Shabani, Mahsima

BACKGROUND: Common genetic variants are associated with risk for hypertrophic cardiomyopathy and dilated cardiomyopathy and with left ventricular (LV) traits. Whether these variants are associated with myocardial fibrosis, an important pathophysiological mediator of cardiomyopathy, is unknown. METHODS: Multi-Ethnic Study of Atherosclerosis participants with T1-mapping cardiac magnetic resonance imaging in-whom extracellular volume was assessed, and genotyping information was available were included (N=1255). Log extracellular volume (%) was regressed on 50 candidate single nucleotide polymorphisms (previously identified to be associated with hypertrophic cardiomyopathy, dilated cardiomyopathy, and LV traits) adjusting for age, sex, diabetes, blood pressure, and principal components of ancestry. Ancestry-specific results were pooled by fixed-effect meta-analyses. Gene knockdown experiments were performed in human cardiac fibroblasts. RESULTS: The SMARCB1 rs2186370 intronic variant (minor allele frequency: 0.18 in White and 0.50 in Black participants), previously identified as a risk variant for dilated cardiomyopathy and hypertrophic cardiomyopathy, was significantly associated with increased extracellular volume ( P= 0.0002) after adjusting for confounding clinical variables. The SMARCB1 rs2070458 locus previously associated with increased LV wall thickness and mass was similarly significantly associated with increased extracellular volume ( P= 0.0002). The direction of effect was similar in all 4 ancestry groups, but the effect was strongest in Black participants. The variants are strong expression quantitative loci in human LV tissue and associated with genotype-dependent decreased expression of SMARCB1 ( P= 7.3×10 -22 ). SMARCB1 knockdown in human cardiac fibroblasts resulted in increased TGF (transforming growth factor)-β1–mediated α-smooth muscle actin and collagen expression. CONCLUSIONS: Common genetic variation in SMARCB1 previously associated with risk for cardiomyopathies and increased LV wall thickness is associated with increased cardiac magnetic resonance imaging–based myocardial fibrosis and increased TGF-β1 mediated myocardial fibrosis in vitro. Whether these findings suggest a pathophysiologic link between myocardial fibrosis and cardiomyopathy risk remains to be proven.

Introduction: Genome-wide association studies have identified common genetic variants associated with hypertrophic (HCM) and dilated cardiomyopathy (DCM) and left ventricular (LV) characteristics. However, the association of these variants with myocardial fibrosis (MF), an important pathophysiological mediator of cardiomyopathy, is unknown. Hypothesis: We used the Multi-Ethnic Study of Atherosclerosis (MESA), to evaluate the association of these loci with interstitial MF measured by cardiac MRI (CMR). Methods: Unrelated MESA participants with T1-mapping CMR and no history of MI or heart failure were included (n=1,235). We used the extracellular volume (ECV) fraction as a well-established surrogate of interstitial MF. Genotyping was performed by Affymetrix Human SNP array 6.0, and imputation was performed using the TOPMED reference panel. Log ECV(%) was regressed on 50 candidate SNPs, in each ethnicity/race group, adjusting for age, sex, and population stratification principal component. The race-specific results were pooled by fixed-effect meta-analyses using METASOFT. Results: The SMARCB1 rs2186370 intronic variant, previously identified as a risk variant for DCM and HCM, was associated with ECV (p=0.0004). The rs2186370 A allele (frequency: 0.18 in Whites, and 0.50 in African Americans) was associated with a 1.6% increase in ECV. The rs2070458 A allele (another SMARCB1 intronic variant), in complete linkage disequilibrium with rs2186370, was known previously to be associated with increased LV wall thickness, mass, and concentricity. The direction of effect was similar in all four ancestry groups, but the effect was strongest in African Americans. These variants are strong expression quantitative loci (eQTLs) in human LV tissue and are associated with genotype-dependent expression of SMARCB1 ( p =7.3х10 -22 ) and MMP11 ( p =2.5х10 -24 ). Conclusions: In conclusion, variants in two linked SMARCB1 loci previously associated with HCM, DCM and LV traits, are associated with CMR-based interstitial MF. These loci are eQTL not only for SMARCB1 but also for MMP11 an important mediator of MF . These findings underscore the important link between MF and CM, and potentially provide novel mechanistic targets in understanding the development of CM.

Introduction: It has been previously demonstrated that cardiomyopathy predisposing common genetic variation in SMARCB1 is associated with interstitial myocardial fibrosis (MF) as determined by cardiac MRI (CMR) linking the development of cardiomyopathy with MF. SMARCB1 is known to be a tumor suppressor, but it’s role in the pathophysiology of MF and fibroblast physiology has not been functionally validated. Hypothesis: SMARCB1 plays an important role in regulating human cardiac fibroblast (HCF) differentiation/activation and collagen deposition, which are the critical steps in the development of MF. Methods: Cultured HCFs were treated with TGF-β1 (10ng/ml) for 48hrs to transdifferentiate into myofibroblasts, which are characterized by increased expression of α-smooth muscle actin (α-SMA, ACTA2) and collagen I (COL1A1). HCFs were transfected by siRNA using Lipofectamine™ RNAiMAX Transfection Reagent to knock-down (KD) the expression of SMARCB1 . Total RNA and protein were extracted from cell lysate followed by qPCR and Western blot (WB) to quantify the expression level of SMARCB1, ACTA2 and COL1A1. Triplicate experiments were performed using biologically independent samples. For statistical analysis, a paired two-tail t-test was used. Results: HCFs treatment with TGF-β1, resulted in a significant decrease in expression of SMARCB1 (p=0.031) with accompanying significant increase in expression of ACTA2 (p=0.025) and COL1A1 (p=0.0015) as compared to control by qPCR and WB. SMARCB1 KD resulted in a further significant increase in expression of ACTA2 (p=0.018) and COL1A1 (p=0.028) as compared to non-target siRNA control group at both the transcript and protein level. Conclusion: We demonstrate for the first time that a gene associated with the development of cardiomyopathy SMARCB1 mediates TGF-β1 induced cardiac fibrosis. These findings suggest SMARCB1 as a potential therapeutic target to attenuate MF in cardiomyopathy.