Hypertrophic cardiomyopathy mutations increase myofilament Ca buffering, alter intracellular Ca handling, and stimulate Ca-dependent signaling.

Mutations in thin filament regulatory proteins that cause hypertrophic cardiomyopathy (HCM) increase myofilament Ca sensitivity. Mouse models exhibit increased Ca buffering and arrhythmias, and we hypothesized that these changes are primary effects of the mutations (independent of compensatory changes) and that increased Ca buffering and altered Ca handling contribute to HCM pathogenesis via activation of Ca-dependent signaling. Here, we determined the primary effects of HCM mutations on intracellular Ca handling and Ca-dependent signaling in a model system possessing Ca-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 of Ca binding, resulting in higher Ca buffering of mutant cardiomyocytes. We observed increased diastolic [Ca] and slowed Ca reuptake, coupled with a significant decrease in basal sarcomere length and slowed relaxation. HCM mutant cells had higher sodium/calcium exchanger activity, sarcoplasmic reticulum Ca load, and sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) activity driven by Ca/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 Ca homeostasis also increased signaling via both calcineurin/NFAT and extracellular signal-regulated kinase pathways. Altered myofilament Ca buffering is the primary initiator of signaling cascades, indicating that directly targeting myofilament Ca sensitivity provides an attractive therapeutic approach in HCM.