Characterising the myotropic and calcitropic effects of the organic carbon monoxide releasing molecule oCOm-21

Acute heart failure patients undergoing cardiopulmonary bypass procedures require inotropic support to improve haemodynamic function and cardiac output. These pharmacological agents are sub-categorised based on the following three myocardial mechanisms: calcitropes which augment intracellular [Ca²⁺]; myotropes which target the muscular motor, the myofilament; and mitotropes, which alter myocardial energetics. Current clinically used inotropes are heavily represented by calcitropic agents such as dobutamine and milrinone that, can inherently, promote arrhythmia formation and increase mortality. Therefore, there is a clinical need to improve the development of new inotropes which have little effect on intracellular Ca²⁺ flux and instead, target the myofilament. Low-doses of carbon monoxide (CO) induce inotropic effects in perfused hearts. Although the mechanism of action remains unclear, CO can interact with the myofilament protein troponin C and decrease Ca²⁺ entry into the cytosol. Using the CO-releasing prodrug, oCOm-21, this research study hypothesised that this positive inotropic effect may result from a CO-mediated increase in myofilament Ca²⁺ sensitivity without a concurrent increase in intracellular Ca²⁺ transient magnitude.

The positive inotropic mechanism of oCOm-21 was investigated at three levels: at the myofilament, in intact cardiomyocytes, and in intact hearts. Chapter 2 explored the positive inotropic mechanism of oCOm-21 at the myofilament using permeabilised cardiomyocytes, and determined whether oCOm-21 alters myofilament phosphorylation or directly interacts with the myofilament to increase force production. Force was measured as a function of pCa (-log [Ca²⁺]) in the range of pCa 9.0-4.5 under five treatment conditions: vehicle, oCOm-21 (3 or 10mM), the CO-depleted drug control BP-21, and the Ca²⁺-sensitising inotrope levosimendan (1 mM). The Ca²⁺ concentration at which 50% of maximal force is produced (pCa₅₀), was used to measure Ca²⁺ sensitivity. Total phosphorylation of the myofilament proteome was measured using the phospho-protein gel stain, ProQ Diamond followed by SYPRO Ruby gel stain to normalise to the total protein. oCOm-21 (10 mM), but not BP-21 significantly increased pCa₅₀ compared to vehicle, respectively (pCa50 5.52 vs. 5.47 vs. 5.44; P < 0.05). Notably, no alterations in myofilament phosphorylation occurred, indicating that oCOm-21 directly interacts with the myofilament to increase force output. Levels of free heme, derived from mitochondria were determined in the permeabilised cardiomyocyte preparations. Incubation with the heme scavenger hemopexin (HPX) (1 mM ) abolished the Ca²⁺ sensitising mechanism of oCOm-21, indicating a heme-dependent mechanism.

In Chapter 3, oCOm-21 was administered to Langendorff-perfused hearts and permeabilised cardiomyocytes were examined to establish if the Ca²⁺ sensitising effect was conserved ex vivo. oCOm-21 (3, 10  mM) increased left ventricular developed force (LVDP), rate of contraction (dP/dt_max), rate of relaxation (dP/dt_min), and rate pressure product without a change in heart rate. These effects occurred without a change in myofilament protein phosphorylation, quantified using ProQ Diamond. The effects of oCOm-21 on Ca²⁺ flux were subsequently examined in isolated, intact, rat cardiomyocytes loaded with the fluorescent Ca²⁺ dye, fluo-4-AM. oCOm-21 at 10  mM but not 3 mM, decreased Ca²⁺transient amplitudes consistent with a negative calcitropic mechanism. Interestingly, this reduction in Ca²⁺ amplitudes was also associated with a reduction in Ca²⁺ spark frequency, indicative of an anti-arrhythmic effect.

These results support the hypothesis that oCOm-21-derived CO directly increases myofilament Ca²⁺ sensitivity. In line with the study hypothesis, this mechanism was dependent on heme but, whether the mechanism is reliant on a post-translational modification or a direct interaction with myofilament proteins remains to be examined. The increased Ca²⁺sensitivity observed at the myofilament level, was also conserved in a pre-treated heart, and occurred without a change in myofilament phosphorylation. Notably, oCOm-21 decreased intracellular Ca²⁺ transient amplitudes and reduced Ca²⁺spark frequency enforcing a negative calcitropic mechanism. This finding supports the hypothesis that oCOm-21 mediated positive inotropy results from a myotropic mechanism and not a calcitropic mechanism. Further analyses will probe the contribution of a mitotropic mechanism by oCOm-21 and confirm specific binding sites with the myofilament.