Low expression of the K280N TNNT2 mutation is sufficient to increase basal myofilament activation in human hypertrophy cardiomyopathy

Vasco Sequeira; Lili Wang; Paul J M Wijnker; Kyungsoo Kim; Jose R Pinto; Cris Dos Remedios; Charles Redwood; Bjorn C Knollmann; Jolanda van der Velden

doi:10.1016/j.jmccpl.2022.100007

Low expression of the K280N TNNT2 mutation is sufficient to increase basal myofilament activation in human hypertrophy cardiomyopathy

J Mol Cell Cardiol Plus. 2022 Sep:1:100007. doi: 10.1016/j.jmccpl.2022.100007.

Authors

Vasco Sequeira^{1

2

3

4}, Lili Wang³, Paul J M Wijnker^{1

2}, Kyungsoo Kim³, Jose R Pinto⁵, Cris Dos Remedios⁶, Charles Redwood⁷, Bjorn C Knollmann³, Jolanda van der Velden^{1

8

9}

Affiliations

¹ Amsterdam UMC, Vrije Universiteit Amsterdam, Physiology, Amsterdam Cardiovascular Sciences, Amsterdam, the Netherlands.
² Netherlands Heart Institute, Utrecht, the Netherlands.
³ Division of Clinical Pharmacology, Vanderbilt School of Medicine, Nashville, United States.
⁴ Comprehensive Heart Failure Center (CHFC) University Clinic Würzburg, Würzburg, Germany.
⁵ Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL, USA.
⁶ Muscle Research Unit, Discipline of Anatomy & Histology, Bosch Institute, The University of Sydney, Sydney, Australia.
⁷ Radcliffe Department of Medicine, University of Oxford, UK.
⁸ Amsterdam UMC location Vrije Universiteit Amsterdam, Physiology, De Boelelaan 1117, Amsterdam, the Netherlands.
⁹ Amsterdam Cardiovascular Sciences, Heart Failure and Arrhythmias, Amsterdam, the Netherlands.

Abstract

Background: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder with patients typically showing heterozygous inheritance of a pathogenic variant in a gene encoding a contractile protein. Here, we study the contractile effects of a rare homozygous mutation using explanted tissue and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to gain insight into how the balance between mutant and WT protein expression affects cardiomyocyte function.

Methods: Force measurements were performed in cardiomyocytes isolated from a HCM patient carrying a homozygous troponin T mutation (cTnT-K280N) and healthy donors. To discriminate between mutation-mediated and phosphorylation-related effects on Ca²⁺-sensitivity, cardiomyocytes were treated with alkaline phosphatase (AP) or protein kinase A (PKA). Troponin exchange experiments characterized the relation between mutant levels and myofilament function. To define mutation-mediated effects on Ca²⁺-dynamics we used CRISPR/Cas9 to generate hiPSC-CMs harbouring heterozygous and homozygous TnT-K280N mutations. Ca²⁺-transient and cell shortening experiments compared these lines against isogenic controls.

Results: Myofilament Ca²⁺-sensitivity was higher in homozygous cTnT-K280N cardiomyocytes and was not corrected by AP- and PKA-treatment. In cTnT-K280N cells exchanged with cTnT-WT, a low level (14%) of cTnT-K280N mutation elevated Ca²⁺-sensitivity. Similarly, exchange of donor cells with 45 ± 2% cTnT-K280N increased Ca²⁺-sensitivity and was not corrected by PKA. cTnT-K280N hiPSC-CMs show elevated diastolic Ca²⁺ and increases in cell shortening. Impaired cardiomyocyte relaxation was only evident in homozygous cTnT-K280N hiPSC-CMs.

Conclusions: The cTnT-K280N mutation increases myofilament Ca²⁺-sensitivity, elevates diastolic Ca²⁺, enhances contractility and impairs cellular relaxation. A low level (14%) of the cTnT-K280N sensitizes myofilaments to Ca²⁺, a universal finding of human HCM.

Keywords: Hypertrophic cardiomyopathy; Myofilament Ca2+-sensitivity; Protein toxic threshold; Troponin T; hiPSC-CM.

Grants and funding

R01 HL128683/HL/NHLBI NIH HHS/United States