Abstract
A reduction in cardiac output is commonly associated with ischaemic heart disease and there is a therapeutic need to design novel inotropic drugs to improve cardiac function. Previous data has shown an inotropic response to our novel organic carbon monoxide (CO)-releasing molecule, oCOm-21, in isolated perfused rat hearts. Current literature suggests that changes in myofilament protein phosphorylation is related to cardiac contractility. Therefore, the aim of this work was to determine whether the inotropic response from oCOm-21 relates to changes in the phosphorylation of myofilament proteins.
An Aurora Scientific Permeabilised Myocyte System 1600A was used to generate force-velocity curves to determine whether this inotropic response is observed in isolated rat cardiomyocytes. Student t-tests were used to conduct statistical analyses between the vehicle and drug group, and results were expressed as mean ± SEM. When compared to vehicle control (n = 6), oCOm-21 (10 μM, n = 3) led to a significant increase in cell shortening velocity (0.90 ± 0.04 vs. 1.50 ± 0.13 cell length / s, respectively, P < 0.01) and power output (P/Po * cell length / s, 15.55 ± 1.19 vs. 21.30 ± 1.46, respectively, P < 0.05).
To further investigate if phosphorylation changes are involved in the contractile and peak power increases obtained to oCOm-21, protein expression was examined using Pro-Q™ Diamond Phosphoprotein gel stain (phosphorylated protein stain) and SYPRO® Ruby gel staining (total protein stain). These experiments were conducted in both membrane-permeabilised and intact cardiomyocytes to examine any change at the myofilament level (confirmed with native protein expression) and whole-cell. These cardiomyocytes were incubated with either vehicle, 3μM, or 10 μM oCOm-21 for 20 minutes. There were, however, no differences seen in either myofilament or total protein phosphorylation between the groups.
These results highlight the ability of oCOm-21 to increase the velocity of contraction and the peak power output of a cardiomyocyte, thereby, potentially improving the ability of the heart to pump blood. However, this observation was found to be unrelated to phosphorylation. Further investigation is warranted to understand the inotropic mechanism of oCOm-21 to further develop this inotropic prodrug as a future therapeutic.