Showing papers by "Benjamin Meder published in 2022"

A human cell atlas of the pressure-induced hypertrophic heart

Abstract: Abstract Pathological cardiac hypertrophy is a leading cause of heart failure, but knowledge of the full repertoire of cardiac cells and their gene expression profiles in the human hypertrophic heart is missing. Here, by using large-scale single-nucleus transcriptomics, we present the transcriptional response of human cardiomyocytes to pressure overload caused by aortic valve stenosis and describe major alterations in cardiac cellular crosstalk. Hypertrophied cardiomyocytes had reduced input from endothelial cells and fibroblasts. Genes encoding Eph receptor tyrosine kinases, particularly EPHB1 , were significantly downregulated in cardiomyocytes of the hypertrophied heart. Consequently, EPHB1 activation by its ligand ephrin (EFN)B2, which is mainly expressed by endothelial cells, was reduced. EFNB2 inhibited cardiomyocyte hypertrophy in vitro, while silencing its expression in endothelial cells induced hypertrophy in co-cultured cardiomyocytes. Our human cell atlas of the hypertrophied heart highlights the importance of intercellular crosstalk in disease pathogenesis and provides a valuable resource.

Reduction of A-to-I RNA editing in the failing human heart regulates formation of circular RNAs

Abstract: Abstract Alterations of RNA editing that affect the secondary structure of RNAs can cause human diseases. We therefore studied RNA editing in failing human hearts. Transcriptome sequencing showed that adenosine-to-inosine (A-to-I) RNA editing was responsible for 80% of the editing events in the myocardium. Failing human hearts were characterized by reduced RNA editing. This was primarily attributable to Alu elements in introns of protein-coding genes. In the failing left ventricle, 166 circRNAs were upregulated and 7 circRNAs were downregulated compared to non-failing controls. Most of the upregulated circRNAs were associated with reduced RNA editing in the host gene. ADAR2, which binds to RNA regions that are edited from A-to-I, was decreased in failing human hearts. In vitro , reduction of ADAR2 increased circRNA levels suggesting a causal effect of reduced ADAR2 levels on increased circRNAs in the failing human heart. To gain mechanistic insight, one of the identified upregulated circRNAs with a high reduction of editing in heart failure, AKAP13, was further characterized. ADAR2 reduced the formation of double-stranded structures in AKAP13 pre-mRNA, thereby reducing the stability of Alu elements and the circularization of the resulting circRNA. Overexpression of circAKAP13 impaired the sarcomere regularity of human induced pluripotent stem cell-derived cardiomyocytes. These data show that ADAR2 mediates A-to-I RNA editing in the human heart. A-to-I RNA editing represses the formation of dsRNA structures of Alu elements favoring canonical linear mRNA splicing and inhibiting the formation of circRNAs. The findings are relevant to diseases with reduced RNA editing and increased circRNA levels and provide insights into the human-specific regulation of circRNA formation.

Functional Therapeutic Target Validation Using Pediatric Zebrafish Xenograft Models

Abstract: Simple Summary Despite the major progress of precision and personalized oncology, a significant therapeutic benefit is only achieved in cases with directly druggable genetic alterations. This highlights the need for additional methods that reliably predict each individual patient’s response in a clinically meaningful time, e.g., through ex vivo functional drug screen profiling. Moreover, patient-derived xenograft (PDX) models are more predictive than cell culture studies, as they reconstruct cell–cell and cell–extracellular matrix (ECM) interactions and consider the tumor microenvironment, drug metabolism and toxicities. Zebrafish PDXs (zPDX) are nowadays emerging as a fast model allowing for multiple drugs to be tested at the same time in a clinically relevant time window. Here, we show that functional drug response profiling of zPDX from primary material obtained through the INdividualized Therapy FOr Relapsed Malignancies in Childhood (INFORM) pediatric precision oncology pipeline provides additional key information for personalized precision oncology. Abstract The survival rate among children with relapsed tumors remains poor, due to tumor heterogeneity, lack of directly actionable tumor drivers and multidrug resistance. Novel personalized medicine approaches tailored to each tumor are urgently needed to improve cancer treatment. Current pediatric precision oncology platforms, such as the INFORM (INdividualized Therapy FOr Relapsed Malignancies in Childhood) study, reveal that molecular profiling of tumor tissue identifies targets associated with clinical benefit in a subgroup of patients only and should be complemented with functional drug testing. In such an approach, patient-derived tumor cells are exposed to a library of approved oncological drugs in a physiological setting, e.g., in the form of animal avatars injected with patient tumor cells. We used molecularly fully characterized tumor samples from the INFORM study to compare drug screen results of individual patient-derived cell models in functional assays: (i) patient-derived spheroid cultures within a few days after tumor dissociation; (ii) tumor cells reisolated from the corresponding mouse PDX; (iii) corresponding long-term organoid-like cultures and (iv) drug evaluation with the corresponding zebrafish PDX (zPDX) model. Each model had its advantage and complemented the others for drug hit and drug combination selection. Our results provide evidence that in vivo zPDX drug screening is a promising add-on to current functional drug screening in precision medicine platforms.

Non-invasive imaging as the cornerstone of cardiovascular precision medicine.

Abstract: AIMS To provide an overview of the role of cardiovascular (CV) imaging in facilitating and advancing the field of precision medicine in CV disease. METHODS AND RESULTS Non-invasive CV imaging is essential to accurately and efficiently phenotype patients with heart disease, including coronary artery disease (CAD) and heart failure (HF). Various modalities, such as echocardiography, nuclear cardiology, cardiac computed tomography (CT), cardiovascular magnetic resonance (CMR), and invasive coronary angiography, and in some cases a combination, can be required to provide sufficient information for diagnosis and management. Taking CAD as an example, imaging is essential for the detection and functional assessment of coronary stenoses, as well as for the quantification of cardiac function and ischaemic myocardial damage. Furthermore, imaging may detect and quantify coronary atherosclerosis, potentially identify plaques at increased risk of rupture, and guide coronary interventions. In patients with HF, imaging helps identify specific aetiologies, quantify damage, and assess its impact on cardiac function. Imaging plays a central role in individualizing diagnosis and management and to determine the optimal treatment for each patient to increase the likelihood of response and improve patient outcomes. CONCLUSIONS Advances in all imaging techniques continue to improve accuracy, sensitivity, and standardization of functional and prognostic assessments, and identify established and novel therapeutic targets. Combining imaging with artificial intelligence, machine learning and computer algorithms, as well as with genomic, transcriptomic, proteomic, and metabolomic approaches, will become state of the art in the future to understand pathologies of CAD and HF, and in the development of new, targeted therapies.

Cardiac sarcoidosis: growing evidence in risk stratification.

Abstract: Clinical management of cardiac sarcoidosis.

VARS2 Depletion Leads to Activation of the Integrated Stress Response and Disruptions in Mitochondrial Fatty Acid Oxidation

Abstract: Mutations in mitochondrial aminoacyl-tRNA synthetases (mtARSs) have been reported in patients with mitochondriopathies: most commonly encephalopathy, but also cardiomyopathy. Through a GWAS, we showed possible associations between mitochondrial valyl-tRNA synthetase (VARS2) dysregulations and non-ischemic cardiomyopathy. We aimed to investigate the possible consequences of VARS2 depletion in zebrafish and cultured HEK293A cells. Transient VARS2 loss-of-function was induced in zebrafish embryos using Morpholinos. The enzymatic activity of VARS2 was measured in VARS2-depleted cells via northern blot. Heterozygous VARS2 knockout was established in HEK293A cells using CRISPR/Cas9 technology. BN-PAGE and SDS-PAGE were used to investigate electron transport chain (ETC) complexes, and the oxygen consumption rate and extracellular acidification rate were measured using a Seahorse XFe96 Analyzer. The activation of the integrated stress response (ISR) and possible disruptions in mitochondrial fatty acid oxidation (FAO) were explored using RT-qPCR and western blot. Zebrafish embryos with transient VARS2 loss-of-function showed features of heart failure as well as indications of CNS and skeletal muscle involvements. The enzymatic activity of VARS2 was significantly reduced in VARS2-depleted cells. Heterozygous VARS2-knockout cells showed a rearrangement of ETC complexes in favor of complexes III2, III2 + IV, and supercomplexes without significant respiratory chain deficiencies. These cells also showed the enhanced activation of the ISR, as indicated by increased eIF-2α phosphorylation and a significant increase in the transcript levels of ATF4, ATF5, and DDIT3 (CHOP), as well as disruptions in FAO. The activation of the ISR and disruptions in mitochondrial FAO may underlie the adaptive changes in VARS2-depleted cells.

Genetic architecture of cardiac dynamic flow volumes

Abstract: Cardiac blood flow is a critical determinant of human health. However, definition of its genetic architecture is limited by the technical challenge of capturing dynamic flow volumes from cardiac imaging at scale. We present DeepFlow, a deep learning system to extract cardiac flow and volumes from phase contrast cardiac magnetic resonance imaging. A mixed linear model applied to 37,967 individuals from the UK Biobank reveals novel genome-wide significant associations across cardiac dynamic flow volumes including aortic forward velocity, total left ventricular stroke volume, forward left ventricular flow and aortic regurgitation fraction. Mendelian randomization using CAUSE reveals a causal role for aortic root size in aortic valve regurgitation. The most significant contributing variants (near ELN, FBN1 and ULK4) are implicated in connective tissue and blood pressure pathways. DeepFlow cardiac flow phenotyping at scale, combined with population-level genotyping data in the UK Biobank, reinforces the contribution of connective tissue genes, blood pressure and root size to aortic valve function in the general population.

Genotype Complements the Phenotype: Identification of the Pathogenicity of an LMNA Splice Variant by Nanopore Long-Read Sequencing in a Large DCM Family

Abstract: Dilated cardiomyopathy (DCM) is a common cause of heart failure (HF) and is of familial origin in 20–40% of cases. Genetic testing by next-generation sequencing (NGS) has yielded a definite diagnosis in many cases; however, some remain elusive. In this study, we used a combination of NGS, human-induced pluripotent-stem-cell-derived cardiomyocytes (iPSC-CMs) and nanopore long-read sequencing to identify the causal variant in a multi-generational pedigree of DCM. A four-generation family with familial DCM was investigated. Next-generation sequencing (NGS) was performed on 22 family members. Skin biopsies from two affected family members were used to generate iPSCs, which were then differentiated into iPSC-CMs. Short-read RNA sequencing was used for the evaluation of the target gene expression, and long-read RNA nanopore sequencing was used to evaluate the relevance of the splice variants. The pedigree suggested a highly penetrant, autosomal dominant mode of inheritance. The phenotype of the family was suggestive of laminopathy, but previous genetic testing using both Sanger and panel sequencing only yielded conflicting evidence for LMNA p.R644C (rs142000963), which was not fully segregated. By re-sequencing four additional affected family members, further non-coding LMNA variants could be detected: rs149339264, rs199686967, rs201379016, and rs794728589. To explore the roles of these variants, iPSC-CMs were generated. RNA sequencing showed the LMNA expression levels to be significantly lower in the iPSC-CMs of the LMNA variant carriers. We demonstrated a dysregulated sarcomeric structure and altered calcium homeostasis in the iPSC-CMs of the LMNA variant carriers. Using targeted nanopore long-read sequencing, we revealed the biological significance of the variant c.356+1G>A, which generates a novel 5′ splice site in exon 1 of the cardiac isomer of LMNA, causing a nonsense mRNA product with almost complete RNA decay and haploinsufficiency. Using novel molecular analysis and nanopore technology, we demonstrated the pathogenesis of the rs794728589 (c.356+1G>A) splice variant in LMNA. This study highlights the importance of precise diagnostics in the clinical management and workup of cardiomyopathies.

NIMA-related kinase 9 regulates the phosphorylation of the essential myosin light chain in the heart

Abstract: To adapt to changing hemodynamic demands, regulatory mechanisms modulate actin-myosin-kinetics by calcium-dependent and -independent mechanisms. We investigate the posttranslational modification of human essential myosin light chain (ELC) and identify NIMA-related kinase 9 (NEK9) to interact with ELC. NEK9 is highly expressed in the heart and the interaction with ELC is calcium-dependent. Silencing of NEK9 results in blunting of calcium-dependent ELC-phosphorylation. CRISPR/Cas9-mediated disruption of NEK9 leads to cardiomyopathy in zebrafish. Binding to ELC is mediated via the protein kinase domain of NEK9. A causal relationship between NEK9 activity and ELC-phosphorylation is demonstrated by genetic sensitizing in-vivo. Finally, we observe significantly upregulated ELC-phosphorylation in dilated cardiomyopathy patients and provide a unique map of human ELC-phosphorylation-sites. In summary, NEK9-mediated ELC-phosphorylation is a calcium-dependent regulatory system mediating cardiac contraction and inotropy.

Targeted Analysis of circRNA Expression in Patient Samples by Lexo-circSeq

Abstract: Recently, circular RNAs (circRNAs) have been extensively studied in animals and plants. circRNAs are generated by backsplicing from the same linear transcripts that are canonically spliced to produce, for example, mature mRNAs. circRNAs exhibit tissue-specific expression and are potentially involved in many diseases, among them cardiovascular diseases. The comprehensive analysis of circRNA expression patterns across larger patient cohorts requires a streamlined and cost-effective workflow designed to meet small input requirements. In this article, we present Lexo-circSeq, a targeted RNA sequencing approach that can profile up to 110 circRNAs and their corresponding linear transcripts in one experiment. We established Lexo-circSeq employing total human heart RNA and show that our protocol can detect depletion of a specific circRNA in hiPSC-derived cardiomyocytes. Finally, Lexo-circSeq was applied to biopsies from patients diagnosed with dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Interestingly, our results indicate that circular-to-linear-ratios for circSLC8A1 and circRBM33 are deregulated in cardiomyopathy.

Content-Aware Differential Privacy with Conditional Invertible Neural Networks

Abstract: , Abstract. Differential privacy (DP) has arisen as the gold standard in protecting an individual’s privacy in datasets by adding calibrated noise to each data sample. While the application to categorical data is straightforward, its usability in the context of images has been limited. Contrary to categorical data the meaning of an image is inherent in the spatial correlation of neighboring pixels making the simple application of noise infeasible. Invertible Neural Networks (INN) have shown excellent generative performance while still providing the ability to quantify the exact likelihood. Their principle is based on transforming a complicated distribution into a simple one e.g. an image into a spherical Gaussian. We hypothesize that adding noise to the latent space of an INN can en-able differentially private image modification. Manipulation of the latent space leads to a modified image while preserving important details. Further, by conditioning the INN on meta-data provided with the dataset we aim at leaving dimensions important for downstream tasks like classification untouched while altering other parts that potentially contain identifying information. We term our method content-aware differential privacy (CADP). We conduct experiments on publicly available bench-marking datasets as well as dedicated medical ones. In addition, we show the generalizability of our method to categorical data. The source code is publicly available at github.com/Cardio-AI/CADP .

Deep phenotyping of two pre-clinical mouse models and a cohort of RBM20 mutation carriers reveals no sex-dependent disease severity in RBM20 cardiomyopathy.

Abstract: Aims RBM20 cardiomyopathy is an arrhythmogenic form of dilated cardiomyopathy caused by mutations in the splicing factor RBM20. A recent study found a more severe phenotype in male RBM20 cardiomyopathy patients than in female patients. Here, we aim to determine sex differences in an animal model of RBM20 cardiomyopathy, and investigate potential underlying mechanisms. Additionally, we aim to determine sex and gender differences in clinical parameters in a novel RBM20 cardiomyopathy patient cohort. Methods and Results We characterized a Rbm20 knockout (KO) mouse model, and show that splicing of key RBM20 targets, cardiac function, and arrhythmia susceptibility do not differ between sexes. Next, we performed deep phenotyping of these mice, and show that male and female Rbm20-KO mice possess transcriptomic and phosphoproteomic differences. Hypothesizing that these differences may influence the heart's ability to compensate for stress, we exposed Rbm20-KO mice to acute catecholaminergic stimulation, and again found no functional differences. We also replicate the lack of functional differences in a mouse model with the Rbm20-R636Q mutation. Lastly, we present a patient cohort of 33 RBM20 cardiomyopathy patients, and show that these patients do not possess sex and gender differences in disease severity. Conclusions Current mouse models of RBM20 cardiomyopathy show more pronounced changes in gene expression and phosphorylation of cardiac proteins in male mice, but no sex differences in cardiac morphology and function. Moreover, other than reported before, male RBM20 cardiomyopathy patients do not present with worse cardiac function in a patient cohort from Germany and the Netherlands.

Die Kardiologie

Abstract: In the majority of cases patients with high-grade tricuspid valve insufficiency (TI) represent a high-risk population and the treatment is particularly complex. Therefore, selection of an appropriate therapeutic pathway for affected individuals first requires precise characterization of the genesis of the vitium. This includes transthoracic and transesophageal echocardiography, right heart and left heart catheterization and additional imaging by CT and MRI as needed. Treatment requires interdisciplinary discussion, including optimization of drug treatment for left heart failure, discussion of antiarrhythmic treatment procedures for atrial genesis of TI, exclusion of pathologies of the heart requiring surgical treatment, and exclusion of indications for targeted therapy for pulmonary arterial hypertension (PAH). Interventional treatment now has an important place, particularly because of the very frequent comorbidity of patients. The 2021 ESC guidelines recommended catheter-based reconstruction for the first time. The still limited experience with this complex form of treatment, the lack of randomized prospective studies on its effectiveness, and challenges in periprocedural imaging have prompted the DGK to formulate these recommendations in anticipation of concrete criteria for future center certification when providing a program for interventional treatment of tricuspid regurgitation. This paper is intended to contribute to the provision of this important treatment to patients in Germany under the premise of the best possible quality.

EPHB4 mutations in dilated cardiomyopathy

Abstract: Cardiac homeostasis relies on the appropriate provision of nutrients and functional specialization of local endothelial cells. Previously we reported in this journal that the endothelial Eph-ephrin signalling, in particular the ligand EphB4, is required for the maintenance of vascular integrity and correct fatty acid transport uptake in the heart via regulating the caveolar trafficking of the fatty acid receptor CD36. In the mouse, endothelial specific loss-of-function of the receptor EphB4, or its ligand ephrin-B2, induces Dilated Cardiomyopathy (DCM) like defects (Luxan et al., 2019). Here, we have identified new rare EPHB4 variants in a cohort of 573 DCM patients. Similar to what we had observed in the EphB4 mutant mice, EPHB4 variants carrying patients show an altered expression pattern of CD36 and CAV1 in the myocardium. Our study confirms a crucial role of the Eph-ephrin signalling pathway, and in particular the receptor EPHB4, in the development of DCM in humans.

Decrease of RNA editing in the failing heart leads to induction of circRNAs

Abstract: Adenosine-to-Inosine (A-to-I) RNA editing is a post-transcriptional modification process that affects the secondary structure of RNAs. Changes in RNA editing have been associated with human diseases. We therefore aimed to analyze editing in the healthy and failing human heart. Transcriptome sequencing of human heart samples of heart failure (HF) patients (n=20) and controls (n=10) revealed A-to-I editing as the major type of editing (>80%). In HF patients, RNA editing was reduced, which was primarily attributable to Alu elements in introns of protein-coding genes. We identified 166 upregulated circRNAs in HF, with the majority showing reduced RNA editing in their parental host gene (88.3%). CircRNA expression did not correlate with their corresponding host gene (R=0.07, P<0.05), suggesting that an alternative splicing mechanism gives rise to the elevated circRNA levels in HF. The RNA editing enzyme ADAR2, which binds to RNA regions that are edited from adenosine to inosine, was decreased in failing human hearts (−68.2%). In vitro, reduction of ADAR2 increased circRNA levels suggesting a causal effect of reduced ADAR2 levels on increased circRNAs in the failing human heart. To gain mechanistic insight, we examined the formation of circRNAs on one exemplary candidate. AKAP13 was among the top edited mRNAs in the human heart and gave rise to a circular transcript, which was elevated in HF. ADAR2 reduced the formation of double-stranded structures in AKAP13 pre-mRNA, thereby reducing the stability of Alu elements and the circularization of the resulting circRNA. Overexpression of circAKAP13 impaired the sarcomere regularity of human induced pluripotent stem cell-derived cardiomyocytes (−31.0%). Our study shows that ADAR2 mediates A-to-I RNA editing in the human heart. We describe an alternative splicing mechanism of circRNAs in the human heart. In the healthy human heart, A-to-I RNA editing represses the formation of dsRNA structures of Alu elements thereby favoring linear mRNA splicing. Our results contribute to a better mechanistic understanding into the human-specific regulation of circRNA formation and are relevant to diseases with reduced RNA editing and increased circRNA levels. Type of funding sources: None.

Structured, Harmonized, and Interoperable Integration of Clinical Routine Data to Compute Heart Failure Risk Scores

Abstract: Risk prediction in patients with heart failure (HF) is essential to improve the tailoring of preventive, diagnostic, and therapeutic strategies for the individual patient, and effectively use health care resources. Risk scores derived from controlled clinical studies can be used to calculate the risk of mortality and HF hospitalizations. However, these scores are poorly implemented into routine care, predominantly because their calculation requires considerable efforts in practice and necessary data often are not available in an interoperable format. In this work, we demonstrate the feasibility of a multi-site solution to derive and calculate two exemplary HF scores from clinical routine data (MAGGIC score with six continuous and eight categorical variables; Barcelona Bio-HF score with five continuous and six categorical variables). Within HiGHmed, a German Medical Informatics Initiative consortium, we implemented an interoperable solution, collecting a harmonized HF-phenotypic core data set (CDS) within the openEHR framework. Our approach minimizes the need for manual data entry by automatically retrieving data from primary systems. We show, across five participating medical centers, that the implemented structures to execute dedicated data queries, followed by harmonized data processing and score calculation, work well in practice. In summary, we demonstrated the feasibility of clinical routine data usage across multiple partner sites to compute HF risk scores. This solution can be extended to a large spectrum of applications in clinical care.

Multi-omics assessment of dilated cardiomyopathy using non-negative matrix factorization

Abstract: Dilated cardiomyopathy (DCM), a myocardial disease, is heterogeneous and often results in heart failure and sudden cardiac death. Unavailability of cardiac tissue has hindered the comprehensive exploration of gene regulatory networks and nodal players in DCM. In this study, we carried out integrated analysis of transcriptome and methylome data using non-negative matrix factorization from a cohort of DCM patients to uncover underlying latent factors and covarying features between whole-transcriptome and epigenome omics datasets from tissue biopsies of living patients. DNA methylation data from Infinium HM450 and mRNA Illumina sequencing of n = 33 DCM and n = 24 control probands were filtered, analyzed and used as input for matrix factorization using R NMF package. Mann-Whitney U test showed 4 out of 5 latent factors are significantly different between DCM and control probands (P<0.05). Characterization of top 10% features driving each latent factor showed a significant enrichment of biological processes known to be involved in DCM pathogenesis, including immune response (P = 3.97E-21), nucleic acid binding (P = 1.42E-18), extracellular matrix (P = 9.23E-14) and myofibrillar structure (P = 8.46E-12). Correlation network analysis revealed interaction of important sarcomeric genes like Nebulin, Tropomyosin alpha-3 and ERC-protein 2 with CpG methylation of ATPase Phospholipid Transporting 11A0, Solute Carrier Family 12 Member 7 and Leucine Rich Repeat Containing 14B, all with significant P values associated with correlation coefficients >0.7. Using matrix factorization, multi-omics data derived from human tissue samples can be integrated and novel interactions can be identified. Hypothesis generating nature of such analysis could help to better understand the pathophysiology of complex traits such as DCM.

Abstract P2044: The Cardioprotective Egr-1 And Maladaptive Sp-1 Zinc Finger Transcription Factor Reciprocally Regulates Expression Of The Cardiomyocyte Performance Enhancing Protein S100a1

Abstract: Background and Hypothesis: Expression of the cardiomyocyte (CM) protein S100A1, which improves contractile performance of the heart, sharply increases during postnatal myocardial maturation but declines rapidly in failing hearts. We therefore hypothesized that CMs are wired with transcriptional factors (TFs) that positively and negatively regulate S100A1’s gene locus activity. Understanding these reciprocal circuits may be relevant for advanced therapeutic modulation of S100A1’s abundance in diseased hearts. Methods and Results: H9C2 rat cardiomyoblasts, an animal-free in vitro tool, displayed a strong concordant rise in S100A1 mRNA and protein levels (8.1+/-1.1 vs. cont.; n=9, p<0.05) amongst other CM markers (i.e., SERCA2a) over a 5-day CM differentiation protocol. Overall TF activity during this process was computationally inferred from TF binding site (TFBS) assessments in promoters of all actively regulated transcripts provided by a time-resolved (undifferentiated, day 0, 2 and 5) transcriptome analysis. From these TFs, EGR1 and SP1 amongst eight others were chosen due to their abundance both in differentiated H9C2-CMs and adult rat hearts and TFBS in the -1000 to +500 bp rat S100A1 gene promoter region due to a 5’ RACE-PCR based transcription start site identification in our model. To capture the relevant TFs from this group, we next co-incubated nuclear extracts from H9C2-CMs with biotinylated fragments of the aforementioned S100A1 promoter region coupled to streptavidin beads. S100A1 promoter-bound TFs were identified by mass spectrometry and only factors with a >2-fold enrichment over control were selected delivering EGR1 and SP1 as top hits. Subsequent siRNA-mediated silencing of both TFs yielded an EGR1 and SP1 knock-down dose-dependent inhibition (80%; p<0.05 vs. contr., n=9) and amplification (126%; p<0.05 vs. contr., n=9) of the rise both in S100A1 mRNA and protein levels in the H9C2-CM model that validated our comp./exp. pipeline and biological relevance of our TF hits, respectively. Conclusion: Our study identified the known cardioprotective EGR1 and maladaptive SP1 as novel positive and negative TF regulators of CM S100A1 expression and targets for therapeutic S100A1 gene locus modulation i.e., by SP1 TFBS gene-editing.