Allele-Specific Suppression of Variant MHC With High-Precision RNA Nuclease CRISPR-Cas13d Prevents Hypertrophic Cardiomyopathy.

BACKGROUND: Familial hypertrophic cardiomyopathy has severe clinical complications of heart failure, arrhythmia, and sudden cardiac death. Heterozygous single nucleotide variants (SNVs) of sarcomere genes such as MYH7 are the leading cause of this type of disease. CRISPR-Cas13 (clustered regularly interspaced short palindromic repeats and their associated protein 13) is an emerging gene therapy approach for treating genetic disorders, but its therapeutic potential in genetic cardiomyopathy remains unexplored. METHODS: We developed a sensitive allelic point mutation reporter system to screen the mutagenic variants of Cas13d. On the basis of Cas13d homology structure, we rationally designed a series of Cas13d variants and obtained a high-precision Cas13d variant (hpCas13d) that specifically cleaves the MYH7 variant RNAs containing 1 allelic SNV. We validated the high precision and low collateral cleavage activity of hpCas13d through various in vitro assays. We generated 2 HCM mouse models bearing distinct MYH7 SNVs and used adenovirus-associated virus serotype 9 to deliver hpCas13d specifically to the cardiomyocytes. We performed a large-scale library screening to assess the potency of hpCas13d in resolving 45 human MYH7 allelic pathogenic SNVs. RESULTS: Wild-type Cas13d cannot distinguish and specifically cleave the heterozygous MYH7 allele with SNV. hpCas13d, with 3 amino acid substitutions, had minimized collateral RNase activity and was able to resolve various human MYH7 pathological sequence variations that cause hypertrophic cardiomyopathy. In vivo application of hpCas13d to 2 hypertrophic cardiomyopathy models caused by distinct human MYH7 analogous sequence variations specifically suppressed the altered allele and prevented cardiac hypertrophy. CONCLUSIONS: Our study unveils the great potential of CRISPR-Cas nucleases with high precision in treating inheritable cardiomyopathy and opens a new avenue for therapeutic management of inherited cardiac diseases.

Fabry disease: development and progression of left ventricular hypertrophy despite long-term enzyme replacement therapy.

BACKGROUND: Enzyme replacement therapy (ERT) may halt or attenuate disease progression in patients with Anderson-Fabry disease (AFD). However, whether left ventricular hypertrophy (LVH) can be prevented by early therapy or may still progress despite ERT over a long-term follow-up is still unclear. METHODS: Consecutive patients with AFD from the Independent Swiss-Fabry Cohort receiving ERT who were at least followed up for 5 years were included. Cardiac progression was defined as an increase of >10 g/m2 in left ventricular mass index (LVMI) between the first and the last available follow-up transthoracic echocardiography. RESULTS: 60 patients (35 (23-48) years, 39 (65%) men) were followed up for 10.5 (7.2-12.2) years. 22 had LVH at ERT start (LVMI of 150±38 g/m2). During follow-up, 22 (36%, 34±15 years) had LVMI progression of 12.1 (7-17.6) g/m2 per 100 patient-years, of these 7 (11%, 29±13 years) with no LVH at baseline. Three of them progressed to LVH. LVMI progression occurred mostly in men (17 of 39 (43%) vs 5 of 21 (24%), p<0.01) and after the age of 30 years (17 of 22 (77%)). LVH at ERT start was associated with LVMI progression (OR 1.3, 95% CI 1.1 to 2.6; p=0.02). A total of 19 (31%) patients experienced a major AFD-related event. They were predominantly men (17 of 19, 89%), older (45±11 vs 32±9 years) with baseline LVH (12 of 19, 63%), and 10 of 19 (52%) presented with LVMI progression. CONCLUSIONS: Over a median follow-up of >10 years under ERT, 36% of the patients still had LVMI cardiac progression, and 32%, predominantly older men, experienced major AFD-related events. LVH at treatment initiation was a strong predictor of LVMI progression and adverse events on ERT.

Machine learning-based detection of sleep-disordered breathing in hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is often concomitant with sleep-disordered breathing (SDB), which can cause adverse cardiovascular events. Although an appropriate approach to SDB prevents cardiac remodelling, detection of concomitant SDB in patients with HCM remains suboptimal. Thus, we aimed to develop a machine learning-based discriminant model for SDB in HCM. METHODS: In the present multicentre study, we consecutively registered patients with HCM and performed nocturnal oximetry. The outcome was a high Oxygen Desaturation Index (ODI), defined as 3% ODI >10, which significantly correlated with the presence of moderate or severe SDB. We randomly divided the whole participants into a training set (80%) and a test set (20%). With data from the training set, we developed a random forest discriminant model for high ODI based on clinical parameters. We tested the ability of the discriminant model on the test set and compared it with a previous logistic regression model for distinguishing SDB in patients with HCM. RESULTS: Among 369 patients with HCM, 228 (61.8%) had high ODI. In the test set, the area under the receiver operating characteristic curve of the discriminant model was 0.86 (95% CI 0.77 to 0.94). The sensitivity was 0.91 (95% CI 0.79 to 0.98) and specificity was 0.68 (95% CI 0.48 to 0.84). When the test set was divided into low-probability and high-probability groups, the high-probability group had a higher prevalence of high ODI than the low-probability group (82.4% vs 17.4%, OR 20.9 (95% CI 5.3 to 105.8), Fisher's exact test p<0.001). The discriminant model significantly outperformed the previous logistic regression model (DeLong test p=0.03). CONCLUSIONS: Our study serves as the first to develop a machine learning-based discriminant model for the concomitance of SDB in patients with HCM. The discriminant model may facilitate cost-effective screening tests and treatments for SDB in the population with HCM.

Frequency of misdiagnosis in hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic Cardiomyopathy (HCM) is characterized by unexplained left ventricle hypertrophy (LVH) ≥15 mm. The condition is often hereditary and family screening is recommended to reduce the risk of adverse disease complications and premature death among relatives. Correct diagnosis of index patients is important to ensure that only relatives at risk of disease development are invited for family screening. PURPOSE: To investigate if patients with ICD-10 codes for HCM (DI421) or hypertrophic obstructive cardiomyopathy (DI422) fulfilled recognised diagnostic criteria. METHODS: All patients with ICD-10 codes for HCM or HOCM at a Department of Cardiology were identified and had their diagnosis validated by a cardiac investigation or a review of their medical records and previous investigations. RESULTS: Two hundred and forty patients had ICD-10 codes for HCM/HOCM, of whom 202 (84%, 202/240) underwent re-examination, while 38 (16%, 38/240) had their hospital notes reviewed. Seventy-six patients (32%, n = 76/240) did not fulfil diagnostic criteria, of whom 39, (51%, n = 39/76) had normal (10 mm) or modest LV wall thickness (11-14 mm). The remaining 37 patients (49%, n = 37/76) had LVH ≥15 mm, which was well-explained by uncontrolled hypertension, (32%, n = 24/76), aortic valve stenosis (19%, n = 7/76) or wild-type amyloidosis (16%, 6/76). CONCLUSION: One-third of patients with ICD-10 codes for HCM or HOCM did not fulfil recognised diagnostic criteria. Incorrect diagnosis of HCM may cause unnecessary family investigations which may be associated with anxiety, and a waste of health care resources. This highlights the need for specialised cardiomyopathy services to ensure correct diagnosis and management of HCM.

Incidence and risk factors for development of left ventricular hypertrophy in Fabry disease.

BACKGROUND: Left ventricular hypertrophy (LVH) is the principal cardiac manifestation of Fabry disease (FD). This study aimed to determine the incidence and predictors of LVH development in a contemporary cohort of patients with FD and no LVH at baseline evaluation. METHODS: Consecutively referred adult (aged ≥16 years) patients with FD were enrolled into an observational cohort study. Patients were prospectively followed in a specialist cardiomyopathy centre and the primary endpoint was the first detection of LVH (left ventricular mass index (LVMi) ≥115 g/m2 in men and ≥95 g/m2 in women). RESULTS: From a cohort of 393 patients, 214 (aged 35.8±13.8 years; 61 (29%) males) had no LVH at first evaluation. During a median follow-up of 9.4 years (IQR 4.7-12.7), 55 patients (24.6%) developed LVH. The estimated incidence of LVH was 11.3% (95% CI 6.5% to 16.1%) at 5 years, 29.1% (95% CI 21.5% to 36.7%) at 10 years and 45.0% (95% CI 33.8% to 62.4%) at 15 years of follow-up. On multivariable analysis, independent predictors for LVH development were age (HR 1.04 (95% CI 1.02 to 1.06) per 1-year increase, p<0.001), male sex (HR 2.90 (95% CI 1.66 to 5.09), p<0.001) and an abnormal ECG (HR 3.10 (95% CI 1.72 to 5.57), p<0.001). The annual rate of change in LVMi was +2.77 (IQR 1.45-4.62) g/m2/year in males and +1.38 (IQR 0.09-2.85) g/m2/year in females (p<0.001). CONCLUSIONS: Approximately one-quarter of patients with FD developed LVH during follow-up. Age, male sex and ECG abnormalities were associated with a higher risk of developing LVH in patients with FD.

Comparison of Early and Mid-Term Outcomes After Classic and Modified Morrow Septal Myectomy in Patients with Hypertrophic Obstructive Cardiomyopathy.

INTRODUCTION: The aim of our study is to compare the early and mid-term outcomes of patients with hypertrophic obstructive cardiomyopathy who underwent classic and modified Morrow septal myectomy. METHODS: Between 2014 and 2019, 48 patients (24 males; mean age 49.27±16.41 years) who underwent septal myectomy were evaluated. The patients were divided into two groups - those who underwent classic septal myectomy (n=28) and those who underwent modified septal myectomy (n=20). RESULTS: Mitral valve intervention was higher in the classic Morrow group than in the modified Morrow group, but there was no significant difference (P=0.42). Mortality was found to be lower in the modified Morrow group than in the classic Morrow group (P=0.01). In both groups, the mean immediate postoperative gradient was significantly higher than the mean of the 3rd and 12th postoperative months. The preoperative and postoperative gradient difference of the modified Morrow group was significantly higher than of the classic Morrow group (P<0.001). CONCLUSION: Classic Morrow and modified Morrow procedures are effective methods for reducing left ventricular outflow tract obstruction. The modified Morrow procedure was found to be superior to the classic Morrow procedure in terms of reducing the incidence of mitral valve intervention with the reduction of the left ventricular outflow tract gradient.

Multiplexed Functional Assessments of MYH7 Variants in Human Cardiomyocytes.

BACKGROUND: Pathogenic autosomal-dominant missense variants in MYH7 (myosin heavy chain 7), which encodes the sarcomeric protein (ß-MHC [beta myosin heavy chain]) expressed in cardiac and skeletal myocytes, are a leading cause of hypertrophic cardiomyopathy and are clinically actionable. However, ≈75% of MYH7 missense variants are of unknown significance. While human-induced pluripotent stem cells (hiPSCs) can be differentiated into cardiomyocytes to enable the interrogation of MYH7 variant effect in a disease-relevant context, deep mutational scanning has not been executed using diploid hiPSC derivates due to low hiPSC gene-editing efficiency. Moreover, multiplexable phenotypes enabling deep mutational scanning of MYH7 variant hiPSC-derived cardiomyocytes are unknown. METHODS: To overcome these obstacles, we used CRISPRa On-Target Editing Retrieval enrichment to generate an hiPSC library containing 113 MYH7 codon variants suitable for deep mutational scanning. We first established that ß-MHC protein loss occurs in a hypertrophic cardiomyopathy human heart with a pathogenic MYH7 variant. We then differentiated the MYH7 missense variant hiPSC library to cardiomyocytes for multiplexed assessment of ß-MHC variant abundance by massively parallel sequencing and hiPSC-derived cardiomyocyte survival. RESULTS: Both the multiplexed assessment of ß-MHC abundance and hiPSC-derived cardiomyocyte survival accurately segregated all known pathogenic variants from synonymous variants. Functional data were generated for 4 variants of unknown significance and 58 additional MYH7 missense variants not yet detected in patients. CONCLUSIONS: This study leveraged hiPSC differentiation into disease-relevant cardiomyocytes to enable multiplexed assessments of MYH7 missense variants for the first time. Phenotyping strategies used here enable the application of deep mutational scanning to clinically actionable genes, which should reduce the burden of variants of unknown significance on patients and clinicians.

Percutaneous intramyocardial septal radiofrequency ablation after 5-year follow-up.

OBJECTIVE: The objective is to evaluate the 5-year follow-up results of percutaneous intramyocardial septal radiofrequency ablation (PIMSRA) for hypertrophic obstructive cardiomyopathy (HOCM), including clinical status, electrocardiographic and echocardiographic characteristics. METHODS: 27 patients (age: 44.3±15.5 years; 67% men, 33% women) with severely symptomatic HOCM who underwent PIMSRA from October 2016 to September 2017 were included. Their clinical status, resting and exercise stress echocardiography, electrocardiography and cardiac MRI (CMRI) after long-term follow-up were assessed. RESULTS: One patient died of intracerebral haemorrhage 1 year post procedurally. The New York Heart Association class, Canadian Cardiovascular Society class and exercise-induced syncopal attacks improved significantly in 26 patients (all p<0.01). Left ventricular (LV) outflow tract gradients revealed sustained reduction (resting: from 95.0 to 9.0 mm Hg, p<0.001; post exercise: from 130.5 to 21.0 mm Hg, p<0.001). The echocardiographic evaluation revealed decreased septal thickness, LV posterior wall thickness and left atrial (LA) diameter (all p<0.001). CMRI data revealed decrease in LV mass index and LA volume index and increase in LV end-diastolic volume index and stroke volume index between baseline and long-term follow-up (all p<0.05). The global longitudinal strain of LV improved from (-11.9%±3.7%) before the procedure to (-13.1%±3.9%) at the last check (p<0.001). Malignant ventricular arrhythmia and heart failure events were not observed. CONCLUSIONS: PIMSRA can effectively alleviate symptoms in patients with HOCM and improve their hemodynamics in the long term. TRIAL REGISTRATION NUMBER: NCT02888132.

Deficiency of Transcription Factor Sp1 Contributes to Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most prevalent monogenic heart disorder. However, the pathogenesis of HCM, especially its nongenetic mechanisms, remains largely unclear. Transcription factors are known to be involved in various biological processes including cell growth. We hypothesized that SP1 (specificity protein 1), the first purified TF in mammals, plays a role in the cardiomyocyte growth and cardiac hypertrophy of HCM. METHODS: Cardiac-specific conditional knockout of Sp1 mice were constructed to investigate the role of SP1 in the heart. The echocardiography, histochemical experiment, and transmission electron microscope were performed to analyze the cardiac phenotypes of cardiac-specific conditional knockout of Sp1 mice. RNA sequencing, chromatin immunoprecipitation sequencing, and adeno-associated virus experiments in vivo were performed to explore the downstream molecules of SP1. To examine the therapeutic effect of SP1 on HCM, an SP1 overexpression vector was constructed and injected into the mutant allele of Myh6 R404Q/+ (Myh6 c. 1211C>T) HCM mice. The human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a patient with HCM were used to detect the potential therapeutic effects of SP1 in human HCM. RESULTS: The cardiac-specific conditional knockout of Sp1 mice developed a typical HCM phenotype, displaying overt myocardial hypertrophy, interstitial fibrosis, and disordered myofilament. In addition, Sp1 knockdown dramatically increased the cell area of hiPSC-CMs and caused intracellular myofibrillar disorganization, which was similar to the hypertrophic cardiomyocytes of HCM. Mechanistically, Tuft1 was identified as the key target gene of SP1. The hypertrophic phenotypes induced by Sp1 knockdown in both hiPSC-CMs and mice could be rescued by TUFT1 (tuftelin 1) overexpression. Furthermore, SP1 overexpression suppressed the development of HCM in the mutant allele of Myh6 R404Q/+ mice and also reversed the hypertrophic phenotype of HCM hiPSC-CMs. CONCLUSIONS: Our study demonstrates that SP1 deficiency leads to HCM. SP1 overexpression exhibits significant therapeutic effects on both HCM mice and HCM hiPSC-CMs, suggesting that SP1 could be a potential intervention target for HCM.

Exploring the Connection Between Relaxed Myosin States and the Anrep Effect.

The Anrep effect is an adaptive response that increases left ventricular contractility following an acute rise in afterload. Although the mechanistic origin remains undefined, recent findings suggest a two-phase activation of resting myosin for contraction, involving strain-sensitive and posttranslational phases. We propose that this mobilization represents a transition among the relaxed states of myosin-specifically, from the super-relaxed (SRX) to the disordered-relaxed (DRX)-with DRX myosin ready to participate in force generation. This hypothesis offers a unified explanation that connects myosin's SRX-DRX equilibrium and the Anrep effect as parts of a singular phenomenon. We underscore the significance of this equilibrium in modulating contractility, primarily studied in the context of hypertrophic cardiomyopathy, the most common inherited cardiomyopathy associated with diastolic dysfunction, hypercontractility, and left ventricular hypertrophy. As we posit that the cellular basis of the Anrep effect relies on a two-phased transition of myosin from the SRX to the contraction-ready DRX configuration, any dysregulation in this equilibrium may result in the pathological manifestation of the Anrep phenomenon. For instance, in hypertrophic cardiomyopathy, hypercontractility is linked to a considerable shift of myosin to the DRX state, implying a persistent activation of the Anrep effect. These valuable insights call for additional research to uncover a clinical Anrep fingerprint in pathological states. Here, we demonstrate through noninvasive echocardiographic pressure-volume measurements that this fingerprint is evident in 12 patients with hypertrophic obstructive cardiomyopathy before septal myocardial ablation. This unique signature is characterized by enhanced contractility, indicated by a leftward shift and steepening of the end-systolic pressure-volume relationship, and a prolonged systolic ejection time adjusted for heart rate, which reverses post-procedure. The clinical application of this concept has potential implications beyond hypertrophic cardiomyopathy, extending to other genetic cardiomyopathies and even noncongenital heart diseases with complex etiologies across a broad spectrum of left ventricular ejection fractions.

Role of Biomarkers of Myocardial Injury to Predict Adverse Outcomes in Hypertrophic Cardiomyopathy.

BACKGROUND: Serum troponins and CK-MB (creatine kinase-MB) are readily detectable and reliable cardiac-specific biomarkers of subclinical myocardial injury. This study explores the roles of cTnI (cardiac troponin I) and CK-MB in hypertrophic cardiomyopathy (HCM). METHODS: This study included 1045 patients with HCM who had baseline cTnI and CK-MB measurements at Fuwai Hospital between 1999 and 2019. Patients were excluded if they had undergone percutaneous coronary intervention or coronary artery bypass grafting, or had renal failure. Five end points were studied: all-cause death, cardiovascular death, noncardiovascular death, sudden cardiac death, and other cardiovascular death. Cox regression was used to assess the associations of cTnI and CK-MB levels with outcomes. RESULTS: Nine hundred seventy patients with available follow-up data were finally analyzed (mean age, 49.3 years; 36.4% female). During the median 4.3-year follow-up period, 87 patients reached the end points. Higher cTnI (per 0.05 ng/mL increase) and CK-MB (per 1 IU/L increase) levels were associated with increased risks of all-cause death (cTnI: adjusted hazard ratio [HR], 1.038, P<0.001; CK-MB: adjusted HR, 1.021, P=0.004), cardiovascular death (cTnI: adjusted HR, 1.040, P<0.001; CK-MB: adjusted HR, 1.025, P=0.006), and sudden cardiac death (cTnI: adjusted HR, 1.045, P<0.001; CK-MB: adjusted HR, 1.032, P=0.001). Patients with elevated levels of both cTnI and CK-MB had worse prognoses than patients with an elevated level of either biomarker alone and patients who did not have an elevated level of either biomarker. Addition of the binary indicator elevation of both cTnI and CK-MB significantly improved the discrimination and reclassification abilities of the standard HCM Risk- sudden cardiac death model (C statistics: P=0.002; net reclassification improvement, 0.652; integrated discrimination improvement, 0.064). CONCLUSIONS: Comprehensive evaluations of biomarkers of myocardial injury, cTnI and CK-MB, have considerable value for predicting adverse outcomes among patients with HCM. Routine cTnI and CK-MB assessments may help to guide implantable cardioverter defibrillator implantation for primary prevention in HCM.

Meta-Analysis of Penetrance and Systematic Review on Transition to Disease in Genetic Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is characterized by unexplained left ventricular hypertrophy and is classically caused by pathogenic or likely pathogenic variants (P/LP) in genes encoding sarcomere proteins. Not all subclinical variant carriers will manifest clinically overt disease because penetrance (proportion of sarcomere or sarcomere-related P/LP variant carriers who develop disease) is variable, age dependent, and not reliably predicted. METHODS: A systematic search of the literature was performed. We used random-effects generalized linear mixed model meta-analyses to contrast the cross-sectional prevalence and penetrance of sarcomere or sarcomere-related genes in 2 different contexts: clinically-based studies on patients and families with HCM versus population or community-based studies. Longitudinal family/clinical studies were additionally analyzed to investigate the rate of phenotypic conversion from subclinical to overt HCM during follow-up. RESULTS: In total, 455 full-text manuscripts and articles were assessed. In family/clinical studies, the prevalence of sarcomere variants in patients diagnosed with HCM was 34%. The penetrance across all genes in nonproband relatives carrying P/LP variants identified during cascade screening was 57% (95% CI, 52%-63%), and the mean age at HCM diagnosis was 38 years (95% CI, 36%-40%). Penetrance varied from ≈32% for MYL3 (myosin light chain 3) to ≈55% for MYBPC3 (myosin-binding protein C3), ≈60% for TNNT2 (troponin T2) and TNNI3 (troponin I3), and ≈65% for MYH7 (myosin heavy chain 7). Population-based genetic studies demonstrate that P/LP sarcomere variants are present in the background population but at a low prevalence of <1%. The penetrance of HCM in incidentally identified P/LP variant carriers was also substantially lower at ≈11%, ranging from 0% in Atherosclerosis Risk in Communities to 18% in UK Biobank. In longitudinal family studies, the pooled phenotypic conversion across all genes was 15% over an average of ≈8 years of follow-up, starting from a mean of ≈16 years of age. However, short-term gene-specific phenotypic conversion varied between ≈12% for MYBPC3 and ≈23% for MYH7. CONCLUSIONS: The penetrance of P/LP variants is highly variable and influenced by currently undefined and context-dependent genetic and environmental factors. Additional longitudinal studies are needed to improve our understanding of true lifetime penetrance in families and in the community and to identify drivers of the transition from subclinical to overt HCM.

Age-dependent association of metabolic dyslipidemia with clinical expression of hypertrophic cardiomyopathy.

BACKGROUND: Previous studies have shown that the clinical expression of hypertrophic cardiomyopathy (HCM) can be determined by obesity and metabolic syndrome. The present study aimed to investigate the association between triglyceride and high-density lipoprotein cholesterol (HDLC) level, the two dyslipidemia-related components of metabolic syndrome, and the incidence of HCM. We also explored an age-dependent association between them. METHODS: Individuals without previous HCM diagnosis who underwent a designated national health examination in 2009 were recruited. Individuals who used lipid-lowering medications within 1-year of the baseline were excluded. The outcome of interest was a newly diagnosed HCM. RESULTS: Our cohort consisted of 8,652,709 individuals (mean 46 years, 55.6% men). During the median 9.3 years of follow-up, 5932 (0.07%) individuals were newly diagnosed with HCM. There was a gradual increase in the incidence of HCM towards higher triglyceride and lower HDL-C levels (log-rank p < 0.001). When stratified by age, the incidence of HCM was highest in individuals aged ≥65 years, followed by those aged 40-64 and 20-39 years (0.22% vs. 0.07% vs. 0.03%, log-rank p < 0.001). In individuals aged 20-39 years, a higher triglyceride level was associated with a higher incidence of HCM (i.e., ≥200 vs. <100 mg/dL: adjusted hazard ratio 2.28, 95% confidence interval 1.89-2.75), whereas there was no significant association in older groups (p-for-interaction<0.001). Similarly, a lower HDL-C level was associated with a higher incidence of HCM, particularly in individuals aged 20-39 years (p-for-interaction = 0.001). CONCLUSIONS: High triglyceride and low HDL-C levels are associated with a higher incidence of HCM, particularly in young individuals.