Chronotropic action of immobilized subtilisins during the perfusion of an isolated rat heart

The pharmacological experiments on isolated organs (ex vivo) are the preferred method for assessing the primary pharmacodynamics of the studied drugs, since this method is completely excluded the systemic influence of neurohumoral regulation. In the last decade, a new group of thrombolytic drugs based on immobilized subtilisins has been formed. At the stage of registrational preclinical and clinical studies, their pleiotropic pharmacological effects have not been studied. Meanwhile, there is a reason to consider that their pharmacological activity in the bloodstream is not limited to thrombolytic action, but may be extended to a systemic effect on the cardiovascular system. 
The aim of the study was to investigate the chronotropic effects of an isolated heart during its perfusion with solutions of immobilized subtilisins at different concentrations. 
Material and methods. The isolated rat heart model according to Langendorff was used in the study. The experiment included 50 Wistar rats, which were divided into 5 groups: isolated hearts perfused only with Krebs – Henseleit solution (control) or with immobilized subtilisins in 4 concentrations (170, 340, 510 и 1020 U/l). 
Results and discussion. The immobilized subtilisins have a negative chronotropic effect. The onset of the effect depends on the drug concentration in the solution: the higher concentration, the earlier effect. From 5 to 10 minutes of perfusion, a negative chronotropic effect is observed using of immobilized subtilisins at any dose. The duration of its increase is manifested up to 10–20 minutes, depending on the drug concentration in solution. After 20 minutes of perfusion, the achieved negative chronotropic effect remains at a plateau level up to 40 minutes. 
Conclusion. The immobilized subtilisins have an independent pharmacological effect on heart rate.

1. Langendorff O. Studies on the surviving mammalian heart. Pflugers Arch. 1895;61:291–332. [In German]. doi: 10.1007/BF01812150

2. Korolev D.V., Minasian S.M., Galagudza M.M. Research equipment for registration of surface electrogram of the isolated heart of laboratory animals. Biotekhnosfera = Biotechnosphere. 2014;(5):49–53. [In Russian].

3. Toropova Ya.G., Mukhamadiyarov R.A., Golovkin A.S. Effect of different concentrations liposomal emoxipine on coronary flow, contractive and pump function of the isolated rat heart after normotermic ischemia and further reperfusion. Rossiyskiy fiziologicheskiy zhurnal imeni Ivana Mikhaylovicha Sechenova = Russian Journal of Physiology. 2013;99(7):869–875. [In Russian].

4. Khisamieva L.I., Chershintseva N.N., Kuptsova A.M., Ziyatdinova N.I., Zefirov T.L. Blockade of α2-adrenergic receptors inhibits functional parameters of langendorff-isolated rat heart. Bull. Exp. Biol. Med. 2021;172:288–291. [In Russian]. doi: 10.47056/0365-9615-2021-172-9-273-276

5. Baikalov G.I., Knyazev R.A., Ershov K.I., Bakhareva K.I., Soldatova M.S., Madonov P.G. Study of the immobilized subtilisins influence on coronary blood flow of an isolated rat heart. Journal of Siberian Medical Sciences. 2021;(3):56–65. [In Russian]. doi: 10.31549/2542-1174-2021-3-56-65

6. Bell R.M., Mocanu M.M., Yellon D.M. Retrograde heart perfusion: the Langendorff technique of isolated heart perfusion. J. Mol. Cell. Cardiol. 2011;50(6):940–950. doi: 10.1016/j.yjmcc.2011.02.018

7. Olejnickova V., Novakova M., Provaznik I. Isolated heart models: cardiovascular system studies and technological advances. Med. Biol. Eng. Comput. 2015;53(7):669–678. doi: 10.1007/s11517-015-1270-2

8. Watanabe M., Okada T. Langendorff perfusion method as an ex vivo model to evaluate heart function in rats. Methods Mol. Biol. 2018;1816:107–116. doi: 10.1007/978-1-4939-8597-5_8

9. Madonov P.G., Mishenina S.V., Kinsht D.N., Kikhtenko N.V. Chemical and pharmacological properties of subtilisins. Sibirskiy nauchnyy meditsinskiy zhurnal = Siberian Scientific Medical Journal. 2016;36(3):13–22. [In Russian].

10. Madonov P.G., Mishenina S.V., Kinsht D.N., Kikhtenko N.V. Targeted pharmacodynamic of subtilisins. Sibirskiy nauchnyy meditsinskiy zhurnal = Siberian Scientific Medical Journal. 2016;36(4):15–23. [In Russian].

11. Madonov P., Leont’ev S., Zotov S., Ufimtsev M., Mishenina S., Kinsht D. Initial results of oral thrombolytic agent clinical application. Proceedings: proc. 25th Biennial international congress on thrombosis, Venice, Italy, May 23–26, 2018. Venice. 2018;2(9):530. doi: 10.3390/proceedings2090530

12. Madonov P.G., Momot A.P., Mamaev A.N., Roitman E.V., Mishenina S.V. Non-plasmin fibrinolysis with immobilized subtilisins. Tromboz, gemostaz i reologiya = Thrombosis, Hemostasis and Rheology. 2019;(3):24–32. [In Russian]. doi: 10.25555/THR.2019.3.0886

13. Mishenina S.V., Baikalov G.I., Baikalova N.E., Makarov V.K., Madonov P.G. Rationale for the use of immobilized subtilisins for targeted therapy of venous thrombosis. Journal of Siberian Medical Sciences. 2020;(1):76–88. doi: 10.31549/2542-1174-2020-1-76-88

14. Madonov P.G., Mishenina S.V., Roitman E.V., Ishchenko N.A. Pharmacological thrombolysis: what’s new? Tromboz, gemostaz i reologiya = Thrombosis, Hemostasis and Rheology. 2020;(2):40–52. [In Russian]. doi: 10.25555/THR.2020.2.0917