Contractile effects of dulaglutide in the human atrium

Figures

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  Fig. 1. Putative mechanism(s) of action of dulaglutide in human cardiomyocytes. Endogenous GLP-1 stimulates (red arrow) the GLP-1 receptor (GLP-1R) to generate cAMP. The GLP-1R is antagonized (blue arrow) by exendin9-39. Isoprenaline also elevates cAMP. The cAMP then activates cAMP-dependent protein kinases (PKA). PKA phosphorylates and activates phospholamban (PLB), the ryanodine receptor (RYR, through which Ca2+ can leave the sarcoplasmic reticulum and thus increase force more rapidly and to a higher extent), the L-type calcium ion channels (LTCC, facilitating Ca2+ entry through the sarcolemma, leading to more generated force), the inhibitory subunit of troponin (TnI) and C-protein, both located in myofilaments. PLB can increase force of contraction in the heart and the muscle relaxation. RYR can also be phosphorylated by the Ca2+ -calmodulin dependent protein kinase (not shown). The cAMP is degraded by phosphodiesterases (PDE) to inactive 5′-AMP. PDE 3 and PDE 4 are inhibited by cilostamide and rolipram, respectively. Dulaglutide can be regarded as two identical peptide chains connected by disulfide bonds (pink arrows). The N-terminal part of dulaglutide starts with a mutated GLP-1…

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  Fig. 2. Dulaglutide failed to increase the beating rate in mouse atria. (A) Original recoding of beating rate of 100 nM dulaglutide alone and then 1 µM isoprenaline in mouse right atrial preparations. (B) Bar diagram of the effects of 100 nM dulaglutide (Dula) alone on beating rate in isolated mouse right atrial preparation. (C) Effects of dulaglutide 100 nM (Dula) or 1 µM isoprenaline (Iso) in the presence of 100 nM rolipram(Roli) on the beating rate in right atrial preparations. Ordinates in the Figure indicate beats per minute (bpm). Numbers in brackets indicate number of experiments. * indicates significant differences (compare p-values next to asterisks) versus Ctr (pre-dulaglutide values) or after 100 nM rolipram (Roli)

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  Fig. 3. Dulaglutide failed to increase the force of contraction in mouse atria. Original recordings in wild type mouse left atrial preparations. Effect of dulaglutide 100 nM alone (A, top) or in the presence of 100 nM rolipram and then 1 µM isoprenaline (A, bottom) on force of contraction in isolated left mouse atrial preparations. Bar diagram on the effects of 100 nM dulaglutide (Dula) versus pre-drig value (Ctr) on force of contraction (B). Bar diagram on the effects of 100 nM dulaglutide (Dula) in the presence of 100 nM rolipram versus pre-drug value (Roli) on force of contraction (B). Arrows in (A) and ordinates in (B) and (C) give force of contraction in milliNewton (mN). Horizontal bar indicates time in minutes (min) (A, top). Numbers in brackets indicate the number of experiments. * indicate significant differences versus Ctr or Roli

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  Fig. 4. Dulaglutide alone increased force of contraction in HAP. Original recordings in HAP (A). Dulaglutide cumulatively applied (10–100 nM), induced a time-dependent and concentration-dependent positive inotropic effect in HAP (Fig. 4A). Subsequently, exendin9-39(exendin) reduced and a maximum effective concentration of isoprenaline (1 µM) increased force of contraction in HAP (A). Effects of 100 nM dulaglutide (Dula) compared to controls (Ctr) on force of contraction in mN are presented in (B). Moreover, the effect of 100 nM dulaglutide in HAP on the rate of tension development (C), on the rate of tension relaxation (D), time to peak tension (E) and time of tension relaxation (F) are plotted Ordinates indicate force in (A) and (B), or the maximum (+ dF/dt) or minimum (− dF/dt) rate of tension development in mN per second (mN/s) in (C) and (C) and in milliseconds (ms) in (E) and (F). * (next to the exact p-values in brackets) indicate significant differences versus Ctr (pre-dulaglutide values)

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  Fig. 5. Dulaglutide in the presence of cilostamide increased force of contraction in HAP. Two different original recordings in HAP from one patient are depicted in (A). Dulaglutide in the presence of 100 nM cilostamide induced a time-dependent-dependent positive inotropic effect in HAP (A). Subsequently, exendin9-39(exendin, vertical arrow) reduced and isoprenaline (1 µM) again increased force of contraction. We display a concentration-response curve for the effect of dulaglutide in the presence of 100 nM cilostamide (Cilo) on force of contraction in % of the effect of Cilo ((B), Cilo = 7. 8 mN ± 0.85 mN). Moreover, we display effect of 100 nm Dulaglutide in the presence of 100 nM cilostamide on force of contraction ((C), in mN), on the maximum rate of tension development (dF/dtmax, (D) in mN/s), on the rate of tension relaxation (dF/dtmin, (E) in mN/s) on the time to peak tension ((F), in milliseconds, ms), and time to relaxation (G). Abscissa in (B) indicates concentrations of dulaglutide in negative decadic logarithm of molar concentrations. First significant differences versus 100 nM cilostamide (Cilo) are indicated in asterisk in (B). Otherwise asterisks (next to exact p-values in…

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  Fig. 6. Dulaglutide acts via GLP-1R in HAP. Antagonism at GLP-1R. Dulaglutide (100 nM) raised force of contraction in the presence of 100 nM cilostamide in HAP to 8.9 ± 0.88 mN (left hand side bar). Subsequently applied 100 nM exendin9-39 reduced force of contraction (right hand side bar). Ordinate gives force of contraction in percentage of the effect of 100 nM dulaglutide before exendin9-39 was added in HAP. Number in brackets means number of experiments. * (next to the exact p-value) indicates a significant decrease versus the inotropic effect of 100 nM dulaglutide

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  Fig. 7. Dulaglutide increases the phosphorylation state of phospholamban in HAP. Both dulaglutide 100 nM (Dula) and 1 µM isoprenaline increased phosphorylation of regulatory proteins in the presence of 100 nM cilostamide (Cilo) in paced (1 Hz) isolated contracting strips from the human right atrium. Effects of 100 nM dulaglutide on phospholamban phosphorylation at serine 16 (PLB Ser16) is seen on the Western blot on the left hand side (A). The phosphorylation of troponin I (pTnI) is depicted on the Western blot on the right hand side (B). Arrows indicated PLB at about 23 kDa and TnI at about 31 kDa. As a loading control we assessed the protein expression of calsequestrin (CSQ) at about 63 kDa (arrow) by cutting the lanes of the blot and incubating the parts with different primary antibodies. Blots were quantified by densitometry to calculate the ratio signal for phospholamban phosphorylated at amino acid 16 and calsequestrin from the same lanes (C). Blots were also quantified by densitometry to calculate the ratio signal for phosphorylated troponin inhibitor (Phospho TnI) at amino acid 16 and calsequestrin (CSQ) from the same lanes (D). Numbers in brackets indicate the number of…