Establishing disease causality for a novel gene variant in familial dilated cardiomyopathy using a functional in-vitro assay of regulated thin filaments and human cardiac myosin

Ethics

All research involving human subjects (including human material and human data) was
performed in accordance with the Declaration of Helsinki. The Institutional Review
Board at Stanford University approved the study. Written informed consent was obtained
from all adult participating subjects, specifically the mother and father of the proband.
For their children, including the proband, all of whom were minors at the time of
the study, informed consent was obtained from the parents as well.

Patient clinical evaluation

All nuclear family members underwent transthoracic echocardiographic screening to
evaluate cardiac function. Standardized measurements of cardiac chamber size and volume,
valvular function, and systolic and diastolic function were carried out according
to American Society of Echocardiography guidelines.

Genetic testing

Genetic testing of the proband was performed by sequencing 23 genes previously associated
with DCM: LMNA, LDB3, TNNT2, DES, SGCD, PLN, ACTC1, MYH7, TPM1, TNNI3, TAZ, TTR, MYBPC3, LAMP2, MTTK, MTTL1, MTTQ, MTTH, MTTS1, MTTS2, MTND1, MTND5, and MTND6 (GeneDx). Targeted PCR and Sanger sequencing of exon 13 of TNNT2 was carried out
in the proband and 5 immediate nuclear family members to confirm the presence or absence
of the variant. Blood samples were obtained and DNA was isolated from buffy coat samples
(Epicentre, MasterPureâ„¢ DNA Purification Kit). The primers used were as follows: forward,
5?-CAGGGGGTTTGGGGAGGGTTAG-3?; reverse, 3?-GTGGGGCACCTGCTCAGTTCTCT-5?.

Cloning of human cardiac troponin mutants and human ?-cardiac myosin

The R205Q mutation was introduced by QuikChange site-directed mutagenesis (Stratagene)
into human adult cardiac TnT (TNNT2) and confirmed by sequencing. For fluorescence experiments, a similar strategy was
used to make the triple mutation of TnC containing C35S, T53C, and C84S. A truncated
version of human cardiac myosin heavy chain 7 (MYY7) corresponding to a short S1 (residues 1–808), with (for ATPase and motility measurements)
and without (for stop flow measurements) an eGFP linker, was constructed and produced
as previously described 11], 26].

Protein expression

Human adult cardiac troponin subunit (TNNT2, TNNI3, TNNC2) expression and purification methods were based on previously published protocols
27]–30]. Tropomyosin was purified from bovine cardiac tissue according to the protocol of
Smillie 31]. Chicken skeletal actin and human ?-cardiac myosin were prepared as previously described
26].

Tropomyosin pyrene binding

Tropomyosin pyrene labeling and binding assays were performed as described previously
11]. All binding experiments were carried out with 100 nM pyrene-tropomyosin at 23 °C
in 20 mM Hepes, pH 7.5, 100 mM KCl, 2 mM MgCl
₂
, and 1 mM DTT.

Coupled actin-activated ATPase assay

We used the NADH-coupled assay at 30 °C to measure the Ca
²⁺
sensitivity of the steady state actin-activated ATPase rate for the human ?-cardiac
S1 11], 32]. The concentrations of freshly prepared proteins used were 3.5 ?M actin, 1 ?M tropomyosin,
2 ?M troponin, and 0.3 to 0.5 ?M S1. The final buffer conditions were 20 mM imidazole,
10 mM KCl, 2 mM MgCl
₂
, 1 mM DTT, 2 mM ATP, the Ca
²⁺
buffers (2 mM EGTA, 4 mM NTA, and varying concentrations of CaCl
₂
), and the NADH-coupling system 32]. The pCa Calculator developed by Dweck et al. was used to determine the composition
of the Ca
²⁺
buffers with EGTA and NTA 33].

ANS troponin complex

The triple TnC mutant was labeled with the fluorescent dye IAANS and ANS-labeled TnC
steady state assays were performed in a Fluorolog fluorimeter (Horiba Scientific)
at 23 °C, similar to previous studies 11], 13], 34]. Final buffer conditions were 200 mM Hepes, pH 7.5, 10 mM KCl, 3 mM MgCl
₂
, 1 mM DTT, 4 mM NTA, and 2 mM EGTA, and the amount of CaCl
₂
added was determined using the pCa calculator 33]. Transient Ca
²⁺
dissociation rates were measured using a HiTech SF-61DX2 (TgK Scientific Ltd., U.K.)
stopped-flow apparatus at 23 °C as previously described 11]. Each data trace was individually fit to a single exponential function, with more
than five individual traces for both WT and R205Q.

ADP release

Actin was labeled similarly to previous methods 35]. ADP release rates were measured using a HiTech SF-61DX2 (TgK Scientific Ltd., U.K.)
stopped-flow apparatus at 23 °C in 25 mM Hepes, pH 7.5, 25 mM KCl, 4 mM MgCl
₂
, 0.2 mM CaCl
₂
, and 1 mM DTT. For WT and R205Q thin filaments, ?6 traces were collected and fit
individually. Regulated thin filaments were prepared with S1-ADP at a final concentration
of 2 ?M actin, 0.5 ?M tropomyosin, 0.5 ?M troponin, 2 ?M S1, and 50 ?M ADP and then
rapidly mixed with 2 mM ATP, 50 ?M ADP.