Familial Dilated Cardiomyopathy Associated With a Novel Combination of Compound Heterozygous TNNC1 Variants

Abstract

Familial dilated cardiomyopathy (DCM), clinically characterized by enlargement and dysfunction of one or both ventricles of the heart, can be caused by variants in sarcomeric genes including TNNC1 (encoding cardiac troponin C, cTnC). Here, we report the case of two siblings with severe, early onset DCM who were found to have compound heterozygous variants in TNNC1: p.Asp145Glu (D145E) and p.Asp132Asn (D132N), which were inherited from the parents. We began our investigation with CRISPR/Cas9 knockout of TNNC1 in Xenopus tropicalis, which resulted in a cardiac phenotype in tadpoles consistent with DCM. Despite multiple maneuvers, we were unable to rescue the tadpole hearts with either human cTnC wild-type or patient variants to investigate the cardiomyopathy phenotype in vivo. We therefore utilized porcine permeabilized cardiac muscle preparations (CMPs) reconstituted with either wild-type or patient variant forms of cTnC to examine effects of the patient variants on contractile function. Incorporation of 50% WT/50% D145E into CMPs increased Ca 2+ sensitivity of isometric force, consistent with prior studies. In contrast, incorporation of 50% WT/50% D132N, which had not been previously reported, decreased Ca 2+ sensitivity of isometric force. CMPs reconstituted 50-50% with both variants mirrored WT in regard to myofilament Ca 2+ responsiveness. Sinusoidal stiffness (SS) (0.2% peak-to-peak) and the kinetics of tension redevelopment (k TR ) at saturating Ca 2+ were similar to WT for all preparations. Modeling of Ca 2+ -dependence of k TR support the observation from Ca 2+ responsiveness of steady-state isometric force, that the effects on each mutant (50% WT/50% mutant) were greater than the combination of the two mutants (50% D132N/50% D145E). Further studies are needed to ascertain the mechanism(s) of these variants.

Eliminating the First Inactive State and Stabilizing the Active State of the Cardiac Regulatory System Alters Behavior in Solution and in Ordered Systems

“…Steady-state isometric force data were analyzed and fit using a two-or three-parameter sigmoidal Hill equation as previously described. 19,36,37 Rate of Tension Redevelopment (k TR ): Briefly, after force levels reached a steady state at different Ca 2+ activation levels, measurements of the rate of tension redevelopment were obtained by shortening a CMP by 20% of its original length (L 0 ), followed by a rapid 25% restretch, and shortened back to L 0 . 36−38 The CMP lengths were controlled using a high-speed servomotor model 322C from Aurora Scientific.…”

“…All experiments were performed at room temperature (21 °C). Steady-state isometric force data were analyzed and fit using a two- or three-parameter sigmoidal Hill equation as previously described. ,, …”

“…The SS data were analyzed using R Studio Fast Fourier transform, and the values were reported in units of megapascals. The data were fit using a four-parameter sigmoidal Hill equation as previously described. ,, Statistical analyses between groups was done with the unpaired Student’s t -test with SigmaPlot v12 (Systat Software, Inc.).…”

Cardiomyocyte Dysfunction in Inherited Cardiomyopathies

“…The double compound heterozygous DCM variant (E59D/D75Y) has also been shown to decrease myofilament Ca 2+ sensitivity and reduce strong actin–myosin binding [ 135 , 136 ]. A recent study by Landim-Vieira et al, 2019 on the missense compound heterozygous DCM-causing variants (D132N and D145E) demonstrated no significant changes in contractile parameters, indicating to the potential involvement of other pathological mechanisms beyond myofilament dysfunction [ 137 ].…”

How to increase precision in Xenopus gene editing and base editing experiments

“…The emphasis should be on accurate quantification of defective phenotypes as they arise in the developing embryo and study of genes whose human orthologs are implicated in congenital disease. X. tropicalis knock-outs have already been used as part of large, multi-species studies that confirm disease causality for candidate genes identified through patient genome sequencing [13,88,89]. The real challenge is to move beyond functional-nulls and to replicate the actual missense mutations found in patients, which are likely to encode far subtler hypomorphic changes to protein activity.…”