OR-1896 increases force of contraction in the isolated human atrium

doi:10.21203/rs.3.rs-2717966/v1

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OR-1896 increases force of contraction in the isolated human atrium

https://doi.org/10.21203/rs.3.rs-2717966/v1

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published 24 Jun, 2023

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OR-1896 ((R)-N-(4-(4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl) phenyl)-acetamide) is the main active metabolite of levosimendan. However, nobody has reported a positive inotropic effect of OR-1896 in isolated human cardiac preparations. The mechanism of action of OR-1896 remains controversial. Hence, we wanted to know whether OR-1896 exerts a positive inotropic effect in humans and what might be the underlying mechanism. Therefore, we measured the contractile effects of OR-1896 (0.01–10 µM cumulatively applied) in isolated electrically stimulated (1 Hz) human right atrial preparations (HAP), obtained during cardiac surgery. OR-1896 given alone exerted time- and concentration-dependent positive inotropic effects. 1 µM OR-1896 increased force by 72 ± 14.7% (p < 0.05, n = 6) and shortened the time of relaxation by 10.6 ± 3.6% (p < 0.05, n = 11) in HAP started at 0.1 µM, plateaued at 1 µM OR-1896 and was antagonized by 1 µM propranolol. The maximum positive inotropic effect of OR-1896 in human right atrial preparations was than that of 10 µM isoprenaline. EMD 57033 (10 µM), a calcium sensitizer, increased force of contraction further in the additional presence of 1 µM OR-1896 by 109 ± 19% (p < 0.05, n = 4). Cilostamide (10 µM), an inhibitor of phosphodiesterase III given before OR-1896 (1 µM) blocked the positive inotropic effect of OR-1896 in HAP. Our data suggest that OR-1896 is indeed a positive inotropic agent in the human heart. OR-1896 acts as a PDE III - inhibitor and OR-1896 is unlikely to act as a calcium sensitizer in the human heart.

OR-1896

human atrium

inotropy

phospholamban

In the heart positive inotropic effects can be achieved by with phosphodiesterase inhibitors (PDE, scheme in Fig. 1A). PDE III inhibitors like milrinone exerted positive inotropic, lusitropic and positive chronotropic effects (reviews: Schmitz et al.1989, 1992, Scholz and Meyer 1986). However, more heart failure patients died in the milrinone group than in the placebo group (Packer et al. 1991).

Hence, new mechanisms of inotropic action independent of an elevation of Ca²⁺ levels in the cytosol of cardiomyocytes (Fig. 1A) have thereafter been sought. For instance, so called “calcium sensitizers” (Rüegg et al. 1984, Ventura et al. 1992), like levosimendan, OR-1896 (Haikala et al. 1995, 1997, structural formula in Fig. 1B), CGP 48506 (Neumann et al. 1996, Zimmermann et al. 1996) or EMD 57033 (Neumann et al. 1995, Uhlmann et al. 1995) were studied. These calcium sensitizers raise the affinity of myofilaments for calcium cations but some of these drugs retained PDE-inhibitory activity.

Levosimendan was marketed as a pure calcium sensitizer (Haikala et al. 1995). In contrast to this view, in guinea-pig cardiac preparations: levosimendan accelerated cardiac relaxation in the guinea pig heart (Boknik et al. 1997), levosimendan exerted a positive chronotropic effect in spontaneously beating right atrial preparations from guinea pig hearts (Boknik et al. 1997), levosimendan elevated cAMP concentrations, increased the phosphorylation state of phospholamban, the inhibitory subunit of troponin and C-protein and enhanced the current through the L-type calcium channel in cardiomyocytes from guinea pig ventricles (Boknik et al. 1997, Virag et al. 1996). In contrast to levosimendan, a pure calcium sensitizer, namely CGP 48506, prolonged cardiac relaxation time and did not increase cAMP content nor phospholamban phosphorylation in guinea pig cardiac preparations (Zimmermann et al. 1996, 1998).

Consistent with the findings in guinea pig cardiac preparations, the phosphodiesterase III inhibitor cilostamide (Fig. 1B) blocked any positive inotropic effect of levosimendan in ventricular muscle strips from failing human hearts (Ørstavik et al. 2014). Thus, the positive inotropic effect of levosimendan seems to result from inhibition of phosphodiesterase III in the failing human ventricle (Ørstavik et al. 2014). Their conclusion was supported by their finding that the β-adrenoceptor antagonist timolol reversed the positive inotropic effect of levosimendan in human ventricular preparations (Ørstavik et al. 2014). More recently, we could confirm these data in human atrial preparations (Rayo-Abella et al. 2022 a, b): levosimendan increased force of contraction in human atrial preparations and this increase was accompanied by an elevation in the phosphorylation state of phospholamban and both effects were abrogated by propranolol. Moreover, cilostamide pretreatment hindered levosimendan to increase force of contraction in isolated human atrial preparations (Rayo-Abella et al. 2022 a, b).

OR-1896, the main metabolite of levosimendan, when given alone increased force of contraction in rat papillary muscles (Ørstavik et al. 2015). One noted that OR-1896 induced a positive inotropic effect starting at 0.1 µM OR-1896 that plateaued at 1 µM (Ørstavik et al. 2015). This effect positive inotropic effect of OR-1896 was augmented by rolipram (10 µM, a phosphodiesterase IV inhibitor) but blocked by the PDE III inhibitors milrinone (1 µM) or cilostamide (1 µM, Ørstavik et al. 2015). The positive inotropic effect of OR-1896 could not by augmented by additionally applied EMD57033 (3 µM, a calcium sensitizer). OR-1896 was less effective than isoprenaline (100 µM, Ørstavik et al. 2015). OR-1896 alone, exerted a lusitropic effect that was potentiated by rolipram (Ørstavik et al. 2015). In the presence of a β-adrenoceptor antagonist, OR failed to increase force of contraction in rat papillary muscles (Ørstavik et al. 2015). OR-1896 did not alter the potency of Ca²⁺ to raise force of contraction rat papillary muscles (Ørstavik et al. 2015). OR-1896 inhibited PDE activity in the rat heart with a similar efficacy as cilostamide (Ørstavik et al. 2015). Consistent with a PDE inhibition, the authors noted that cAMP was increased after addition of OR-1896 to rat ventricular cardiomyocytes (Ørstavik et al. 2015). The authors concluded that in rat papillary muscles OR-1896 increased force of contraction by inhibition of PDE III (Ørstavik et al. 2015). However, they did not test human tissue.

In patients, OR-1896 has a longer half-life than levosimendan, which amounts to about one hour, in contrast, the half-life of OR-1896 is about 70–80 hours (Koskinen et al. 2008, Grześk et al. 2022). Plasma levels of OR-1896 are higher in rapid acetylators of OR-1896 than in slow acetylators of OR-1896 (Antila et al. 2004). Hence, in some patients OR-1896 might be clinically especially relevant in their response to levosimendan treatment. There are data from skinned fibers that OR-1896 is a calcium sensitizer also in human ventricle (Papp et al. 2004). Usually OR-1895 on a molar basis is less potent than levosimendan. For instance, levosimendan was more potent to increase the rate of force development in living rats (Segreti et al. 2008). Likewise, OR-1896 was less potent than levosimendan to inhibit PDE III (levosimendan: 2.5 nM, OR-1896: 94 nM) and PDE IV (levosimendan: 25 µM, OR-1896: 286 µM) in the guinea pig heart. However, levosimendan and OR-1896 were about equipotent (levosimendan 15 nM, OR-1896: 25 nM) to raise intraventricular developed pressure in Langendorff perfused guinea pig hearts (Szilágyi et al. 2004). Both levosimendan and OR-1896 raised the rate of pressure development by about 25% and were thus equieffective (Szilagyi et al. 2004). OR-1896 is assumed to contribute to the clinical effect of levosimendan in heart failure patients. OR-1896 has also vasodilatory properties that are explained in part by opening of potassium channel and a cAMP increase in the vasculature (review: Burkhoff et al. 2021). These vasodilatory ancillatory effects are thought to be beneficial in patients with heart failure.

The group of Masao Endoh generated data that OR-1896 in dog papillary muscles OR-1896 increased force of contraction at least in part by elevating cytosolic free calcium ions (Takahashi et al. 2000). Moreover, the positive inotropic effect of OR-1896 was antagonized by carbachol, suggesting to the authors that also a cAMP dependent component contributes to the positive inotropic effect of OR-1896 in the dog heart (Takahashi et al. 2000).

We find it important to understand better the mechanism of action of the active metabolite of levosimendan namely OR-1896 in order to better understand how the long term actions of levosimendan on the human heart come about mechanistically. However, the effects of OR-1896 in human atrium or ventricle on force of contraction are unknown. For comparison, we performed similar experiments on cardiac atrial preparations from mice. These mouse data have the potential benefit to study the effect of OR-1896 on sinus node function in isolated preparations which is not readily feasible in human hearts. Thence, we studied the hypothesis that OR-1896 increased contractile function in isolated electrically paced human right atrial muscle strips. Furthermore, we asked whether this effect is altered by concomitant application of a PDE III inhibitor and is accompanied by increased phospholamban phosphorylation.

Contractile studies in mice

In brief, wild type mice were sacrificed, the thorax was opened the heart was mobilized and cut from the ascending aorta to make sure the right atrium was not damaged. Then the whole heart was transferred to a dissection chamber filled with gassed Tyrode´s solution at room temperature. Right or left atrial preparations were isolated and mounted in organ baths as described by Gergs et al. (2013, 2017, 2019) and Neumann et al. (2003). Force was detected under isometric conditions, amplified and fed into a digitizer and quantified by a commercial software (LabChart 8, AD Instruments, Spechbach, Germany).

Contraction Studies In Human Atrium

These experiments was performed as reported repeatedly (e.g. Gergs et al. 2009; Neumann et al. 2021a, b). In brief, during cardiac surgery, at the site where the cannula for extracorporeal circulation entered the heart, small muscle strips were obtained from the right atrium. Patients were aged between 48 and 72 years. Medication included acetylsalicylic acid, nitrates, diuretics, β-adrenoceptor blockers and anticoagulants. Atrial trabeculae were dissected and mounted in organ bath and electrically stimulated (1 Hz) and processed like mouse preparations (see above).

Western Blotting

Western blotting

The process of sample homogenization, protein concentration measurement, electrophoresis, antibodies incubation and signal quantification were performed following our previously published protocols with slight modifications (Boknik et al. 2018; Gergs et al. 2009; Gergs et al. 2019a). Electrophoresis was performed in Novex™ 4–20% “Tris-Glycine Plus Midi Protein Gels” (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA), The run was performed at 4°C for approximately one hour at 120 V in the “NuPAGE MES SDS Running Buffer” (Thermo Fisher Scientific, Waltham, Massachusetts, USA) using a XCell4 SureLock™ Midi-Cell chamber (Life Technologies by Thermo Fisher Scientific, Waltham, Massachusetts, USA). Protein transfer into membranes (Amersham™ Protran, GE Healthcare, Chicago, Illinois, USA) was performed at 2 A for 2 h at 4°C. Membrane blocking for one hour at room temperature was followed by overnight incubation at 4°C with the primary antibody for serine 16 - phosphorylated phospholamban (1:5000; catalogue number: A010-12AP; PLB Ser16; Badrilla, Leeds, UK), SERCA2 ATPase (1:20.000; catalogue number: ab2861; abcam, Cambridge, UK), phospho-troponin I (1:5000; catalogue number: 4004; Ser23/24; cell signaling technology, Leiden, The Netherlands). While calsequestrin antibody was used as loading control (1:20.000; product number: ab3516; abcam, Cambridge, UK). Visualization of the signals was performed by using chemiluminescent HRP substrate (Immobilon™ Western, Millipore, Merck; Darmstadt, Germany) and a digital imaging system (Amersham ImageQuant 800; Cytiva Europe GmbH, Freiburg im Breisgau, Germany).

Data analysis

Data were treated as in most our previous studies (e.g. Gergs et al. 2019, Neumann et al. 2021 a, b). Shown are means ± standard error of the mean. Statistical significance was estimated using the analysis of variance (ANOVA) followed by Bonferroni’s t-test or the Student’s t-test as appropriate. A P-value of less than 0.05 was considered significant. Experimental data for agonist-induced positive inotropic and chronotropic effects were analyzed by fitting sigmoidal curves to the experimental data with GraphPad Prism 5.0. All other statistical analyses were performed as indicated in the figures and tables.

Drugs And Materials

(-)-Isoprenaline (+)-bitartrate, rolipram, propranolol and cilostamide were purchased from Sigma-Aldrich (Deisenhofen, Germany). OR-1896 ((R)-N-(4-(4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl) phenyl)acetamide) was from Biozol (Munich. Germany). All other chemicals were of the highest purity grade commercially available. Deionized water was used throughout the experiments. Stock solutions were freshly prepared daily.

1. Mouse

1.1. Force of contraction in left atrial preparations:

OR-1896 cumulatively applied, when given alone, from 10 nM to 10 µM (the highest concentration tested in this study) did not raise increase contractility in electrically driven (1 Hz) left atrial preparations of mouse hearts compared to control conditions (OR-1896 alone: original recording: Fig. 2A). However, when first 0.1 µM rolipram (a phosphodiesterase IV inhibitor, Fig. 1A, B) was applied, rolipram itself increased force of contraction to some extent (Fig. 2A), and additionally applied OR-1896 augmented force of contraction further (Fig. 2A), in left atrial (LA) preparations from mice. The positive inotropic effects OR-1896 in the presence of rolipram were diminished by 10 µM propranolol (Fig. 2A). The effects of OR-1896 were time-dependent and concentration (10 nM to 10 µM)-dependent (Fig. 2A). The positive inotropic effect reached its maximum at 1 µM OR-1896 (Fig. 2A). However, subsequently applied 10 µM isoprenaline increased force of contraction in LA further indicating that OR-1896 under our experimental conditions is less effective than isoprenaline to raise force of contraction (Fig. 2A). In several initial experiments, we gave rolipram and then non-cumulatively only one concentration of OR-1896 (Fig. 2B). An effect of rolipram was apparent, but OR-1896, did not increase force further. We decided to construct concentration response curves for OR-1896, hoping to detect at least a small positive inotropic effect. But as seen in Fig. 2C, the effects were too small to gain significance if under these more favorite conditions. Several such experiments are summarized in Fig. 2B.

1.2. Beating rate in right atrial preparations:

One could argue, that our concentrations of OR-1896 or rolipram were too low to detect functional alterations in the mouse atrium. However, this does not seem to be an acceptable generalization: OR-1896 alone does not affect spontaneously beating right atrial preparations (Fig. 2E). However, in the additional presence of 0.1 µM rolipram, which exercised a positive chronotropic effect of its own (Fig. 2E), OR-1896 non-cumulatively applied (Fig. 2D) and cumulatively applied (Fig. 2E) exerted an increase in beating rate.

2. Human studies:

2.1. Force of contraction in isolated human atrial preparations:

In contrast to murine atrial preparations, (Fig. 2C), cumulatively applied OR-1896 alone exerted a concentration-dependent positive inotropic effect in human atrial preparations (Fig. 3A). In these human atrial preparations, OR-1896 concentration dependently reduced time to peak tension (Fig. 3B) and time of relaxation (Fig. 3B). Moreover, OR-1896 concentration dependently increase the rate of tension development and the rate of tension relaxation (Fig. 3C). In addition, we detected no significant calcium sensitizing properties: while increasing Ca²⁺ concentration raised force of contraction these effects were not potentiated by OR-1896 (Fig. 3D).

In contrast, while 1 µM cilostamide increased force of contraction in human atrial preparations to some extent and OR-1896 exerted a small addition positive inotropic effect (Fig. 4C), 10 µM cilostamide impaired any positive inotropic effect of subsequently added 1 µM OR-1896 (Fig. 4B). This is in line with findings by others in rat ventricular strips (Ørstavik et al. 2015). The positive inotropic effects of OR-1896 (1 µM) were abrogated by 1 µM propranolol in human atrial preparations consistent with the action OR-1896 as a PDE III inhibitor in human atrial tissue (Fig. 1A). After maximal stimulation by 1 µM OR-1896, force of contraction was augmented by additionally applied EMD 57033 (Fig. 4B, C), a calcium sensitizer, suggesting that in human atrium, EMD 57033 and OR-1896 have different mechanisms of action (Fig. 1A). These data combined argue against a calcium sensitizing effect of OR-1896, at least under our experimental conditions in human atrium. Moreover, the maximum inotropic effect to OR-1896 was lower in than that of 10 µM isoprenaline (Fig. 4A), in line with reports for levosimendan in human ventricular preparations (Ørstavik et al. 2014) and in human atrial preparation (Rayo-Abella et al. 2022 a,b).

2.2. Phospholamban phosphorylation in the human atrium:

1 µM OR1896 alone (that is: non cumulatively applied) increased the phosphorylation state of phospholamban at serine 16 in contracting human atrial strips that were freezed-clamped at the maximum of the positive inotropic effect (Fig. 5). The positive inotropic effects of OR1896 in human atrial strips were completely reversed by 1 µM propranolol (Fig. 4A). In the same atrial preparations where force was recorded and that were freeze-clamped, 1 µM propranolol reduced the increase in the phosphorylation state of phospholamban induced by 1 µM OR1896 (Fig. 5), arguing against an action of OR-1896 as a pure calcium sensitizer in the human atrium.

Contractile data in mouse left atrium:

The present data on mouse left atrium are somewhat divergent from those in rat ventricular preparations by others (Ørstaviket al. 2015). These authors unlike ourselves noted that OR-1896 when given alone was effective to raise force of contraction in rat ventricular muscle strips (Ørstavik et al. 2015). We would argue that this observation is concordant with the interpretation that OR-1896 heightens force of contraction in the mouse heart through inhibition of phosphodiesterase III, keeping in mind that PDE III is relatively unimportant for force regulation in the mouse heart. OR-1896 alone in rat ventricular cardiomyocytes has been shown to increase cAMP levels (Ørstavik et al. 2015), thus it is plausible that they observed a small positive inotropic effect with OR-1896 alone. Interestingly, in their study on rat ventricular cardiomyocytes, in the additional presence of cilostamide, OR-1896 did not increase cAMP levels further, supporting the assumption that OR-1896 increased cAMP levels already via PDE III inhibition in rat ventricle (Ørstavik et al. 2015). Moreover, the authors failed to detect Ca²⁺ sensitizing properties of OR 18956 in rat ventricle, but noted that OR-1896 could inhibit the activity of PDE III in rat ventricular preparations (Ørstavik et al. 2015).

Chronotropic Effects Of Or-1896 In Mouse Right Atrium

The present work clearly shows that OR-1896 increased the beating rate in the mouse right atrium. These positive chronotropic effects in mouse strongly argue against an action of OR-1896 as a calcium sensitizer. Moreover, the effect in the sinus node also seemed to result from amplification of cAMP levels. Endogenous noradrenaline may activate the β-adrenoceptor in the mouse sinus node. These findings are consistent with the view that OR-1896 acts via elevation of cAMP also in sinus node cells. This observation concurs with the hypothesis that also in the mouse sinus node, OR-1896 operates as a phosphodiesterase III inhibitor: fittingly, pre-incubation with rolipram amplified the positive chronotropic effect of OR-1896, because now PDE III and PDE IV are inhibited and only then a functional effect on the mouse sinus node can occur.

Effects Of Or-1896 In Human Atrium

In contrast to mouse atrial preparations, in isolated electrically stimulated human atrial preparations, OR-1896 alone escalated force of contraction and shortened, for instance, relaxation time. We assume this is probably due to a surge in the phosphorylation state of phospholamban that we reported in Fig. 5. Cardiac relaxation is thought to be enhanced by phosphorylation of phospholamban (Hamstra et al. 2020) and phosphorylation of the inhibitory subunit of troponin (Vetter et al. 2018). Positive inotropic effects of OR-1896 in human atrial preparations have, to the best of our knowledge, not been reported before. On the other hand, our data are fostered by the fact that OR-1896 shortened time of relaxation in rat ventricular muscle strips (Ørstavik et al. 2015).

Finally, our observation that propranolol countermands the positive inotropic effect of OR-1896 and reduces the OR-1896 induced increase in phospholamban phosphorylation in human atrial preparations also contradicts the view that OR-1896 is a calcium sensitizer in the human heart.

The present data strongly suggest that the positive inotropic and relaxant effects of OR-1896 in the isolated human right atrium result from lowered activity of phosphodiesterase III.

Acknowledgements:

The technical assistance of Pia Willmy is greatly appreciated. This work contains parts of the Ph. D. thesis of LMRA.

Authors´s Contributions: designed project: JN and UG. Supplied reagents and clinical data: BH. Performed experiments: LMRA, PG, MNB, JN. Supplied samples and clinical data: BH. Performed statistics and plotted data: LMRA, UG. Wrote paper: UG and JN. All authors have read and agree with the submission of the present version of the work. The authors declare that all data were generated in-house and that no paper mill was used.

Funding There was no external funding.

Availability of data and materials The data of this study are available from the corresponding author upon reasonable request.

Ethical Approval Animals: The investigation conformed to the Guide for the Care and Use of Laboratory Animals as published by the National Research Council (2011). The animals were handled and maintained according to the approved protocols of the Animal Welfare Committee of the University of Halle-Wittenberg, Halle, Germany. Humans: This study complies with the Declaration of Helsinki and has been approved by the local ethics committee (hm-bü 04.08.2005).

Consent to Participate Informed written consent was obtained from all patients included in the study.

Consent to Publish All authors declare that they have seen and approved the submitted version of this manuscript.

Competing Interests The authors declare no competing interests of financial or personal nature.

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OR-1896 increases force of contraction in the isolated human atrium

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Journal Publication

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Abstract

Figures

Introduction

Methods

Contractile studies in mice

Contraction Studies In Human Atrium

Western Blotting

Data analysis

Drugs And Materials

Results

1. Mouse

1.1. Force of contraction in left atrial preparations:

1.2. Beating rate in right atrial preparations:

2. Human studies:

2.1. Force of contraction in isolated human atrial preparations:

2.2. Phospholamban phosphorylation in the human atrium:

Discussion

Contractile data in mouse left atrium:

Chronotropic Effects Of Or-1896 In Mouse Right Atrium

Effects Of Or-1896 In Human Atrium

Conclusion

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1