Inotropic responses to human gene 2 (B29) relaxin in a rat model of myocardial infarction (MI): effect of pertussis toxin

Abstract

Relaxin produces powerful inotropic and chronotropic responses in isolated atria. The effect of relaxin has been examined in a rat model of cardiac failure, induced by myocardial infarction (MI). Maximum inotropic responses to isoprenaline (sham 5.4+/-0.3 mN; MI 2.6+/-0.3 mN; P<0.001) and relaxin (sham 5.1+/-0.6 mN; MI 2.8+/-0.5 mN; P=0.013) were reduced in left atria following MI. No change in chronotropic responsiveness was observed in right atria. Pertussis toxin (PTX) treatment restored inotropic responses to isoprenaline (sham 5.5+/-1.3 mN; MI 5.8+/-1.0 mN; P=0.850) but not to relaxin. Instead, PTX reduced inotropic responses to relaxin in sham animals to the same level seen in the MI group (sham 3.2+/-1.7 mN; MI 2.8+/-0.6 mN; P=0.847). In right atria, PTX treatment did not affect the maximum chronotropic response to isoprenaline, but reduced responses to relaxin in both sham and MI animals. R3 relaxin and relaxin receptor (LGR7) mRNA was present in atria and left ventricle (LV) from sham and MI animals. R3 relaxin mRNA expression was increased in atria but not LV from MI animals. LGR7 mRNA expression was reduced in atria and LV from MI animals. PTX treatment in unoperated rats increased chronotropic responses (vehicle 184.3+/-5.3 beats min(-1); PTX 211.3+/-9.5 beats min(-1); P=0.029) and produced a rightward shift in the concentration-response curve to isoprenaline in left atria. PTX reduced inotropic (vehicle 3.3+/-0.7 mN; PTX 0.8+/-0.2 mN; P=0.005) and chronotropic (vehicle 130.2+/-8.1 beats min(-1); PTX 90.6+/-11.1 beats min(-1); P=0.012) responses to relaxin. 6 In left atria, relaxin produced a small increase in cAMP compared to those produced by isoprenaline and forskolin. However, PTX treatment significantly reduced relaxin-, isoprenaline- and forskolin-stimulated cAMP accumulation. Cardiac failure in MI animals caused a reduced inotropic response to both relaxin and (-)-isoprenaline. In non-MI animals, PTX treatment also reduced inotropic responses to relaxin. Differences between responses to (-)-isoprenaline and relaxin can be explained by changes in coupling efficiency occurring at the level of adenylate cyclase.

Figure 1

Inotropic responses in electrically stimulated left atria. (a–c) Responses to isoprenaline (ISO) and relaxin (h2R1x (B29)) in sham (filled symbols) and MI (open symbols) rats. (d–f) Responses to isoprenaline and h2R1x (B29) in sham and MI rats after PTX treatment. P values on c/r curves are from a two-way ANOVA performed to determine differences between curves. P values on bar graphs indicate significance using an unpaired Student's t-test.

Figure 2

Chronotropic responses in spontaneously beating right atria. (a–c) Responses to isoprenaline (ISO) and relaxin (h2R1x (B29)) in sham (filled symbols) and MI (open symbols) rats. (d–f) Responses to isoprenaline and h2R1x (B29) in sham and MI rats after PTX treatment. P values on c/r curves are from a two-way ANOVA performed to determine differences between curves. P values on bar graphs indicate significance using an unpaired Student's t-test.

Figure 3

RT–PCR of R1 relaxin, R3 relaxin and rat LGR7 mRNA expression in atrial (a) and LV (b) tissue from sham (lanes 2–5) and MI (lanes 6–9) animals. Ethidium bromide-stained PCR products of R1 relaxin (396 bp), R3 relaxin (292 bp) and rat LGR7 (850 bp) are shown, while GAPDH products were used as controls for quality and equal loading of the cDNA. Samples consist of a molecular weight marker (lane 1), and gene transcripts from four separate sham atria (a, lanes 2–5), four separate MI atria (a, lanes 6–9), four separate sham LV (b, lanes 2–5) and four separate MI LV (b, lanes 6–9). cDNA from a late pregnant ovary was used as a positive control for R1 relaxin (b, lane 10), while cDNA from the pons/medulla and cortex were used as positive controls for R3 relaxin and rat LGR7 mRNA expression, respectively. Water replaced cDNA in negative control reactions for each PCR (b, lane 11).

Figure 4

(a–c) Effect of PTX treatment on inotropic responses to (a) isoprenaline, (b) hR1x (B29) and (c) single consecutive doses of isoprenaline (ISO, 300 nM), adenosine (30 μM) and calcium (7 mM) after the relaxin c/r curve. (d–f) Effect of PTX treatment on chronotropic responses to (d) isoprenaline, (e) hR1x (B29) and (f) single dose of isoprenaline (ISO, 300 nM) after the relaxin c/r curve. Filled symbols represent vehicle-treated animals, whereas open symbols represent PTX-treated animals. P values on c/r curves are from a two-way ANOVA performed to determine differences between curves. P values on bar graphs indicate significance using an unpaired Students' t-test.