Divergent Molecular Phenotypes in Point Mutations at the Same Residue in Beta-Myosin Heavy Chain Lead to Distinct Cardiomyopathies.

In genetic cardiomyopathies, a frequently described phenomenon is how similar mutations in one protein can lead to discrete clinical phenotypes. One example is illustrated by two mutations in beta myosin heavy chain (ß-MHC) that are linked to hypertrophic cardiomyopathy (HCM) (Ile467Val, I467V) and left ventricular non-compaction (LVNC) (Ile467Thr, I467T). To investigate how these missense mutations lead to independent diseases, we studied the molecular effects of each mutation using recombinant human ß-MHC Subfragment 1 (S1) in in vitro assays. Both HCM-I467V and LVNC-I467T S1 mutations exhibited similar mechanochemical function, including unchanged ATPase and enhanced actin velocity but had opposing effects on the super-relaxed (SRX) state of myosin. HCM-I467V S1 showed a small reduction in the SRX state, shifting myosin to a more actin-available state that may lead to the "gain-of-function" phenotype commonly described in HCM. In contrast, LVNC-I467T significantly increased the population of myosin in the ultra-slow SRX state. Interestingly, molecular dynamics simulations reveal that I467T allosterically disrupts interactions between ADP and the nucleotide-binding pocket, which may result in an increased ADP release rate. This predicted change in ADP release rate may define the enhanced actin velocity measured in LVNC-I467T, but also describe the uncoupled mechanochemical function for this mutation where the enhanced ADP release rate may be sufficient to offset the increased SRX population of myosin. These contrasting molecular effects may lead to contractile dysregulation that initiates LVNC-associated signaling pathways that progress the phenotype. Together, analysis of these mutations provides evidence that phenotypic complexity originates at the molecular level and is critical to understanding disease progression and developing therapies.

Targeting the sarcomere in inherited cardiomyopathies.

Variants in >12 genes encoding sarcomeric proteins can cause various cardiomyopathies. The two most common are hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Current therapeutics do not target the root causes of these diseases, but attempt to prevent disease progression and/or to manage symptoms. Accordingly, novel approaches are being developed to treat the cardiac muscle dysfunction directly. Challenges to developing therapeutics for these diseases include the diverse mechanisms of pathogenesis, some of which are still being debated and defined. Four small molecules that modulate the myosin motor protein in the cardiac sarcomere have shown great promise in the settings of HCM and DCM, regardless of the underlying genetic pathogenesis, and similar approaches are being developed to target other components of the sarcomere. In the setting of HCM, mavacamten and aficamten bind to the myosin motor and decrease the ATPase activity of myosin. In the setting of DCM, omecamtiv mecarbil and danicamtiv increase myosin activity in cardiac muscle (but omecamtiv mecarbil decreases myosin activity in vitro). In this Review, we discuss the therapeutic strategies to alter sarcomere contractile activity and summarize the data indicating that targeting one protein in the sarcomere can be effective in treating patients with genetic variants in other sarcomeric proteins, as well as in patients with non-sarcomere-based disease.

Chronic Calmodulin-Kinase II Activation Drives Disease Progression in Mutation-Specific Hypertrophic Cardiomyopathy.

BACKGROUND: Although the genetic causes of hypertrophic cardiomyopathy (HCM) are widely recognized, considerable lag in the development of targeted therapeutics has limited interventions to symptom palliation. This is in part attributable to an incomplete understanding of how point mutations trigger pathogenic remodeling. As a further complication, similar mutations within sarcomeric genes can result in differential disease severity, highlighting the need to understand the mechanism of progression at the molecular level. One pathway commonly linked to HCM progression is calcium homeostasis dysregulation, though how specific mutations disrupt calcium homeostasis remains unclear. METHODS: To evaluate the effects of early intervention in calcium homeostasis, we used 2 mouse models of sarcomeric HCM (cardiac troponin T R92L and R92W) with differential myocellular calcium dysregulation and disease presentation. Two modes of intervention were tested: inhibition of the autoactivated calcium-dependent kinase (calmodulin kinase II [CaMKII]) via the AC3I peptide and diltiazem, an L-type calcium channel antagonist. Two-dimensional echocardiography was used to determine cardiac function and left ventricular remodeling, and atrial remodeling was monitored via atrial mass. Sarcoplasmic reticulum Ca2+ATPase activity was measured as an index of myocellular calcium handling and coupled to its regulation via the phosphorylation status of phospholamban. RESULTS: We measured an increase in phosphorylation of CaMKII in R92W animals by 6 months of age, indicating increased autonomous activity of the kinase in these animals. Inhibition of CaMKII led to recovery of diastolic function and partially blunted atrial remodeling in R92W mice. This improved function was coupled to increased sarcoplasmic reticulum Ca2+ATPase activity in the R92W animals despite reduction of CaMKII activation, likely indicating improvement in myocellular calcium handling. In contrast, inhibition of CaMKII in R92L animals led to worsened myocellular calcium handling, remodeling, and function. Diltiazem-HCl arrested diastolic dysfunction progression in R92W animals only, with no improvement in cardiac remodeling in either genotype. CONCLUSIONS: We propose a highly specific, mutation-dependent role of activated CaMKII in HCM progression and a precise therapeutic target for clinical management of HCM in selected cohorts. Moreover, the mutation-specific response elicited with diltiazem highlights the necessity to understand mutation-dependent progression at a molecular level to precisely intervene in disease progression.

Biophysical Derangements in Genetic Cardiomyopathies.

This article focuses on three "bins" that comprise sets of biophysical derangements elicited by cardiomyopathy-associated mutations in the myofilament. Current therapies focus on symptom palliation and do not address the disease at its core. We and others have proposed that a more nuanced classification could lead to direct interventions based on early dysregulation changing the trajectory of disease progression in the preclinical cohort. Continued research is necessary to address the complexity of cardiomyopathic progression and develop efficacious therapeutics.

Atomic resolution probe for allostery in the regulatory thin filament.

Calcium binding and dissociation within the cardiac thin filament (CTF) is a fundamental regulator of normal contraction and relaxation. Although the disruption of this complex, allosterically mediated process has long been implicated in human disease, the precise atomic-level mechanisms remain opaque, greatly hampering the development of novel targeted therapies. To address this question, we used a fully atomistic CTF model to test both Ca(2+) binding strength and the energy required to remove Ca(2+) from the N-lobe binding site in WT and mutant troponin complexes that have been linked to genetic cardiomyopathies. This computational approach is combined with measurements of in vitro Ca(2+) dissociation rates in fully reconstituted WT and cardiac troponin T R92L and R92W thin filaments. These human disease mutations represent known substitutions at the same residue, reside at a significant distance from the calcium binding site in cardiac troponin C, and do not affect either the binding pocket affinity or EF-hand structure of the binding domain. Both have been shown to have significantly different effects on cardiac function in vivo. We now show that these mutations independently alter the interaction between the Ca(2+) ion and cardiac troponin I subunit. This interaction is a previously unidentified mechanism, in which mutations in one protein of a complex indirectly affect a third via structural and dynamic changes in a second to yield a pathogenic change in thin filament function that results in mutation-specific disease states. We can now provide atom-level insight that is potentially highly actionable in drug design.