Physiologic Studies of Familial Hypertrophic Cardiomyopathy-Related Mutations in Cardiac Troponin T

Abstract

Thick and thin filament mutations of the cardiac sarcomere have been linked to Familial Hypertrophic Cardiomyopathy (FHC), an autosomal dominant disease generally characterized by left ventricular hypertrophy and sudden cardiac death. Actin, tropomyosin, and the trimeric troponin complex (TnT, TnC, TnI) form a functional unit of the thin filament, chiefly operating as a Ca2+-responsive apparatus responsible for regulating the interactions with the thick filament to form force-generating cross-bridges. FHC patients demonstrate diastolic dysfunction and our transgenic murine models containing independent cTnT mutations R92L, R92Q, or Delta160E also exhibit diastolic dysfunction observed by increased Ca2+ sensitivity and impaired myocellular and whole heart relaxation. We hypothesize that cTnT mutations disrupt protein-protein interactions within the thin filament leading to impairment of beta-adrenergic responsiveness and heart rate regulation.;R92L mice demonstrate impaired mechanical properties, including impaired peak rate of relaxation and time to 90% relaxation in adult cardiac myocytes contraction transients. Measurements of Ca2+ transients in these myocytes and sarcoplasmic reticulum Ca2+-ATPase (SERCA2a)-uptake measurements of ventricular homogenates demonstrate no discernable differences from non-transgenic (Non-Tg) mice. Quantitation of Ca2+ handling proteins such as phospholamban (PLB), phosphorylated PLB, Na+/Ca 2+ exchanger, SERCA2a as well as levels of the myofilament protein, cTnI were normal. However, protein kinase A (PKA)-mediated phosphorylated TnI (P-TnI) levels were decreased at baseline and with beta-receptor agonist stimulation. Normal P-PLB levels in R92L suggest that the Ca2+ axis of the beta-adrenergic signaling cascade is intact while the myofilament axis is impaired via decreased accessibility to the PKA phosphorylation sites on cTnI. Protein quantification in Delta160E mice revealed no differences from Non-Tg, highlighting a specific impairment in R92L.;To investigate decreased physical accessibility to PKA-mediated cTnI phosphorylation sites at residues Ser-22/23, we crossed phosphomimetic cTnI S22D/S23D (D22D23) mice with our cTnT R92L mice to generate double transgenic R92L/D22D23 mice. Myocyte contraction and relaxation measurements in R92L/D22D23 demonstrated a significant recovery to Non-Tg levels. Ca2+ transient measurements in R92L/D22D23 were normal. Whole heart isovolumic studies demonstrated impaired contractility and relaxation in R92L; however, R92L/D 22D23 mice demonstrated a complete rescue to Non-Tg levels. To test whether acquisition of the D22D23 mutation was simply a gain of function mutation, we also generated the double mutant Delta160E/D 22D23 and found that contraction and relaxation in Delta160E were impaired but Delta160E/D22D23 demonstrated limited improvement. These results demonstrate that impaired accessibility by PKA to the cTnI phosphorylation sites is specific to cTnT R92L mice and the blunted beta-adrenergic responsiveness can partially be restored via a phosphomimetic genetic switch of Ser-22/23 to Asp-22/23 in cTnI.;With demonstrable impairment of the beta-adrenergic responsiveness in our cTnT mutant mice, we also investigated autonomic function via assessment of heart rate variability (HRV). Autonomic dysfunction has been suggested in FHC patients that exhibit abnormal blood pressure responses such as a paradoxical drop in pressure after exercise. Continuous 24-hr baseline electrocardiograph recordings were taken from Delta160E and R92Q, phenotypically related to R92L. While Delta160E did not differ from Non-Tg, R92Q demonstrated decreased HRV indicating an increased sympathetic drive that was assessed from a decreased standard deviation of the R-R interval, decreased low frequency, and increased high frequency. Decreased beta-adrenergic receptor density could provide a mechanism for the purported increase in sympathetic drive but density measurements were normal. Arrhythmia analysis revealed an increase in heart block and premature ventricular contractions in R92Q but not in Delta160E. These results demonstrate altered autonomic function in cTnT R92Q mice that was assessed from non-invasive HR monitoring which may provide a potential prognostic screen in patients with FHC. Collectively, these studies highlight the need to investigate independent sarcomeric protein mutations to discover a link between genotype and phenotype.