Contractile effects of retatrutide in isolated mouse atrial preparations

Figures

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  Fig. 1. Conceivable actions of retatrutide in cardiomyocytes. Glucagon stimulates glucagon receptors (GCGR antagonized by adomeglivant). GLP-1 stimulates GLP-1 receptors (GLP-1R, antagonized by exendin9-39) and GIP the GIP receptors (GIPR, antagonized by ProGIP). Then via stimulatory GTP-binding proteins, adenylyl cyclase (AC) catalyzes the formation of cAMP which could directly activate hyperpolarization-activated cyclic nucleotide gated channels (HCN). The cAMP activates a cAMP-dependent protein kinase (PKA). This PKA activates by phosphorylation (P) cardiac regulatory proteins which are reversed by protein phosphatases (PP). The cAMP is degraded by phosphodiesterases IV (PDE) that can be inhibited by rolipram. PKA (inhibited by H89) can phosphorylate phospholamban (PLB) or HCN. SERCA pumps Ca2+ into the sarcoplasmic reticulum. Ca2+ binds to calsequestrin (CSQ). RyR indicates the ryanodine receptor. LTCC means the L-type Ca2+ channel. PIA (R-PIA) is an agonist at A1-adenosine receptors (A1-R) which is antagonized by DPCPX. Carbachol stimulates the M2-muscarinic receptor (M2-R) which is antagonized by atropine. The effect of carbachol and PIA are mediated by inhibitory GTP-binding…

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  Fig. 2. Retatrutide increases the beating rate. A Original recording of FOC in mouse left electrically stimulated (top, LA) and right spontaneously beating (bottom, RA) isolated atrial preparations. Additions of retatrutide (cumulatively applied) and isoprenaline are indicated with vertical lines. Vertical bar as beginning of (A) (top) gives force of contraction in milli Newton (mN). Ordinate (bottom) gives beating rate in beats per minute (bpm). Horizontal bars give time in minutes (min). B Summarized effects of retatrutide and 1 µM isoprenaline on beating rate in RA. Ordinate gives beats per minute (bpm). Abscissa: negative decadic logarithm of concentration of retatrutide or effect of isoprenaline (Iso) or pre-drug value (Ctr). Number in brackets indicate number of experiments

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  Fig. 3. Retatrutide fails to increase force of contraction. Force of contraction was measured in isolated spontaneously beating right atrial mouse preparations (A) or in electrically stimulated mouse left atrial preparations (B). Ordinates indicates force of contraction in percentage of pre-drug value (Ctr). Abscissa gives concentration of retatrutide in nM concentrations. Number in brackets indicates number of experiments. * indicate p < 0.05 against Ctr

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  Fig. 4. Propranolol does not block the effect of retatrutide. A An original recording of the beating rate in the spontaneously beating right atrium. Horizontal bar indicates time in 10 min. Arrows indicates where 1 µM propranolol was added. Concentration of retatrutide in decadic logarithm of molar concentration in is indicated by a horizonal line above the original recording. B Summarized concentration dependent effects of retatrutide on beating rate. Abscissa indicated the concentration of retatrutide in negative decadic logarithm of molar concentrations or control values (before any drug application: Ctr). Number in brackets indicates number of experiments. + indicate p < 0.05 against Ctr. Ordinates indicates beating rate in beats per minute

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  Fig. 5. Effect of retatrutide on beating rate is mediated by GCGR. The beating rate before the exendin 9–39 (exendin) or ProGIP or adomeglivant was defined as 100% and regarded as control (Ctr) as seen in Fig. 4. Thereafter the antagonist was added, and its maximim negative inotropic effect was measured. Several such experiments are summarized. Numbers in bars: top numbers: arithmetic mean values of beating rate; bottom numbers: number of experiments. *p < 0.05 vs. Ctr

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  Fig. 6. Carbachol reduces beating rate after retatrutide. A Original recording of beating rate in isolated spontaneously beating right atrial mouse preparations. Ordinate: beating rate in beats per minute. Horizontal bar indicates time in minutes (min). Arrows indicate addition of 100 nM retatrutide, 1 µM carbachol or 1 µM atropine, respectively. B Bar diagram of force of contraction in right atrial preparations. Ordinate (F) in mN. * indicates p < 0.05 versus retatrutide. Ctr: pre-drug value. Reta (100 nM retatrutide). Carba (1 µM carbachol). C Bar diagram of the maximum rate of tension development. Ordinate in mN/s. * indicates p < 0.05 versus retatrutide. Ctr: pre-drug value. pre-drug value. Reta (100 nM retatrutide). Carba (1 µM carbachol). D Bar diagram of rate of relaxation. Ordinate in mN/s. * indicates p < 0.05 versus retatrutide. Ctr: pre-drug value. pre-drug value. Reta (100 nM retatrutide). Carba (1 µM carbachol). E Bar diagram of the beating rate. Ordinate in beats per minute (Bpm). pre-drug value. Reta (100 nM retatrutide). Carba (1 µM carbachol). * indicates p < 0.05 versus retatrutide (Reta). Number in brackets indicates number of experiments

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  Fig. 7. PIA reduces beating rate after retatrutide. A Original recording of beating rate in isolated spontaneously beating right atrial mouse preparations. Ordinate: beating rate in beats per minute (BPM). Horizontal bar indicates time in minutes (min). Arrows indicate addition of 100 nM retatrutide, 1 µM PIA and DPCPX, respectively. B Bar diagram of the beating rate. Ordinate gives beats per minute (Bpm). Ctr: pre-drug value. Reta (100 nM retatrutide). PIA (1 µM PIA). * indicates p < 0.05 versus (Reta)

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  Fig. 8. H89 attenuates the effect of retatrutide on beating rate. A Concentration response curve for the effect of isoprenaline (Iso) on beating rate in isolated mouse right atrial preparations (WT) in the presence (circles) or in the absence of 3 µM H89 (triangles). B Concentration response curve for the effect of isoprenaline on beating rate) in isolated right atrial preparations. Ordinates indicate beating rate in beats per minutes. * indicates the first significant (p < 0.05) difference versus Ctr (pre-drug value). Numbers in brackets indicate the number of experiments. Abscissae control conditions (Ctr: pre-drug) or after 3 µM H89 or indicate negative decadic logarithm of isoprenaline or retatrutide in molar concentration, which were subsequently applied. Number in brackets indicates number of experiments. # indicate p < 0.05 versus Ctr. + indicate p < 0.05 against in the absence of H89

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  Fig. 9. Rolipram potentiates the effect of retatrutide on beating rate. A Original recording of beating rate in isolated right atrial mouse preparations. Horizontal bar indicates time in 10 min. Arrows indicate addition of 10 µM propranolol then 100 nM rolipram, then retatrutide was cumulatively applied thereafter exendin9-39 (Exen), then ProGIP, and finally, 10 µM adomeglivant (Ado). At the end, increasing concentrations of isoprenaline were cumulatively applied. Please note that isoprenaline was here less effective than in previous figures, because the bath still contained 10 µM propranolol from the initial incubation. The concentration of retatrutide is given in negative decadic logarithm of molar concentrations as indicated by a horizontal line above the tracing. Horizontal line indicates time in minutes (min). B Summarized effect of retatrutide on beating rate in the absence (squares) and the presence of 100 nM rolipram (circles). Ctr indicates basal conditions, Prop means in the presence of 10 µM propranolol, Rol means in the presence of 100 nM rolipram. Abscissa otherwise indicates the negative logarithmic concentrations of retatrutide. Then, the effect of 100 nM exendin 9–39…

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  Fig. 10. Signalling scheme for retatrutide. To summarize our interpretation of our findings, retatrutide binds to glucagon receptors (GCGR). Then, GCGR via stimulatory GTP-binding (Gs) the activity of adenylyl cyclase (AC). Hereby, cAMP is generated. This cAMP activates cAMP-dependent protein kinase (PKA). This activates HCN and this depolarizes the cardiomyocyte and as a consequence, then the beating rate in the mouse atrium increases (PCE)