HFD increases heart rate and arrhythmia index in Drosophila

(A) Schematic illustration of the experimental design. Adult flies (female) were fed either a normal fat diet (NFD) or a high-fat diet (HFD) for 7 days following eclosion from pupa, then subjected to heart functional analysis using optical coherence tomography (OCT) to determine the heart period and the arrhythmia index. (B) Representative images obtained from OCT videos of w1118 flies that were fed NFD or HFD as indicated. Scale bars: 2 seconds. (C,D) Quantitation of heart period (C; n=15 NFD, n=11 HFD) and arrhythmia index (D; n=14 NFD, n=12 HFD). Statistical analysis was performed using t-test corrected with Welch; **, P<0.01.

HFD up-regulates adipokinetic hormone gene Akh and activates Akh neurons

(A) Representative confocal image of Akh producing cells (APCs; green, GFP) in Akh- Gal4>10xUAS-GFP (Akh-Gal4>GFP) transgenic flies (3-day-old females). The image shows the anterior section of the digestive system, including the crop, esophagus, and proventriculus. The APCs are located at the corpora cardiaca, which attaches to the esophagus, just anterior to the proventriculus. Phalloidin stains actin filaments red; DAPI stains DNA in nucleus blue. Scale bar: 100 μm. (B) RT-qPCR analysis of Akh expression in w1118 flies (female) that were fed either a normal fat diet (NFD) or a high-fat diet (HFD) for 7 days following eclosion from pupa. Ten flies per group, repeated three times. Statistical analysis was performed with unpaired t-test corrected with Welch; *, P<0.05. (C) Representative confocal images of Akh- Gal4>CaLexA APCs from female adult flies fed an NFD or HFD (for 7 days following eclosion from pupa). CaLexA is a transcription-based genetically encoded calcium indicator for neuronal activity. CaLexA fluorescence has been presented as heatmap; scale from 0 (blue), no CaLexA detected, to 255 (red) high levels CaLexA. Scale bars: 20 μm. (D) Quantitation of CaLexA fluorescence in C (7-day-old females; NFD, n=14; HFD, n=10). Statistical analysis was performed with unpaired t-test corrected with Welch; ***, P<0.001.

Akh regulates the heartbeat

(A) Representative images obtained from optical coherence tomography (OCT) videos of control (Akh-Gal4) and Akh-RNAi (Akh-Gal4>UAS-Akh-RNAi) flies (females, 7 days old) that were fed a normal fat diet (NFD) or a high-fat diet (HFD), as indicated, for seven days starting at eclosion from pupa. Scale bars: 2 seconds. (B) Quantitation of the arrhythmia index in control (n=15 NFD, n=13 HFD) and Akh-RNAi (n=13 NFD, n=14 HFD) flies. Statistical analysis was performed with two-way ANOVA corrected with Sidak. Statistical significance: *, p<0.05; ****, p<0.0001; ns, not significant.

AkhR regulates the heartbeat

(A) RT-qPCR analysis of AkhR expression in w1118 flies (female) that were fed either a normal fat diet (NFD) or a high-fat diet (HFD) for 7 days following eclosion from pupa. Ten flies per group, repeated three times. Statistical analysis was performed with unpaired t-test corrected with Welch; *, P<0.05. (B) Quantitation of arrhythmia index in control (n=14 NFD, n=12 HFD) and AkhRnull(n=11 NFD, n=13 HFD) flies. Statistical analyses were performed with two-way ANOVA corrected with Sidak; **, P<0.01; ***, P<0.001; ns, not significant. (C) Representative images obtained from optical coherence tomography (OCT) videos of control (w1118) and AkhRnull mutant flies flies (females, 7 days old) that were fed NFD or HFD, as indicated, for seven days starting at eclosion from pupa. Scale bars: 2 seconds.

AkhR cardiac neurons (ACNs) form synaptic connections with the posterior heart

(A) Graphic depiction of the body (no legs or wings) of an adult fly with the head oriented towards the top. The heart tube (red) is located along the midline in the abdomen (bottom of graphic), with the alary muscles that connect the heart to the exoskeleton represented in light grey. (B) A representative confocal image of an AkhR-Gal4>10xUAS-GFP adult fly (7-day-old, female). The dorsal region of an abdomen corresponding to the boxed region in (A) is shown. AkhR>GFP labels AkhR in green. ACN, AkhR cardiac neuron. Phalloidin stains actin filaments red. Scale bar: 100 μm. (C) Schematic illustration of the posterior fly heart. Corresponds to boxed region in (A). (D) A representative confocal image of the posterior heart of an AkhR- Gal4>10xUAS-GFP fly (7-day-old, female) corresponding to the boxed region in (C). AkhR>GFP labels AkhR in green. Phalloidin stains actin filaments red. Scale bar: 50 μm. (E) Schematic illustration of the posterior end of an adult fly heart. The neurites of the cardiac neurons are depicted in green. Relates to the boxed region in (C). (F) Confocal image of the posterior heart of an AkhR-Gal4>10xUAS-GFP fly (7-day-old, female) corresponding to the boxed region in (E). AkhR>GFP labels AkhR in green. Anti-Bruchpilot (Brp), a marker for the active zone, is shown in magenta. Scale bar: 10 μm.

Partial elimination of ACNs causes arrhythmia

(A) A representative confocal image of an adult AkhR-Gal4>rpr, GFP (AkhR-Gal4>UAS-rpr, UAS-GFP) fly (7-day-old, female). AkhR>GFP labels AkhR in green. AkhR, adipokinetic hormone receptor; rpr, reaper. Phalloidin stains actin filaments red. Scale bar: 100 μm. (B) Representative images obtained from optical coherence tomography (OCT) videos of control (w1118) and AkhR-Gal4>rpr (AkhR-Gal4>UAS-rpr, UAS-GFP) flies (7-day-old, female). Scale bar: 2 seconds. (B) Quantitation of arrhythmia index in control (n=14 NFD) and AkhR-Gal4>rpr (n=23 NFD). Statistical analysis was performed using t-test corrected with Welch; ****, P<0.0001.

Model: Akh/AkhR mediates the HFD-induced cardiac arrhythmia

A high-fat diet (HFD) upregulates adipokinetic hormone (Akh) levels in the Akh producing cells (APCs) located at the corpora cardiaca adjacent to the anterior esophagus. HFD also increases APC activity, this results in increased Akh section into the circulation. Akh acts on the Akh receptors (AkhR) expressed by the AkhR cardiac neurons (ACN). The ACN are a pair of neurons located at the posterior end of the adult fly heart, that regulate the heartbeat. Through this signaling pathways, increased Akh under HFD conditions leads to increased arrhythmia.

HFD increases crop size

(A) Representative confocal images of crops from female adults (w1118) fed a normal fat diet (NFD) or a high-fat diet (HFD) for 7 days following eclosion from pupa. DAPI stains DNA in blue. Scale bar: 100 μm. (B) Quantitation of crop area. n=8 NFD and n=6 HFD. Statistical analysis was performed using t-test corrected with Welch; ***, P<0.001.