Mitochondrial DNA: Hotspot for Potential Gene Modifiers Regulating Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a prevalent and untreatable cardiovascular disease with a highly complex clinical and genetic causation. HCM patients bearing similar sarcomeric mutations display variable clinical outcomes, implying the involvement of gene modifiers that regulate disease progression. As individuals exhibiting mutations in mitochondrial DNA (mtDNA) present cardiac phenotypes, the mitochondrial genome is a promising candidate to harbor gene modifiers of HCM. Herein, we sequenced the mtDNA of isogenic pluripotent stem cell-cardiomyocyte models of HCM focusing on two sarcomeric mutations. This approach was extended to unrelated patient families totaling 52 cell lines. By correlating cellular and clinical phenotypes with mtDNA sequencing, potentially HCM-protective or -aggravator mtDNA variants were identified. These novel mutations were mostly located in the non-coding control region of the mtDNA and did not overlap with those of other mitochondrial diseases. Analysis of unrelated patients highlighted family-specific mtDNA variants, while others were common in particular population haplogroups. Further validation of mtDNA variants as gene modifiers is warranted but limited by the technically challenging methods of editing the mitochondrial genome. Future molecular characterization of these mtDNA variants in the context of HCM may identify novel treatments and facilitate genetic screening in cardiomyopathy patients towards more efficient treatment options.

P5269The impact of sarcomeric mutations on myocardial fibrosis and ventricular diastolic function in hypertrophic cardiomyopathy (SADS-TW HCM registry study)

Background Hypertrophic cardiomyopathy (HCM) may manifest as diastolic dysfunction. The degree of myocardial fibrosis quantified by late gadolinium enhancement (LGE) in cardiac magnetic resonance (CMR) imaging is positively correlated with the risk of sudden cardiac death, whereas the impact of sarcomeric mutations on the ventricular diastolic function and myocardial fibrosis is unclear. Purpose We aimed to investigate the difference of the ventricular diastolic function and the degree of myocardial fibrosis between the HCM patients with and without sarcomeric mutation. Methods From 2014 to 2018, we prospectively enrolled 55 unrelated patients with HCM as defined by the 2014 European Society of Cardiology guideline. All enrolled patients underwent next-generation sequencing screening of 20 sarcomeric genes and CMR examination for the evaluation of left ventricular (LV) function, mass and LGE. Results After comparing the results with several public databases (Taiwan Biobank, gnomAD, HGMD, ClinVar) and performing in silico analyses (SIFT, Polyphen-2, PROVEAN, REVEL, CADD), 24 pathogenic variants were identified in 22 HCM patients. Although there were no differences in demographic data and clinical presentations between the mutation-positive and mutation-negative groups, the degree of LGE (14.2±14.3 vs 6.2±8.9%, p=0.015) and left atrial diameter (4.54±0.63 vs 4.00±0.49 cm, p<0.001) were significantly higher in the mutation-positive group, whereas the LV ejection fraction, mass, strain rates, peak ejection and filling rates, peak intra-LV and tricuspid regurgitation pressure gradient were similar in both groups. Table 1. Comparisons of the CMR parameters of HCM patients with and without sarcomeric mutation Sarcomeric mutation (+, M+), Sarcomeric mutation (−, M−), Control (C), M+ vs M− M+ vs C M− vs C n=21 n=34 n=36 P-value P-value P-value PER/LVEDV, L/s −4.73±1.18 −5.03±1.19 −3.49±0.78 0.361 <0.001* <0.001* PFR/LVEDV, L/s 4.05±1.00 3.74±1.25 5.52±1.20 0.341 <0.001* <0.001* Global radial diastolic strain rate, /s −2.49±0.99 −2.74±1.51 −2.68±0.88 0.527 0.210 0.846 Global circumferential diastolic strain rate, /s 0.89±0.19 0.92±0.30 1.49±0.32 0.684 <0.001* <0.001* Global longitudinal diastolic strain rate, /s 0.64±0.19 0.62±0.24 0.95±0.27 0.744 <0.001* <0.001* C: control; LVEDV: left ventricular end-diastolic volume; M+: sarcomeric mutation (+); M−: sarcomeric mutation (−); PER: peak ejection rate; PFR: peak filling rate. *p<0.05. Conclusions The HCM patients with sarcomeric mutations had a higher degree of LV myocardial fibrosis than patients without mutations, which may imply that these mutations accelerate myocardial fibrosis in HCM. Nonetheless, there was no difference in diastolic function between the patients with and without sarcomeric mutation. Acknowledgement/Funding None

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

Sudden unexpected death of an infant (SUDI) is a devastating occurrence for families. To investigate the genetic pathogenesis of SUDI, we sequenced >70 genes from 191 autopsy-negative SUDI victims. Ten infants sharing a previously unknown variant in troponin I (TnI) were identified. The mutation (TNNI1R37C+/−) is in the fetal/neonatal paralog of TnI, a gene thought to be expressed in the heart up to the first 24 months of life. Using phylogenetic analysis and molecular dynamics simulations, it was determined that arginine at residue 37 inTNNI1may play a critical functional role, suggesting that the variant may be pathogenic. We investigated the biophysical properties of theTNNI1R37C mutation in human reconstituted thin filaments (RTFs) using fluorometry. RTFs reconstituted with the mutant R37C TnI exhibited reduced Ca2+-binding sensitivity due to an increased Ca2+off-rate constant. Furthermore, we generatedTNNI1R37C+/−mutants in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) using CRISPR-Cas9. In monolayers of hiPSC-CMs, we simultaneously monitored voltage and Ca2+transients through optical mapping and compared them to their isogenic controls. We observed normal intrinsic beating patterns under control conditions inTNNI1R37C+/−at stimulation frequencies of 55 beats/min (bpm), but these cells showed no restitution with increased stimulation frequency to 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 critical physiological and mechanistic bases to investigate sarcomeric mutations in the pathogenesis of SUDI.

Which way to grow? Force over time may be the heart’s Dao de jing

Genetic cardiomyopathy manifests as either a hypertrophic or dilated phenotype. However, molecular mechanisms that determine which disease pathway emerges in patients is largely unknown. Work from the Molkentin laboratory published in the May issue of the journal Cell provides novel insights into this fundamental question. The investigators found that sarcomeric mutations associated with a reduced muscle contraction-time integral resulted in a dilated cardiomyopathy, while mutations associated with an increase in this parameter were associated with a hypertrophic phenotype. The molecular cellular cues that orchestrate which cardiomyopathic pathway ensues appear to be the signal transduction pathways involving the molecules MEK1 and ERK1/2. The identified signals driving overall growth of the heart, on the either hand, were found to involve Calcineurin and NFAT. These findings may help improve treatment strategies aimed to combat familial cardiopathy and, moreover, pave the way to the development of novel personalized medicine based therapy by using cardiac cells that are derived from individual patient’s induced pluripotent stem (iPS) cells.