Glucagon Can Increase Force of Contraction via Glucagon Receptors in the Isolated Human Atrium

Figures

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  Figure 1. Putative mechanism(s) of action of glucagon in cardiomyocytes. Glucagon stimulates the glucagon receptor (GR). Then, via stimulatory GTP-binding proteins, the adenylyl cyclase (AC) catalyzes the formation of cAMP. This cAMP activates a cAMP-dependent protein kinase (PKA) and PKA activates by phosphorylation (P) cardiac regulatory proteins such as the L-type Ca2+ channel (LTCC), the ryanodine receptor (RyR), phospholamban (PLB) or the inhibitory subunit of troponin (TnI). The cAMP is degraded by phosphodiesterases (PDE) that can be inhibited in part by cilostamide. If-currents are indicated by HCN, which is directly activated by cAMP. The sarcoendoplasmatic Ca2+-ATPase (SERCA) pumps Ca2+ into the sarcoplasmic reticulum, where the Ca2+ binds to calsequestrin (CSQ). The myofibrils are responsible for the generation of force, which is symbolized here by a single muscle contraction over time. SC203972 acts as a competitive antagonist at the glucagon receptor.

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  Figure 2. Original recording of the force of contraction of a human right atrial preparation. Glucagon alone, cumulatively applied at concentrations ranging from 1 to 10 micromoles per liter, did not exert any effects. The vertical bar indicates the developed force in millinewtons (mN), while the horizontal bar indicates the time in minutes (min).

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  Figure 3. Positive inotropic effects of cilostamide on force of contraction. (A) Original recording of the force of contraction of a human right atrial preparation. Cilostamide alone induced a positive inotropic effect. The vertical bar indicates the developed force in millinewtons (mN), while the horizontal bar indicates the time in minutes (min). (B–E) Summarized effects of cilostamide alone on the force of contraction (B), the rate of tension development (dF/dtmax, (C)), the time to peak tension (TTP, (D)) and the time to 90% relaxation (TR90, (E)). * p < 0.05 vs. control (Ctr = predrug value). The numbers in the bars indicate the number of experiments.

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  Figure 4. Original recordings of human right atrial preparations. In cilostamide pre-stimulated preparations, glucagon increased the force of contraction when cumulatively applied (A) or when, alternatively, only a discrete concentration of glucagon was applied (B,C). The glucagon receptor antagonist SC203972 (SC) diminished the effects of glucagon (A,C). The β-adrenoceptor agonist isoprenaline was much more efficacious in increasing the force of contraction compared to glucagon (C). The vertical bars indicate the developed force in millinewtons (mN), while the horizontal bars indicate the time in minutes (min).

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  Figure 5. A discrete dose of glucagon exerted a positive inotropic effect after pre-stimulation of the human right atrial preparation by 1 nM isoprenaline. The vertical bar indicates the developed force in millinewtons (mN), while the horizontal bar indicates the time in minutes (min).

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  Figure 6. Summarized effects of cumulatively applied glucagon in presence of cilostamide on (A) the force of contraction (glucagon −6: p = 0.002, test power (1 − β) = 0.9895), (B) the rate of tension development (dF/dtmax) and the rate of tension relaxation (dF/dtmin) (glucagon −6: p = 0.003, test power (1 − β) = 0.867), (C) the time to peak tension (TTP) (glucagon −5.5: p = 0.007, test power (1 − β) = 0.952), and (D) the time to 90% relaxation (TR90) (glucagon −5.5: p = 0.007, test power (1 − β) = 0.943) in human right atrial preparations. # p < 0.05 vs. cilostamide (Cilo). The numbers in the brackets indicate the number of experiments.

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  Figure 7. Stimulation of GLP-1 receptors and glucagon receptors in human right atrial preparations, with (bottom) and without (top) prior stimulation with cilostamide. As indicated, exenatide, exendin(9-39), glucagon, or SC203972 were added at the specified concentrations. At the end, a maximal concentration of isoprenaline (Iso) was added. All compounds were applied without washout steps. The vertical bars indicate the developed force in millinewtons (mN), while the horizontal bars indicate the time in minutes (min).

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  Figure 8. Glucagon-dependent phosphorylation of phospholamban (PLB). (A) Through Western blotting, the effect of 1 µM glucagon in presence of 0.1 µM cilostamide on PLB serine-16 phosphorylation (PS16-PLB) in human right atrial preparation could be demonstrated (sample 1). The effect was antagonized by additional application of 1 µM SC203972 (sample 2). PLB can usually be detected in its polymeric form (p) by Western blotting when loading non-heated samples. Boiling of the samples shifts the polymeric form to its monomeric form (m), which is not easily detectable. Here, we used an isoprenaline-treated mouse ventricular homogenate as positive control (+Ctr) that was loaded either non-heated (n) or after boiling (b) to demonstrate this shift and to identify the PLB band. The human samples were not heated. To ensure a comparable protein loading, the protein expression of calsequestrin (CSQ) was utilized as a cardiac myocytes-specific loading control. A pre-stained molecular weight marker (RM, rainbow marker) was used to identify the molecular weight range for cutting the membrane. (B) Quantification of the band intensities from four independent experiments, normalized to CSQ (PS16-PLB/CSQ). C,…