A high-resolution map of human RNA translation.

Translated small open reading frames (smORFs) can have important regulatory roles and encode microproteins, yet their genome-wide identification has been challenging. We determined the ribosome locations across six primary human cell types and five tissues and detected 7,767 smORFs with translational profiles matching those of known proteins. The human genome was found to contain highly cell-type- and tissue-specific smORFs and a subset that encodes highly conserved amino acid sequences. Changes in the translational efficiency of upstream-encoded smORFs (uORFs) and the corresponding main ORFs predominantly occur in the same direction. Integration with 456 mass-spectrometry datasets confirms the presence of 603 small peptides at the protein level in humans and provides insights into the subcellular localization of these small proteins. This study provides a comprehensive atlas of high-confidence translated smORFs derived from primary human cells and tissues in order to provide a more complete understanding of the translated human genome.

The penetrance of rare variants in cardiomyopathy-associated genes: A cross-sectional approach to estimating penetrance for secondary findings.

Understanding the penetrance of pathogenic variants identified as secondary findings (SFs) is of paramount importance with the growing availability of genetic testing. We estimated penetrance through large-scale analyses of individuals referred for diagnostic sequencing for hypertrophic cardiomyopathy (HCM; 10,400 affected individuals, 1,332 variants) and dilated cardiomyopathy (DCM; 2,564 affected individuals, 663 variants), using a cross-sectional approach comparing allele frequencies against reference populations (293,226 participants from UK Biobank and gnomAD). We generated updated prevalence estimates for HCM (1:543) and DCM (1:220). In aggregate, the penetrance by late adulthood of rare, pathogenic variants (23% for HCM, 35% for DCM) and likely pathogenic variants (7% for HCM, 10% for DCM) was substantial for dominant cardiomyopathy (CM). Penetrance was significantly higher for variant subgroups annotated as loss of function or ultra-rare and for males compared to females for variants in HCM-associated genes. We estimated variant-specific penetrance for 316 recurrent variants most likely to be identified as SFs (found in 51% of HCM- and 17% of DCM-affected individuals). 49 variants were observed at least ten times (14% of affected individuals) in HCM-associated genes. Median penetrance was 14.6% (±14.4% SD). We explore estimates of penetrance by age, sex, and ancestry and simulate the impact of including future cohorts. This dataset reports penetrance of individual variants at scale and will inform the management of individuals undergoing genetic screening for SFs. While most variants had low penetrance and the costs and harms of screening are unclear, some individuals with highly penetrant variants may benefit from SFs.

IL11 (Interleukin-11) Causes Emphysematous Lung Disease in a Mouse Model of Marfan Syndrome.

BACKGROUND: Marfan Syndrome (MFS) is an inherited connective tissue disorder caused by mutations in the FBN1 (fibrillin-1) gene. Lung abnormalities are common in MFS, but their pathogenesis is poorly understood. IL11 (interleukin-11) causes aortic disease in a mouse model of MFS and was studied here in the lung. METHODS: We examined histological and molecular phenotypes in the lungs of Fbn1C1041G/+ mice (mouse model of Marfan Syndrome [mMFS]), an established mouse model of MFS. To identify IL11-expressing cells, we used immunohistochemistry on lungs of 4- and 16-week-old Fbn1C1041G/+:Il11EGFP/+ reporter mice. We studied the effects of IL11 inhibition by RT-qPCR, immunoblots and histopathology in lungs from genetic or pharmacologic models: (1) 16-week-old IL11 receptor (IL11RA) knockout mMFS mice (Fbn1C1041G/+:Il11ra1-/- mice) and (2) in mMFS mice administered IgG control or interleukin-11 receptor antibodies twice weekly from 4 to 24 weeks of age. RESULTS: mMFS lungs showed progressive loss and enlargement of distal airspaces associated with increased proinflammatory and profibrotic gene expression as well as matrix metalloproteinases 2, 9, and 12. IL11 was increased in mMFS lungs and localized to smooth muscle and endothelial cells in young mMFS mice in the Fbn1C1041G/+:Il11EGFP/+ reporter strain and in fibroblasts, in older mice. In mMFS mice, genetic (Fbn1C1041G/+:Il11ra1-/-) or pharmacologic (anti-interleukin-11 receptor) inhibition of IL11 signaling reduced lung emphysema, fibrosis, and inflammation. This protective effect was associated with reduced pathogenic ERK1/2 signaling and lower metalloproteinase 2, 9, and 12 expression. CONCLUSIONS: IL11 causes lung disease in mMFS. This reveals a shared IL11-driven disease mechanism in lung and aorta in MFS and suggests inhibition of IL11 signaling as a holistic approach for treating multiorgan morbidity in MFS.

IL-11 is a crucial determinant of cardiovascular fibrosis.

Fibrosis is a common pathology in cardiovascular disease. In the heart, fibrosis causes mechanical and electrical dysfunction and in the kidney, it predicts the onset of renal failure. Transforming growth factor ß1 (TGFß1) is the principal pro-fibrotic factor, but its inhibition is associated with side effects due to its pleiotropic roles. We hypothesized that downstream effectors of TGFß1 in fibroblasts could be attractive therapeutic targets and lack upstream toxicity. Here we show, using integrated imaging-genomics analyses of primary human fibroblasts, that upregulation of interleukin-11 (IL-11) is the dominant transcriptional response to TGFß1 exposure and required for its pro-fibrotic effect. IL-11 and its receptor (IL11RA) are expressed specifically in fibroblasts, in which they drive non-canonical, ERK-dependent autocrine signalling that is required for fibrogenic protein synthesis. In mice, fibroblast-specific Il11 transgene expression or Il-11 injection causes heart and kidney fibrosis and organ failure, whereas genetic deletion of Il11ra1 protects against disease. Therefore, inhibition of IL-11 prevents fibroblast activation across organs and species in response to a range of important pro-fibrotic stimuli. These results reveal a central role of IL-11 in fibrosis and we propose that inhibition of IL-11 is a potential therapeutic strategy to treat fibrotic diseases.

Disease-specific variant pathogenicity prediction significantly improves variant interpretation in inherited cardiac conditions.

PURPOSE: Accurate discrimination of benign and pathogenic rare variation remains a priority for clinical genome interpretation. State-of-the-art machine learning variant prioritization tools are imprecise and ignore important parameters defining gene-disease relationships, e.g., distinct consequences of gain-of-function versus loss-of-function variants. We hypothesized that incorporating disease-specific information would improve tool performance. METHODS: We developed a disease-specific variant classifier, CardioBoost, that estimates the probability of pathogenicity for rare missense variants in inherited cardiomyopathies and arrhythmias. We assessed CardioBoost's ability to discriminate known pathogenic from benign variants, prioritize disease-associated variants, and stratify patient outcomes. RESULTS: CardioBoost has high global discrimination accuracy (precision recall area under the curve [AUC] 0.91 for cardiomyopathies; 0.96 for arrhythmias), outperforming existing tools (4-24% improvement). CardioBoost obtains excellent accuracy (cardiomyopathies 90.2%; arrhythmias 91.9%) for variants classified with >90% confidence, and increases the proportion of variants classified with high confidence more than twofold compared with existing tools. Variants classified as disease-causing are associated with both disease status and clinical severity, including a 21% increased risk (95% confidence interval [CI] 11-29%) of severe adverse outcomes by age 60 in patients with hypertrophic cardiomyopathy. CONCLUSIONS: A disease-specific variant classifier outperforms state-of-the-art genome-wide tools for rare missense variants in inherited cardiac conditions ( https://www.cardiodb.org/cardioboost/ ), highlighting broad opportunities for improved pathogenicity prediction through disease specificity.

Beyond fitness tracking: The use of consumer-grade wearable data from normal volunteers in cardiovascular and lipidomics research.

The use of consumer-grade wearables for purposes beyond fitness tracking has not been comprehensively explored. We generated and analyzed multidimensional data from 233 normal volunteers, integrating wearable data, lifestyle questionnaires, cardiac imaging, sphingolipid profiling, and multiple clinical-grade cardiovascular and metabolic disease markers. We show that subjects can be stratified into distinct clusters based on daily activity patterns and that these clusters are marked by distinct demographic and behavioral patterns. While resting heart rates (RHRs) performed better than step counts in being associated with cardiovascular and metabolic disease markers, step counts identified relationships between physical activity and cardiac remodeling, suggesting that wearable data may play a role in reducing overdiagnosis of cardiac hypertrophy or dilatation in active individuals. Wearable-derived activity levels can be used to identify known and novel activity-modulated sphingolipids that are in turn associated with insulin sensitivity. Our findings demonstrate the potential for wearables in biomedical research and personalized health.

The application of exercise stress cardiovascular magnetic resonance in patients with suspected dilated cardiomyopathy.

OBJECTIVES: The imaging features of dilated cardiomyopathy (DCM) overlap with physiological exercise-induced cardiac remodeling in active and otherwise healthy individuals. Distinguishing the two conditions is challenging. This study examined the diagnostic and prognostic roles of exercise stress imaging in asymptomatic patients with suspected DCM. METHODS: Exercise stress cardiovascular magnetic resonance (CMR) was performed in 60 asymptomatic patients with suspected DCM (dilated left ventricle and/or impaired systolic function on CMR), who also underwent DNA sequencing for DCM-causing genetic variants. Confirmed DCM was defined as genotype- and phenotype-positive (G+P+). Another 100 healthy subjects were recruited to establish normal exercise capacities (peak exercise cardiac index; PeakCI). The primary outcome was a composite of all-cause mortality, cardiac decompensation and ventricular arrhythmic events. RESULTS: No patients with confirmed G+P+ DCM had PeakCI exceeding the 35th percentile specific for age and sex. Applying this threshold in G-P+ patients, those with PeakCI below 35th percentile had characteristics similar to confirmed DCM while patients with higher PeakCI were younger, more active and higher longitudinal strain. Adverse cardiovascular events occurred only in patients with low exercise capacity (P = 0.004). CONCLUSIONS: In individuals with suspected DCM, exercise stress CMR demonstrates diagnostic and prognostic potential in distinguishing between pathological DCM and physiological exercise-induced cardiac remodeling.

Early Regenerative Capacity in the Porcine Heart.

BACKGROUND: The adult mammalian heart has limited ability to repair itself after injury. Zebrafish, newts, and neonatal mice can regenerate cardiac tissue, largely by cardiac myocyte (CM) proliferation. It is unknown whether hearts of young large mammals can regenerate. METHODS: We examined the regenerative capacity of the pig heart in neonatal animals (ages 2, 3, or 14 days postnatal) after myocardial infarction or sham procedure. Myocardial scar and left ventricular function were determined by cardiac magnetic resonance imaging and echocardiography. Bromodeoxyuridine pulse-chase labeling, histology, immunohistochemistry, and Western blotting were performed to study cell proliferation, sarcomere dynamics, and cytokinesis and to quantify myocardial fibrosis. RNA-sequencing was also performed. RESULTS: After myocardial infarction, there was early and sustained recovery of cardiac function and wall thickness in the absence of fibrosis in 2-day-old pigs. In contrast, older animals developed full-thickness myocardial scarring, thinned walls, and did not recover function. Genome-wide analyses of the infarct zone revealed a strong transcriptional signature of fibrosis in 14-day-old animals that was absent in 2-day-old pigs, which instead had enrichment for cytokinesis genes. In regenerating hearts of the younger animals, up to 10% of CMs in the border zone of the myocardial infarction showed evidence of DNA replication that was associated with markers of myocyte division and sarcomere disassembly. CONCLUSIONS: Hearts of large mammals have regenerative capacity, likely driven by cardiac myocyte division, but this potential is lost immediately after birth.

Defining the genetic architecture of hypertrophic cardiomyopathy: re-evaluating the role of non-sarcomeric genes.

AIM: Hypertrophic cardiomyopathy (HCM) exhibits genetic heterogeneity that is dominated by variation in eight sarcomeric genes. Genetic variation in a large number of non-sarcomeric genes has also been implicated in HCM but not formally assessed. Here we used very large case and control cohorts to determine the extent to which variation in non-sarcomeric genes contributes to HCM. METHODS AND RESULTS: We sequenced known and putative HCM genes in a new large prospective HCM cohort (n = 804) and analysed data alongside the largest published series of clinically genotyped HCM patients (n = 6179), previously published HCM cohorts and reference population samples from the exome aggregation consortium (ExAC, n = 60 706) to assess variation in 31 genes implicated in HCM. We found no significant excess of rare (minor allele frequency < 1:10 000 in ExAC) protein-altering variants over controls for most genes tested and conclude that novel variants in these genes are rarely interpretable, even for genes with previous evidence of co-segregation (e.g. ACTN2). To provide an aid for variant interpretation, we integrated HCM gene sequence data with aggregated pedigree and functional data and suggest a means of assessing gene pathogenicity in HCM using this evidence. CONCLUSION: We show that genetic variation in the majority of non-sarcomeric genes implicated in HCM is not associated with the condition, reinforce the fact that the sarcomeric gene variation is the primary cause of HCM known to date and underscore that the aetiology of HCM is unknown in the majority of patients.

Sacubitril/valsartan versus valsartan in regressing myocardial fibrosis in hypertension: a prospective, randomized, open-label, blinded endpoint clinical trial protocol.

Background: Diffuse interstitial myocardial fibrosis is a key common pathological manifestation in hypertensive heart disease (HHD) progressing to heart failure (HF). Angiotensin receptor-neprilysin inhibitors (ARNi), now a front-line treatment for HF, confer benefits independent of blood pressure, signifying a multifactorial mode of action beyond hemodynamic regulation. We aim to test the hypothesis that compared with angiotensin II receptor blockade (ARB) alone, ARNi is more effective in regressing diffuse interstitial myocardial fibrosis in HHD. Methods: Role of ARNi in Ventricular Remodeling in Hypertensive LVH (REVERSE-LVH) is a prospective, randomized, open-label, blinded endpoint (PROBE) clinical trial. Adults with hypertension and left ventricular hypertrophy (LVH) according to Asian sex- and age-specific thresholds on cardiovascular magnetic resonance (CMR) imaging are randomized to treatment with either sacubitril/valsartan (an ARNi) or valsartan (an ARB) in 1:1 ratio for a duration of 52 weeks, at the end of which a repeat CMR is performed to assess differential changes from baseline between the two groups. The primary endpoint is the change in CMR-derived diffuse interstitial fibrosis volume. Secondary endpoints include changes in CMR-derived left ventricular mass, volumes, and functional parameters. Serum samples are collected and stored to assess the effects of ARNi, compared with ARB, on circulating biomarkers of cardiac remodeling. The endpoints will be analyzed with reference to the corresponding baseline parameters to evaluate the therapeutic effect of sacubitril/valsartan vs. valsartan. Discussion: REVERSE-LVH will examine the anti-fibrotic potential of sacubitril/valsartan and will offer mechanistic insights into the clinical benefits of sacubitril/valsartan in hypertension in relation to cardiac remodeling. Advancing the knowledge of the pathophysiology of HHD will consolidate effective risk stratification and personalized treatment through a multimodal manner integrating complementary CMR and biomarkers into the conventional care approach.Clinical Trial Registration: ClinicalTrials.gov, identifier, NCT03553810.

Impact of Diabetes on Myocardial Fibrosis in Patients With Hypertension: The REMODEL Study.

BACKGROUND: Compared with patients with hypertension only, those with hypertension and diabetes (HTN/DM) have worse prognosis. We aimed to characterize morphological differences between hypertension and HTN/DM using cardiovascular magnetic resonance; and compare differentially expressed proteins associated with myocardial fibrosis using high throughput multiplex assays. METHODS: Asymptomatic patients underwent cardiovascular magnetic resonance: 438 patients with hypertension (60±8 years; 59% males) and 167 age- and sex-matched patients with HTN/DM (60±10 years; 64% males). Replacement myocardial fibrosis was defined as nonischemic late gadolinium enhancement on cardiovascular magnetic resonance. Extracellular volume fraction was used as a marker of diffuse myocardial fibrosis. A total of 184 serum proteins (Olink Target Cardiovascular Disease II and III panels) were measured to identify unique signatures associated with myocardial fibrosis in all patients. RESULTS: Despite similar left ventricular mass (P=0.344) and systolic blood pressure (P=0.086), patients with HTN/DM had increased concentricity and worse multidirectional strain (P<0.001 for comparison of all strain measures) compared to hypertension only. Replacement myocardial fibrosis was present in 28% of patients with HTN/DM compared to 16% of those with hypertension (P<0.001). NT-proBNP (N-terminal pro-B-type natriuretic peptide) was the only protein differentially upregulated in hypertension patients with replacement myocardial fibrosis and independently associated with extracellular volume. In patients with HTN/DM, GDF-15 (growth differentiation factor 15) was independently associated with replacement myocardial fibrosis and extracellular volume. Ingenuity Pathway Analysis demonstrated a strong association between increased inflammatory response/immune cell trafficking and myocardial fibrosis in patients with HTN/DM. CONCLUSIONS: Adverse cardiac remodeling was observed in patients with HTN/DM. The novel proteomic signatures and associated biological activities of increased immune and inflammatory response may partly explain these observations.

Genotype-Phenotype Taxonomy of Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is an important cause of sudden cardiac death associated with heterogeneous phenotypes, but there is no systematic framework for classifying morphology or assessing associated risks. Here, we quantitatively survey genotype-phenotype associations in HCM to derive a data-driven taxonomy of disease expression. METHODS: We enrolled 436 patients with HCM (median age, 60 years; 28.8% women) with clinical, genetic, and imaging data. An independent cohort of 60 patients with HCM from Singapore (median age, 59 years; 11% women) and a reference population from the UK Biobank (n=16 691; mean age, 55 years; 52.5% women) were also recruited. We used machine learning to analyze the 3-dimensional structure of the left ventricle from cardiac magnetic resonance imaging and build a tree-based classification of HCM phenotypes. Genotype and mortality risk distributions were projected on the tree. RESULTS: Carriers of pathogenic or likely pathogenic variants for HCM had lower left ventricular mass, but greater basal septal hypertrophy, with reduced life span (mean follow-up, 9.9 years) compared with genotype negative individuals (hazard ratio, 2.66 [95% CI, 1.42-4.96]; P<0.002). Four main phenotypic branches were identified using unsupervised learning of 3-dimensional shape: (1) nonsarcomeric hypertrophy with coexisting hypertension; (2) diffuse and basal asymmetrical hypertrophy associated with outflow tract obstruction; (3) isolated basal hypertrophy; and (4) milder nonobstructive hypertrophy enriched for familial sarcomeric HCM (odds ratio for pathogenic or likely pathogenic variants, 2.18 [95% CI, 1.93-2.28]; P=0.0001). Polygenic risk for HCM was also associated with different patterns and degrees of disease expression. The model was generalizable to an independent cohort (trustworthiness, M1: 0.86-0.88). CONCLUSIONS: We report a data-driven taxonomy of HCM for identifying groups of patients with similar morphology while preserving a continuum of disease severity, genetic risk, and outcomes. This approach will be of value in understanding the causes and consequences of disease diversity.

Markers of Focal and Diffuse Nonischemic Myocardial Fibrosis Are Associated With Adverse Cardiac Remodeling and Prognosis in Patients With Hypertension: The REMODEL Study.

BACKGROUND: The prognostic significance of focal and diffuse myocardial fibrosis in patients with cardiovascular risk factors is unclear. METHODS: REMODEL (Response of the Myocardium to Hypertrophic Conditions in the Adult Population) is an observational cohort of asymptomatic patients with essential hypertension. All participants underwent cardiovascular magnetic resonance to assess for myocardial fibrosis: nonischemic late gadolinium enhancement (LGE), native myocardial T1, postcontrast myocardial T1, extracellular volume fraction including/excluding LGE regions, interstitial volume (extracellular volume×myocardial volume), and interstitial/myocyte ratio. Primary outcome was a composite of first occurrence acute coronary syndrome, heart failure hospitalization, strokes, and cardiovascular mortality. Patients were recruited from February 2016 and followed until June 2021. RESULTS: Of the 786 patients with hypertension (58±11 years; 39% women; systolic blood pressure, 130±14 mm Hg), 145 (18%) had nonischemic LGE. Patients with nonischemic LGE were more likely to be men, have diabetes, be current smokers, and have higher blood pressure (P<0.05 for all). Compared with those without LGE, patients with nonischemic LGE had greater left ventricular mass (66±22 versus 49±9 g/m2; P<0.001), worse multidirectional strain (P<0.001 for all measures), and elevated circulating markers of myocardial wall stress and myocardial injury, adjusted for potential confounders. Twenty-four patients had primary outcome over 39 (30-50) months of follow-up. Of all the cardiovascular magnetic resonance markers of myocardial fibrosis assessed, only nonischemic LGE (hazard ratio, 6.69 [95% CI, 2.54-17.60]; P<0.001) and indexed interstitial volume (hazard ratio, 1.11 [95% CI, 1.04-1.19]; P=0.002) demonstrated independent association with primary outcome. CONCLUSIONS: In patients with hypertension, myocardial fibrosis on cardiovascular magnetic resonance is associated with adverse cardiac remodeling and outcomes.

Precision Phenotyping of Dilated Cardiomyopathy Using Multidimensional Data.

BACKGROUND: Dilated cardiomyopathy (DCM) is a final common manifestation of heterogenous etiologies. Adverse outcomes highlight the need for disease stratification beyond ejection fraction. OBJECTIVES: The purpose of this study was to identify novel, reproducible subphenotypes of DCM using multiparametric data for improved patient stratification. METHODS: Longitudinal, observational UK-derivation (n = 426; median age 54 years; 67% men) and Dutch-validation (n = 239; median age 56 years; 64% men) cohorts of DCM patients (enrolled 2009-2016) with clinical, genetic, cardiovascular magnetic resonance, and proteomic assessments. Machine learning with profile regression identified novel disease subtypes. Penalized multinomial logistic regression was used for validation. Nested Cox models compared novel groupings to conventional risk measures. Primary composite outcome was cardiovascular death, heart failure, or arrhythmia events (median follow-up 4 years). RESULTS: In total, 3 novel DCM subtypes were identified: profibrotic metabolic, mild nonfibrotic, and biventricular impairment. Prognosis differed between subtypes in both the derivation (P < 0.0001) and validation cohorts. The novel profibrotic metabolic subtype had more diabetes, universal myocardial fibrosis, preserved right ventricular function, and elevated creatinine. For clinical application, 5 variables were sufficient for classification (left and right ventricular end-systolic volumes, left atrial volume, myocardial fibrosis, and creatinine). Adding the novel DCM subtype improved the C-statistic from 0.60 to 0.76. Interleukin-4 receptor-alpha was identified as a novel prognostic biomarker in derivation (HR: 3.6; 95% CI: 1.9-6.5; P = 0.00002) and validation cohorts (HR: 1.94; 95% CI: 1.3-2.8; P = 0.00005). CONCLUSIONS: Three reproducible, mechanistically distinct DCM subtypes were identified using widely available clinical and biological data, adding prognostic value to traditional risk models. They may improve patient selection for novel interventions, thereby enabling precision medicine.

Critical Conditions for Studying Interleukin-11 Signaling In Vitro and Avoiding Experimental Artefacts.

Interleukin (IL) 11 is a member of the IL6 family of cytokines which require the ubiquitous gp130 receptor to activate canonical (JAK/STAT) and non-canonical (e.g., ERK) signaling pathways. The IL11 cytokine is upregulated in a number of fibro-inflammatory diseases and cancer, where it binds the cognate IL11 receptor alpha subunit (IL11RA) to form a hexameric IL11:IL11RA:gp130 signaling complex. The specific IL11RA receptor is highly expressed on cells of the stromal and parenchymal niche but expressed at low levels on immune cells, highly passaged cells, or transformed cell lines. Consequently, primary cells such as hepatic stellate cells, fibroblasts, and hepatocytes are ideal experimental systems to study IL11 signaling in vitro. In contrast to immortalized cell lines, primary cells better display relevant cellular physiology and pathobiology. This collection of protocols details experimental and culturing conditions for primary cells that preserve meaningful cellular states and physiological responses ex vivo in conventional 2D cell culture systems. Readouts of cellular activity are chosen carefully to capture the non-canonical, post-transcriptional activity of IL11 signaling. Our data suggest that cell type, cell culture conditions, passage number, concentrations of stimuli, timing, and other factors have major implications for studies of IL11 signaling. In vitro experiments with primary cell material need to be planned and executed with great caution. Otherwise, physiologically relevant mechanisms may become dysfunctional and reproducible experimental artefacts can obscure our view of true cytokine biology. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expansion of primary human hepatic stellate cells (HSCs) and human renal proximal tubular epithelial cells (HRPTEpiCs) Basic Protocol 2: Expansion of primary human lung fibroblasts (HLFs) Alternate Protocol 1: Isolation and expansion of primary mouse lung fibroblasts Support Protocol 1: Freezing and thawing of primary cells Support Protocol 2: Operetta high-content imaging-based phenotyping Support Protocol 3: Colorimetric assay of solubilized collagen Support Protocol 4: Quantification of fibrosis marker secretion Support Protocol 5: Western blotting studies of IL11 signaling in HSCs, HLFs, and HRPTEpiCs Basic Protocol 3: IL11 stimulation of primary human hepatocytes Alternate Protocol 2: IL11 stimulation of primary mouse hepatocytes Support Protocol 6: Alanine transaminase (ALT) secretion by human and mouse hepatocytes.

Lowering the Recommended Maximal Wall Thickness Threshold Improves Diagnostic Sensitivity in Asians With Hypertrophic Cardiomyopathy.

Background: Hypertrophic cardiomyopathy (HCM) is defined as left ventricular end-diastolic maximal wall thickness (WTMax) ≥15.0 mm, without accounting for ethnicity, sex, and body size. It is well-established that Asians have smaller hearts than do Caucasians. Objectives: This study aims to examine the implications of this single absolute WTMax threshold on the diagnosis of HCM in Asians. Methods: The study consisted of 360 healthy volunteers (male: n = 174; age: 50 ± 12 years) and 114 genetically characterized patients with HCM (male: n = 83; age: 52 ± 13 years; genotype-positive, n = 39). All participants underwent cardiovascular magnetic resonance. WTMax was measured semiautomatically at end-diastole according to the standard 16 myocardial segments. Results: Healthy male volunteers had increased WTMax compared with that of female volunteers (8.4 ± 1.2 mm vs 6.6 ± 1.1 mm, respectively; P < 0.001). Conversely, WTMax was similar between male and female patients with HCM (15.2 ± 3.4 mm vs 14.7 ± 3.0 mm, respectively; P = 0.484) and between those with and without a pathogenic gene variant (P = 0.828). Using the recommended diagnostic threshold of 15.0 mm, 56 patients with HCM had WTMax <15.0 mm and no healthy volunteers had WTMax >15.0 mm (specificity of 100% and sensitivity of 51%). Lowering WTMax thresholds to 10.0 mm in female patients and 12.0 mm in male patients did not affect specificity (100%) but significantly improved sensitivity (84%). Despite lower left ventricular mass, female patients with HCM demonstrated more features of adverse cardiac remodeling than did male patients: increased myocardial fibrosis, higher asymmetric ratio, and disproportionately worse myocardial strain. Conclusions: The study highlights cautious application of guideline-recommended WTMax to diagnose HCM in Asians. Lowering WTMax to account for ethnicity and sex improves diagnostic sensitivity without compromising specificity.

Interleukin-11 is important for vascular smooth muscle phenotypic switching and aortic inflammation, fibrosis and remodeling in mouse models.

Transforming growth factor beta-1 (TGFß1) is a major driver of vascular smooth muscle cell (VSMC) phenotypic switching, an important pathobiology in arterial disease. We performed RNA-sequencing of TGFß1-stimulated human aortic or arterial VSMCs which revealed large and consistent upregulation of Interleukin 11 (IL11). IL11 has an unknown function in VSMCs, which highly express the IL11 receptor alpha, suggestive of an autocrine loop. In vitro, IL11 activated ERK signaling, but inhibited STAT3 activity, and caused VSMC phenotypic switching to a similar extent as TGFß1 or angiotensin II (ANGII) stimulation. Genetic or therapeutic inhibition of IL11 signaling reduced TGFß1- or ANGII-induced VSMC phenotypic switching, placing IL11 activity downstream of these factors. Aortas of mice with Myh11-driven IL11 expression were remodeled and had reduced contractile but increased matrix and inflammatory genes expression. In two models of arterial pressure loading, IL11 was upregulated in the aorta and neutralizing IL11 antibodies reduced remodeling along with matrix and pro-inflammatory gene expression. These data show that IL11 plays an important role in VSMC phenotype switching, vascular inflammation and aortic pathobiology.

Genetic Studies of Hypertrophic Cardiomyopathy in Singaporeans Identify Variants in TNNI3 and TNNT2 That Are Common in Chinese Patients.

BACKGROUND: To assess the genetic architecture of hypertrophic cardiomyopathy (HCM) in patients of predominantly Chinese ancestry. METHODS: We sequenced HCM disease genes in Singaporean patients (n=224) and Singaporean controls (n=3634), compared findings with additional populations and White HCM cohorts (n=6179), and performed in vitro functional studies. RESULTS: Singaporean HCM patients had significantly fewer confidently interpreted HCM disease variants (pathogenic/likely pathogenic: 18%, P<0.0001) but an excess of variants of uncertain significance (24%, P<0.0001), as compared to Whites (pathogenic/likely pathogenic: 31%, excess of variants of uncertain significance: 7%). Two missense variants in thin filament encoding genes were commonly seen in Singaporean HCM (TNNI3:p.R79C, disease allele frequency [AF]=0.018; TNNT2:p.R286H, disease AF=0.022) and are enriched in Singaporean HCM when compared with Asian controls (TNNI3:p.R79C, Singaporean controls AF=0.0055, P=0.0057, genome aggregation database-East Asian AF=0.0062, P=0.0086; TNNT2:p.R286H, Singaporean controls AF=0.0017, P<0.0001, genome aggregation database-East Asian AF=0.0009, P<0.0001). Both these variants have conflicting annotations in ClinVar and are of low penetrance (TNNI3:p.R79C, 0.7%; TNNT2:p.R286H, 2.7%) but are predicted to be deleterious by computational tools. In population controls, TNNI3:p.R79C carriers had significantly thicker left ventricular walls compared with noncarriers while its etiological fraction is limited (0.70 [95% CI, 0.35-0.86]) and thus TNNI3:p.R79C is considered variant of uncertain significance. Mutant TNNT2:p.R286H iPSC-CMs (induced pluripotent stem cells derived cardiomyocytes) show hypercontractility, increased metabolic requirements, and cellular hypertrophy and the etiological fraction (0.93 [95% CI, 0.83-0.97]) support the likely pathogenicity of TNNT2:p.R286H. CONCLUSIONS: As compared with Whites, Chinese HCM patients commonly have low penetrance risk alleles in TNNT2 or TNNI3 but exhibit few clinically actionable HCM variants overall. This highlights the need for greater study of HCM genetics in non-White populations.

Author Correction: High-Sensitivity cardiac Troponins in Cardio-Healthy Subjects: A Cardiovascular Magnetic Resonance Imaging Study.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Digital phenotyping by consumer wearables identifies sleep-associated markers of cardiovascular disease risk and biological aging.

Sleep is associated with various health outcomes. Despite their growing adoption, the potential for consumer wearables to contribute sleep metrics to sleep-related biomedical research remains largely uncharacterized. Here we analyzed sleep tracking data, along with questionnaire responses and multi-modal phenotypic data generated from 482 normal volunteers. First, we compared wearable-derived and self-reported sleep metrics, particularly total sleep time (TST) and sleep efficiency (SE). We then identified demographic, socioeconomic and lifestyle factors associated with wearable-derived TST; they included age, gender, occupation and alcohol consumption. Multi-modal phenotypic data analysis showed that wearable-derived TST and SE were associated with cardiovascular disease risk markers such as body mass index and waist circumference, whereas self-reported measures were not. Using wearable-derived TST, we showed that insufficient sleep was associated with premature telomere attrition. Our study highlights the potential for sleep metrics from consumer wearables to provide novel insights into data generated from population cohort studies.