Interplay between SERCA, 4E-BP, and eIF4E in the Drosophila heart

Abstract

Appropriate cardiac performance depends on a tightly controlled handling of Ca2+ in a broad range of species, from invertebrates to mammals. The role of the Ca2+ ATPase, SERCA, in Ca2+ handling is pivotal, and its activity is regulated, inter alia, by interacting with distinct proteins. Herein, we give evidence that 4E binding protein (4E-BP) is a novel regulator of SERCA activity in Drosophila melanogaster during cardiac function. Flies over-expressing 4E-BP showed improved cardiac performance in young individuals associated with incremented SERCA activity. Moreover, we demonstrate that SERCA interacts with translation initiation factors eIF4E-1, eIF4E-2 and eIF4E-4 in a yeast two-hybrid assay. The specific identification of eIF4E-4 in cardiac tissue leads us to propose that the interaction of elF4E-4 with SERCA may be the basis of the cardiac effects observed in 4E-BP over-expressing flies associated with incremented SERCA activity.

Fig 1. Overexpression of 4E-BP improves cardiac performance.

Average values of intracellular Ca²⁺ cycling parameters. Black bars, control flies (tinC-Gal4, UAS-GCaMP3/+). Green bars, flies overexpressing 4E-BP in cardiac tissue (tinC-Gal4, UAS-GCaMP3 / UAS-4E-BP). Overexpression of 4E-BP did not produce significant changes in heart rate (A). Instead, significantly increased Ca²⁺ transient amplitude (B), maximum rate of Ca²⁺ transient increase (ΔF/dt_max) and decay (ΔF/dt_min) (C–D) were observed in 7-day-old flies. Time constant of Ca²⁺ transient decay—Tau (E)—was not significantly changed, whereas the estimated SERCA activity (F) was incremented. Overexpression of 4E-BP in 40-day-old flies did not increase these parameters significantly, compared to control flies. All results are expressed as mean ± SEM. * p<0.05, ** p<0.01 and **** p< 0.001.

Fig 2. Intracellular Ca²⁺ transient is not affected by augmented dTOR expression, but it is altered during starvation in young wild type flies.

Tor overexpression did not change heart rate (A), Ca²⁺ transient amplitude (B), maximum rate of Ca²⁺ transient increase (ΔF/dt_max) and decay (ΔF/dt_min) (C–D), time constant of Ca²⁺ transient decay, Tau (E) or SERCA activity (F) in 7-day-old flies. Instead, 48 hr of starvation drastically accelerated maximum rate of Ca²⁺ transient increase (ΔF/dt_max) and decay (ΔF/dt_min) (C–D) that provoked reduction in the Ca²⁺ transient amplitude (B) probably due to incremented heart rate (A) in control flies (tinC-Gal4, UAS-GCaMP3 / +). These changes were accompanied by augmented SERCA activity. All results are expressed as mean ± SEM. * p<0.05, ** p<0.01 and **** p< 0.001.

Fig 3. Starvation alters intracellular Ca²⁺ transient in 4E-BP OE flies.

Forty-eight hours of starvation increased heart rate (A) in 7-day-old 4E-BP overexpression flies (A) and decreased the amplitude of the Ca²⁺ transient (B). Maximal rates of Ca²⁺ transient increase (ΔF/dt_max) and decay (ΔfFdt_min) (C–D) were reduced. The time constant of Ca²⁺ transient decay—Tau (E)—remained without changes (E). SERCA activity was augmented (F) compared to control non-starved flies. All results are expressed as mean ± SEM. * p<0.05 with respect to control group; *** p<0.001 **** p<0.0001; # p<0.05 with respect to UAS-4E-BP flies, #### p<0.0001.

Fig 4. SERCA interacts with eIF4E-4, eIF4E-1 and eIF4E-2 in the yeast two-hybrid system.

A) “Prey” SERCA (amino acids 1–250)-activator domain (AD; upper panel) interacts with “baits” eIF4E-4, eIF4E-1 and eIF4E-2 in a yeast two-hybrid assay. eIF4E-4 exhibits the strongest interaction. “Prey” 4E-BP-AD (lower panel) was used as a positive control. Empty vector (BD) was used as a negative control. B) pSERCA(51–250)-AD, but not pSERCA(200–250)-AD, interacts with eIF4E-4-BD. L, leucine; W, tryptophan; H, histidine; 3AT, 3-amino-1,2,4-triazole.–(L,W), growth control. Interactions were tested under increasing astringency conditions (3 to 30 mM 3-amino-1,2,4-triazole, 3AT).

Fig 5. mRNA of eIF4E-4 is expressed in cardiac tissue of Drosophila melanogaster.

RT-PCR using specific primers to amplify a fragment of tubulin and of eIF4E-4 was carried out using isolated cardiac tissue. The size of each respective amplicon corresponds to the expected size (tubulin: 182 bp; eIF4E-4: 166 bp).

Fig 6. Representative diagram of a wildtype cardiomyocyte and one that overexpresses 4E-BP.

This model proposes that overexpressed 4E-BP competes with SERCA for eIF4E-4, thereby reducing eIF4E-4 binding to SERCA and causing increased SERCA activity and improved cardiac performance. Ca²⁺ handling-proteins are indicated: Voltage gated Ca²⁺ channel, eukaryotic translation initiation factor, variant 4 (eIF4E-4), Ca^2+-ATPase (SERCA), ryanodine receptor, Na⁺/Ca²⁺ exchanger (NCX), calcium ion (Ca²⁺), sodium ion (Na⁺).