Fluorescent Labeling of &lt;em&gt;Drosophila&lt;/em&gt; Heart Structures

Alayari, Nakissa N.; Vogler, Georg; Taghli-Lamallem, Ouarda; Ocorr, Karen; Bodmer, Rolf; Cammarato, Anthony

doi:10.3791/1423

Cited by 53 publications

(54 citation statements)

References 10 publications

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“…Confocal microscopy was performed as detailed by Alayari et al (2009) 34 with an Olympus FluoView FV10i Confocal Microscope system at 10× magnification.…”

Section: Methodsmentioning

confidence: 99%

A Drosophila melanogaster Model of Diastolic Dysfunction and Cardiomyopathy Based on Impaired Troponin-T Function

Viswanathan

Kaushik

Engler

et al. 2014

Circulation Research

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Rationale Regulation of striated muscle contraction is achieved by Ca2+-dependent steric modulation of myosin cross-bridge cycling on actin by the thin filament troponin-tropomyosin complex. Alterations in the complex can induce contractile dysregulation and disease. For example, mutations between or near residues 112–136 of cardiac troponin-T, the crucial N-terminal TnT1 tropomyosin-binding region, cause cardiomyopathy. The Drosophila up101 Glu/Lys amino acid substitution lies C-terminally adjacent to this phylogenetically conserved sequence. Objective Using a highly integrative approach, we sought to determine the molecular trigger of up101 myofibrillar degeneration, to evaluate contractile performance in the mutant cardiomyocytes, and to examine the effects of the mutation on the entire Drosophila heart to elucidate regulatory roles for conserved TnT1 regions and provide possible mechanistic insight into cardiac dysfunction. Methods and Results Live video imaging of Drosophila cardiac tubes revealed the troponin-T mutation prolongs systole and restricts diastolic dimensions of the heart, due to increased numbers of actively cycling myosin cross-bridges. Elevated resting myocardial stiffness, consistent with up101 diastolic dysfunction, was confirmed by an atomic force microscopy-based nanoindentation approach. Direct visualization of mutant thin filaments via electron microscopy and three-dimensional reconstruction resolved destabilized tropomyosin positioning and aberrantly exposed myosin binding sites under low Ca2+ conditions. Conclusions As a result of troponin-tropomyosin dysinhibition, up101 hearts exhibit cardiac dysfunction and remodeling comparable to that observed during human restrictive cardiomyopathy. Thus, reversal of charged residues about the conserved tropomyosin-binding region of TnT1 may perturb critical intermolecular associations required for proper steric regulation, which likely elicits myopathy in our Drosophila model.

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“…Confocal microscopy was performed as detailed by Alayari et al (2009) 34 with an Olympus FluoView FV10i Confocal Microscope system at 10× magnification.…”

Section: Methodsmentioning

confidence: 99%

A Drosophila melanogaster Model of Diastolic Dysfunction and Cardiomyopathy Based on Impaired Troponin-T Function

Viswanathan

Kaushik

Engler

et al. 2014

Circulation Research

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“…according to standard procedures as previously described (ALAYARI et al 2009;YU et al 2010) using 5% BSA in TBS as the blocking reagent with a rabbit αGFP antibody (Invitrogen, Inc.), and Alexa Fluor488 goat αrabbit secondary antibody (Invitrogen, Inc.). After immuno--staining, the Drosophila heart was visualized under a Zeiss LSM510 confocal microscope.…”

Section: Discussionmentioning

confidence: 99%

Galactokinase Is a Novel Modifier of Calcineurin-Induced Cardiomyopathy in Drosophila

Lee

Wolf

et al. 2014

Genetics

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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 based on previous calcineurin studies in the fly and identified galactokinase as a novel modifier of calcineurin-induced cardiomyopathy. Genomic deficiencies spanning the galactokinase locus, transposable elements that disrupt galactokinase, and cardiac-specific RNAi knockdown of galactokinase suppressed constitutively active calcineurin-induced cardiomyopathy. In addition, in flies expressing constitutively active calcineurin using the Gal4/UAS system, a transposable element in galactokinase suppressed the increase in heart wall thickness. Finally, genetic disruption of galactokinase suppressed calcineurin-induced wing vein abnormalities. Collectively, 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.A CTIVATED/uninhibited calcineurin is both necessary and sufficient to induce cardiac hypertrophy (Molkentin et al. 1998;Wilkins and Molkentin 2002;Van Berlo et al. 2013). Transgenic mice expressing constitutively active calcineurin (CanA act ) display cardiac hypertrophy (Molkentin et al. 1998) and the genetic or pharmacological inhibition of calcineurin suppresses agonist and pressure overloadinduced cardiac hypertrophy (Sussman et al. 1998;Taigen et al. 2000;Wilkins and Molkentin 2002;Van Berlo et al. 2013). Prolonged cardiac hypertrophy is a known risk factor for dilated cardiomyopathy, heart failure, and sudden death (Levy et al. 1990;Messerli and Ketelhut 1991;Drazner et al. 2004;George 2013;Grossman and Paulus 2013). In contrast, cardiac hypertrophy stimulated by exercise is physiological, is not typically associated with abnormal cardiac function, and does not stimulate calcineurin/nuclear factor of activated T cells (NFAT) signaling , supporting the concept that calcineurin promotes pathological cardiac hypertrophy.Calcineurin acts as a calcium/calmodulin-dependent protein phosphatase that consists of two subunits: a large CanA subunit (60 kDa) and a small CanB subunit (19 kDa). In the mouse, there are three CanA genes (Ppp3ca, Ppp3cb, and Ppp3cc) and two CanB genes (Ppp3r1 and Ppp3r2); in the fly, there are three CanA genes (CanA1, CanA-14F, and Pp2B-14D) and two CanB genes (CanB and CanB2) (NCBI Gene, http://www.ncbi.nlm.nih.gov/gene). The large CanA subunit has phosphatase activity and consists of several domains: ...

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“…Fluorescent labeling was performed using a modified protocol as previously described for Figures 2, S1, S2, S3, and S4 [51]. Briefly, ventrally-exposed beating hearts were relaxed with 10 mM EGTA, then fixed with 3.7% paraformaldehyde in hemolymph, and then permeabalized in 0.1% Triton X in PBS (PBST).…”

Section: Methodsmentioning

confidence: 99%

Extracellular matrix downregulation in the Drosophila heart preserves contractile function and improves lifespan

et al. 2017

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Aging is associated with extensive remodeling of the heart, including basement membrane (BM) components that surround cardiomyocytes. Remodeling is thought to impair cardiac mechanotransduction, but the contribution of specific BM components to age-related lateral communication between cardiomyocytes is unclear. Using a genetically tractable, rapidly aging model with sufficient cardiac genetic homology and morphology, e.g. Drosophila melanogaster, we observed differential regulation of BM collagens between laboratory strains, correlating with changes in muscle physiology leading to cardiac dysfunction. Therefore, we sought to understand the extent to which BM proteins modulate contractile function during aging. Cardiac-restricted knockdown of ECM genes Pericardin, Laminin A, and Viking in Drosophila prevented age-associated heart tube restriction and increased contractility, even under viscous load. Most notably, reduction of Laminin A expression correlated with an overall preservation of contractile velocity with age and extension of organismal lifespan. Global heterozygous knockdown confirmed these data, which provides new evidence of a direct link between BM homeostasis, contractility, and maintenance of lifespan.

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Fluorescent Labeling of <em>Drosophila</em> Heart Structures

Cited by 53 publications

References 10 publications

A Drosophila melanogaster Model of Diastolic Dysfunction and Cardiomyopathy Based on Impaired Troponin-T Function

A Drosophila melanogaster Model of Diastolic Dysfunction and Cardiomyopathy Based on Impaired Troponin-T Function

Galactokinase Is a Novel Modifier of Calcineurin-Induced Cardiomyopathy in Drosophila

Extracellular matrix downregulation in the Drosophila heart preserves contractile function and improves lifespan

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