The human subjects research in this study was approved by the Institutional Review Board of Yale University School of Medicine. Xenopus were maintained and cared for in an aquatics facility in accordance with Yale University Institutional Animal Care and Use Committee protocols.

Genomic DNA was isolated from either venous blood or saliva samples. Whole exome sequencing was performed by using IDT xGen capture kit followed by Illumina DNA sequencing (HiSeq 4000). Paired end sequence reads were converted to FASTQ format and were aligned to the reference human genome (hg19). SNVs and indels were called using a GATK pipeline and annotated using AnnoVar.

CRISPR/Cas9-mediated genome editing in Xenopus tropicalis tadpoles was used as previously described (Bhattacharya et al., 2015). Briefly, two non-overlapping, independent CRISPR sgRNAs targeting tnnc1 were designed to generate knockdowns.

CRISPR 1 oligo (targets exon 4):

and CRISPR 2 oligo (targets exon 5):

The sgRNAs were synthesized using the EnGen sgRNA synthesis kit (NEB), and underwent subsequent purification and concentration using the RNA Clean & Concentrator-5 kit (Zymo). Individual sgRNAs were injected at 400 pg/embryo with 1.6 ng of Cas9 protein into single celled embryos according to standard methods (Ran et al., 2013). Uninjected control and CRISPR tadpoles were raised in 10 cm dishes until stage 42 to stage 45 of development. To visualize beating hearts, these tadpoles were embedded in low melt agarose in 1/9 X MR and images were obtained using a Thorlabs Telesto 1325 nm spectral domain optical coherence tomography system as previously described (Deniz et al., 2018).

Wild-type (WT) and mutant (either D132N or D145E) human cTnC proteins were cloned, expressed, and purified as previously described (Landstrom et al., 2008; Dweck et al., 2010). Site-directed mutagenesis by PCR was used to engineer point mutations into the pET3-d vector. Sequences were verified prior to expression and purification of the cTnC mutants.

Different Ca²⁺ solutions ranging from pCa 8.0 (relaxing) to 4.0 (activating) were calculated utilizing a pCa Calculator (Dweck et al., 2005). All solutions contained: 20 mM 3-[N-morpholino]propanesulfonic acid (MOPS), 7 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 15 mM phosphocreatine, 15 units mL^–1 creatine phosphokinase, 2.5 mM MgATP^2–, 1 mM free Mg²⁺, ionic strength maintained constant at 150 mM by adding Kpropionate (KPr), varying [Ca²⁺], pH 7.0. Anion of choice for the pCa solutions was propionate (e.g., CaPr₂, MgPr₂, and KPr). Solutions were prepared at room temperature (20–21°C).

Left ventricular papillary muscles were harvested from porcine hearts obtained from a local abattoir and dissected into small muscle bundles (Landstrom et al., 2008). Dissected cardiac tissue was exposed to pCa 8.0 relaxation solution (10^–8 M free [Ca²⁺]) containing non-ionic detergent Triton X-100 (1% v:v). After incubating for 4 h at 4°C, skinned cardiac muscle preparations (CMPs) were transferred to 51% glycerol-relaxing pCa 8.0 solution (v:v), stored at −20°C, and utilized within 1 month. Strips of muscle, 0.8–1.0 mm in length and 0.15–0.25 mm in diameter, were mounted using aluminum foil T-clips to a force transducer on one end and a motor on the other end. CMPs were immersed in pCa 8.0 relaxation solution and the initial length (L₀) was set at 2.1 μm sarcomere length measured by HeNe laser diffraction. Initial maximal Ca²⁺-activated tension was measured at pCa 4.0 (10^–4 M free [Ca²⁺]).

Native (endogenous) cTnC was extracted by incubating CMPs in 5 mM CDTA solution (pH 8.4) for ∼1.5 h at 21°C as previously described (Landstrom et al., 2008). To evaluate the efficacy of cTnC extraction, residual Ca²⁺-activated tension was measured by immersing the CMPs in pCa 4. CMPs exhibiting residual force >40% (n = 2) were excluded from analysis. CMPs with residual force ranging from 8.4 to 39.4% were reconstituted by incubation for ∼5 min total (five incubations of ∼1 min each) at 21°C with one of four conditions: drops of pCa 8.0 solution containing 1.0 mg mL^–1 of either 100% WT cTnC, 50% WT/50% D132N cTnC mix, 50% WT/50% D145E cTnC mix or 50% D132N/50% D145E cTnC mix. Tension recovery was evaluated by comparing the reconstituted maximal tension at pCa 4.0 to the initial maximal Ca²⁺-activated tension measured prior to cTnC extraction (P/P₀) and ranged from 81% to 123%.

pCa-Force: Ca²⁺-dependence of steady-state, isometric force was measured by incubating reconstituted CMPs in a series of Ca²⁺ solutions ranging from pCa 8.0 to 4.0 at 21°C. Normalized [Ca²⁺]-force data for each CMP were fit using non-linear least squares regression to a 2-parameter Hill equation to obtain parameter estimates for pCa₅₀ and n_Hill, as described (Veltri et al., 2017a).

Kinetics of Tension Redevelopment (k_TR): After force reached steady-state in each pCa solution, measurement of the rate of tension redevelopment was obtained by shortening CMPs by 20% L₀, followed by rapid, 25% re-stretch, then release back to L₀ (Gonzalez-Martinez et al., 2018). The apparent rate constant (k_TR) was obtained from each tension recovery time course as described previously (Chase et al., 1994; Regnier et al., 1999). Conditions where steady-state, isometric force was below 15% of the maximal force were excluded from the k_TR analysis because of the relatively low signal-to-noise at the lowest levels of isometric force. Estimations of the 3-state model parameters (f, g, and k_OFF) were computed in MatLab as previously described (Gonzalez-Martinez et al., 2018) with the exceptions that force for each condition was assumed to be 1.0, and also because there are no direct measurements of Ca²⁺ binding to or dissocation from the mutant cTnCs, the value for k_ON for all simulations was assumed to be that derived from measurements on WT, 1.84 × 10⁸ (Pinto et al., 2011a). In addition to estimates for parameters f, g, and k_OFF, the modeling also provides an estimate for pCa₅₀; the modified least-squares criterion is weighted such that the best fit, predicted pCa₅₀ is essentially the same (well within experimental error) as that measured experimentally for the same condition.

Statistical analyses including non-linear regression were performed using SigmaPlot v.12.0. Data were tested for significant statistical differences using one-way ANOVA with post hoc Student-Newman–Keuls test. Data are shown as mean ± S.E.

The proband was a female delivered full term following an uncomplicated pregnancy that included two normal fetal echocardiograms. She had initially been well without symptoms referable to the cardiovascular system. She demonstrated no feeding intolerance, premature fatigue, irritability, pallor, cyanosis, increased work of breathing, tachypnea, excessive diaphoresis, or lethargy. She had a good appetite and breast fed without difficulty. Screening echocardiography was done at 18 days of age due to an older sibling with neonatal cardiomyopathy of presumed idiopathic etiology. This showed borderline enlargement of the left ventricle with a left ventricular end diastolic diameter (LVEDD) of 2.18 cm (Z score of 0.8) and a low decreased M-mode left ventricular ejection fraction (LVEF) of 48%. Given the family history, she was admitted to the hospital briefly with a diagnosis of DCM for initiation of medical therapy with captopril, furosemide and aspirin. She was followed closely as an outpatient, with electrocardiogram having signs of left ventricular enlargement and serial echocardiograms demonstrating gradually worsening left ventricular function, necessitating a steady escalation of her medical management (Figure 1). Notably, right ventricular size and function remained normal throughout. By 7 months of age both dilation and function of her left ventricle had worsened with echocardiography showing LVEDD 3.40 cm (Z score of 4.09) and M-mode LVEF of 32%. At 13 months of age, echocardiography showed LVEDD 4.50 cm (Z score of 8.55) and M-mode LVEF of 33%. She was being managed with enalapril, carvedilol, digoxin, furosemide and aspirin, and had been listed for cardiac transplantation due to her severely depressed heart function. She suffered a cardiac arrest while awaiting a suitable donor and expired at 14 months of age.

Family history was notable for an 11 year-old brother who presented in the neonatal period with poor feeding and growth, and was diagnosed with severe DCM. He was managed initially with medications then received a cardiac transplantation at 13 months of age. He has done well since, with good function of his transplanted heart, and without any signs of skeletal muscle problems. Both parents, currently in their early 40′s, have had normal echocardiograms and do not have any signs of cardiovascular disease. There is no other family history of cardiomyopathy, congenital heart defects or sudden unexpected cardiac death.

Due to the strong family history, the proband underwent genetic testing with whole exome sequencing, which identified two variants of uncertain significance, D132N (c.394G>A, p.Asp132Asn) and D145E (c.435C>A, p.Asp145Glu), in the TNNC1 gene (NM_003280.2). Subsequent analysis of the family (proband, brother and both parents) revealed that these variants were inherited in trans (Figures 2A,B). Both variants are located in the region of divalent cation binding site IV in the C-domain of cTnC (Figure 3). Both of these aspartate residues, as well as the surrounding amino acids, are highly conserved among vertebrates (Figure 3D). In the Genome Aggregation Database (gnomAD), D145E has a frequency of 1.28 × 10^–4, whereas D132N has not been previously reported in a healthy population. These were also noted to be the only two rare (<0.5% minor allele frequency in the general population) single gene variants across the entire exome that were shared by both affected siblings, and thus no other rare or pathogenic variants were found in other known cardiomyopathy genes in either of the siblings. The proband was sequenced to a mean depth of 102 independent reads per targeted base and her affected brother and their unaffected parents were sequenced to a mean depth of 44–49X. Greater than 10X coverage was achieved for 98% of the proband’s exome and 94% for the rest of family members’ exomes. Greater than 20X coverage was achieved for 96% of the proband’s exome and 88% for her other family members.

To assess the consequences of altered cTnC in an in vivo system, we began by using CRISPR/Cas9-mediated gene editing to knockout TNNC1 in Xenopus. Loss of the TNNC1 gene did not prevent the early stages of development, and resulted in a dramatic cardiac phenotype in tadpoles consistent with DCM (Figure 4 and Supplementary Video S1). Notably, the knockout tadpoles demonstrated ventricular dilation, wall thinning and almost imperceptible cardiac motion. With the ultimate goal of introducing the two mutations separately and together, we then attempted to rescue this cardiomyopathy phenotype by expression of human cTnC in depleted frog embryos. Unpredictably, we were unable to obtain expression of cTnC in the tadpole hearts despite employing various techniques injecting either TNNC1 mRNA or TNNC1 plasmid DNA under control of a CMV promoter (Table 1).

It was previously shown that cTnC-depleted CMPs reconstituted with cTnC-D145E increased Ca²⁺ sensitivity of steady-state isometric force generation (Landstrom et al., 2008; Pinto et al., 2011a; Veltri et al., 2017a). However, to our knowledge, the effects of cTnC-D132N or mixtures of these cTnCs have not been previously reported. Considering that both siblings were found to be compound heterozygous for D145E and D132N in cTnC, we sought to explore their potential functional significance in vitro.

Native, endogenous cTnC was extracted from porcine CMPs using CDTA incubations and reconstituted with recombinant expressed human cTnC-WT (control) or mixtures of cTnCs (section “Experimental Procedures”). The range of average of residual tension post-extraction was 18% ∼ 35%, with individual values ranging from 8.4 to 39.4%, indicating that the majority of native cTnC had been extracted from the myofilaments (Table 2, where force values are reported as specific force). Upon reconstitution, the average tension recovery was at least 87%, indicating that the majority of troponin molecules had been functionally reconstituted with exogenous cTnC and that both WT and mutant recombinant cTnCs were competent for Ca²⁺-activation of contraction.

CMPs reconstituted with a mixture of 50% WT and 50% D132N variant (WT/D132N) displayed significantly reduced myofilament Ca²⁺ sensitivity of force generation (pCa₅₀ = 5.32 ± 0.03), with a rightward shift of 0.11 pCa units compared with CMPs reconstituted with 100% WT cTnC (pCa₅₀ = 5.43 ± 0.02) (Table 2 and Figures 5A,B). Consistent with previous reports that compared 100% D145E with 100% WT (Landstrom et al., 2008; Pinto et al., 2009; Veltri et al., 2017a), we found that reconstitution with a mixture of 50% WT and 50% D145E (WT/D145E) significantly increased myofilament Ca²⁺ sensitivity of tension (pCa₅₀ = 5.58 ± 0.04), with a leftward shift of 0.15 pCa units (Table 2 and Figures 5A,B). Interestingly, we observed no significant difference in myofilament Ca²⁺ sensitivity of tension upon reconstitution of CMPs with a mixture of 50% D132N and 50% D145E (D132N/D145E) (Table 2 and Figures 5A,B). Although the D132N variant in 50%:50% combination with either WT or D145E tended to reduce maximal tension recovery, the differences were not statistically significant (Table 2 and Figure 5B). The Hill coefficient (n_Hill) is an indicator of cooperativity of thin filament activation. Compared to WT (n_Hill = 1.70 ± 0.20), only the WT/D132N mixture exhibited a significant change – an increase – in the apparent cooperativity (n_Hill = 2.60 ± 0.45) (Table 2).

Because we observed no significant difference in myofilament Ca²⁺ sensitivity of tension for the 50%:50% combination of the two variant cTnCs (D132N/D145E) compared with 100% WT (Table 2 and Figures 5A,B), we investigated two additional indices of contractile function: SS and kinetics of tension redevelopment (k_TR). SS is used as a metric of the overall number of cross-bridges, whereas k_TR is used a metric of the kinetics of cross-bridge cycling at maximal Ca²⁺-activation and also informs about thin filament regulatory unit dynamics at submaximal Ca²⁺-activation. We found no significant difference in maximum SS values when comparing CMPs reconstituted with the variants (WT/D132N; WT/D145E; D132N/D145E) to those reconstituted with 100% WT (Table 3 and Figures 6A,B), suggesting that force per cross-bridge was unchanged. In addition, there was little or no difference in any of the relationships between SS and isometric force (Figure 6B).

Maximum k_TR values were not significantly different when comparing CMPs reconstituted with the variants (WT/D132N; WT/D145E; D132N/D145E) to those reconstituted with 100% WT (Table 3 and Figures 7A,B). This result informs us that, according to Brenner (1988), the sum of the sum of the rates of cross-bridge attachment and detachment (f+g) is approximately constant for all four conditions. To explore the possibility that the variants may have altered cross-bridge attachment (f) and detachment (g) rates even though the sum remained constant, we used a 3-state model of muscle regulation to evaluate the Ca²⁺-dependence of k_TR (Landesberg and Sideman, 1994; Hancock et al., 1997; Loong et al., 2013; Gonzalez-Martinez et al., 2018). The force-k_TR data for CMPs containing the mutants (WT/D132N, WT/D145E or D132N/D145E) appear to be less curvilinear than for WT (Figure 7B). The fits of the 3-state model capture the curvature of the data from CMPs containing mutants, but does not fully capture the greater curvature of the WT data (Figure 7B). Best fit values for f, g, and k_OFF corresponding to the lines in Figure 7B are reported in Table 4. The 3-state model’s four parameters (f, g, k_ON, and k_OFF) are all lumped parameters that primarily inform about cross-bridge cycling (f and g) and thin filament regulatory unit dynamics including Ca²⁺ binding to and dissociation from cTnC (k_ON and k_OFF). Computational modeling indicates that k_OFF is, as expected, generally faster as Ca²⁺ sensitivity decreases (Table 4), although this relationship is modulated by additional factors because k_OFF reflects more than just Ca²⁺ dissociation from cTnC. Furthermore, the modeling indicates that f was slower than WT and g was faster for both WT/D132N and WT/D145E (Table 4). For the two mutants together (D132N/D145E), f and g changed in the same directions as for the individual variants (WT/D132N and WT/D145E), but the magnitudes of the changes were less; in other words, the combination of the two variants appeared to partially ameliorate the effects of the individual variants, as was also found for Ca²⁺ sensitivity of steady state isometric force (Table 2 and Figures 5A,B).

Although multiple in silico predictors of variant effects – including SIFT, PolyPhen2 and CADD – predict that each of these variants is likely to be detrimental, they are both classified as variants of uncertain significance in ClinVar. The D132N variant has not been reported in the Genome Aggregation Database (gnomAD). The population frequency of the D145E variant is reported as 0.0001228 in gnomAD, and the Atlas of Cardiac Genetic Variations classifies this uncommon variant as “unlikely to be pathogenic.” This classification of D145E is consistent with individuals such as the proband’s mother, who is heterozygous and asymptomatic. The available evidence, therefore, suggests that D145E could possibly be a risk factor for cardiomyopathy in some cases, but does not appear to be disease-causing when present in the heterozygous state. The two affected siblings presented here, in contrast to the parents, have D145E in trans with the D132N variant. When viewed in this light, the D145E population frequency is well within an expected range for a recessive disease.

To assess the combined effects of two potentially dysfunctional TNNC1 alleles, we performed knockout experiments in Xenopus, revealing a dramatic DCM phenotype in tadpoles (Figure 4 and Supplementary Video S1) that is consistent with a previous report in zebrafish (Ho et al., 2009). Our multiple attempts to rescue this phenotype with human cTnC protein expression (Table 1) were unsuccessful, however, as the protein could be found expressed in other tissues but not in the heart (data not shown). The reason for this remains unclear to us at this time, though the fact that we have extensive experience obtaining wide expression – including in the heart – of multiple other proteins in tadpoles, suggests that this may be an issue specific to TNNC1.

Interestingly, the D145E variant was first described in a patient with hypertrophic cardiomyopathy (HCM) and it was the only variant found in a screen of fifteen HCM-susceptibility genes (Landstrom et al., 2008). In contrast, a subsequently described proband with DCM also had a rare variant in the MYBPC3 gene, which was hypothesized to combine with cTnC D145E to manifest as DCM (Hershberger et al., 2010; Pinto et al., 2011b). Based on these opposing findings, it was suggested that this second patient may have initially developed HCM that rapidly progressed to DCM (Freeman et al., 2001; Fujino et al., 2001, 2002; Nanni et al., 2003). The proband described here, however, was followed closely since birth given her brother’s diagnosis, and she never had any echocardiographic or other evidence of HCM, suggesting that this combination of cTnC alleles results purely in DCM. Of significance is another example of compound heterozygosity where the D145E variant was again associated with inherited cardiomyopathy (Ploski et al., 2016). In this case, two pediatric patients were diagnosed with autosomal recessive restrictive cardiomyopathy which was fatal during infancy. Sanger sequencing revealed that both of the affected individuals were compound heterozygous for genetic variants in TNNC1 corresponding to D145E and A8V in the cTnC protein.

Variants in TNNC1 can affect skeletal muscle function because cTnC is also expressed in slow skeletal muscle. The D145E mutant was reported to increase slow skeletal muscle ATPase activity at low and high calcium concentrations in reconstituted myofibrils (Veltri et al., 2017b). Although it is known that D145E does not affect myofilament calcium sensitivity in slow skeletal skinned fibers (Veltri et al., 2017b), it is unclear whether D132N has any effect on slow skeletal muscle function. Importantly, no skeletal muscle weakness was reported in the patient’s history.

Both variants are located in the C-domain of cTnC (Figure 3). The missense D132N variant is located within the G-helix that is part of EF-hand site IV, and D145E is one of the divalent cation (Ca²⁺ or Mg²⁺) coordinating residues (+Z position) in site IV. While the regulatory site for Ca²⁺ binding is site II in the N-domain of cTnC, our prior results indicate that there is communication between the C- and N-domains (Badr et al., 2016) such that changes in the C-domain can influence the regulatory N-domain (Marques et al., 2017; Veltri et al., 2017a). The results of this study demonstrated that the D145E variant (WT/D145E) increases myofilament Ca²⁺ sensitivity even when WT is also present, while the D132N mutation (WT/D132N) had the opposite effect of decreasing Ca²⁺ sensitivity (Table 2 and Figures 5A,B) even though the two affected residues are relatively close in the primary sequence and the 3D structure (Figure 3). In general, myofilament incorporation of troponin variants that cause opposing effects on thin filament Ca²⁺ responsiveness are expected to normalize the myofilament Ca²⁺ sensitivity (Li et al., 2010; Alves et al., 2014, 2017; Dieseldorff Jones et al., 2018), and that was what was found in this study. Compared to WT, the 50%:50% mix of D132N/D145E showed similar myofilament Ca²⁺ sensitivity and Hill coefficient (n_Hill) values, the latter generally reflecting cooperative processes associated with isometric force generation. While incorporation of the two mutants appeared to normalize opposite effects of the two variants on Ca²⁺ dependence of isometric force, what is difficult to reconcile is the severe DCM pathology associated with the presence of the two mutations.

A paradigm associating changes in myofilament Ca²⁺ sensitivity commonly found in HCM (increased) and DCM (decreased) is generally well-accepted (Willott et al., 2010; van der Velden and Stienen, 2019). This study, however, presents a unique clinical case in which we observed no significant changes in myofilament Ca²⁺ sensitivity due to a combination of variants in patients with DCM. How can this finding be explained? After depleting native cTnC from CMPs, each available unfilled cTnC site was presumably reconstituted, depending on the experiment, with either exogenous WT, WT/D132N mix, WT/D145E mix, or D132N/D145E mix in an attempt to mimic what is expected to be found in the patients (D132N/D145E) or the parents (WT/D132N or WT/D145E), along with the control condition (100% WT). Previous studies showed that D145E cTnC dissociates more slowly from the thin filament compared to WT cTnC indicating that D145E has a higher binding affinity for the thin filament (Marques et al., 2017), although the functional data (Table 2) do not suggest that this difference in affinity influenced the results.

As with our finding, there has been some variation in prior reports on variants in TNNC1 with regard to the hypothesis that HCM is always associated with increased Ca²⁺ sensitivity and DCM with decreased Ca²⁺ sensitivity. The first DCM-associated variant identified in TNNC1, G159D (Mogensen et al., 2004), has been explored by several groups with conflicting findings. It was initially reported that G159D did not alter myofilament Ca²⁺ sensitivity in Tn-exchanged skinned rat trabeculae (Biesiadecki et al., 2007). In contrast, both cTnC-depleted skinned porcine papillary muscle and in vitro regulated thin filaments reconstituted with cTnC-G159D exhibited reduced myofilament Ca²⁺ sensitivity (Robinson et al., 2007; Dweck et al., 2008). Conversely, G159D exhibited increased myofilament Ca²⁺ sensitivity in both human skinned ventricular myocytes obtained from a patient bearing G159D, and thin filaments reconstituted with skeletal muscle actin, human cardiac tropomyosin, and troponin complex containing cTnC G159D (Dyer et al., 2009).

Loss of the lusitropic response (cardiac muscle relaxation) is considered a common feature of DCM and HCM (Wang et al., 2012; Dweck et al., 2014; Messer and Marston, 2014). In the case of thin filament mutants, this can be partially attributed to uncoupling of cTnI phosphorylation from modulation of Ca²⁺ sensitivity (Memo et al., 2013; Messer and Marston, 2014). Studies using TnC-extracted porcine papillary muscle fibers reconstituted with cTnC DCM-associated mutants (Y5H, M103I or I148V) exhibited diminished or abolished PKA-mediated Ca²⁺ desensitization (Pinto et al., 2011b). Although we did not test for PKA-mediated myofilament Ca²⁺ desensitization in our current study, we speculate that the presence of both mutant proteins could interfere with cTnI phosphorylation levels and functional consequences. This could cause a loss of downstream β-adrenergic stimulation which is crucial to effect the lusitropic response and diastolic ventricular filling.

Alternative possibilities exist because the two mutations are not present in the same protein. In the affected siblings, the two variant proteins are expected to be distributed randomly in thin filaments, and differences in function might affect communication between adjacent regulatory units when they contain different cTnCs. The lack of change in n_Hill for D132N/D145E (Table 2 and Figures 5A,B) suggests this might not be a plausible explanation, although the Hill coefficient represents the overall response and does not inform about highly localized effects when two variant cTnCs are present. Mechanical kinetics provides information beyond steady-state force measurement, and cooperative interactions among regulatory units do not appear to be required for fast rates of tension redevelopment, k_TR (Chase et al., 1994). Both the force-k_TR data (Figure 7B) and the 3-state model analyses of those force-k_TR relations (Table 4) suggest that the presence of either variant (WT/D132N or WT/D145E) shifts the force-k_TR relation, and alters contractile kinetics (k_TR in Figure 7B, and f and g in Table 4) in the same direction. However, both the kinetic data (Figure 7B) and modeling (Table 4) support the conclusion from analysis of the steady-state force-pCa data (Table 2 and Figures 5A,B) that the combination of the two variants, D132N/D145E, in thin filaments may partially ameliorate the effects of either mutant on contractile function. This is because D132N/D145E yields results more similar to 100% WT than either WT/D132N or WT/D145E (Table 4 and Figure 7B). Thus it appears to be necessary to seek alternative explanations, beyond changes in biomechanical function, to explain the deleterious effects of the two variants in combination in the proband and her brother.

We and others have previously suggested that troponin subunits may participate in cellular functions that extend beyond contractile regulation in the sarcomere (Asumda and Chase, 2012; Wu et al., 2015; Zhang et al., 2016; Pinto et al., 2017). In fact, cTnC has been previously detected in cardiomyocyte nuclei, as well as the nuclei of certain cancer cells (Johnston et al., 2018). In addition, cTnC has been identified in the mitochondrial compartment of some cancer cell lines. Therefore, a potential mechanistic explanation for the severe disease phenotype associated with troponin mutants may be attributed to perturbations in its putative nuclear/mitochondrial function, whereby gene expression or bioenergetics could be altered by cTnC variants such as those reported here.

In summary, we report the association of compound heterozygous variants in TNNC1 in siblings with early onset familial DCM. We provide functional evidence that both variants separately lead to abnormal myofilament Ca²⁺ sensitivity in which the variants D132N and D145E are, respectively, associated with reduced or increased myofilament Ca²⁺ sensitivity relative to WT, while these effects compensated for no change relative to WT when the two variants were combined. Analyses of activation-dependence of k_TR suggest that both variants have similar effects on contractile kinetics, although there was no significant change in maximal k_TR, and that the combination of the two variants again partially ameliorated the effects of the individual variants. None of the observed changes in mechanics, however, can explain the clinical outcomes in this family. Further studies are needed to understand precisely how these variants combine to result in a severe DCM phenotype.