The HCM-associated cardiac Troponin T mutation K280N increases the energetic cost of tension generation in human cardiac myofibrils

A novel homozygous mutation in the TNNT2 gene encoding cardiac troponin T (cTnT K280N) was identified in one HCM patient undergoing cardiac transplantation. mRNA and Mass Spectrometry analyses revealed expression of the mutant alleles without evidence of haploinsufficiency. Kinetics of contraction and relaxation of myofibrils from a frozen left ventricular sample of the K280N HCM patient were compared to those of ‘‘control’’ myofibrils (from donor hearts, from aortic stenosis patients, and from HCM patients negative for sarcomeric protein mutations). Preparations, mounted in a force recording apparatus (15 C), were maximally Ca2þ -activated (pCa 4.5) and fully relaxed (pCa 9) by rapid (<10 ms) solution switching. The rate constant of active tension generation following maximal Ca2þ activation (kACT) was markedly faster in K280N myofibrils (ca. 1.7 s 1) compared to controls (0.7-1 s 1). The rate constant of isometric relaxation (slow kREL) was 2-3-fold faster in K280N myofibrils than in controls, evidence that the apparent rate with which cross-bridges leave the force generating states is accelerated in the mutant preparations. The results suggest that the energy cost of tension generation is increased in the K280N sarcomeres. Simultaneous measurements of maximal isometric ATPase and Ca2þ -activated force in Triton-permeabilized left ventricular muscle strips from the K280N sample demonstrated that tension cost was much higher in the K280N than in control myocardium. Replacement of the mutant protein by exchange with wild-type recombinant human Tn in the K280N myofibrils reduced both kACT and slow kREL close to control values. This indicates that the HCM-associated TNNT2 mutation K280N primarily alters apparent cross-bridge kinetics and impairs sarcomere energetics. Supported by the 7th Framework Program of the European Union, ‘‘BIG-HEART’’ grant agreement 241577.