Video Protocol: Fly models of heart function

J Vis Exp. 2022 Aug 25;(186). doi: 10.3791/63939.

Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function

Gracheva E, Wang F, Matt A, Liang H, Fishman M, Zhou C

Abstract:

Using Drosophila melanogaster (fruit fly) as a model organism has ensured significant progress in many areas of biological science, from cellular organization and genomic investigations to behavioral studies. Due to the accumulated scientific knowledge, in recent years, Drosophila was brought to the field of modeling human diseases, including heart disorders. The presented work describes the experimental system for monitoring and manipulating the heart function in the context of a whole live organism using red light (617 nm) and without invasive procedures. Control over the heart was achieved using optogenetic tools. Optogenetics combines the expression of light-sensitive transgenic opsins and their optical activation to regulate the biological tissue of interest. In this work, a custom integrated optical coherence tomography (OCT) imaging and optogenetic stimulation system was used to visualize and modulate the functioning D. melanogaster heart at the 3rd instar larval and early pupal developmental stages. The UAS/GAL4 dual genetic system was employed to express halorhodopsin (eNpHR2.0) and red-shifted channelrhodopsin (ReaChR), specifically in the fly heart. Details on preparing D. melanogaster for live OCT imaging and optogenetic pacing are provided. A lab-developed integration software processed the imaging data to create visual presentations and quantitative characteristics of Drosophila heart function. The results demonstrate the feasibility of initiating cardiac arrest and bradycardia caused by eNpHR2.0 activation and performing heart pacing upon ReaChR activation.

DOI: 10.3791/63939
PMID: 36094265

Drosophila used to define a "priority list" of genes associated with albuminuria

Teumer A, et al. Genome-wide association meta-analyses and fine-mapping elucidate pathways influencing albuminuria. Nat Commun. 2019 Sep 11;10(1):4130. PMID: 31511532.

Abstract: "Increased levels of the urinary albumin-to-creatinine ratio (UACR) are associated with higher risk of kidney disease progression and cardiovascular events, but underlying mechanisms are incompletely understood. Here, we conduct trans-ethnic (n = 564,257) and European-ancestry specific meta-analyses of genome-wide association studies of UACR, including ancestry- and diabetes-specific analyses, and identify 68 UACR-associated loci. Genetic correlation analyses and risk score associations in an independent electronic medical records database (n = 192,868) reveal connections with proteinuria, hyperlipidemia, gout, and hypertension. Fine-mapping and trans-Omics analyses with gene expression in 47 tissues and plasma protein levels implicate genes potentially operating through differential expression in kidney (including TGFB1, MUC1, PRKCI, and OAF), and allow coupling of UACR associations to altered plasma OAF concentrations. Knockdown of OAF and PRKCI orthologs in Drosophila nephrocytes reduces albumin endocytosis. Silencing fly PRKCI further impairs slit diaphragm formation. These results generate a priority list of genes and pathways for translational research to reduce albuminuria."

Fly research contributes to studies related to obesity -- 2 reports

Guida MC, Birse RT, Dall'Agnese A, Toto PC, Diop SB, Mai A, Adams PD, Puri PL, Bodmer R. Intergenerational inheritance of high fat diet-induced cardiac lipotoxicity in Drosophila. Nat Commun. 2019 Jan 14;10(1):193. PMID: 30643137; PMCID: PMC6331650.

From the abstract: "Obesity is strongly correlated with lipotoxic cardiomyopathy, heart failure and thus mortality. ... and increasing evidence suggests that the parents' nutritional status may predispose their offspring to lipotoxic cardiomyopathy. ... Here we report that cardiac dysfunction induced by high-fat-diet (HFD) persists for two subsequent generations in Drosophila and is associated with reduced expression of two key metabolic regulators, adipose triglyceride lipase (ATGL/bmm) and transcriptional cofactor PGC-1. We provide evidence that targeted expression of ATGL/bmm in the offspring of HFD-fed parents protects them, and the subsequent generation, from cardio-lipotoxicity. Furthermore, we find that intergenerational inheritance of lipotoxic cardiomyopathy correlates with elevated systemic H3K27 trimethylation. Lowering H3K27 trimethylation genetically or pharmacologically in the offspring of HFD-fed parents prevents cardiac pathology. This suggests that metabolic homeostasis is epigenetically regulated across generations."

Justice AE, Karaderi T, Highland HM, et al. Protein-coding variants implicate novel genes related to lipid homeostasis contributing to body-fat distribution. Nat Genet. 2019 Feb 18. PMID: 30778226.

Abstract: "Body-fat distribution is a risk factor for adverse cardiovascular health consequences. We analyzed the association of body-fat distribution, assessed by waist-to-hip ratio adjusted for body mass index, with 228,985 predicted coding and splice site variants available on exome arrays in up to 344,369 individuals from five major ancestries (discovery) and 132,177 European-ancestry individuals (validation). We identified 15 common (minor allele frequency, MAF ≥5%) and nine low-frequency or rare (MAF <5%) coding novel variants. Pathway/gene set enrichment analyses identified lipid particle, adiponectin, abnormal white adipose tissue physiology and bone development and morphology as important contributors to fat distribution, while cross-trait associations highlight cardiometabolic traits. In functional follow-up analyses, specifically in Drosophila RNAi-knockdowns, we observed a significant increase in the total body triglyceride levels for two genes (DNAH10 and PLXND1). We implicate novel genes in fat distribution, stressing the importance of interrogating low-frequency and protein-coding variants."

TREK-1 potassium channel in heart health: From Drosophila genetic screen to mammalian follow-up studies

Abraham DM, Lee TE, Watson LJ, Mao L, Chandok GS, Wang HG, Frangakis S, Pitt GS, Shah SH, Wolf MJ, Rockman HA. The two-pore-domain potassium channel TREK-1 mediates cardiac fibrosis and diastolic dysfunction. J Clin Invest. 2018 Aug 28. PMID: 30153110.

From the abstract: "Cardiac two pore domain potassium channels (K2P) exist in organisms from Drosophila to humans ... We identified a K2P gene, CG8713 (sandman), in a Drosophila genetic screen and show that sandman is critical to cardiac function. Mice lacking an ortholog of sandman, TWIK related potassium channel (TREK-1 or Kcnk2), exhibit exaggerated pressure overload induced concentric hypertrophy and alterations in fetal gene expression ... These findings indicate a central role for cardiac fibroblast TREK-1 in the pathogenesis of pressure overload-induced cardiac dysfunction and serve as a conceptual basis for its inhibition for as a potential therapy."

Drosophila model points to involvement of potassium channel in aging hearts

Klassen MP, Peters CJ, Zhou S, Williams HH, Jan LY, Jan YN. Age-dependent diastolic heart failure in an in vivo Drosophila model. Elife. 2017 Mar 22;6. pii: e20851. PMID: 28328397; PMCID: PMC5362267.

The abstract: "While the signals and complexes that coordinate the heartbeat are well established, how the heart maintains its electromechanical rhythm over a lifetime remains an open question with significant implications to human health. Reasoning that this homeostatic challenge confronts all pulsatile organs, we developed a high resolution imaging and analysis toolset for measuring cardiac function in intact, unanesthetized Drosophila melanogaster. We demonstrate that, as in humans, normal aging primarily manifests as defects in relaxation (diastole) while preserving contractile performance. Using this approach, we discovered that a pair of two-pore potassium channel (K2P) subunits, largely dispensable early in life, are necessary for terminating contraction (systole) in aged animals, where their loss culminates in fibrillatory cardiac arrest. As the pumping function of its heart is acutely dispensable for survival, Drosophila represents a uniquely accessible model for understanding the signaling networks maintaining cardiac performance during normal aging."

Protocol for testing of candidate heart-disease genes in Drosophila

Zhu JY, Fu Y, Richman A, Han Z. Validating Candidate Congenital Heart Disease Genes in Drosophila. Bio Protoc. 2017 Jun 20;7(12). pii: e2350. PMID: 29276722; PMCID: PMC5739056.

From the abstract: "... A high throughput in vivo model system to validate candidate gene association with pathology is ... useful. We present such a system employing Drosophila to validate candidate congenital heart disease (CHD) genes. The protocols exploit comprehensive libraries of UAS-GeneX-RNAi fly strains ... These protocols were recently used to evaluate more than 100 candidate CHD genes implicated by patient whole-exome sequencing (Zhu et al., 2017)."

Fly research & heart disease -- a review

Ma L. Can the Drosophila model help in paving the way for translational medicine in heart failure? Biochem Soc Trans. 2016 Oct 15;44(5):1549-1560. Review. PMID: 27911738.

Treatment of Drosophila model of DM1 with pentamidine reverses cellular and organismal phenotypes

Chakraborty M, Selma-Soriano E, Magny E, Couso JP, Pérez-Alonso M, Charlet-Berguerand N, Artero R, Llamusi B. Pentamidine rescues contractility and rhythmicity in a Drosophila model of myotonic dystrophy heart dysfunction. Dis Model Mech. 2015 Dec;8(12):1569-78. PMID: 26515653; PMCID: PMC4728315.

From the abstract: "Up to 80% of individuals with myotonic dystrophy type 1 (DM1) will develop cardiac abnormalities at some point during the progression of their disease, the most common of which is heart blockage ... Such blockage is characterized by conduction defects and supraventricular and ventricular tachycardia, and carries a high risk of sudden cardiac death. ... Here, we describe the characterization of the heart phenotype in a Drosophila model expressing pure expanded CUG repeats under the control of the cardiomyocyte-specific driver GMH5-Gal4. ... As a proof of concept that the fly heart model can be used for in vivo testing of promising therapeutic compounds, we fed flies with pentamidine, a compound previously described to improve DM1 phenotypes. Pentamidine not only released Muscleblind from the CUG RNA repeats and reduced ribonuclear formation in the Drosophila heart, but also rescued heart arrhythmicity and contractility, and improved fly survival in animals expressing 250 CUG repeats."

Can we outrun aging? Fly study of impact of fatiguing exercise on aging

Zheng L, Feng Y, Wen DT, Wang H, Wu XS. Fatiguing exercise initiated later in life reduces incidence of fibrillation and improves sleep quality in Drosophila. Age (Dordr). 2015 Aug;37(4):9816. PMID: 26206392; PMCID: PMC4512962.

From the abstract: "As the human body ages, the risk of heart disease and stroke greatly increases. While there is evidence that lifelong exercise is beneficial to the heart's health, the effects of beginning exercise later in life remain unclear. This study aimed to investigate whether exercise training started later in life is beneficial to cardiac aging in Drosophila. ... We found that 2.0 and 2.5 h of exercise caused exercise-induced fatigue, and fatiguing exercise is beneficial for cardiac and healthy aging overall. This study provides a basis for further study in humans on the impact of beginning an exercise regimen later in life on cardiac health."

Review discusses fly and other models of heart failure

Kooij V, Venkatraman V, Tra J, Kirk JA, Rowell J, Blice-Baum A, Cammarato A, Van Eyk JE. Sizing up models of heart failure: Proteomics from flies to humans. Proteomics Clin Appl. 2014 Oct;8(9-10):653-64. PMID: 24723306; PMCID: PMC4282793.

From the abstract: "Cardiovascular disease is the leading cause of death in the western world. Heart failure is a heterogeneous and complex syndrome... The ability to utilize genetic manipulation and biochemical experimentation in animal models has made them indispensable ... Similarly, proteomics has been helpful for elucidating complicated cellular and molecular phenotypes and has the potential to identify circulating biomarkers and drug targets ... In this review, the use of human samples and animal model systems (pig, dog, rat, mouse, zebrafish, and fruit fly) in cardiac research is discussed. Additionally, the protein sequence homology between these species and the extent of conservation at the level of the phospho-proteome in major kinase signaling cascades involved in heart failure are investigated."

Drosophila myotonic dystrophy model responds to drug used to treat human patients with the condition

Chakraborty M, Selma-Soriano E, Magny E, Couso JP, Pérez-Alonso M, Charlet-Berguerand N, Artero R, Llamusi B. Pentamidine rescues contractility and rhythmicity in a Drosophila model of myotonic dystrophy heart dysfunction. Dis Model Mech. 2015 Oct 29. PMID: 26515653.

From the abstract: "Up to 80% of myotonic dystrophy type 1 (DM1) patients will develop cardiac abnormalities ... very few animal model studies have focused on the heart dysfunction in DM1. Here, we describe the characterization of the heart phenotype in a Drosophila model expressing pure expanded CUG repeats under the control of the cardiomyocyte-specific driver GMH5-Gal4. ... Similarly to what has been reported in human DM1 patients, heart-specific expression of toxic RNA resulted in reduced survival, increased arrhythmia, altered diastolic and systolic function ... As a proof of concept that the fly heart model can be used for in vivo testing of promising therapeutic compounds, we fed flies with pentamidine, a compound previously described to improve DM1 phenotypes. Pentamidine not only released Muscleblind and reduced ribonuclear formation in the Drosophila heart but rescued heart arrhythmicity and contractility, and improved fly survival in animals expressing 250 CUG repeats."

Mitochondrial disease and the heart explored in fly study

Martínez-Morentin L, Martínez L, Piloto S, Yang H, Schon EA, Garesse R, Bodmer R, Ocorr K, Cervera M, Arredondo JJ. Cardiac deficiency of single cytochrome oxidase assembly factor scox induces p53-dependent apoptosis in a Drosophila cardiomyopathy model. Hum Mol Genet. 2015 Jul 1;24(13):3608-22. PMID: 25792727; PMCID: PMC4459388.

From the abstract: “... most patients with mitochondrial disease produced by defects in the oxidative phosphorylation (OXPHOS) system are susceptible to cardiac involvement. ... but the molecular mechanisms involved in cardiac impairment are unknown. One of the most frequent OXPHOS defects in humans frequently associated with cardiomyopathy is cytochrome c oxidase (COX) deficiency caused by mutations in COX assembly factors such as Sco1 and Sco2. ... we have heart specifically interfered scox expression, the single Drosophila Sco orthologue. Cardiac-specific knockdown of scox reduces fly lifespan, and it severely compromises heart function and structure, producing dilated cardiomyopathy. ... Genetic and molecular evidence strongly suggest that dp53 is directly involved in the development of the cardiomyopathy induced by scox deficiency. Remarkably, apoptosis is enhanced in the muscle and liver of Sco2 knock-out mice, clearly suggesting that cell death is a key feature of the COX deficiencies produced by mutations in Sco genes in humans.”

Run, sleep and thrive

Zheng L, Feng Y, Wen DT, Wang H, Wu XS. Fatiguing exercise initiated later in life reduces incidence of fibrillation and improves sleep quality in Drosophila. Age (Dordr). 2015 Aug;37(4):9816. PMID: 26206392; PMCID: PMC4512962.

From the abstract: "As the human body ages, the risk of heart disease and stroke greatly increases. While there is evidence that lifelong exercise is beneficial to the heart's health, the effects of beginning exercise later in life remain unclear. This study aimed to investigate whether exercise training started later in life is beneficial to cardiac aging in Drosophila. ... We found that 2.0 and 2.5 h of exercise caused exercise-induced fatigue, and fatiguing exercise is beneficial for cardiac and healthy aging overall. This study provides a basis for further study in humans on the impact of beginning an exercise regimen later in life on cardiac health. "

Review highlights usefulness of Drosophila as a model for study of congenital heart disease

Vogler G, Bodmer R. Cellular Mechanisms of Drosophila Heart Morphogenesis. J Cardiovasc Dev Dis. 2015 Mar 1;2(1):2-16.  PMID: 26236710

From the abstract: "Many of the major discoveries in the fields of genetics and developmental biology have been made using the fruit fly, Drosophila melanogaster. With regard to heart development, the conserved network of core cardiac transcription factors that underlies cardiogenesis has been studied in great detail in the fly, and the importance of several signaling pathways that regulate heart morphogenesis, such as Slit/Robo, was first shown in the fly model. ... The specific limitations of the various cardiac model systems currently employed (mammalian and fish models) provide a niche for the fly model ... Here, we review recent advances made using the Drosophila embryo that identify factors relevant for heart formation. ..."

New fly model of obesity-related heart disease

Hardy CM, Birse RT, Wolf MJ, Yu L, Bodmer R, Gibbs AG. Obesity-associated Cardiac Dysfunction in Starvation-Selected Drosophila melanogaster. Am J Physiol Regul Integr Comp Physiol. 2015 Jul 1:ajpregu.00160.2015. PMID: 26136533.

Vinculin involvement in regulation of heart function during aging

Kaushik G, Spenlehauer A, Sessions AO, Trujillo AS, Fuhrmann A, Fu Z, Venkatraman V, Pohl D, Tuler J, Wang M, Lakatta EG, Ocorr K, Bodmer R, Bernstein SI, Van Eyk JE, Cammarato A, Engler AJ. Vinculin network-mediated cytoskeletal remodeling regulates contractile function in the aging heart. Sci Transl Med. 2015 Jun 17;7(292):292ra99. PMID: 26084806.

From the abstract: "The human heart is capable of functioning for decades despite minimal cell turnover or regeneration, suggesting that molecular alterations help sustain heart function with age. However, identification of compensatory remodeling events in the aging heart remains elusive. We present the cardiac proteomes of young and old rhesus monkeys and rats, from which we show that certain age-associated remodeling events within the cardiomyocyte cytoskeleton are highly conserved and beneficial rather than deleterious. Targeted transcriptomic analysis in Drosophila confirmed conservation and implicated vinculin as a unique molecular regulator of cardiac function during aging. ... Moreover, cardiac-specific vinculin overexpression increased median life span by more than 150% in flies. ... These findings suggest that the heart has molecular mechanisms to sustain performance and promote longevity, which may be assisted by therapeutic intervention to ameliorate the decline of function in aging patient hearts."

Huntington's and the heart

Melkani GC, Trujillo AS, Ramos R, Bodmer R, Bernstein SI, Ocorr K. Huntington's disease induced cardiac amyloidosis is reversed by modulating protein folding and oxidative stress pathways in the Drosophila heart. PLoS Genet. 2013;9(12):e1004024. PMID: 24367279; PMCID: PMC3868535.

From the abstract: "... We conclude that modulation of both protein unfolding and oxidative stress pathways in the Drosophila heart model can ameliorate the detrimental PolyQ effects, thus providing unique insights into the genetic mechanisms underlying amyloid-induced cardiac failure in HD patients."

Fly model of cardiomyopathy--role for galactokinase

Lee TE, Yu L, Wolf MJ, Rockman HA. Galactokinase Is a Novel Modifier of Calcineurin-Induced Cardiomyopathy in Drosophila. Genetics. 2014 Jul 31. PMID: 25081566.

From the abstract: "Activated/uninhibited calcineurin is both necessary and sufficient to induce cardiac hypertrophy, a condition that often leads to dilated cardiomyopathy, heart failure, and sudden cardiac death. We expressed constitutively active calcineurin in the adult heart of Drosophila melanogaster and identified enlarged cardiac chamber dimensions and reduced cardiac contractility. In addition, expressing constitutively active calcineurin in the fly heart using the Gal4/UAS system induced an increase in heart wall thickness. We performed a targeted genetic screen for modifiers of calcineurin-induced cardiac enlargement ... we generated a model for discovering novel modifiers of calcineurin-induced cardiac enlargement in the fly and identified galactokinase as a previously unknown regulator of calcineurin-induced cardiomyopathy in adult Drosophila."

Methods review--use of Drosophila for heart-disease related studies

Ocorr K, Vogler G, Bodmer R. Methods to assess Drosophila heart development, function and aging. Methods. 2014 Apr 12. pii: S1046-2023(14)00133-9. PMID: 24727147.

From the abstract: "In recent years the Drosophila heart has become an established model for many different aspects of human cardiac disease. This model has allowed identification of disease-causing mechanisms underlying congenital heart disease and cardiomyopathies and has permitted the study of underlying genetic, metabolic and age-related contributions to heart function. In this review we discuss methods currently employed in the analysis of the Drosophila heart structure and function ..."

Drosophila and the heart--SERCA study

Abraham DM, Wolf MJ. Disruption of sarcoendoplasmic reticulum calcium ATPase function in Drosophila leads to cardiac dysfunction. PLoS One. 2013 Oct 3;8(10):e77785. PMID: 24098595; PMCID: PMC3789689.

From the abstract: "... we show that disruption of CaP60A in a cardiac specific manner results in cardiac dilation and dysfunction ... In addition, the co-expression of a calcium release channel mutation with CaP60A (kum170) is sufficient to rescue the cardiac phenotype but not paralysis. Finally, we show that CaP60A overexpression is able to rescue cardiac function in a model of Drosophila cardiac dysfunction similar to what is observed in mammals ..."