Abstract
Reperfusion of the myocardium, a necessary procedure attendant in coronary artery bypass graft surgeries, is paradoxically associated with deleterious ischaemia reperfusion injury. Improved strategies such as the cardioprotective carbon monoxide, which can be used peri operatively, are required to attenuate this injury. The novel pro-drug oCOm-21 releases low doses of carbon monoxide and is currently being investigated for its cardioprotective and positive inotropic effects. Previous studies in our laboratory group have demonstrated improved contractility and recovery of haemodynamic parameters in a Langendorff perfused rat heart model. This present study examines the action of oCOm-21 on contractility parameters of Triton X-100-skinned/permeabilised cardiomyocytes and on the post-translational modification phosphorylation of key myofilament proteins.
Force-velocity curves were examined using skinned cardiomyocytes through utilising a microphysiological apparatus. oCOm-21 (10 µM) increased cell shortening velocity (1.35 ± 0.09 cell length shortening/second vs control: 0.90 ± 0.04 cell length shortening/second, P < 0.05) and increased power output (0.20 ± 0.01 force/maximum force vs control: 0.16 ± 0.01 force/maximum force, P < 0.05) at varying loads. This finding provides valuable evidence of the ability of oCOm-21 to directly modify the myofilament in the absence of an intact myocardium and associated signalling mechanisms to exert positive inotropic effects.
Four groups were tested for protein phosphorylation levels: direct oCOm-21 treatment to skinned and intact cardiomyocytes (in vitro), and oCOm-21 pre-treated Langendorff perfused skinned and intact cardiomyocytes (ex vivo). This protocol was conducted using Pro-Q™ Diamond Phosphoprotein staining and Western blotting. oCOm-21 treatment did not change cardiac troponin I phosphorylation levels in all groups, except for the pre-treated intact cardiomyocyte group showing an increase in phosphorylation staining with 10 µM drug treatment, which was not supported by the Western blotting results. oCOm-21 also did not result in any changes in phosphorylation levels of cardiac troponin T or cardiac myosin binding protein C in any groups. oCOm-21 (10 µM) decreased myosin light chain phosphorylation in the pre-treated intact cardiomyocyte group, however, no changes were observed in the other three cardiomyocyte groups. Myosin light chain phosphorylation was not confirmed by Western blot.
These results provide evidence of an inotropic effect occurring at the myofilament level with oCOm-21, however, the mechanism remains elusive. It is uncertain that phosphorylation of the II key myofilament proteins investigated are involved in the positive inotropic response of oCOm-21. Further investigation is required to gain a better understanding of oCOm-21.