The Effects Of Cardiac Troponin Activator CK-358 On Cardiac Contractility In A Mouse Model Of Genetic Dilated Cardiomyopathy

Genetic dilated cardiomyopathy (DCM) is one of the most common causes of heart failure. However, most treatments do not address the underlying contractile deficit caused by the disease. In an effort to create more targeted DCM therapeutics, novel small molecule drugs known as sarcomeric modulators have been of recent interest. In a recent study, a cardiac troponin activator was shown to improve cardiac muscle contractility without adversely affecting energetics or diastolic function. Here, we aim to elucidate the mechanism by which the cardiac troponin activator CK-3827358 (CK-358) is able to recover contractile function in a mouse model of DCM with a mutation at residue 61 of cardiac troponin C (cTnC I61Q). We measured steady state force and k tr , a measure of crossbridge cycling kinetics, in demembranated ventricular tissue from control and cTnC I61Q mice in solutions with increasing concentrations of calcium, ranging from pCa 9 to pCa 4. The cTnC I61Q mutation significantly decreased calcium sensitivity versus control (pCa 50 : 5.48 ± 0.06 vs 5.75 ± 0.01). Compared to controls, CK-358 (1 μM) significantly increased calcium sensitivity (pCa 50 ) in tissue from both cTnC I61Q and control mice (6.03 ± 0.06 and 6.50 ± 0.18, P=0.005) without any change in maximum force. CK-358 also significantly increased k tr , possibly suggesting activation of thin filaments. Next, we used a fluorescently labeled human cardiac troponin C (HcTnC) conjugated to N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) combined with subunits T and I to make a full HcTn complex. We performed calcium titrations with the HcTn-IANBD and saw no change in fluorescence in the control protein with and without CK-358. Finally, we performed HcTnC titrations with HcTnI and saw no change in fluorescence at maximum, sub-maximum, and minimum calcium concentrations. Overall, we demonstrate that cardiac troponin activator CK-358 did not alter calcium binding to HcTnC or its interaction with HcTnI. However, it restores the calcium sensitivity of cTnC I61Q DCM ventricular sarcomeres. Future studies will examine the effects of CK-358 on calcium binding in decorated thin filaments and measure activation using stopped flow spectroscopy in this model of DCM.