In Vivo Analysis of Troponin C Knock-In (A8V) Mice: Evidence that TNNC1 is a Hypertrophic Cardiomyopathy Susceptibility Gene

Abstract

Recently, the TNNC1 gene that encodes cardiac troponin C (cTnC) was found as a target for many hypertrophic cardiomyopathy (HCM) mutations in humans, eliciting alterations in the Ca2+ binding properties of the N-domain of cTnC. We genetically engineered knock-in mice containing the HCM-associated A8V mutation in cTnC (heterozygote = KI-TnC-A8V+/-; homozygote = KI-TnC-A8V+/+) in order to characterize its in vivo, molecular and cellular effects. ECHO revealed that at 3 months old (mo) KI-TnC-A8V+/+ mice display increased IVRT and E/A ∼1 compared to WT, suggesting diastolic dysfunction; whereas KI-TnC-A8V+/- showed signs of cardiac restriction at 14 mo. Histopathology of both genotyped hearts revealed papillary muscle hypertrophy, interstitial fibrosis, and myofibrilar disarray. Real-time PCR analysis at 3 mo demonstrated increases in BNP, α-MHC and β-MHC mRNA levels in the right ventricles of the KI-TnC-A8V mice (only ANP increased in the left ventricle). We identified in intact KI-TnC-A8V+/- and KI-TnC-A8V+/+ cardiomyocytes: a significant decrease in the sarcomere length at several stimulation frequencies; prolonged Ca2+ and contractile transient kinetics at 4Hz; uncoupling between Ca2+ decay (delayed) and contractile (no change) transients at 6Hz; suggesting a mechanical frequency-dependent uncoupling from the Ca2+ transient. Furthermore, a decrease in the baseline Ca2+ fluorescence and in Ca2+ peak percentage was also detected in KI-TnC-A8V+/- and KI-TnC-A8V+/+, indicating increased myofilament Ca2+ buffering. The calcium sensitivity of contraction in skinned fibers increased in a gene dose fashion: KI-TnC-A8V+/+ > KI-TnC-A8V+/- > WT. The rate of relaxation in KI-TnC-A8V+/+ cardiac skinned fibers investigated by flash photolysis was found increased, compared to WT. These results suggest that the A8V mutation in cTnC increases Ca2+ binding affinity to its N-domain eliciting changes in intracellular Ca2+ homeostasis and cellular mechanical function, ultimately leading to diastolic dysfunction.