attP40, BDSC: 36304). Cadiac-specific driver Hand-Gal4 was obtained from Dr. Olson's laboratory, (University of Texas Southwestern Medical Center, Dallas, TX, USA) and another one for panneuronal expression (Elav-Gal4; BL#458). The RNAi system uses small interfering RNA (siRNA) strands that are complementary to a gene of interest to experimentally silence its expression. The Shaker knockdown was accomplished through UAS-RNAi lines targeting Shaker transcripts BL#53347(attP40 insertion) and BL#31680 (attP2 insertion). The siRNA fragments bind to the mRNA of Shaker gene and degrade them, thereby preventing mRNA translation (Ichim et al. 2004). We used BDSC: 36303 control line carrying an empty attP2 vector inserted in the 3rd chromosome, while the BDSC: 36304 control line carrying an empty attP40 vector in the 2nd chromosome, which ensures the phenotypes observed in the experiments appeared from Shaker gene silencing and not from insertional effects at specific sites. The flies from each UAS-RNAi Shaker and control line were crossed with virgin female flies from the Hand-Gal4 and Elav-Gal4 for cardiac and panneuronal driver lines respectively. The F1 progeny flies were collected (Villanueva et al. 2019; Guo et al. 2024). Male and female progeny flies were separated and maintained in a standard food at 25 °C and transferred onto fresh food every 3–4 days throughout the study. Using three-week-old (mid age) and five-week-old (early aging) male and female flies we have performed all the experiments with indicated number of flies in each experiment.

Progeny from each line was collected at three-weeks or five-weeks of age for heart physiology analysis. Semi-intact heart dissections were performed to make the heart visible for recording (Fink et al. 2009; Guo et al. 2024). Flies were made unconscious with CO₂ gas and fixed on their backs to petri dishes with petroleum jelly. Under a light microscope, the head and thorax were removed, followed by a small incision at the apex of the abdomen. Plates were then flooded with warmed, aerated artificial hemolymph (Trehalose 5 mM, supplemented with physiological saline containing 108 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 8 mM MgCl2, 1 mM NaH₂PO₄, 4 mM NaHCO₃, 15 mM HEPES and sucrose 10 mM with pH 7.2) to ensure hearts maintained myogenic activity during recording. which is continuously aerated to maintain myogenic contraction. The top of the abdomen was then removed, followed by the intestine and fat, exposing the heart. After that recordings were taken using an immersion microscope lens with an attached high-speed camera. Heart physiology was recorded between the second and third body segments to ensure consistency across flies. Recordings were made using a Promon u750 microscope camera in B&W at 200 frames/second, for 30 seconds (Fink et al. 2009; Gill et al. 2015; Guo et al. 2024). Videos were analyzed using semi-automated heart analysis (SOHA) software and data output was organized in excel files with M-mode records (Cammarato et al. 2015; Gill et al. 2015). The SOHA data we analyzed included heart period, arrhythmicity index, systolic and diastolic intervals, systolic and diastolic diameters and fractional shortening (Guo et al. 2024). The sample size for heart analysis is 15–20 per group. The SOHA analysis performed blind conditions. Using Prism 10 software, the SOHA output variables for experimental lines were statistically compared to the control lines using one-way analysis of variance (ANOVA). Significance was set at p < 0.05, and Tukey's multiple comparisons test was used for each data set.

As previously described, the fly bodies were maintained for 20 min in a 4% paraformaldehyde (PFA) solution after the heads, legs, and wings were removed for the cytological test (Guo et al. 2024; Abou Daya et al. 2025). The fixed samples were then incubated for 15 min between each of the three PBS washes. The thoraces were longitudinally oriented in a cryomold using OCT (Fisher Scientific #4585) and flash-frozen on dry ice. Following cryosectioning to reach a thickness of 30 µm, three further washes in 1 × PBS were carried out, each requiring a 15-min incubation period. Samples were rinsed three times in PBS to assess structural abnormalities following a 30-min staining process with 0.1-µm Alexa-594-Phalloidin to detect actin-containing myofibrils. The quantification of myofibril stained with phalloidin was performed using Image J using images from multiple sections for each group (Guo et al. 2024). In disorganized hearts the F-actin -containing myofibrils found to have more gaps than organized hearts in controls. Sample size n = 5 flies per genotype.

As previously used, individual flies were anesthetized with CO₂ and placed in glass tubes with food at one end and cotton plug at another end for aeration. These tubes were placed in the Drosophila activity monitor system (DAMS, Trikinetics) (Abou Daya et al. 2025). These monitors were then placed in incubators to regulate temperature and humidity (25 °C and 50% relative humidity). The data analysis was performed using Clock lab software, DAM analysis, outliers were removed with modified Z-score method in Microsoft Excel, and final graphs were plotted using Graph Pad Prism l0 software. We measured sleep, activity during daytime, nighttime and overall (24 h). Raw data was used to analyze sleep (immobility of a fly > 5 min), activity (fly crossing of an infrared beam at the center of DAM), Average sleep/activity behavior of individual fly from each genotype and condition in 24 h (ZT0-ZT24) was calculated over 5 days (Yadav et al. 2025; Abou Daya et al. 2025). Data from the day the individual flies were introduced to the DAMS monitors was not included in the average to allow for adaptation. Sample size n = 25–30 flies per genotype and age group. One-Way ANOVA is used for statistical analysis.

Shaker^mns is a specific mutant allele of the Shaker gene responsible for shortened sleep duration (mini sleep mutation). These flies need significantly less sleep compared to wild-type flies (Cirelli et al. 2005). This occurs because K⁺ channels play a critical role in regulating neuronal excitability, and their dysfunction leads to hyperactivity and reduced sleep (Kim et al. 2020). The cardiac physiology and sleep/circadian activity of flies were analyzed for the Sh^mns and Sh⁵ (Shaker gene point mutation) and compared to that of the age and sex-matched Drosophila control line w¹¹¹⁸. As shown in Fig. 1 A-H different variables of cardiac physiological data including: A. Heart rate (HR) B. Heart period (HP), C. Arrhythmicity index (AI), D, E. Diastolic (DI) and Systolic intervals (SI), F, G. Diastolic diameter (DD) and Systolic diameter (SD), and H. Fractional shortening (FS) were collected from three-week and five-week-old male flies. Sh^mns showed an increase in HP, AI, DI, and SI than w¹¹¹⁸ and Sh⁵, as well as significantly decreased FS and HR compared to w¹¹¹⁸. Sh⁵ shows a significant reduction in FS and an increase in HR compared to w¹¹¹⁸. DD and SD did not find any significant difference between the control and mutants. The Sh^mns five-week flies showed an increased HP, DI, and SI than w¹¹¹⁸, and Sh⁵ and AI, DD, and SD showed no change, but FS was significantly reduced compared to w¹¹¹⁸ in both Sh^mns and Sh⁵flies. The HR was reduced in Sh^mns compared to w¹¹¹⁸ and Sh⁵. Whereas Sh⁵ showed reduced SI, and FS compared to w¹¹¹⁸. In males at three weeks (Fig. 1 I–J) phalloidin staining of the heart showed a significant difference between w¹¹¹⁸ and Sh^mns, where Sh^mns possesses more disorganization of actin-containing myofibrils, compared to age-matched control w¹¹¹⁸. (Fig. 1 K–P), showed sleep/circadian activity analysis of w¹¹¹⁸ and Sh^mns, we observed a significant increase in activity and reduced sleep in Sh^mns compared to w¹¹¹⁸ at three-weeks and five-weeks of age. Sh^mns severely impairs cardiac function and reduces sleep, with age-related worsening in males. Sh⁵ shows milder, age-dependent cardiac physiology changes. These results highlight Shaker K⁺ channels' critical role in regulating cardiac function and sleep behavior.

We have investigated the impact of both Sh^mns and Sh⁵ alleles on female flies. (Fig. 2 A-H) represents cardiac data collected from three and five-week-old females. Sh^mns showed a significant increase in HP, DI, and SI compared to controls and Sh^mnsx Sh⁵ flies in both three weeks and five weeks of age. AI decreased in Sh^mnsx Sh⁵ than Sh^mns flies at five weeks and DD increased in Sh^mnsx Sh⁵ than Sh^mns flies at three weeks. Sh⁵ showed an increase in DI in five weeks compared to w¹¹¹⁸. Also, Sh⁵ showed a significant increase in HP in three weeks, SI at three weeks and five weeks of age in Sh^mns. At three weeks and five weeks of age, Sh^mns flies showed increased HP, DI and SI and in AI only in five weeks than w¹¹¹⁸. In five weeks Sh⁵ flies AI, SI, and FS are reduced, whereas DD and SD increased than Sh^mns. Sh^mnsx Sh⁵ flies showed decreased HP, DI, and SI at three weeks and five weeks of age and decreased AI than Sh^mns flies only by five weeks observed. We also found reduced SD with increased FS in Sh^mnsx Sh⁵ flies compared to Sh⁵ flies at five weeks. HR was reduced in Sh^mns flies at both ages, and there was no significant difference in Sh^mnsx Sh⁵ and Sh⁵ flies, but Sh^mnsx Sh⁵ flies showed a significant increase than Sh^mns flies at three weeks and five weeks of age. Sh^mnsx Sh⁵ mutants showing improved HP, DI, and SI, compared to Sh^mns alone at three weeks and five weeks of age, suggesting a non-additive or compensatory genetic interaction between Shaker alleles. Our data suggested that trans-heterozygous Sh^mnsx Sh⁵ mutants do not exacerbate Sh^mns-induced cardiac defects, instead, they are partially rescuing Sh^mns-induced heart dysfunctions.

Light significantly impacts sleep-circadian cycles and cardiac function. Since the Sh^mns mutant flies are genetically predisposed to have shorter sleep cycles compared to w¹¹¹⁸ flies, we performed cardiac physiology and sleep/circadian activity analysis to understand the impact of constant light on cardiac physiology both in male and female flies and sleep-health using male flies. (Fig. 3 A-H) represents the impact of light/dark (LD) and light/light (LL) cues-induced circadian cycle disruption on cardiac physiology on three-week-old flies. HR, HP, AI, DI, SI, DD, and SD did not show any statistically significant difference in LD vs LL condition in w¹¹¹⁸ as well as in Sh^mns male flies. We only observed reduced FS in LL w¹¹¹⁸ than LD w¹¹¹⁸. During LD condition Sh^mns flies showed a significant increase in HP, AI, DI, SI, DD, and SD than LD w¹¹¹⁸. During LL condition Sh^mns flies showed a significant increase in HP, DI, and SI compared to LL w¹¹¹⁸, but FS was reduced.

In females (Fig. 3 I–P) at three weeks LL condition showed significantly reduced HR, FS, and increased HP, AI in w¹¹¹⁸ flies compared to LD. In Sh^mns flies SD was reduced and FS increased significantly in LL compared to the LD condition. During LD condition Sh^mns flies showed increased HP, DI, SI, and reduced HR compared to LD w¹¹¹⁸. During LL Sh^mns flies showed increased HP, DI, SI, FS, and decreased DD and SD were observed compared to LL w¹¹¹⁸. The HR was reduced in LD Sh^mns flies and LL Sh^mns flies compared to respective controls in males and females, suggesting the light-light cycles regulate heart functions differently in mutant flies than w¹¹¹⁸.

Phalloidin staining (Fig. 3 Q, R) indicates reduced organization of F-actin containing myofibrillar percent in heart muscles of LL w¹¹¹⁸ and LL Sh^mns than LD w¹¹¹⁸, at three weeks age, with no significant difference with LD Sh^mns genotype. Further we have checked the impact of heart physiology on sleep/circadian activity parameters (Fig. 3 S–X), we found day activity, night activity, and total activity were increased, and day sleep, night sleep, and total sleep were reduced in Sh^mns than w¹¹¹⁸ flies during LD condition. During LL condition, day activity, night, and total activity increased, and sleep significantly decreased in Sh^mns flies than w¹¹¹⁸ flies at three weeks of age. We have not observed any significant difference during LL and LD conditions between w¹¹¹⁸ and Sh^mns flies, respectively. Sh^mns flies under LL conditions showed elevated HP, DI, SI and FS, reduced HR, in both male and female (Fig. 3) and sleep, (in males) with increased activity levels compared to LL w¹¹¹⁸. These results indicate that disrupted light cues worsen Sh^mns-associated physiological and behavioral impairments, highlighting the interaction between circadian regulation and Shaker channel function.

To understand the impact of feeding-fasting rhythms on heart physiology and sleep/circadian activity, on shaker mutant, we employed well-studied paradigm, known as time-restricted feeding (TRF) which is shown to annotate cardiac, and sleep-dysfunction linked with aging (Gill et al. 2015). Cardiac physiology under TRF and ad libitum feeding (ALF) of Sh^mns are shown in Fig. 4 A–H in males, and Fig. 4 I–P in females flies at three-week age. The male heart physiology did not show a significant difference between ALF Sh^mns and TRF Sh^mns male flies. Whereas in female flies, HP, AI, and DI decreased, and HR was increased in TRF Sh^mns flies, and SI, DD, SD, and FS did not show a significant difference compared with ALF Sh^mns flies. TRF partially rescues cardiac dysfunction and sleep/circadian activity disruptions in Sh^mns mutant flies. TRF improved heart function in females. Based on our observation in female flies, TRF improved cardiac parameters such as reduced HP, AI, and DI, and increased HR. In males, TRF did not show major changes in cardiac physiology. The sleep/circadian activity analysis (Fig. 4 Q–V) in male flies at three weeks of age are showed increased day activity, night activity and total activity (Fig. 4 Q–S), whereas day sleep, night sleep and total sleep was reduced significantly in TRF than in ALF Sh^mns flies (Fig. 4 T–V). These findings suggest that feeding timing can partially rescue Shaker-linked cardiac and sleep-circadian behavioral impairments through circadian-aligned interventions.

Drosophila is a well-established model for studying the human sleep–circadian cycle and age or disease-related changes in cardiac physiology. However, Drosophila cardiac function is influenced primarily by glutamatergic and neuropeptidergic signals, whereas the mammalian heart relies on autonomic regulation, a specialized conduction system, and multiple chambers. Likewise, although the A-type Shaker channel in flies regulates repolarization, its role is not directly comparable to Kv1.1 loss in mice, and there is no clear anatomical equivalent between species. Therefore, cross-species comparisons are useful for identifying conserved principles of ion-channel regulation but do not represent mechanistic equivalence. Consequently, findings from fly models should be interpreted with caution when relating them to mammalian physiology, and despite their mechanistic value, direct clinical significance cannot be drawn.