In vitro analyses of suspected arrhythmogenic thin filament variants as a cause of sudden cardiac death in infants

Sanam Shafaattalaba,b, Alison Yueh Lia,c, Eric Lina,b, Charles M. Stevensb, Laura J. Dewara,b, Francis C. Lynnb, Shubhayan Sanatanib, Zachary Laksmana,d, Ryan D. Morinc, Filip van Petegeme, Leif Hove-Madsena,f, D. Peter Tielemang,h, Jonathan P. Davisi, and Glen F. Tibbitsa,b,c,1

aMolecular Cardiac Physiology Group, Simon Fraser University, Burnaby, BC V5A 1S6, Canada; bBC Children’s Hospital Research Institute, Vancouver, BC V5Z 4H4, Canada; cDepartment of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada; dCardiology, The University of British Columbia, Vancouver, BC V6T 1Z3, Canada; eBiochemistry, The University of British Columbia, Vancouver, BC V6T 1Z3, Canada; fConsejo Superior de Investigaciones Científicas, 08025 Barcelona, Spain; gCentre for Molecular Simulation, University of Calgary, Calgary, AB T2N 1N4, Canada; hDepartment of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada; and iPhysiology and Cell Biology, The Ohio State University, Columbus, OH 43210

Edited by Christine E. Seidman, Howard Hughes Medical Institute, Brigham and Women’s Hospital, and Harvard Medical School, Boston, MA, and approved January 23, 2019 (received for review November 7, 2018) Sudden unexpected death of an infant (SUDI) is a devastating the cause of SUDI is challenging. We sought to develop a “plat- occurrence for families. To investigate the genetic pathogenesis form” to determine the pathogenicity and the possible arrhyth- of SUDI, we sequenced >70 from 191 autopsy-negative mogenic nature of such mutations in infants by using the TNNI1 + − SUDI victims. Ten infants sharing a previously unknown variant R37C / mutation, which is a variant of unknown significance, as + − in I (TnI) were identified. The mutation (TNNI1 R37C / ) an example. is in the fetal/neonatal paralog of TnI, a thought to be In a recent study (3), next-generation sequencing was per- expressed in the heart up to the first 24 months of life. Using phy- formed on tissues from 191 autopsy-negative SUDI cases to logenetic analysis and molecular dynamics simulations, it was de- identify potentially pathogenic genetic variants resulting in termined that arginine at residue 37 in TNNI1 mayplayacritical inherited arrhythmia syndromes. Strikingly, 10 out of 191 in- functional role, suggesting that the variant may be pathogenic. fants who died in the first 24 mo of life, 7 of whom were un- MEDICAL SCIENCES We investigated the biophysical properties of the TNNI1 R37C mu- der 4 mo of age, exhibited an identical variant in the neonatal + − tation in human reconstituted thin filaments (RTFs) using fluorometry. form of the cardiac gene (TNNI1 R37C / ,Chr1: RTFs reconstituted with the mutant R37C TnI exhibited reduced 201383726-201383726 G>A GRCh37/hg19). This gene is the 2+ 2+ Ca -binding sensitivity due to an increased Ca off-rate constant. predominantly expressed troponin I paralog in the heart until at +/− Furthermore, we generated TNNI1 R37C mutants in human in- least 9–24 mo of age, at which point the cardiac troponin I encoded duced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) us- by TNNI3 becomes dominant. As a matter of clarification, TNNI1 ing CRISPR-Cas9. In monolayers of hiPSC-CMs, we simultaneously 2+ encodesaTnIproteinfirstfoundtobeexpressedinslowskeletal monitored voltage and Ca transients through optical mapping muscle (commonly referred to as ssTnI); however, it is the domi- and compared them to their isogenic controls. We observed normal nant form of TnI expressed in the fetal/neonatal/infant heart. TNNI1 +/− intrinsic beating patterns under control conditions in R37C Thus, we refer to the gene product of TNNI1 in this study as the at stimulation frequencies of 55 beats/min (bpm), but these cells showed no restitution with increased stimulation frequency to Significance 65 bpm and exhibited alternans at >75 bpm. The WT hiPSC-CMs did not exhibit any sign of arrhythmogenicity even at stimulation frequencies of 120 bpm. The approach used in this study provides A major aim of this study was to develop an experimental critical physiological and mechanistic bases to investigate sarcomeric pipeline for the analysis and assessment of variants potentially causal in sudden cardiac death of infants (SCDI). The TNNI mutations in the pathogenesis of SUDI. + − R37C / variant in this study was reliably and reproducibly dis- cardiac troponin | thin filament | hiPSC-CM | sudden cardiac death | ruptive to the normal physiology, in contrast to that in isogenic arrhythmia controls, across a battery of in silico and in vitro assays. This strengthens the evidence of pathogenicity and implicates a neonatal gene paralog encoding a sarcomeric contractile udden unexpected death in infants (SUDI) is a devastating as having likely contributed to SCDI through a proarrhythmic Soccurrence for parents and health care practitioners. SUDI is pathway. This platform has the potential to be applied broadly particularly challenging when the cause of death is not identified as part of a standardized and clinically relevant death in- despite a thorough investigation including autopsy, toxicology, vestigation in cases of SCDI where novel variants are identified. microbiology, and other analyses. Postmortem molecular genetic testing has provided clues to the cause of death in a portion of Author contributions: S. Shafaattalab, E.L., L.J.D., L.H.-M., J.P.D., and G.F.T. designed re- autopsy-negative SUDI victims. A significant number of SUDI search; S. Shafaattalab, A.Y.L., and C.M.S. performed research; F.C.L., D.P.T., and J.P.D. cases are likely due to sudden cardiac arrest caused by lethal contributed new reagents/analytic tools; S. Shafaattalab, A.Y.L., E.L., C.M.S., L.J.D., F.C.L., S. Sanatani, Z.L., R.D.M., F.v.P., L.H.-M., D.P.T., J.P.D., and G.F.T. analyzed data; and arrhythmias. However, proof of causality when a variant of in- S. Shafaattalab, L.J.D., S. Sanatani, Z.L., F.v.P., L.H.-M., D.P.T., J.P.D., and G.F.T. wrote terest is identified in postmortem testing is often lacking (1). the paper. While there have been cases of SUDI observed in the absence The authors declare no conflict of interest. of the typical phenotype, the arrhythmogenic mechanism has usu- This article is a PNAS Direct Submission. ally been linked to the phenotype (2). In general, lethal arrhythmias Published under the PNAS license. caused by dysfunctional ion channels (i.e., channelopathies) 1To whom correspondence should be addressed. Email: [email protected]. are better understood than those caused by mutations in genes This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. that encode sarcomeric . Thus, determining whether 1073/pnas.1819023116/-/DCSupplemental. this variant in a gene that encodes a cardiac contractile protein is

www.pnas.org/cgi/doi/10.1073/pnas.1819023116 PNAS Latest Articles | 1of6 Downloaded by guest on September 27, 2021 fetal/neonatal cardiac form of TnI (fcTnI) and the gene product (TNNI1) is a paralog of the adult cardiac (TNNI3) gene in which of TNNI3 as the adult cardiac form of TnI (acTnI). 31 amino acids on the N terminus are truncated and the rest of Our strategy involved several approaches: (i) phylogenetic the coding region exhibits ∼65% identity. Fig. 1A shows the analysis, (ii) molecular dynamics (MD) simulations of the tro- superimposition of the WT neonatal Tn complex (cTnC, light ponin complex, (iii) biochemical assays of reconstituted neonatal gray; fcTnI, dark gray; cTnT, black) with the TNNI1 R37C mu- human thin filaments (RTFs), and (iv) optical mapping of tant (cTnC, green; fcTnI, red; cTnT, blue). Fig. 1B (same color + monolayers of human induced pluripotent stem cell-derived scheme as Fig. 1A) depicts the MD simulation-derived H bond cardiomyocytes (hiPSC-CMs). While hiPSC-CMs have been network around the R37 residue in the WT neonatal complex. used as a tool for modeling human genetic diseases, including The 500-ns molecular dynamics simulation predicts that fcTnI + sarcomeric cardiomyopathies (4, 5), the experiments have been R37 forms H bonds with neighboring H34 on fcTnI and resi- subjected to the criticism that hiPSC-CMs lack the maturity of an dues D249 and E252 on cTnT. Fig. 1C (same color scheme as adult cardiomyocyte. However, because these hiPSC-CMs ex- Fig. 1A) illustrates how the TNNI1 R37C mutation severely press the fetal/neonatal form of TnI at a higher level than the disrupts the H bond network observed in in the WT simulations + adult paralogs (3) (SI Appendix, Fig. S7) and for other reasons (blue). Thus, the R37C variant is predicted to disrupt the H described in this study, they represent an ideal tool to study bonding between troponin I and and is predicted to sudden unexpected death in the young (SUDY) in many re- impact not only the position of TnI with respect to TnT but also spects. In the present study, we used CRISPR-Cas9 to generate the position of TnC with respect to the rest of the Tn complex. + − heterozygous TNNI R37C / hiPSC-CMs, a mutation that we Both in silico approaches described above are predictive of the observed in 10 infants, all of whom died within 24 mo of age. The importance of fcTnI (ssTnI) R37 in the structure of the neonate approach described in this study was able to provide mechanistic troponin complex, but to test the hypothesis that the R37C insights into the pathogenicity of the TNNI1 R37C and suggests mutation has pathogenic implications, two different experimental that it was likely the cause of death in the infants who harbored approaches were used. The first experimental protocol involved this mutation. the use of reconstituted human cardiac troponin complexes, and the second involved RTFs made from recombinant human pro- Results teins for the WT adult, WT neonate, and R37C mutant neonate. To determine the evolutionary importance of the conserved ar- Two parameters were determined for each construct: (i)thesteady- + ginine at residues 37 and 68 encoded in the human paralogs state dissociation constant of Ca2 for both the Tn and RTF com- 2+ TNNI1 and TNNI3, respectively, a sequence alignment was plexes (Kd,inμM) and (ii) the off-rate constant of Ca from both 2+ −1 performed using a broad spectrum of species ranging from hu- the Tn and RTF complexes (koff Ca ,ins ) using a stopped-flow mans to teleosts, as shown in SI Appendix, Table S1. The arginine apparatus (SI Appendix,TableS4).ThedataaredisplayedinFig.2. 2+ at that position or its equivalent was conserved in every species The Kd Ca was significantly (P < 0.05) higher in WT adult vs. WT examined, spanning an evolutionary scale of more than 400 My. neonate in both Tn (0.35 ± 0.02 vs. 0.20 ± 0.02 μM, n = 3) and RTF Molecular dynamics simulations were performed on the tro- complexes (2.80 ± 0.20 vs. 0.90 ± 0.20 μM, n = 5). The higher Kd ponin complex as described in the Methods section. The X-ray found in the WT adult was attributable in large part to a signifi- −1 diffraction-derived crystal structure by Takeda et al. (6) of the cantly higher koff found in both Tn (45.0 ± 1.0 vs. 10.4 ± 0.6 s ) − adult human troponin complex [Protein Data Bank (PDB) ID and RTFs (104.0 ± 5.0 vs. 76.0 ± 1.0 s 1). The R37C mutant had a + code 1J1E] was used as a template. The sequence of neonate significant effect on Ca2 handling in both Tn and RTF complexes cTnC is identical to that of the adult, while the neonate cTnT in comparison with the WT neonate complexes, as shown in Fig. 2. sequence is a splice variant of the adult form with the addition of In a direct comparison of WT neonate vs. the R37C mutant, Kd exon 5, which encodes 10 additional amino acids. The fcTnI gene was significantly higher in both the Tn (0.20 ± 0.2 vs. 0.36 ± 0.02 μM,

Fig. 1. MD simulations of the adult and neonate cardiac troponin complex. Three replicated 500-ns MD simulations were conducted on models based on the X-ray structure of the core domain of the human adult troponin complex (PDB ID code 1J1E). A shows the superimposed representative models of the WT (cTnC is light, fcTnI is dark gray, and cTnT is black) and the fcTnI R37C (cTnC is green, fcTnI is red, and cTnT is blue). The representative structures were calculated by clustering the backbone and Cβ atoms of regions surrounding the Arg or Cys at position 37 of fcTnI (TnI: 12–48; TnT: 255–270; TnC: 94–157) over the combined final 100 ns of the replicated trajectories. The range of motion of the N domain of cTnC is similar for the WT and mutant models over the course of the simulations, despite differences in the position in these representative structures. B shows the inter- acting side chains at the interface between the neo- nate WT (i.e., TNNI R37) fcTnI, cTnT, and cTnC. Hydrogen bonds are shown as dotted yellow lines. R37 is highlighted with a red arrow. C shows the same region in the mutant model with the R37C mutation highlighted with a black arrow. The absence of argi- nine at position 37 disrupts the hydrogen bonding pattern substantially, which suggests that the in- terface at the fcTnI/cTnT/cTnC junction is weakened when cysteine is present in comparison with the WT neonate troponin complex.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1819023116 Shafaattalab et al. Downloaded by guest on September 27, 2021 + n = 3) and RTF (0.9 ± 0.2 vs. 1.3 ± 0.1 μM, n = 5) complexes. This Ca2 transient decay rate (Fig. 4C) to two different field stim- effect was largely due to a significant increase in koff in the ulation frequencies, 55 bpm (solid bars) and 65 bpm (stippled − R37C mutant in both Tn (10.4 ± 0.6 vs. 14.3 ± 1.4 s 1)andRTF bars). These frequencies were chosen because 55 bpm was higher − (76 ± 1vs.104± 6s 1) complexes. than the intrinsic beating frequency and 65 bpm was below a To understand how R37C impacts neonatal cardiomyocyte frequency that elicited alternans or any other form of arrhyth- TNNI1 function, genome-edited R37C heterozygous hiPSCs mia. In WT both the APD50 and CaTD50 show a significant (P < + − (R37C / ) were generated and differentiated to CMs. Briefly, 0.05) decrease of about 11% with an increase in stimulation both WT and mutated hiPSC lines were differentiated to ven- frequency rate, a normal phenomenon related to restitution. A tricular cardiomyocytes (SI Appendix, Fig. S6) using the protocol full restitution curve for APD and CaTD in WT (partial one for + − described by Lian et al. (7) as monolayers in 35-mm petri dishes. R37C / ) is shown in SI Appendix, Fig. S9. At 55 bpm, the +/− Based on fluorescence-activated cell sorting (FACS) analyses R37C hiPSC-CM APD50 and CaTD50 were significantly using cardiac- and ventricular-specific antibodies, cTnC and shorter in duration than those of WT hiPSC-CMs by 14 and light chain ventricular isoform 2 (MLC2v), respectively, 26%, respectively. However, neither APD50 nor CaTD50 values + − + − both WT and R37C / hiPSC-CMs were found to be >80% in R37C / hiPSC-CMs showed any indication of shortening with cardiac-specific and >70% ventricular-specific (SI Appendix, Fig. increased stimulation frequency. 2+ S8). Membrane voltage (Vm) and cytosolic Ca transients were Representative traces of the effects of 500 nM isoproterenol + determined from the fluorescence emitted by RH-237 and Rhod- (ISO) on the voltage and Ca2 transients in hiPSC-CMs are + − 2, respectively. Fig. 3A (WT) and Fig. 3B (R37C / ) show rep- shown in SI Appendix, Fig. S10. The β-adrenergic agonists, such 2+ resentative traces of Vm (red) and Ca transients (blue) from as ISO, are commonly used to unmask arrhythmogenic condi- one region of interest (ROI) from monolayers of hiPSC-CMs tions (8, 9). SI Appendix, Fig. S10 A and B show the WT and + − stimulated at 55 beats/min (bpm). The data clearly demon- R37C / mutant hiPSC-CMs, respectively, “beating” intrinsically + strate that the action potentials and Ca2 transients entrained in normal Tyrode’s solution. Both appear to have normal pat- normally to that stimulation frequency. As shown in Fig. 4C, the terns of excitation. SI Appendix, Fig. S10 C and D show the effect + + − maximum rate of Ca2 transient decay is significantly (P < 0.05) of 500 nM ISO on the WT and R37C / hiPSC-CMs, re- − faster in R37C than in WT (1.4 ± 0.1 vs. 1.0 ± 0.1 F·ms 1). In Fig. spectively. Predictably, in the WT hiPSC-CMs, ISO increased the 3 C and D, the field stimulation rate was increased to 75 bpm. frequency of beating slightly. However, as seen in SI Appendix, + − The WT shows normal entrainment to the stimulation frequency Fig. S10D, 500 nM ISO was clearly arrhythmogenic in R37C /

at both 75 and 120 bpm (Fig. 3E) and never showed any signs of hiPSC-CMs in 80% of the plates tested (SI Appendix, Fig. S10F). MEDICAL SCIENCES alternans over the range of stimulation frequencies used in this On the other hand, WT hiPSC-CMs beat synchronously in doses study. Fig. 3D illustrates the alternan arrhythmia that develops in up to and exceeding 1,000 nM ISO, without any indication of + − the R37C / mutant when stimulated to 75 bpm and higher. arrhythmogenicity (SI Appendix, Fig. S10E). Each plate was tested for 20 s at any given field stimulation rate. Fig. 3F illustrates the deterioration of the alternans at 80 bpm. Discussion None of eight WT plates exhibited any alternans, whereas seven One of the overall goals of this study was to develop an approach + − of eight R37C / plates were in alternans for 100% (i.e., 20 s) of that could determine if cardiac contractile protein mutations of the time. unknown pathogenicity could be determined to be likely causal or + − Fig. 4 illustrates the response of both WT and R37C / with not in the SUDY cases examined. To determine the evolutionary respect to the action potential duration at 50% (APD50) (Fig. importance of the conserved arginine at residue 37 in human 2+ 4A), Ca transient duration at 50% (CaTD50) (Fig. 4B), and TNNI1 (corresponding to residue 68 in the TNNI3 paralog), the

2+ 2+ Fig. 2. Ca dissociation (Kd)(A and B) and Ca off-rate (koff)(C and D) constants determined in human cardiac: troponin (Tn) (A and C) and reconstituted −1 thin filament (B and D) complexes. Shown are the stopped-flow determined koff (s ) and steady-state determined Kd (μM) Tns and RTFs comprised of recombinant proteins: (i) adult Tn complexes, (Tm), and ; (ii) neonate WT comprising Tn complexes, Tm, and actin; and (iii) neonate R37C comprising R37C Tn complexes, Tm, and actin (see SI Appendix, Table S3 for detailed protein composition). *P < 0.05.

Shafaattalab et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 27, 2021 A WT TNNI1 B R37C+/- TNNI1 55 bpm

C D 75 bpm

E F 80 bpm 120 bpm

1 sec Voltage Calcium

Fig. 3. The hiPSC-CMs being field stimulated at different frequencies. Monolayers of hiPSC-CMs (both WT and TNNI1 R37C+/−) were loaded with voltage- (RH- 237, red) and Ca2+-dependent (Rhod-2 AM, blue) dyes and optically mapped while being field stimulated at (A–D) 55 and 75 bpm and (E) 120 bpm (WT only)

for ≥20 s. In the representative traces shown, the TNNI1 WT hiPSC-CMs exhibited clear and significant shortening in both the APD50 and CaTD50 with increased + − + − stimulation frequency (A vs. C) while TNNI1 R37C / hiPSC-CMs did not. Furthermore, at frequencies of 75 bpm, the TNNI1 R37C / hiPSC-CMs displayed + − alternans (arrows) (D). The alternans in the R37C / hiPSC-CMs were observed for a collective 87.5% of time in eight plates measured for 20 s each (140/160 s) + − that were stimulated at 75 bpm. (F) At 80 bpm the alternans in R37C / hiPSC-CMs deteriorated (green line represents each stimulation point). We did not observe any irregular voltage and Ca2+ transients in WT TNNI1 hiPSC-CMs even at stimulation frequencies of 120 bpm (E). (TNNI1 R37C+/− hiPSC-CMs, n = 8; TNNI1 WT hiPSC-CMs, n = 5).

phylogenetic sequence alignment of the relevant regions of the dynamics of the TnC molecule are also similar for the WT and troponin I genes (as shown in SI Appendix,TableS1) clearly illus- R37C models (SI Appendix, Figs. S2 and S3); however, two ca- trates the importance of the arginine at that relative position. The veats about the simulations should be noted: (i) the crystal alignment, which spans from humans to a variety of teleost fish, structure of the human troponin complex as published by Takeda including Danio rerio, represents more than 450 My of evolution et al. (6) is not complete, and (ii) while a structure of the entire (10) in which the arginine at residue 37 of the neonate paralog of thin filament with the troponin complex in place would be ex- fcTnI (or equivalent residue) has been very highly conserved. tremely valuable to answer these questions, we cannot confi- To predict the impact of the TNNI1 R37C mutation on the dently model the neonatal thin filament without an empirical structure of the Tn complex, we used molecular dynamics sim- structure to use as a template. While we do not know the mo- ulations. The homology models were based on the structure of lecular details of the interactions between the troponin complex the human adult troponin complex core domain (6). After 500 ns and the thin filament or how the destabilizing effect of the R37C of simulation, the destabilizing effect of the mutation on the Tn mutation would affect these interactions, we are confident that complex is visible, despite only minor changes in the overall the modeled effect of the mutation on the stability of the com- structure of the complex (Fig. 1A and SI Appendix, Fig. S1). The plex in the context of calcium binding is consistent with the

2+ +/− Fig. 4. Influence of stimulation frequency on (A) APD50,(B) CaTD50 and (C)Ca transient decay rate in WT TNNI1 and R37C TNNI1 hiPSC-CMs. The impact of increasing the stimulation frequency from 55 bpm (solid bars) to 65 bpm (stippled bars) on each of these parameters is shown. A illustrates the effect of +/− increasing the stimulation rate on the APD50 in both WT TNNI1 (red) and R37C TNNI1 (coral) hiPSC-CMs. B exhibits the effect of stimulation frequency of the + + − duration of the Ca2 transient in both WT (blue) and R37C / TNNI1 (azure) hiPSC-CMs. C shows the impact of increasing the stimulation rate on the maximum rate of Ca2+ transient decay in WT (purple) and R37C+/− TNNI1 (lilac) hiPSC-CMs. Data are represented as mean ± SEM (n = 5 per condition). *P < 0.05.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1819023116 Shafaattalab et al. Downloaded by guest on September 27, 2021 available experimental data and the current understanding of the 10 bpm increments. As seen in Fig. 3, both WT and TNNI1 + + − nature of the TnC-Ca2 interaction. The simulations demon- R37 / hiPSC-CMs beat “normally” at 55 and 65 bpm. However, + − strate the key role of the R37 side chain in the hydrogen bond Fig. 4 indicates that at 55 bpm, the TNNI1 R37 / exhibited + network at the interface of TnI/TnT and the C-terminal domain significantly shorter action potential and Ca2 transient durations + of TnC (Fig. 1 B and C). The disruption of the H bond network compared with the isogenic controls. Furthermore, also shown + at this interface is expected to destabilize the complex and pro- in Fig. 4C, the rate of decay of the Ca2 transient was signifi- + − + vides the basis for a molecular explanation of the deleterious cantly greater in TNNI1 R37C / hiPSC-CMs. Ca2 -dependent 2+ effect of the R37C mutation (SI Appendix, Fig. S4). inactivation (CDI) of L-type Ca channel (CaV1.2)iswell Recent work by our group (11) and by Lindert and coworkers documented (22–24) and occurs within 100 ms of channel 2+ (12) has demonstrated that mutations that alter the energetic opening after the Ca levels in the nanodomain around CaV1.2 cost of exposing the hydrophobic patch of TnC are correlated within the dyadic cleft rise to achieve values which are estimated + + with pathogenicity, without directly changing the Ca2 coordi- to be at least an order of magnitude higher than global [Ca2 ] nation. We hypothesize that the weakened interaction may lead (22, 24). While access to this nanodomain is restricted in an to a change in the position or orientation of the TnC protein with adult cardiomyocyte with a fully developed transverse tubular (T- respect to the thin filament, which would alter the competition tubules) network, it is unknown whether this is the case in the between actin and TnC for the TnI switch peptide. This change neonatal cardiomyocyte or hiPSC-CMs, which both lack T-tubules + would primarily affect the off-rate of Ca2 from the N-terminal (25–28). Thus, there remains a strong possibility that the faster 2+ +/− domain of TnC (N-TnC), as the switch peptide stabilizes the koff (Ca )inTNNI1 R37C hiPSC-CMs impacts CaV1.2 CDI, + + Ca2 bound (open) form of N-TnC and prevents Ca2 release. thereby shortening the action potential duration compared with + As shown in SI Appendix, Fig. S2, based on the rmsd of the C- WT hiPSC-CMs. At 55 bpm the Ca2 transient duration is also + − terminal domain of TnC (residues 89–161) with respect to the IT significantly shorter in TNNI1 R37C / hiPSC-CMs compared arm helices of the Tn complex (TnI residues 4–131 and TnT with isogenic WT controls. Because there are several cardiac ion + + residues 226–271), the mutant model TnC C domain appears to channels that are regulated by Ca2 and/or Ca2 /calmodulin be similarly mobile to the WT model over the course of these [e.g., CaV1.2,NaV1.5, ryanodine receptor 2 (RyR2), KV7.1, + + simulations. However, the modeling predicts that the impact of small-conductance Ca2 -activated K channels], it is possible the R37C heterozygous mutation on the structure of the neonate that the altered calcium dissociation rate constant has the po- Tn complex is quite striking, as shown in Fig. 1 B and C. tential to impact multiple channels. However, given that CDI of While both the phylogenetic analyses and the molecular dy- CaV1.2 plays a key role in determining the duration of the namics simulations point to the critical importance of the argi- plateau phase of the action potential, it is likely a critical target MEDICAL SCIENCES nine at residue 37 of the neonate paralog of TnI, they indicate for the effect observed in this study. Thus, we hypothesize that very little about the functional impact. Thus, we undertook a this arrhythmogenic effect is due to (i) shortening of the plateau 2+ series of experiments to determine the dynamics of Ca in- phase of the AP by acting on CaV1.2 and (ii) alteration of the teraction with adult and neonate protein complexes as described RyR2 inactivation kinetics, perhaps due to an increased rate of + in SI Appendix, Tables S2 and S3. Ca2 off-loading from the thin filament. + The Ca2 handling differences between WT adult and WT The WT hiPSC-CMs exhibit the classic APD and CaTD res- neonate were very striking despite a relative conservation of the titution patterns by shortening significantly in response to in- Tn complex structure predicted by the molecular dynamics creasing the stimulation frequency from 45 to 65 bpm, whereas +/− simulations. The sensitivity (as reflected by Kd) of Tn and RTF TNNI1 R37C hiPSC-CMs do not. However, to accurately + for Ca2 was 1.7- and 3.1-fold, respectively, higher in the neonate measure APD and CaTD restitution, a broad range of cycle constructs. These striking differences were virtually entirely due lengths must be used. APD restitution is dependent on many to a significant decrease in the koff values in both Tn and RTF factors, but perhaps most prominent is the rate of recovery of the 2+ complexes in the WT neonates in comparison with those from L-type Ca channel (CaV1.2) because (i) it recovers more slowly + adults (Fig. 2). These differences between neonate and adult than the cardiac Na channel (NaV1.5) [but faster than the current contractile function have been well documented in the literature, carried by the slowly activating delayed rectifier potassium channel in which the cardiac contractile apparatus transitions from being (IKs) and the current carried by the rapidly activating delayed 2+ highly sensitive to Ca with slower kinetics of activation and rectifier potassium channel (IKr)], (ii) it provides the major in- relaxation in fetal/neonatal cardiomyocyte to being less sensitive ward current maintaining the action potential plateau, and (iii) 2+ to Ca with higher rates of activation and relaxation in adult APD is more sensitive to CaV1.2 (29). CaTD is dependent on + myocardium (13–17). Our steady-state and kinetic data directly several factors that relate to Ca2 -dependent ion channels, in- 2+ support this observation, that higher Ca sensitivity (lower Kd) cluding the RyR2 activation and inactivation kinetics (30, 31). 2+ +/− and lower Ca dissociation rate (lower koff) are associated with At 75 bpm, more than 85% (7/8) of the TNNI1 R37C + the WT fetal/neonatal Tn and RTF compared with the WT adult hiPSC-CM plates exhibited both AP and Ca2 alternans, whereas + counterparts (SI Appendix, Fig. S5 and Table S4). none of the isogenic WT controls did. Both AP and Ca2 transient + The direct implication of this observation is that a larger Ca2 alternans occurred simultaneously in every instance (Fig. 3D), and + transient would be required to produce the same amount of thus, it is not likely that AP alternans was driving Ca2 alternans. force in the contraction of a cardiomyocyte. This biophysical Because our optical mapping system allows one to examine mul- property would also be predicted to have a strong impact on the tiple ROIs simultaneously, we could determine that the alternans + Ca2 transient in intact cardiomyocytes and could potentially be were spatially concordant (29). Occasionally, we observed tem- arrhythmogenic. The changes in koff, and hence the changes in poral discordance, implying that some hiPSC-CM monolayers + Ca2 sensitivity, have the potential to alter the elec- were not totally functional syncytia. While there is a relationship trical activity of the heart, which could result in varying degrees of between restitution and alternans, the excitation history makes + − arrhythmogenicity. This is well demonstrated in several hypertro- it complicated (29, 32); however, both TNNI1 R37C / and WT phic cardiomyopathy-associated Tn mutations (18–21). To test this hiPSC-CM plates were subjected to identical stimulation para- + hypothesis specifically related to the TNNI1 R37C mutation, we digms. Recently, it was demonstrated in mice that Ca2 alternans used genome-edited and optically mapped hiPSC-CMs. was attributable to RyR2 inactivation kinetics and not SERCA2a + To compare action potential and Ca2 transient durations, it (31). The most parsimonious explanation for these findings is that + was important to entrain the monolayers to the same stimulation the increased Ca2 off-rate constant from the cardiac thin filament +/− rate, which we started at 55 bpm and subsequently increased in in the TNNI1 R37C hiPSC-CMs causes early CDI of CaV1.2 and

Shafaattalab et al. PNAS Latest Articles | 5of6 Downloaded by guest on September 27, 2021 alteration of RyR2 activation and inactivation kinetics, but clearly, Methods further research is required. Evolutionary conservation of relevant troponin I sequences was conducted as As the stimulation frequency was elevated to 75 to 120 bpm, the described in the SI Appendix, Supplemental Methods section. 2+ WT hiPSC-CM plates exhibited no electrical or Ca transient “ir- Molecular dynamics simulations were conducted as described in the SI +/− regularities,” while the TNNI1 R37C hiPSC-CM plates demon- Appendix, Supplemental Methods section. strated progressively aberrant patterns. Initially, there was a decrease Generation of adult and neonatal human cardiac RTFs was as described in in the amplitude of the alternate “beat” until a total block was ob- the SI Appendix, Supplemental Methods section. served. At slightly higher frequencies, this pattern devolved into a Determination of Ca2+ kinetics of neonate RTF measured by steady-state + chaotic pattern for both electrical and Ca2 transients in TNNI1 and stopped-flow fluorometry was as described in the SI Appendix, Sup- + − R37C / hiPSC-CM plates that one might consider reminiscent of plemental Methods section. an in vitro equivalent of ventricular fibrillation. While it has been Human iPSC maintenance and cardiomyocyte differentiation were con- SI Appendix Supplemental Methods well documented that hiPSC-CMs are immature functionally and ducted as described in the , section. morphologically in comparison with native adult cardiomyocytes Preparation and design of CRISPR-Cas9 and the donor template were SI Appendix Supplemental Methods (26), the expression profile of the two paralogs TNNI1 and TNNI3 conducted as described in the , section. SI Appendix and the developmentally regulated splice variants of TNNT2 in these Transfection and FACS were conducted as described in the , Supplemental Methods section. hiPSC-CMs coincide with those of the infant heart (SI Appendix,Fig. Quantitative real-time PCR was conducted as described in the SI Appendix, S7). These data strongly suggest that the 10 infants harboring the Supplemental Methods TNNI1 +/− section. R37C mutation from our previous study and who died Optical mapping for action potential and calcium transient recordings was within 24 mo of age (3) suffered from sudden cardiac death (SCD). conducted as described in the SI Appendix, Supplemental Methods section. One of the major aims of this study was to develop an ex- Data throughout this study were analyzed using the JMP software package perimental pipeline for the analysis and assessment of variants (SAS Institute) as described in the SI Appendix, Supplemental Methods section. identified in sudden infant death syndrome (SIDS) cases. The +/− TNNI R37C variant under investigation was reliably and re- ACKNOWLEDGMENTS. Dr. Miguel Alcaide Torres contributed to the analysis producibly disruptive to the normal expected physiology and in of the TNNI1 variant, and Marvin Gunawan assisted with the genome editing of distinct contrast to results from their controls across a battery of in the hiPSC. The generous support of the Canadian Institutes of Health Research (CIHR) to G.F.T. (Grant PJT-148964) and D.P.T. (Project Program FRN-CIHR: silico and in vitro assays. This strengthens the evidence of path- 156236) is gratefully acknowledged. The Stem Cell Network also made a signif- ogenicity and implicates a neonatal paralog of a gene encoding a icant contribution (to G.F.T.) to support this work (Grant SCN FY19CTA15). This sarcomeric contractile protein as having likely contributed to the work was undertaken, in part, thanks to funding from the Canada Research sudden death of infants through a proarrhythmic pathway. The Chairs program for Tier 1 Chairs (G.F.T. and D.P.T.). D.P.T. holds the Alberta Innovates Technology Futures Strategic Chair in (Bio) Molecular Simulation. platform that we have employed has the potential to be applied A.Y.L. holds a doctoral fellowship from the Natural Sciences and Engineering broadly as a part of a standardized and clinically relevant death Research Council of Canada. The molecular dynamics simulations were carried investigation in cases of SCD where novel variants are identified. out on the Westgrid Complex under the aegis of Compute Canada.

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