Blunted Length-Dependent Activation Caused by the Homozygous TNNT2 Mutation K280N

Abstract

Background Recently, we observed reduced myofilament length-dependent activation in cardiomyocytes from a patient harboring a homozygous mutation (K280N) in the TNNT2 gene encoding cardiac troponin T (cTnT) compared to non-failing donors. Protein kinase A (PKA)-mediated phosphorylation of cardiac troponin I (cTnI) did not rescue impaired length-dependence. Moreover, cTnI phosphorylation did not differ between TNNT2mut and donor myocardium. In this study we present direct evidence that the K280N mutation in TNNT2 blunts length-dependent activation.Methods Force measurements were performed in single permeabilized cardiomyocytes isolated from the TNNT2mut heart at various [Ca2+] and sarcomere lengths of 1.8 and 2.2 μm, with and without PKA-pretreatment. To investigate if mutant cTnT underlies impaired length-dependent activation, the endogenous mutant troponin complex was partially exchanged with recombinant whole human wild-type troponin (Tnwt) complex. TNNT2mut sample was exchanged with 0.25, 0.5 and 1 mg/mL Tnwt complex, which in accordance with previous studies from our group is predicted to yield ∼40%, ∼50% and ∼70% of troponin exchange, respectively.Results The length-dependent increase in myofilament Ca2+-sensitivity (ΔEC50) was not significantly different between TNNT2mut, TNNT2∼40%, TNNT2∼50%, TNNT2∼70%, (ΔEC50=0.35±0.16, 0.23±0.06, −0.02 ±0.02 and 0.22±0.06 μmol/L, respectively), but was significantly reduced compared to donor (ΔEC50=0.77±0.06 μmol/L). PKA-pretreatment did not restore the reduced length-dependent activation of TNNT2mut and TNNT2∼40% (ΔEC50=0.26±0.19 and 0.32±0.15), but did recover the blunted length-dependent activation of TNNT2∼50% and TNNT2∼70% (ΔEC50=0.71±0.04 and 0.79±0.14) to control values (ΔEC50=0.82±0.09).Conclusions Length-dependent activation of myofilaments is corrected to donor values only when ∼50% of mutant cTnT is replaced by wild-type troponin and subsequent phosphorylation with PKA. Our data show that the TNNT2 mutation K280N underlies impaired length-dependent activation in a dose-dependent manner.