SIFamide peptides modulate cardiac activity differently in two species of Cancer crab

Abstract

The SIFamides are a broadly conserved arthropod peptide family characterized by the C-terminal motif -SIFamide. In decapod crustaceans, two isoforms of SIFamide are known, GYRKPPFNGSIFamide (Gly¹-SIFamide), which is nearly ubiquitously conserved in the order, and VYRKPPFNGSIFamide (Val¹-SIFamide), known only from members of the astacidean genus Homarus. While much work has focused on the identification of SIFamide isoforms in decapods, there are few direct demonstrations of physiological function for members of the peptide family in this taxon. Here, we assessed the effects of Gly¹- and Val¹-SIFamide on the cardiac neuromuscular system of two closely related species of Cancer crab, Cancer borealis and Cancer irroratus. In each species, both peptides were cardioactive, with identical, dose-dependent effects elicited by both isoforms in a given species. Threshold concentrations for bioactivity are in the range typically associated with hormonal delivery, i.e., 10^-9 to 10^-8 M. Interestingly, and quite surprisingly, while the predicted effects of SIFamide on cardiac output are similar in both C. borealis and C. irroratus, frequency effects predominate in C. borealis, while amplitude effects predominate in C. irroratus. These findings suggest that, while SIFamide is likely to increase cardiac output in both crabs, the mechanism through which this is achieved is different in the two species. Immunohistochemical/mass spectrometric data suggest that SIFamide is delivered to the heart hormonally rather than locally, with the source of hormonal release being midgut epithelial endocrine cells in both Cancer species. If so, midgut-derived SIFamide may function as a regulator of cardiac output during the process of digestion.

Keywords: Cardiac neuromuscular system; Cardiotropic peptide; Central pattern generator; Crustacea; Midgut epithelial endocrine signaling; Peptide hormone.

Figure 1.. Diagrammatic sketch of the crab heart and recording of force, indicating the parameters that were measured in each recording.

The cardiac ganglion (green), consisting of 9 neurons, lies inside the heart. The sternal artery was cannulated, and peptides were introduced through the cannula. The five anterior arteries were tied to a force transducer at an angle of 30–45°. Parameters measured included cycle period (used to calculate heartbeat frequency), contraction amplitude (force), contraction duration (measured at half-maximal contraction amplitude), rise time, and relaxation time, as indicated on the diagram.

Figure 2.. Baseline heartbeat frequency was lower in C. borealis than in C. irroratus.

Although heartbeat frequency measured in control saline varied over a greater than 2-fold range in both species, it was lower (unpaired t-test, p<0.0001, n=13 for each species) in C. borealis (mean 0.31 Hz) than in C. irroratus (mean 0.61 Hz). Coefficients of variation were similar in the two species (32.9% in C. borealis; 22.6% in C. irroratus). Error bars indicate standard error (s.e.m.).

Figure 3.. Gly¹-SIFamide increased contraction amplitude and frequency in both Cancer borealis and Cancer irroratus.

Recordings of contractions in control saline and in 10⁻⁷ M Gly¹-SIFamide illustrate the substantial increase in contraction frequency in C. borealis (A) and in contraction amplitude in C. irroratus (B). Smaller increases in contraction amplitude in C. borealis and in contraction frequency in C. irroratus can also be seen. Heartbeat frequency in control saline was higher in C. irroratus than in C. borealis.

Figure 4.. Both Gly¹-SIFamide and Val¹-SIFamide elicited dose-dependent effects on the frequency, amplitude, and duration of heart contractions in C. borealis and in C. irroratus.

For all parameters in both species, there were no significant differences between the effects of Gly¹-SIFamide and Val¹-SIFamide (3-way ANOVA, p>0.05). Thresholds for effects of both peptides were less than 10⁻⁸ M, with both peptides eliciting significant effects on each species at a concentration of 10⁻⁸ M or less. In C. borealis, threshold appeared to be somewhat lower for Gly¹-SIFamide. Gly¹-SIFamide, but not Val¹-SIFamide, elicited significant effects at concentrations of 10⁻⁹ M for both frequency (A1) and duration (C1). (A1, A2) The SIFamides elicited dose-dependent increases in contraction frequency in both species; the increase was greater in C. borealis than in C. irroratus (3-way ANOVA: concentrations, p<0.001; species, p<0.001). (B1, B2) The SIFamides elicited dose-dependent increases in contraction amplitude in both species; the increase was greater in C. irroratus than in C. borealis (3-way ANOVA: concentrations, p<0.001; species, p<0.001). (C1, C2) The SIFamides elicited dose-dependent decreases in contraction duration in both species; this decrease did not differ significantly between the two species (3-way ANOVA: concentrations, p<0.001; species, p>0.05). * indicates values significantly different from 0, single sample t-test. Sample sizes: ≥ 9; see Table 1 for details. Error bars indicate standard error (s.e.m.).

Figure 5.. The decrease in contraction duration elicited by the SIFamides was due largely to decreases in the duration of relaxation from peak contraction back to baseline.

(A1, A2) Rise time decreased significantly only in C. borealis during perfusion of 10⁻⁶ M Val¹-SIFamide. There were no significant differences between the effects of the two peptides or across peptide concentrations; there were, however, differences between the two species (3-way ANOVA: peptides, p>0.05; concentrations, p>0.05; species, p=0.020). (B1, B2) Relaxation time decreased significantly during SIFamide perfusion in nearly all concentrations of both peptides, although neither peptide elicited a significant change when perfused at a concentration of 10⁻⁹ M in C. borealis. Although these decreases in relaxation time (like those in duration) are relatively small, with all means being less than 15% change, the effects of the peptides on relaxation time were dose-dependent and differed between the two species (3-way ANOVA: peptides, p>0.05; concentrations, p=0.004; species, p=0.012). Sample sizes: ≥ 9; see Table 1 for details. Error bars indicate standard error (s.e.m.).

Figure 6.. Area/unit time, used here as an indirect indication of cardiac output, increased similarly in the two Cancer species in response to the native crab SIFamide, Gly¹-SIFamide.

Increases in area/unit time, which is influenced by contraction amplitude, frequency, and duration, were dose-dependent (2-way ANOVA; p <0.0001). This parameter increased at all concentrations tested, from 10⁻⁹ M to 10⁻⁶ M, but did not differ between the two species (2-way ANOVA, p>0.05). * indicates values significantly different from 0, single sample t-test Sample size: n = 9. Error bars indicate standard error (s.e.m.)

Figure 7.. Direct MALDI-FTMS analysis of C. irroratus midgut tissues suggests the presence of Gly¹-SIFamide in the anterior midgut caecum (AMC), but not in the posterior midgut caecum (PMC).

C. irroratus midgut tissues were analyzed by MALDI-FTMS, using DHB as the matrix, with conditions optimized for the accumulation of m/z 1500. (A) Spectrum of a small piece of anterior midgut caecum (AMC) tissue, showing a peak at m/z 1381.74 corresponding to GYRKPPFNGSIFamide (Gly¹-SIFamide). (B) Spectrum from a small piece of posterior midgut caecum (PMC), showing an intense peak appearing at m/z 934.49 identified as APSGFLGMRamide (CabTRP Ia); no signal for Gly¹-SIFamide was detected. (C) Spectrum of an AMC tissue extract, representing peptides extracted from a larger piece of tissue, again showing a peak at m/z 1381.74 corresponding to GYRKPPFNGSIFamide (Gly¹-SIFamide). (D) Spectrum of a PMC tissue extract, representing peptides extracted from a larger piece of tissue, again showing a peak at m/z 934.49 corresponding to CabTRP 1a; no peak for Gly¹-SIFamide was detected. (E) MS/MS spectrum showing product ions produced by the isolation and dissociation of m/z 1381.74 from the AMC tissue extract, providing confirmation of peptide identity via the production of b-type ions (containing the N-terminus) and y-type ions (containing the C-terminus) in a fragmentation pattern characteristic of Gly¹-SIFamide.