Establishing the role of structural elements in cardiac muscle contraction

About This Grant

Project Summary Hereditary cardiomyopathies including dilated (DCM) and hypertrophic (HCM) forms can lead to increased mortality and morbidity. A new class of drugs called myotropes has been developed to target thick filament dysfunction in the heart, and one has recently been FDA-approved for a specific cardiomyopathy. This small molecule drug, however, does not possess the same efficacy for cardiomyopathies that arise from pathogenic variants that disrupt cardiac thin filament (cTF) function. Based upon this strong precedence, we seek to identify cTF structural elements that modulate cardiac performance that are within “cardiomyopathy mutation hotspots” and may serve as new actionable targets for the treatment of cardiac diseases. This proposal addresses key understudied functional regions of cardiac troponin C (cTnC), the Ca2+ binding subunit of the troponin complex, which regulates Ca2+ dependent myofilament activation. These regions include the N-helix of the N-domain (which is only present in vertebrate cardiac and skeletal TnC and absent in all other members of the EF-hand Ca2+ binding protein family) and the C-domain that contains high affinity Ca2+ binding sites. Aim 1 will utilize newly developed knock-in (KI) cTnC -I4M (DCM) and -ΔN (rationally engineered N-helix deletion) mice to evaluate the specific hypothesis that the N-helix of cTnC promotes the transition from the Ca2+-free to Ca2+-bound partially activated and Ca2+-bound fully activated state during cardiac muscle activation. Relatedly in Aim 2, newly developed KI cTnC -D105A and -D141A (rationally engineered to abolish Ca2+ binding to sites 3 and 4, respectively) mice and -D145E reconstituted assays will test the hypothesis that Ca2+ ions bound to the high affinity sites of TnC C-domain stabilize its structure and, therefore, contribute to the allosteric coupling that exists between the two TnC domains. Additionally, this Aim will investigate the physiological role of these cTnC Ca2+ binding sites in myofilament Ca2+ buffering function. Preliminary data with cTnC -I4M and -ΔN mice revealed a reduction in heart contractility and impaired myofilament activation, suggesting that the N-helix acts as a regulator that augments cardiac contraction by slowing down crossbridge kinetics. Echocardiographic analysis of cTnC - D105A and -D141A mice also showed reduced cardiac contractility and pathological remodeling. Embryonic studies will be performed for mouse models that homozygous pups cannot be obtained. These studies will be augmented with reconstituted assays containing the HCM cTnC-D145E protein, which was shown to virtually abolish Ca2+ binding to the high affinity sites. Preliminary studies utilizing Cryo-Electron Microscopy (cryo-EM) show that the cTnC N-helix and the C-domain are directly involved in the activation pathway of native cTF. We will leverage multiple physiology-based and state-of-the-art structural biology techniques, such as cryo-EM and small angle X-ray diffraction. We have improved our cryo-EM capabilities to achieve the highest resolution ever (3.8Å) for cardiac troponin within the native cTF. These studies will allow us to uncover new mechanisms governing contractile regulation in the heart and identify novel specific targets to modulate cardiac contractility.