Raf-mediated cardiac hypertrophy in adultDrosophila

Abstract

SUMMARYIn response to stress and extracellular signals, the heart undergoes a process called cardiac hypertrophy during which cardiomyocytes increase in size. If untreated, cardiac hypertrophy can progress to overt heart failure that causes significant morbidity and mortality. The identification of molecular signals that cause or modify cardiomyopathies is necessary to understand how the normal heart progresses to cardiac hypertrophy and heart failure. Receptor tyrosine kinase (RTK) signaling is essential for normal human cardiac function, and the inhibition of RTKs can cause dilated cardiomyopathies. However, neither investigations of activated RTK signaling pathways nor the characterization of hypertrophic cardiomyopathy in the adult fly heart has been previously described. Therefore, we developed strategies using Drosophila as a model to circumvent some of the complexities associated with mammalian models of cardiovascular disease. Transgenes encoding activated EGFRA887T, Ras85DV12 and Ras85DV12S35, which preferentially signal to Raf, or constitutively active human or fly Raf caused hypertrophic cardiomyopathy as determined by decreased end diastolic lumen dimensions, abnormal cardiomyocyte fiber morphology and increased heart wall thicknesses. There were no changes in cardiomyocyte cell numbers. Additionally, activated Raf also induced an increase in cardiomyocyte ploidy compared with control hearts. However, preventing increases in cardiomyocyte ploidy using fizzy-related (Fzr) RNAi did not rescue Raf-mediated cardiac hypertrophy, suggesting that Raf-mediated polyploidization is not required for cardiac hypertrophy. Similar to mammals, the cardiac-specific expression of RNAi directed against MEK or ERK rescued Raf-mediated cardiac hypertrophy. However, the cardiac-specific expression of activated ERKD334N, which promotes hyperplasia in non-cardiac tissues, did not cause myocyte hypertrophy. These results suggest that ERK is necessary, but not sufficient, for Raf-mediated cardiac hypertrophy.

Cardiac hypertrophy induced by active Raf depends on Yorkie-mediated transcription

“…3, A to C). Cardiac-specific expression of hRaf Act causes abnormal cardiomyocyte fiber morphology (9). tinC-GFP –positive cardiomyocytes in hearts dissected from tinC>hRaf Act flies lacked well-defined circumferential fibers, whereas cardiomyocytes in tinC>hRaf Act + yki RNAi flies had partially rescued fiber abnormalities (Fig.…”

“…Cardiac-specific expression of RNAi targeting ERK partially rescued Yki S168A -induced cardiac hypertrophy in flies. We previously found that cardiac-specific expression of ERK RNAi completely rescues Raf Act -induced cardiac hypertrophy (9). Thus, ERK may be involved in the crosstalk between Raf and Yki in the heart, but further studies are required to determine the potential involvement of other kinases.…”

“…Cardiac function in Drosophila was measured using a custom-built OCT microscopy system (Bioptigen Inc.) as previously described (9, 30, 53). EDD and ESD were determined from three consecutive heartbeats.…”

“…Adult Drosophila corresponding to the F1 offspring of p{tinC-GFP}; p{tinC-Gal4} stocks crossed to specific p{UAS-transgenes} or w 1118 (controls) were collected at 2 to 3 days of age, post-eclosion, to examine adult cardiac morphology as previously described (9, 57). Dissected specimens were stained with an antibody against GFP (1:500) (Invitrogen) and a secondary antibody conjugated to Alexa Fluor 488 (1:500) (Invitrogen).…”

“…Within each abdominal segment, denoted A1 to A6, the heart has eight tinC -expressing myocytes that are laterally paired and comprise the adult fly heart, which has a thickness of single-cell layer. Similar to mammals, cardiac-specific expression of activated human Raf in Drosophila decreases the size of cardiac lumens, increases heart wall thicknesses without inducing hyperplasia, and causes abnormalities in cardiomyocyte contractile fibers (9). Similar to pharmacological inhibition of MEK in mice, the cardiac-specific knockdown of MEK or ERK in flies rescues Raf-mediated hypertrophy (9).…”