De Novo Missense Mutations in TNNC1 and TNNI3 Causing Severe Infantile Cardiomyopathy Affect Myofilament Structure and Function and Are Modulated by Troponin Targeting Agents

Abstract

Rare pediatric non-compaction and restrictive cardiomyopathy are usually associated with a rapid and severe disease progression. While the non-compaction phenotype is characterized by structural defects and is correlated with systolic dysfunction, the restrictive phenotype exhibits diastolic dysfunction. The molecular mechanisms are poorly understood. Target genes encode among others, the cardiac troponin subunits forming the main regulatory protein complex of the thin filament for muscle contraction. Here, we compare the molecular effects of two infantile de novo point mutations in TNNC1 (p.cTnC-G34S) and TNNI3 (p.cTnI-D127Y) leading to severe non-compaction and restrictive phenotypes, respectively. We used skinned cardiomyocytes, skinned fibers, and reconstituted thin filaments to measure the impact of the mutations on contractile function. We investigated the interaction of these troponin variants with actin and their inter-subunit interactions, as well as the structural integrity of reconstituted thin filaments. Both mutations exhibited similar functional and structural impairments, though the patients developed different phenotypes. Furthermore, the protein quality control system was affected, as shown for TnC-G34S using patient’s myocardial tissue samples. The two troponin targeting agents levosimendan and green tea extract (-)-epigallocatechin-3-gallate (EGCg) stabilized the structural integrity of reconstituted thin filaments and ameliorated contractile function in vitro in some, but not all, aspects to a similar degree for both mutations.

Cardiomyopathies in Children: Genetics, Pathomechanisms and Therapeutic Strategies

“…Mutations in MYBPC3 or in genes of interacting proteins might impair the interplay between these proteins and thereby the regulation of contraction. Hereby, an impaired interaction of different proteins with cardiac troponin might also play a role, though the role of the MyBPC-cardiac troponin interaction as well as of its interaction with titin remains to be elucidated [104,114]. Two fascinating studies revealed that MyBPC regulates sarcomeric contractile oscillations, which might be based on its interplay with all sarcomeric partners [115,116].…”

“…They showed that different mutations incorporate differently into the actin filament and destabilize the filaments. A destabilization of actin filaments has also been described by Hassoun et al [114], and pediatric RCM mutations in TNNI3 have been demonstrated to largely affect the integrity of reconstituted thin filament structure [104].…”

Cardiomyocyte Dysfunction in Inherited Cardiomyopathies

“…These variants were shown to either increase the stability of cNTnC open conformation, thereby enhancing Ca 2+ -binding affinity, or to directly increase the binding with cTnI [ 122 , 123 , 124 , 125 ]. In a recent study by us, we provided evidence on structural and functional alterations in thin filaments containing the novel cTnC G34S variant [ 126 ]. We found the TnC G34S mutation to increase Ca 2+ -binding affinity, decrease the binding affinity towards cTnT, and to cause structural impairments in the reconstituted thin filaments.…”

“…It has been also reported that HCM mutations, such as K206Q and R145G, uncouple the effect of TnI phosphorylation by PKA [ 144 ]. Furthermore, we showed the D127Y variant to increase the Ca 2+ sensitivity of reconstituted thin filaments, induce structural perturbations, and cause RCM phenotype [ 126 ]. In contrast to HCM and RCM mutations, DCM mutations in TNNI3 are less frequent.…”

“…Furthermore, EGCg improved the diastolic function in an HCM mouse model [ 195 ]. In a recent study by us, we found levosimendan and EGCg to interact directly with actin and to improve the structural integrity of thin filaments reconstituted with troponin variants [ 126 ].…”