The origins of unpredictability in life outcome prediction tasks.

Why are some life outcomes difficult to predict? We investigated this question through in-depth qualitative interviews with 40 families sampled from a multidecade longitudinal study. Our sampling and interviewing process was informed by the earlier efforts of hundreds of researchers to predict life outcomes for participants in this study. The qualitative evidence we uncovered in these interviews combined with a mathematical decomposition of prediction error led us to create a conceptual framework. Our specific evidence and our more general framework suggest that unpredictability should be expected in many life outcome prediction tasks, even in the presence of complex algorithms and large datasets. Our work provides a foundation for future empirical and theoretical work on unpredictability in human lives.

Transcriptome studies of congenital heart diseases: identifying current gaps and therapeutic frontiers.

Congenital heart disease (CHD) are genetically complex and comprise a wide range of structural defects that often predispose to - early heart failure, a common cause of neonatal morbidity and mortality. Transcriptome studies of CHD in human pediatric patients indicated a broad spectrum of diverse molecular signatures across various types of CHD. In order to advance research on congenital heart diseases (CHDs), we conducted a detailed review of transcriptome studies on this topic. Our analysis identified gaps in the literature, with a particular focus on the cardiac transcriptome signatures found in various biological specimens across different types of CHDs. In addition to translational studies involving human subjects, we also examined transcriptomic analyses of CHDs in a range of model systems, including iPSCs and animal models. We concluded that RNA-seq technology has revolutionized medical research and many of the discoveries from CHD transcriptome studies draw attention to biological pathways that concurrently open the door to a better understanding of cardiac development and related therapeutic avenue. While some crucial impediments to perfectly studying CHDs in this context remain obtaining pediatric cardiac tissue samples, phenotypic variation, and the lack of anatomical/spatial context with model systems. Combining model systems, RNA-seq technology, and integrating algorithms for analyzing transcriptomic data at both single-cell and high throughput spatial resolution is expected to continue uncovering unique biological pathways that are perturbed in CHDs, thus facilitating the development of novel therapy for congenital heart disease.

Mitochondrial MICOS complex genes, implicated in hypoplastic left heart syndrome, maintain cardiac contractility and actomyosin integrity.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with a likely oligogenic etiology, but our understanding of the genetic complexities and pathogenic mechanisms leading to HLHS is limited. We performed whole genome sequencing (WGS) on 183 HLHS patient-parent trios to identify candidate genes, which were functionally tested in the Drosophila heart model. Bioinformatic analysis of WGS data from an index family of a HLHS proband born to consanguineous parents prioritized 9 candidate genes with rare, predicted damaging homozygous variants. Of them, cardiac-specific knockdown (KD) of mitochondrial MICOS complex subunit dCHCHD3/6 resulted in drastically compromised heart contractility, diminished levels of sarcomeric actin and myosin, reduced cardiac ATP levels, and mitochondrial fission-fusion defects. These defects were similar to those inflicted by cardiac KD of ATP synthase subunits of the electron transport chain (ETC), consistent with the MICOS complex's role in maintaining cristae morphology and ETC assembly. Five additional HLHS probands harbored rare, predicted damaging variants in CHCHD3 or CHCHD6. Hypothesizing an oligogenic basis for HLHS, we tested 60 additional prioritized candidate genes from these patients for genetic interactions with CHCHD3/6 in sensitized fly hearts. Moderate KD of CHCHD3/6 in combination with Cdk12 (activator of RNA polymerase II), RNF149 (goliath, E3 ubiquitin ligase), or SPTBN1 (ß-Spectrin, scaffolding protein) caused synergistic heart defects, suggesting the likely involvement of diverse pathways in HLHS. Further elucidation of novel candidate genes and genetic interactions of potentially disease-contributing pathways is expected to lead to a better understanding of HLHS and other CHDs.

SARS-CoV-2 spike protein-mediated cardiomyocyte fusion may contribute to increased arrhythmic risk in COVID-19.

BACKGROUND: SARS-CoV-2-mediated COVID-19 may cause sudden cardiac death (SCD). Factors contributing to this increased risk of potentially fatal arrhythmias include thrombosis, exaggerated immune response, and treatment with QT-prolonging drugs. However, the intrinsic arrhythmic potential of direct SARS-CoV-2 infection of the heart remains unknown. OBJECTIVE: To assess the cellular and electrophysiological effects of direct SARS-CoV-2 infection of the heart using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). METHODS: hiPSC-CMs were transfected with recombinant SARS-CoV-2 spike protein (CoV-2 S) or CoV-2 S fused to a modified Emerald fluorescence protein (CoV-2 S-mEm). Cell morphology was visualized using immunofluorescence microscopy. Action potential duration (APD) and cellular arrhythmias were measured by whole cell patch-clamp. Calcium handling was assessed using the Fluo-4 Ca2+ indicator. RESULTS: Transfection of hiPSC-CMs with CoV-2 S-mEm produced multinucleated giant cells (syncytia) displaying increased cellular capacitance (75±7 pF, n = 10 vs. 26±3 pF, n = 10; P<0.0001) consistent with increased cell size. The APD90 was prolonged significantly from 419±26 ms (n = 10) in untransfected hiPSC-CMs to 590±67 ms (n = 10; P<0.05) in CoV-2 S-mEm-transfected hiPSC-CMs. CoV-2 S-induced syncytia displayed delayed afterdepolarizations, erratic beating frequency, and calcium handling abnormalities including calcium sparks, large "tsunami"-like waves, and increased calcium transient amplitude. After furin protease inhibitor treatment or mutating the CoV-2 S furin cleavage site, cell-cell fusion was no longer evident and Ca2+ handling returned to normal. CONCLUSION: The SARS-CoV-2 spike protein can directly perturb both the cardiomyocyte's repolarization reserve and intracellular calcium handling that may confer the intrinsic, mechanistic substrate for the increased risk of SCD observed during this COVID-19 pandemic.

Autologous Umbilical Cord Blood-Derived Mononuclear Cell Therapy Promotes Cardiac Proliferation and Adaptation in a Porcine Model of Right Ventricle Pressure Overload.

Congenital heart diseases, including single ventricle circulations, are clinically challenging due to chronic pressure overload and the inability of the myocardium to compensate for lifelong physiological demands. To determine the clinical relevance of autologous umbilical cord blood-derived mononuclear cells (UCB-MNCs) as a therapy to augment cardiac adaptation following surgical management of congenital heart disease, a validated model system of right ventricular pressure overload due to pulmonary artery banding (PAB) in juvenile pigs has been employed. PAB in a juvenile porcine model and intramyocardial delivery of UCB-MNCs was evaluated in three distinct 12-week studies utilizing serial cardiac imaging and end-of-study pathology evaluations. PAB reproducibly induced pressure overload leading to chronic right ventricular remodeling including significant myocardial fibrosis and elevation of heart failure biomarkers. High-dose UCB-MNCs (3 million/kg) delivered into the right ventricular myocardium did not cause any detectable safety issues in the context of arrhythmias or abnormal cardiac physiology. In addition, this high-dose treatment compared with placebo controls demonstrated that UCB-MNCs promoted a significant increase in Ki-67-positive cardiomyocytes coupled with an increase in the number of CD31+ endothelium. Furthermore, the incorporation of BrdU-labeled cells within the myocardium confirmed the biological potency of the high-dose UCB-MNC treatment. Finally, the cell-based treatment augmented the physiological adaptation compared with controls with a trend toward increased right ventricular mass within the 12 weeks of the follow-up period. Despite these adaptations, functional changes as measured by echocardiography and magnetic resonance imaging did not demonstrate differences between cohorts in this surgical model system. Therefore, this randomized, double-blinded, placebo-controlled pre-clinical trial establishes the safety of UCB-MNCs delivered via intramyocardial injections in a dysfunctional right ventricle and validates the induction of cardiac proliferation and angiogenesis as transient paracrine mechanisms that may be important to optimize long-term outcomes for surgically repaired congenital heart diseases.

Uncoupling of Proliferative Capacity from Developmental Stage During Directed Cardiac Differentiation of Pluripotent Stem Cells.

Lineage-specific differentiation of human-induced pluripotent stem cells (hiPSCs) into cardiomyocytes (CMs) offers a patient-specific model to dissect development and disease pathogenesis in a dish. However, challenges exist with this model system, such as the relative immaturity of iPSC-derived CMs, which evoke the question of whether this model faithfully recapitulates in vivo cardiac development. As in vivo cardiac developmental stage is intimately linked with the proliferative capacity (or maturation is inversely correlated to proliferative capacity), we sought to understand how proliferation is regulated during hiPSC CM differentiation and how it compares with in vivo mouse cardiac development. Using standard Chemically Defined Media 3 differentiation, gene expression profiles demonstrate that hiPSC-derived cardiomyocytes (hiPSC-CMs) do not progress past the equivalent of embryonic day 14.5 of murine cardiac development. Throughout differentiation, overall DNA synthesis rapidly declines with <5% of hiPSC-CMs actively synthesizing DNA at the end of the differentiation period despite their immaturity. Bivariate cell cycle analysis demonstrated that hiPSC-CMs have a cell cycle profile distinct from their non-cardiac counterparts from the same differentiation, with significantly fewer cells within G1 and a marked accumulation of cells in G2/M than their non-cardiac counterparts throughout differentiation. Pulse-chase analysis demonstrated that non-cardiac cells progressed completely through the cell cycle within a 24-h period, whereas hiPSC-CMs had restricted progression with only a small proportion of cells undergoing cytokinesis with the remainder stalling in late S-phase or G2/M. This cell cycle arrest phenotype is associated with abbreviated expression of cell cycle promoting genes compared with expression throughout murine embryonic cardiac development. In summary, directed differentiation of hiPSCs into CMs uncouples the developmental stage from cell cycle regulation compared with in vivo mouse cardiac development, leading to a premature exit of hiPSC-CMs from the cell cycle despite their relative immaturity.

Identifying gaps in parental support for families of children with hypoplastic left heart syndrome.

PURPOSE: The purpose of this study is to identify gaps in support for parents of children with Hypoplastic Left Heart Syndrome. DESIGN AND METHODS: Using a mixed-methods approach, the researchers first studied the parental and care team experience through interviews of Hypoplastic Left Heart Syndrome mothers and members of the inter-professional care team and then conducted an international survey of 690 Hypoplastic Left Heart Syndrome primary caregivers to validate the qualitative findings. RESULTS: Parental and care team interviews revealed three main gaps in parental support, including lack of open communication, unrealistic parental expectations, and unclear inter-professional team roles. Survey results found that parents whose children were diagnosed with Hypoplastic Left Heart Syndrome after birth indicated significant dissatisfaction with the care team's open communication and welcoming of feedback (p = 0.008). As parents progress through the stages of surgical intervention, they also indicate significant dissatisfaction with the care team's anticipation of parental emotional needs and provision of coping resources (p = 0.003). CONCLUSIONS: Parental support interventions should focus on providing resources to help parents cope, helping the care team model open communication, and welcoming feedback on the parental experience. PRACTICE IMPLICATIONS: Interventions should be piloted with parents who are in the later stages of the surgical intervention timeline or whose children were diagnosed after birth as they are the populations who perceived the least support within this study.

From Safety to Benefit in Cell Delivery During Surgical Repair of Ebstein Anomaly: Initial Results.

BACKGROUND: The objective of this study is to assess the safety and early impact of intramyocardial delivery of autologous bone marrow-derived mononuclear cells (BM-MNC) at time of surgical Ebstein repair. METHODS: Patients with Ebstein anomaly (ages 6 months to 30 years) scheduled to undergo repair of the tricuspid valve were eligible to participate in this open-label, non-randomized phase I clinical trial. BM-MNC target dose was 1-3 million cells/kg. Ten patients have undergone surgical intervention and cell delivery to the right ventricle (RV) and completed 6-month follow-up. RESULTS: All patients underwent surgical tricuspid valve repair and uneventful BM-MNC delivery; there were no ventricular arrhythmias and no adverse events related to study product or delivery. Echocardiographic RV myocardial performance index improved and RV fractional area change showed an initial decline and then through study follow-up. There was no evidence of delayed myocardial enhancement or regional wall motion abnormalities at injection sites on 6-month follow-up magnetic resonance imaging. CONCLUSIONS: Intramyocardial delivery of BM-MNC after surgical repair in Ebstein anomaly can be performed safely. Echocardiography variables suggest a positive impact of cell delivery on the RV myocardium with improvements in both RV size and wall motion over time. Additional follow-up and comparison to control groups are required to better characterize the impact of cell therapy on the myopathic RV in Ebstein anomaly.

Shotgun Immunoproteomics for Identification of Nonhuman Leukocyte Antigens Associated With Cellular Dysfunction in Heart Transplant Rejection.

BACKGROUND: The International Society for Heart and Lung Transplant consensus panel notes that too little data exist regarding the role of non-HLA in allograft rejection. We developed a novel shotgun immunoproteomic approach to determine the identities and potential roles non-HLA play in antibody-mediated rejection (AMR) in heart transplant recipients. METHODS: Serum was collected longitudinally from heart transplant recipients experiencing AMR in the absence of donor-specific anti-HLA antibodies (n = 6) and matched no rejection controls (n = 7). Antidonor heart affinity chromatography columns were formed by recipient immunoglobulin G immobilization at transplantation, acute rejection, and chronic postrejection time points. Affinity chromatography columns were used to capture antigens from individual patient's donor heart biopsies collected at transplantation. Captured proteins were subjected to quantitative proteomic analysis and the longitudinal response was calculated. RESULTS: Overlap in antigen-specific response between AMR and non-AMR patients was only 8.3%. In AMR patients, a total of 155 non-HLAs were identified, with responses toward 43 high prevalence antigens found in ≥50% of patients. Immunofluorescence staining for representative high prevalence antigens demonstrated that their abundance increased at acute rejection, correlating with their respective non-HLA antibody response. Physiological changes in cardiomyocyte and endothelial cell function, following in vitro culture with patient immunoglobulin G, correlated with response toward several high prevalence antigens. CONCLUSIONS: This work demonstrates a novel high-throughput strategy to identify clinically relevant non-HLA from donor endomyocardial biopsy. Such a technique has the potential to improve understanding of longitudinal timing of antigen-specific responses and their cause and effect relationship in graft rejection.

Activation of P53 Via Nutlin-3a Reveals Role for P53 In ROS Signaling During Cardiac Differentiation of hiPSCs.

Activation of the transcription factor P53 within cancer cells is a well-characterized pathway, whereas the effects of P53 activation during development remain largely unexplored. Previous research has indicated that increased levels of P53 protein during key murine developmental stages cause defects in multiple embryonic tissues, including the heart. These findings were confirmed in several different mouse models of congenital heart defects, but P53 activation in a human system of cardiovascular development is not available. Utilizing human induced pluripotent stem cells (hiPSCs), we characterized the normal levels of P53 during cardiac differentiation and showed that levels of P53 are high in hiPSCs and decrease upon cardiac lineage commitment. We also observed P53 localization changed from mainly cytoplasmic in iPS colonies to the nucleus in the Nkx2-5 + cardiac progenitor stage. Pharmacological-mediated increase of P53 protein levels with the Mdm2 inhibitor Nutlin-3a during early (mesoderm to cardiac mesoderm) stages of cardiogenesis resulted in a sizeable loss of cardiomyocytes due to increased apoptosis and cell cycle arrest. Interestingly, increasing P53 levels did not result in apoptosis at later (cardiac progenitor to beating cardiomyocytes) stages of the cardiac differentiation. These results illustrate the temporal sensitivity to increased P53 levels during cardiogenesis. We conducted RNA-Seq on these cells with or without Nutlin-3a to ascertain transcriptional differences due to increased P53 at the different stages during the differentiation. Our results from the RNA-Seq revealed up-regulation of Sestrins after Nutlin-3a treatment suggesting a new role for P53 in the metabolism of cardiac regeneration.

Cone Reconstruction for Ebstein Anomaly: Ventricular Remodeling and Preliminary Impact of Stem Cell Therapy.

OBJECTIVE: To define the impact of tricuspid valve cone reconstruction (CR) on ventricular performance in Ebstein anomaly, both independently and after stem cell therapy. PATIENTS AND METHODS: The control group included 257 patients who had CR between June 2007 and December 2019. Ten subjects of a phase I stem cell therapy trial (May 2017 - March 2019) were compared with the controls to assess the echocardiographic impact on ventricular remodeling. RESULTS: After CR, right ventricular (RV) size decreased and left ventricular (LV) volume increased in all patients. Apical and biplane RV fractional area change (FAC) initially decreased, but rebounded by 6 months postoperation. Short-axis FAC increased early and was maintained at 6 months post-CR in the control group. At 6 months post-CR, cell therapy patients showed a significantly larger increase in short-axis FAC (24.4% vs 29.9%, P=.003). In addition, whereas LV ejection fraction (EF) was unchanged at 6 months post-CR in controls, cell therapy patients showed a significant increase in EF relative to baseline and to controls (55.6% vs 65.0%, P=.007). CONCLUSION: Cone reconstruction reduces tricuspid regurgitation and RV size, but is also associated with increased RV FAC and LV volume. Furthermore, injection of bone marrow-derived stem cells augmented the increase in RV FAC and was associated with improved LV EF at 6 months post-CR. This is evidence of a favorable interventricular interaction. These findings provide motivation for continued investigation into the potential benefits of stem cell therapy in Ebstein anomaly and other congenital cardiac malformations. TRIAL REGISTRATION: clinicaltrials.gov identifier: NCT02914171.

Clinical Impact of Autologous Cell Therapy on Hypoplastic Left Heart Syndrome After Bidirectional Cavopulmonary Anastomosis.

Preservation of right ventricle function (RV) is a key to favorable outcome in Hypoplastic Left Heart Syndrome (HLHS), but methods to preserve or improve RV function are limited. Our goal was to assess the clinical and functional impact of autologous umbilical cord blood-derived mononuclear cells (UCB-MNC) therapy when given to patients with HLHS at Stage II surgery. UCB-MNC patients were enrolled prospectively in a phase I, FDA monitored trial as previously described (Burkhart et al., 2019). Matched retrospective controls were identified by review of clinical databases. Growth and RV echocardiographic variables were assessed in both groups prestage II through the first 6 months postoperatively. Statistical comparisons between the groups at similar postoperative time points were made to define potential impact of the cell therapy. There were 7 UCB-MNC patients and 17 controls. Prestage II, most parameters showed no differences between groups, although median fractional area change (FAC) was slightly greater in the controls (FAC: controls = 45% vs UCB-MNC = 41% P= 0.02). At dismissal, FAC and estimated Ejection Fraction (EF) decreased in controls, while both were unchanged from baseline in UCB-MNC patients (ΔFAC: -5% vs -1%, P < 0.01; ΔEF: -8% vs 0%, P = 0.03, respectively). Subsequently, median FAC increased slightly in UCB-MNC patients over the 6 month follow-up period, while it decreased in controls (ΔFAC: UCB-MNC +3% vs control -5%, P = 0.03). Preoperative weight percentiles were similar in both groups (UCB-MNC 34%ile vs controls 22%ile, P = 0.93). However, by 6 months postoperative, median weight percentile improved to 63% in the UCB-MNC treated group, but declined to 8% in controls (P = 0.02). UCB-MNC therapy appears to limit the initial negative impact on RV FAC and EF seen after stage II surgery. During early follow up, FAC and weight percentile improved in UCB-MNC patients relative to controls, suggesting a beneficial effect of UCB-MNC therapy.

Dysregulated ribonucleoprotein granules promote cardiomyopathy in RBM20 gene-edited pigs.

Ribonucleoprotein (RNP) granules are biomolecular condensates-liquid-liquid phase-separated droplets that organize and manage messenger RNA metabolism, cell signaling, biopolymer assembly, biochemical reactions and stress granule responses to cellular adversity. Dysregulated RNP granules drive neuromuscular degenerative disease but have not previously been linked to heart failure. By exploring the molecular basis of congenital dilated cardiomyopathy (DCM) in genome-edited pigs homozygous for an RBM20 allele encoding the pathogenic R636S variant of human RNA-binding motif protein-20 (RBM20), we discovered that RNP granules accumulated abnormally in the sarcoplasm, and we confirmed this finding in myocardium and reprogrammed cardiomyocytes from patients with DCM carrying the R636S allele. Dysregulated sarcoplasmic RBM20 RNP granules displayed liquid-like material properties, docked at precisely spaced intervals along cytoskeletal elements, promoted phase partitioning of cardiac biomolecules and fused with stress granules. Our results link dysregulated RNP granules to myocardial cellular pathobiology and heart failure in gene-edited pigs and patients with DCM caused by RBM20 mutation.

Patient-specific genomics and cross-species functional analysis implicate LRP2 in hypoplastic left heart syndrome.

Congenital heart diseases (CHDs), including hypoplastic left heart syndrome (HLHS), are genetically complex and poorly understood. Here, a multidisciplinary platform was established to functionally evaluate novel CHD gene candidates, based on whole-genome and iPSC RNA sequencing of a HLHS family-trio. Filtering for rare variants and altered expression in proband iPSCs prioritized 10 candidates. siRNA/RNAi-mediated knockdown in healthy human iPSC-derived cardiomyocytes (hiPSC-CM) and in developing Drosophila and zebrafish hearts revealed that LDL receptor-related protein LRP2 is required for cardiomyocyte proliferation and differentiation. Consistent with hypoplastic heart defects, compared to patents the proband's iPSC-CMs exhibited reduced proliferation. Interestingly, rare, predicted-damaging LRP2 variants were enriched in a HLHS cohort; however, understanding their contribution to HLHS requires further investigation. Collectively, we have established a multi-species high-throughput platform to rapidly evaluate candidate genes and their interactions during heart development, which are crucial first steps toward deciphering oligogenic underpinnings of CHDs, including hypoplastic left hearts.

Intrinsic Endocardial Defects Contribute to Hypoplastic Left Heart Syndrome.

Hypoplastic left heart syndrome (HLHS) is a complex congenital heart disease characterized by abnormalities in the left ventricle, associated valves, and ascending aorta. Studies have shown intrinsic myocardial defects but do not sufficiently explain developmental defects in the endocardial-derived cardiac valve, septum, and vasculature. Here, we identify a developmentally impaired endocardial population in HLHS through single-cell RNA profiling of hiPSC-derived endocardium and human fetal heart tissue with an underdeveloped left ventricle. Intrinsic endocardial defects contribute to abnormal endothelial-to-mesenchymal transition, NOTCH signaling, and extracellular matrix organization, key factors in valve formation. Endocardial abnormalities cause reduced cardiomyocyte proliferation and maturation by disrupting fibronectin-integrin signaling, consistent with recently described de novo HLHS mutations associated with abnormal endocardial gene and fibronectin regulation. Together, these results reveal a critical role for endocardium in HLHS etiology and provide a rationale for considering endocardial function in regenerative strategies.

Loss of Ventricular Function After Bidirectional Cavopulmonary Connection: Who Is at Risk?

Decline of single ventricle systolic function after bidirectional cavopulmonary connection (BDCPC) is thought to be a transient phenomenon. We analyzed ventricular function after BDCPC according to ventricular morphology and correlated this evolution to long-term prognosis. A review from Mayo Clinic databases was performed. Visually estimated ejection fraction (EF) was reported from pre-BDCPC to pre-Fontan procedure. The last cardiovascular update was collected to assess long-term prognosis. A freedom from major cardiac event survival curve and a risk factor analysis were performed. 92 patients were included; 52 had left ventricle (LV) morphology and 40 had right ventricle (RV) morphology (28/40 had hypoplastic left heart syndrome (HLHS)). There were no significant differences in groups regarding BDCPC procedure or immediate post-operative outcome. EF showed a significant and relevant decrease from baseline to discharge in the HLHS group: 59 ± 4% to 49 ± 7% or - 9% (p < 0.01) vs. 58 ± 3% to 54 ± 6% or - 4% in the non-HLHS RV group (p = 0.04) and 61 ± 4% to 60 ± 4% or - 1% in the LV group (p = 0.14). Long-term recovery was the least in the HLHS group: EF prior to Fontan 54 ± 2% vs. 56 ± 6% and 60 ± 4%, respectively (p < 0.01). With a median follow-up of 8 years post-BDCPC, six patients had Fontan circulation failure, four died, and three had heart transplantation. EF less than 50% at hospital discharge after BDCPC was strongly correlated to these major cardiac events (HR 3.89; 95% Cl 1.04-14.52). Patients with HLHS are at great risk of ventricular dysfunction after BDCPC. This is not a transient phenomenon and contributes to worse prognosis.

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.