Identification and In Vivo Characterisation of Cardioactive Peptides in Drosophila melanogaster

Abstract

Neuropeptides and peptide hormones serve as critical regulators of numerous biological processes, including development, growth, reproduction, physiology, and behaviour. In mammals, peptidergic regulatory systems are complex and often involve multiple peptides that act at different levels and relay to different receptors. To improve the mechanistic understanding of such complex systems, invertebrate models in which evolutionarily conserved peptides and receptors regulate similar biological processes but in a less complex manner have emerged as highly valuable. Drosophila melanogaster represents a favoured model for the characterisation of novel peptidergic signalling events and for evaluating the relevance of those events in vivo. In the present study, we analysed a set of neuropeptides and peptide hormones for their ability to modulate cardiac function in semi-intact larval Drosophila melanogaster. We identified numerous peptides that significantly affected heart parameters such as heart rate, systolic and diastolic interval, rhythmicity, and contractility. Thus, peptidergic regulation of the Drosophila heart is not restricted to chronotropic adaptation but also includes inotropic modulation. By specifically interfering with the expression of corresponding peptides in transgenic animals, we assessed the in vivo relevance of the respective peptidergic regulation. Based on the functional conservation of certain peptides throughout the animal kingdom, the identified cardiomodulatory activities may be relevant not only to proper heart function in Drosophila, but also to corresponding processes in vertebrates, including humans.

Keywords: dorsal vessel; endocrine signalling; heart function; heart physiology; neuropeptides; peptide hormones; peptide signalling.

Figure 1

Heart rate is modulated by peptide signalling in semi-intact Drosophila larvae. Individual effects of the tested peptides (1 × 10⁻⁷ M) on heart rate. Data are presented as mean values (± S.E.M.) of the relative changes in heart rate in relation to the individual control preparations (prior to peptide application). A minimum of ten animals per peptide were measured. Significance levels are indicated by asterisks (paired sample Student´s t-test, * p < 0.05; ** p < 0.01; *** p < 0.001). Application of Allatostatin A4 resulted in a complete heartbeat arrest (heart rate not determined, n.d.).

Figure 2

Dose–response curves for the effects of chronotropic peptides in semi-intact Drosophila larvae. Individual effects of the tested peptides at 1 × 10⁻¹¹ M, 1 × 10⁻⁹ M, 1 × 10⁻⁷ M, and 1 × 10⁻⁵ M are shown. Data are presented as mean values (± S.E.M.) of the relative changes in heart rate in relation to the individual control preparations (prior to peptide application). A minimum of ten animals per peptide were measured. Significance levels are indicated by asterisks (paired sample Student´s t-test, * p < 0.05; ** p < 0.01; *** p < 0.001). n.d. indicates “not determined” (due to heartbeat arrest).

Figure 3

Diastolic and systolic intervals, fractional shortening and rhythmicity are modulated by peptide signalling in semi-intact Drosophila larvae. Individual effects of the tested peptides (1 × 10⁻⁷ M) on: (A) diastolic interval; (B) systolic interval; (C) fractional shortening; and (D) rhythmicity. Data are presented as mean values (± S.E.M.) of the relative changes in the respective parameters in relation to the individual control preparations (prior to peptide application). A minimum of ten animals per peptide were measured. Significance levels are indicated by asterisks (paired sample Student´s t-test, * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 4

Representative M-modes of semi-intact larval Drosophila hearts incubated with distinct peptides (1 × 10⁻⁷ M). The movement of the heart walls over time (10 s) is depicted. Application of Corazonin, DH31, Proctolin, or Tachykinins 1, 3, or 5 has a cardioacceleratory effect. Application of Allatostatin A4 results in cardiac arrest. Upper panels represent control hearts (prior to peptide application); lower panels depict the same hearts 1 min after peptide application. Scale bars indicate 1 s.

Figure 5

Knockdown of distinct peptide precursor proteins significantly affects heart function in intact Drosophila larvae. Depicted are the individual effects of peptide precursor protein knockdown on (A) heart rate and (B) rhythmicity. daughterless-Gal4 (da-Gal4) was used as a ubiquitous driver. Data are presented as mean values (± S.E.M.) of the relative changes in the respective parameters in relation to the depicted controls. At least ten animals per genotype were measured. Significance levels are indicated by asterisks (Student´s t-test, * p < 0.05; ** p < 0.01; *** p < 0.001).