Figures and data in Cardiac neurons expressing a glucagon-like receptor mediate cardiac arrhythmia induced by high-fat diet in Drosophila

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7 figures, 1 table and 1 additional file

Figures

Figure 1

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High-fat diet (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 an 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. The numerical data used to generate the figure are provided in Figure 1—source data 1.

Figure 1—source data 1 The numerical data used to generate the Figure 1.: https://cdn.elifesciences.org/articles/94512/elife-94512-fig1-data1-v1.xlsx
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Figure 2 with 1 supplement

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High-fat diet (HFD) upregulates 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. Error bars represent SD. (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 a heatmap; scale from 0 (blue), no CaLexA detected, to 255 (red) high levels CaLexA. Scale bar: 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. (E) Representative confocal images of w¹¹¹⁸ APCs from female adult flies fed an NFD or HFD (for 7 days following eclosion from pupa). Anti-Akh is in red. DAPI stains DNA in blue. Scale bar: 20 μm. (F) Quantitation of anti-Akh fluorescence in E (7-day-old females; NFD, n=8; HFD, n=7). Statistical analysis was performed with unpaired t-test corrected with Welch; **p<0.01. The numerical data used to generate the figure are provided in Figure 2—source data 1.

Figure 2—source data 1 The numerical data used to generate the Figure 2.: https://cdn.elifesciences.org/articles/94512/elife-94512-fig2-data1-v1.xlsx
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Figure 2—figure supplement 1

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High-fat diet (HFD) increases crop size.

(A) Representative confocal images of crops from female adults (w¹¹¹⁸) fed a normal fat diet (NFD) or a 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. Error bars represent SD. Statistical analysis was performed using t-test corrected with Welch; ***p<0.001.

Figure 3 with 1 supplement

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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. The numerical data used to generate the figure are provided in Figure 3—source data 1.

Figure 3—source data 1 The numerical data used to generate the Figure 3.: https://cdn.elifesciences.org/articles/94512/elife-94512-fig3-data1-v1.xlsx
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Figure 3—figure supplement 1

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*Akh*-Gal4>*Akh* IR-depleted Akh.

(A) Representative confocal images of APCs from female adults. Control, *Akh*-Gal4>w¹¹¹⁸. Anti-Akh is in red. DAPI stains DNA in blue. Scale bar: 50 μm. (B) Quantitation of the relative fluorescence intensity of anti-Akh in A. n=4. Statistical analysis was performed using t-test corrected with Welch; **p<0.01.

Figure 4

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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. Error bars represent SD. (B) Quantitation of arrhythmia index in control (n=14 NFD, n=12 HFD) and *AkhR^null* (n=13 NFD, n=11 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 *AkhR^null* mutant 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. The numerical data used to generate the figure are provided in Figure 4—source data 1.

Figure 4—source data 1 The numerical data used to generate the Figure 4.: https://cdn.elifesciences.org/articles/94512/elife-94512-fig4-data1-v1.xlsx
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Figure 5 with 1 supplement

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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 toward 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 gray. (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.

Figure 5—figure supplement 1

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AkhR neuron receives Akh.

(A) Representative confocal images of AkhR neuron from female adults (*AkhR*-Gal4>GFP). GFP is in green. Anti-Akh is in red. DAPI stains DNA in blue. Scale bar: 20 μm.

Figure 6

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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. The numerical data used to generate the figure are provided in Figure 6—source data 1.

Figure 6—source data 1 The numerical data used to generate the Figure 6.: https://cdn.elifesciences.org/articles/94512/elife-94512-fig6-data1-v1.xlsx
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Figure 7

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Model: Akh/AkhR mediates the high-fat diet (HFD)-induced cardiac arrhythmia.

An 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 secretion 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 these signaling pathways, increased Akh under HFD conditions leads to increased arrhythmia.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Antibody	Mouse monoclonal anti-Brp	Developmental Studies Hybridoma Bank	Cat# Nc82 RRID:AB_2314866	IF (1:100)
Antibody	Goat polyclonal anti-mouse Alexa Fluor 647	Jackson Immunoresearch Laboratories	Cat# 115-605-003 RRID:AB_2338902	IF (1:500)
Antibody	Rabbit polyclonal anti-Akh	Prof. Jae Park	N.A.	IF (1:1000)
Antibody	Goat polyclonal anti-rabbit Alexa Fluor 568	Thermo Fisher	Cat# A-11011 RRID:AB_143157	IF (1:500)
Other	Alexa Fluor 647 Phalloidin	Thermo Fisher	Cat# A22287	IF (1:1000)
Other	Normal goat serum	Jackson Immunoresearch Laboratories	Cat# 102643-594	1% in 1xPBST
Genetic reagent (Drosophila melanogaster)	w¹¹¹⁸	Bloomington Drosophila Stock Center	RRID:BDSC_3605
Genetic reagent (D. melanogaster)	Akh-Gal4	Bloomington Drosophila Stock Center	RRID:BDSC_25684
Genetic reagent (D. melanogaster)	UAS-Akh-RNAi	Bloomington Drosophila Stock Center	RRID:BDSC_34960
Genetic reagent (D. melanogaster)	w¹¹¹⁸ P{UAS-rpr.C}27; P{UAS-2xEGFP}AH3	Bloomington Drosophila Stock Center	RRID:BDSC_ 91417
Genetic reagent (D. melanogaster)	AkhR^null	Bloomington Drosophila Stock Center	RRID:BDSC_80937
Genetic reagent (D. melanogaster)	w[]; P{w[+mC]=LexAop-CD8-GFP-2A-CD8-GFP}2; P{w[+mC]=UAS-mLexA-VP16-NFAT*}H2, P{w[+mC]=lexAop-rCD2-GFP}3/TM6B, Tb[1]	Bloomington Drosophila Stock Center	RRID:BDSC_66542
Genetic reagent (D. melanogaster)	10xUAS-GFP	Bloomington Drosophila Stock Center	RRID:BDSC_32185
Genetic reagent (D. melanogaster)	AkhR-T2A-Gal4	Bloomington Drosophila Stock Center	RRID:BDSC_78877
Chemical compound, drug	4% paraformaldehyde (PFA)	Thermo Fisher Scientific	Cat# J19943.K2
Chemical compound, drug	Triton X-100	Sigma-Aldrich	Cat# 9002-93-1	0.2% in 1xPBS
Chemical compound, drug	4′,6-Diamidino-2-phenylindole (DAPI)	Thermo Fisher Scientific	Cat# D1306 RRID:AB_2629482	(0.5 µg/ml)
Commercial assay or kit	10x phosphate buffered saline	Quality Biological	Cat# 119-069-131
Commercial assay or kit	TRIzol	Thermo Fisher Scientific	Cat# 15596026
Commercial assay or kit	EcoDry Premix kit	Takara Bio	Cat# 639549
Commercial assay or kit	PowerSYBR Green	Applied Biosystems, Thermo Fisher Scientific	Cat# 4367659
Commercial assay or kit	VECTASHIELD	Vector Laboratories	Cat# H-1000
Software, algorithm	Adobe Illustrator 2022	Adobe Inc.	RRID:SCR_010279
Software, algorithm	ImageJ	National Institutes of Health	RRID:SCR_003070
Software, algorithm	GraphPad Prism9	GraphPad	RRID:SCR_002798