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

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 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: ...

Reduced Cardiac Calcineurin Expression Mimics Long-Term Hypoxia-Induced Heart Defects in Drosophila

“…A previous study reported that excess calcineurin activity causes cardiac hypertrophy in Drosophila , 31 similar to observations in the mouse heart. 22–25,31 We wondered if CH exposure could counteract calcineurin-induced cardiac hypertrophy.…”

“…41 CanA14F was overall the most downregulated gene in HS hearts (25-fold; Figure 2C; Table I in the Data Supplement). Given calcineurin’s known role in hypertrophy and metabolism, 23,31,32 we chose to focus on the putative effector targets within the cal-cineurin network, CanA14F and Pp2B, for further studies. We hypothesized that cardiac downregulation of calcineurin, as observed with long-term hypoxia exposure, results in cardiac restriction under normoxic conditions in hypoxia-naïve flies.…”

“…26,30 A constitutively active form of the Drosophila Pp2B gene induced cardiac widening in the conical chamber under normoxic conditions, and inhibition of calcineurin in mammalian models is well known to reduce pathological hypertrophy. 24,25,31 However, a role for calcineurin in long-term hypoxia exposures, where hypertrophy can be a defining feature of CH-related diseases, has not yet been identified. 31,32 …”

“…24,25,31 However, a role for calcineurin in long-term hypoxia exposures, where hypertrophy can be a defining feature of CH-related diseases, has not yet been identified. 31,32 …”

“…Under hypoxia, CanA14F or Pp2B knockdown resulted in further loss of cardiac performance. Interestingly, gain of Pp2B function, known to increase heart size under normoxia, 31 was lethal under CH. This suggests that elevating the activity of a hypertrophy inducer, such as calcineurin A, in a hypoxic environment may cause cardiomyocyte instability, resulting in cardiac death and preventing long-term organismal survival.…”