Hypertrophic cardiomyopathy mutations increase myofilament Ca2+ buffering, alter intracellular Ca2+ handling, and stimulate Ca2+-dependent signaling

Abstract

Mutations in thin filament regulatory proteins that cause hypertrophic cardiomyopathy (HCM) increase myofilament Ca²⁺ sensitivity. Mouse models exhibit increased Ca²⁺ buffering and arrhythmias, and we hypothesized that these changes are primary effects of the mutations (independent of compensatory changes) and that increased Ca²⁺ buffering and altered Ca²⁺ handling contribute to HCM pathogenesis via activation of Ca²⁺-dependent signaling. Here, we determined the primary effects of HCM mutations on intracellular Ca²⁺ handling and Ca²⁺-dependent signaling in a model system possessing Ca²⁺-handling mechanisms and contractile protein isoforms closely mirroring the human environment in the absence of potentially confounding remodeling. Using adenovirus, we expressed HCM-causing variants of human troponin-T, troponin-I, and α-tropomyosin (R92Q, R145G, and D175N, respectively) in isolated guinea pig left ventricular cardiomyocytes. After 48 h, each variant had localized to the I-band and comprised ∼50% of the total protein. HCM mutations significantly lowered the <i>K_d</i> of Ca²⁺ binding, resulting in higher Ca²⁺ buffering of mutant cardiomyocytes. We observed increased diastolic [Ca²⁺] and slowed Ca²⁺ reuptake, coupled with a significant decrease in basal sarcomere length and slowed relaxation. HCM mutant cells had higher sodium/calcium exchanger activity, sarcoplasmic reticulum Ca²⁺ load, and sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) activity driven by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of phospholamban. The ryanodine receptor (RyR) leak/load relationship was also increased, driven by CaMKII-mediated RyR phosphorylation. Altered Ca²⁺ homeostasis also increased signaling via both calcineurin/NFAT and extracellular signal-regulated kinase pathways. Altered myofilament Ca²⁺ buffering is the primary initiator of signaling cascades, indicating that directly targeting myofilament Ca²⁺ sensitivity provides an attractive therapeutic approach in HCM.