‘(De-)sensitization’ vs. ‘Uncoupling’: what drives cardiomyopathies in the thin filament? Reply

Dr Hwang's recent letter concerning our paper raises some very interesting points about the molecular mechanism of familial cardiomyopathy (HCM and DCM) due to mutations in the proteins of the thin filament and the key role of troponin. In principle, such mutations could cause defects in force generation or force transmission, but so far all the mutations in thin filament proteins that have been investigated in detail indicate abnormal force production linked to abnormal Ca2+ regulation. Troponin, in concert with tropomyosin and actin, comprises the Ca2+-dependent switch of muscle, and the properties of this switch are modulated by PKA phosphorylating troponin I (TnI); therefore, troponin abnormalities are likely to play a central role in cardiomyopathies. The case has been made that HCM-causing mutations increase myofilament Ca2+ sensitivity, and this is sufficient to trigger the symptoms of HCM. On the other hand, DCM-causing mutations uncouple the normal relationship between Ca2+ sensitivity and PKA-dependent phosphorylation of TnI, and this has been shown to blunt responses to adrenergic stimulation and predispose the heart to systolic dysfunction under stress.1,2 Our recently published work highlights the importance of troponin still more, since we have demonstrated that small molecule Ca2+ sensitizers (EMD57033 and Bepridil) can mimic the uncoupling effect of mutations, while the Ca2+ desensitizer epigallocatechin-3-gallate (EGCG) can restore the modulation of Ca2+ sensitivity by TnI phosphorylation to thin filaments containing DCM or HCM mutations that uncouple this relationship.3 All of these reagents act by binding to troponin; thus understanding how Ca2+ and phosphorylation control contractility through troponin is the key to determining how thin filament cardiomyopathies are initiated. There are severe practical problems in linking troponin structure with function, because the most important parts of the complex are intrinsically disordered and cannot be defined in terms of a static structure. These include the cardiac-specific N-terminus of cTnI (amino acids 1–30) that contains the PKA phosphorylation sites (Serines 22 and 23), the so-called switch peptide of cTnI (148–164), that plays a key role in the Ca2+-dependent switching on and off of the thin filament, and the C-terminus of troponin T (cTnT3 280–298) whose function is not known although it is likely to be located close to the regulatory region of troponin and may interact with the other two peptides. Protein nuclear magnetic resonance has been used to determine the structure of the missing peptides and their binary complexes with troponin C (TnC) in several states.4–9 When TnI is unphosphorylated, there is a weak ionic bond between the N-terminal peptide and the regulatory Ca2+-binding EF hand of TnC. When Ser 22 and 23 are phosphorylated, the binding is further weakened. We have proposed that the unphosphorylated state can also be disrupted by mutations or other alterations in any component of the thin filament or by small molecules resulting in uncoupling. However, while it can account for all our experimental observations, we do not believe that this hypothesis can be tested until the structure and dynamics of the whole troponin structure are known. This question is now being addressed by molecular dynamics simulations, but many practical and technical problems need to be solved before a believable structural and dynamic description of the regulatory elements of troponin becomes possible. In his letter to the editors, Dr Hwang presents a model based on his extensive experience of investigating troponin by protein nuclear magnetic resonance protocols6 that is proposed to be able to explain the structural basis of the results we obtained. In this model, an alternative mechanism of Ca2+-dependent activation is proposed in addition to the current consensus mechanism that accounts for Ca2+ switching of muscle and its modulation by cTnI phosphorylation (see for instance, Solaro et al.10). It is proposed that the helical segment of cTnI (39–60), downstream of the phosphorylatable peptide, that normally interacts strongly with the C-terminal domain of TnC can become dissociated. This allows a different interaction between the two domains of TnC that would have a higher Ca2+ sensitivity and not involve cTnI 1–30, therefore, it would be uncoupled. The rationale for proposing this is the observation that we measure uncoupling and the effects of EMD57033 and EGCG in the in vitro motility assay (IVMA), where the Ca2+ sensitivity of filament activation is very high (EC50 0.1–0.3 µM), whereas the Ca2+ sensitivity of contractility in skinned cardiac muscle in the phosphorylated and unphosphorylated states is ∼10 times lower. However, there seems no justification for a two-mechanism model. As described in our papers, Ca2+ regulation of thin filaments occurs normally when measured by the IVMA and all the effects of phosphorylation, mutants, Ca2+ sensitizers and desensitizers, and especially uncoupling and re-coupling on Ca2+ sensitivity were consistently observed using the IVMA technique, and in the case of the small molecules, effects were shown to be fully reversible. In these studies, Ca2+ sensitivity ranged between 0.04 and 0.2 µM so there is no reason to invoke two mechanisms based on different Ca2+ sensitivity ranges. In fact, it would be very hard indeed to explain how drugs such as EGCG could reverse uncoupling if they were acting through a mechanism separate from the one that modulates Ca2+ sensitivity by phosphorylation or mutations. The issue of absolute Ca2+ sensitivity, and even the experimental system studied, is irrelevant since we have clearly shown that exactly the same modulation of Ca2+ sensitivity of contraction by phosphorylation, drugs and mutations, and the recoupling due to EGCG can be demonstrated in myofibrils, where the Ca2+ sensitivity is in the 0.4–2 µM range.3,11 What we undoubtedly agree upon is the central role of troponin abnormalities in cardiomyopathy and the need to know much more about the structure and dynamics of the intrinsically disordered regulatory segments of the molecule. Not only is this necessary to provide a molecular mechanism for the physiological effects of TnI phosphorylation and mutations in thin filaments that cause cardiomyopathy, it is also essential to understand how small molecules can interact with this system.12 This is clinically relevant since Ca2+ sensitizers acting on TnC have been investigated as inotropic drugs, and our discovery of the ability of EGCG and similar compounds to reverse the uncoupling effect of DCM-causing mutations (and some HCM-causing mutations) points to potential therapies for familial cardiomyopathies.