Periostin regulates lysyl oxidase through ERK1/2 MAPK-dependent serum response factor in activated cardiac fibroblasts.

Collagen crosslinking, mediated by lysyl oxidase, is an adaptive mechanism of the cardiac repair process initiated by cardiac fibroblasts postmyocardial injury. However, excessive crosslinking leads to cardiac wall stiffening, which impairs the contractile properties of the left ventricle and leads to heart failure. In this study, we investigated the role of periostin, a matricellular protein, in the regulation of lysyl oxidase in cardiac fibroblasts in response to angiotensin II and TGFß1. Our results indicated that periostin silencing abolished the angiotensin II and TGFß1-mediated upregulation of lysyl oxidase. Furthermore, the attenuation of periostin expression resulted in a notable reduction in the activity of lysyl oxidase. Downstream of periostin, ERK1/2 MAPK signaling was found to be activated, which in turn transcriptionally upregulates the serum response factor to facilitate the enhanced expression of lysyl oxidase. The periostin-lysyl oxidase association was also positively correlated in an in vivo rat model of myocardial infarction. The expression of periostin and lysyl oxidase was upregulated in the collagen-rich fibrotic scar tissue of the left ventricle. Remarkably, echocardiography data showed a reduction in the left ventricular wall movement, ejection fraction, and fractional shortening, indicative of enhanced stiffening of the cardiac wall. These findings shed light on the mechanistic role of periostin in the collagen crosslinking initiated by activated cardiac fibroblasts. Our findings signify periostin as a possible therapeutic target to reduce excessive collagen crosslinking that contributes to the structural remodeling associated with heart failure.

Cardiac Fibroblastic Niches in Homeostasis and Inflammation.

Fibroblasts are essential for building and maintaining the structural integrity of all organs. Moreover, fibroblasts can acquire an inflammatory phenotype to accommodate immune cells in specific niches and to provide migration, differentiation, and growth factors. In the heart, balancing of fibroblast activity is critical for cardiac homeostasis and optimal organ function during inflammation. Fibroblasts sustain cardiac homeostasis by generating local niche environments that support housekeeping functions and by actively engaging in intercellular cross talk. During inflammatory perturbations, cardiac fibroblasts rapidly switch to an inflammatory state and actively communicate with infiltrating immune cells to orchestrate immune cell migration and activity. Here, we summarize the current knowledge on the molecular landscape of cardiac fibroblasts, focusing on their dual role in promoting tissue homeostasis and modulating immune cell-cardiomyocyte interaction. In addition, we discuss potential future avenues for manipulating cardiac fibroblast activity during myocardial inflammation.

Revisiting Cardiac Biology in the Era of Single Cell and Spatial Omics.

Throughout our lifetime, each beat of the heart requires the coordinated action of multiple cardiac cell types. Understanding cardiac cell biology, its intricate microenvironments, and the mechanisms that govern their function in health and disease are crucial to designing novel therapeutical and behavioral interventions. Recent advances in single-cell and spatial omics technologies have significantly propelled this understanding, offering novel insights into the cellular diversity and function and the complex interactions of cardiac tissue. This review provides a comprehensive overview of the cellular landscape of the heart, bridging the gap between suspension-based and emerging in situ approaches, focusing on the experimental and computational challenges, comparative analyses of mouse and human cardiac systems, and the rising contextualization of cardiac cells within their niches. As we explore the heart at this unprecedented resolution, integrating insights from both mouse and human studies will pave the way for novel diagnostic tools and therapeutic interventions, ultimately improving outcomes for patients with cardiovascular diseases.

Intersection of Immunology and Metabolism in Myocardial Disease.

Immunometabolism is an emerging field at the intersection of immunology and metabolism. Immune cell activation plays a critical role in the pathogenesis of cardiovascular diseases and is integral for regeneration during cardiac injury. We currently possess a limited understanding of the processes governing metabolic interactions between immune cells and cardiomyocytes. The impact of this intercellular crosstalk can manifest as alterations to the steady state flux of metabolites and impact cardiac contractile function. Although much of our knowledge is derived from acute inflammatory response, recent work emphasizes heterogeneity and flexibility in metabolism between cardiomyocytes and immune cells during pathological states, including ischemic, cardiometabolic, and cancer-associated disease. Metabolic adaptation is crucial because it influences immune cell activation, cytokine release, and potential therapeutic vulnerabilities. This review describes current concepts about immunometabolic regulation in the heart, focusing on intercellular crosstalk and intrinsic factors driving cellular regulation. We discuss experimental approaches to measure the cardio-immunologic crosstalk, which are necessary to uncover unknown mechanisms underlying the immune and cardiac interface. Deeper insight into these axes holds promise for therapeutic strategies that optimize cardioimmunology crosstalk for cardiac health.

Dissecting and Visualizing the Functional Diversity of Cardiac Macrophages.

Cardiac macrophages represent a functionally diverse population of cells involved in cardiac homeostasis, repair, and remodeling. With recent advancements in single-cell technologies, it is possible to elucidate specific macrophage subsets based on transcriptional signatures and cell surface protein expression to gain a deep understanding of macrophage diversity in the heart. The use of fate-mapping technologies and parabiosis studies have provided insight into the ontogeny and dynamics of macrophages identifying subsets derived from embryonic and adult definitive hematopoietic progenitors that include tissue-resident and bone marrow monocyte-derived macrophages, respectively. Within the heart, these subsets have distinct tissue niches and functional roles in the setting of homeostasis and disease, with cardiac resident macrophages representing a protective cell population while bone marrow monocyte-derived cardiac macrophages have a context-dependent effect, triggering both proinflammatory tissue injury, but also promoting reparative functions. With the increased understanding of the clinical relevance of cardiac macrophage subsets, there has been an increasing need to detect and measure cardiac macrophage compositions in living animals and patients. New molecular tracers compatible with positron emission tomography/computerized tomography and positron emission tomography/ magnetic resonance imaging have enabled investigators to noninvasively and serially visualize cardiac macrophage subsets within the heart to define associations with disease and measure treatment responses. Today, advancements within this thriving field are poised to fuel an era of clinical translation.

Exploring T Cell Receptor Repertoires in Myocardial Diseases.

Mounting experimental and clinical evidence has revealed that adaptive immune mechanisms targeting myocardial antigens are triggered by different forms of cardiac injury and impact disease progression. B and T lymphocytes recognize specific antigens via unique adaptive immune receptors generated through a somatic rearrangement process that generates a potential repertoire of 1019 unique receptors. While the adaptive immune receptor repertoire diversity provides the basis for immunologic specificity, making sense of it can be a challenging task. In the present review, we discuss key aspects underlying the generation of TCRs (T cell receptors) and emerging tools for their study in the context of myocardial diseases. Moreover, we outline how exploring TCR repertoires could lead to a deeper understanding of myocardial pathophysiological principles and potentially serve as diagnostic tools.

Myocardial Inflammation in Heart Failure With Reduced and Preserved Ejection Fraction.

Heart failure (HF) is characterized by a progressive decline in cardiac function and represents one of the largest health burdens worldwide. Clinically, 2 major types of HF are distinguished based on the left ventricular ejection fraction (EF): HF with reduced EF and HF with preserved EF. While both types share several risk factors and features of adverse cardiac remodeling, unique hallmarks beyond ejection fraction that distinguish these etiologies also exist. These differences may explain the fact that approved therapies for HF with reduced EF are largely ineffective in patients suffering from HF with preserved EF. Improving our understanding of the distinct cellular and molecular mechanisms is crucial for the development of better treatment strategies. This article reviews the knowledge of the immunologic mechanisms underlying HF with reduced and preserved EF and discusses how the different immune profiles elicited may identify attractive therapeutic targets for these conditions. We review the literature on the reported mechanisms of adverse cardiac remodeling in HF with reduced and preserved EF, as well as the immune mechanisms involved. We discuss how the knowledge gained from preclinical models of the complex syndrome of HF as well as from clinical data obtained from patients may translate to a better understanding of HF and result in specific treatments for these conditions in humans.

Repair of the Infarcted Heart: Cellular Effectors, Molecular Mechanisms and Therapeutic Opportunities.

The adult mammalian heart has limited endogenous regenerative capacity and heals through the activation of inflammatory and fibrogenic cascades that ultimately result in the formation of a scar. After infarction, massive cardiomyocyte death releases a broad range of damage-associated molecular patterns that initiate both myocardial and systemic inflammatory responses. TLRs (toll-like receptors) and NLRs (NOD-like receptors) recognize damage-associated molecular patterns (DAMPs) and transduce downstream proinflammatory signals, leading to upregulation of cytokines (such as interleukin-1, TNF-α [tumor necrosis factor-α], and interleukin-6) and chemokines (such as CCL2 [CC chemokine ligand 2]) and recruitment of neutrophils, monocytes, and lymphocytes. Expansion and diversification of cardiac macrophages in the infarcted heart play a major role in the clearance of the infarct from dead cells and the subsequent stimulation of reparative pathways. Efferocytosis triggers the induction and release of anti-inflammatory mediators that restrain the inflammatory reaction and set the stage for the activation of reparative fibroblasts and vascular cells. Growth factor-mediated pathways, neurohumoral cascades, and matricellular proteins deposited in the provisional matrix stimulate fibroblast activation and proliferation and myofibroblast conversion. Deposition of a well-organized collagen-based extracellular matrix network protects the heart from catastrophic rupture and attenuates ventricular dilation. Scar maturation requires stimulation of endogenous signals that inhibit fibroblast activity and prevent excessive fibrosis. Moreover, in the mature scar, infarct neovessels acquire a mural cell coat that contributes to the stabilization of the microvascular network. Excessive, prolonged, or dysregulated inflammatory or fibrogenic cascades accentuate adverse remodeling and dysfunction. Moreover, inflammatory leukocytes and fibroblasts can contribute to arrhythmogenesis. Inflammatory and fibrogenic pathways may be promising therapeutic targets to attenuate heart failure progression and inhibit arrhythmia generation in patients surviving myocardial infarction.

The Role of Scintigraphy with Bone Radiotracers in Cardiac Amyloidosis.

Cardiac amyloidosis (CA) is caused by the myocardial deposition of misfolded proteins, either amyloid transthyretin (ATTR) or immunoglobulin light chains (AL). The paradigm of this condition has transformed, since CA is increasingly recognized as a relatively prevalent cause of heart failure. Cardiac scintigraphy with bone tracers is the unique noninvasive technique able to confirm CA without performing tissue biopsy or advanced imaging tests. A moderate-to-intense myocardial uptake (Perugini grade ≥2) associated with the absence of a monoclonal component is greater than 99% specific for ATTR-CA, while AL-CA confirmation requires tissue biopsy.

Pathophysiology of Cardiac Amyloidosis.

Amyloidosis refers to a heterogeneous group of disorders sharing common pathophysiological mechanisms characterized by the extracellular accumulation of fibrillar deposits consisting of the aggregation of misfolded proteins. Cardiac amyloidosis (CA), usually caused by deposition of misfolded transthyretin or immunoglobulin light chains, is an increasingly recognized cause of heart failure burdened by a poor prognosis. CA manifests with a restrictive cardiomyopathy which progressively leads to biventricular thickening, diastolic and then systolic dysfunction, arrhythmias, and valvular disease. The pathophysiology of CA is multifactorial and includes increased oxidative stress, mitochondrial damage, apoptosis, impaired metabolism, and modifications of intracellular calcium balance.

Curcumin-loaded nanoparticles effectively prevent T4-induced oxidative stress in rat heart.

In this study, we report the cardioprotective effect of the glycerol monooleate (GMO) based nanocurcumin in both in vitro and in vivo conditions under a hyperthyroid state. The heart is one of the primary target organs sensitive to the action of thyroid hormone, and slight variations in the thyroid hormone serum concentrations result in measurable changes in cardiac performance. Hyperthyroidism-induced hypermetabolism is associated with oxidative stress and is an important mechanism responsible for the progression of heart failure. Curcumin has been known to play a protective role against oxidative stress-related diseases like Alzheimer's, asthma, and aging due to its antioxidant properties. Nevertheless, its potent biological activity has been hindered due to its poor bioavailability. To overcome this drawback, a GMO-based biodegradable nanoparticle (NP) formulation loaded with curcumin has been developed, and the protective effect of curcumin-loaded NPs was compared against the native drug. Oxidative stress parameters like reactive oxygen species (ROS) release, change in mitochondrial membrane permeability, lipid peroxidation (LPx), lactate dehydrogenase (LDH) release, and the activity and protein expression of the endogenous antioxidant enzymes like superoxide dismutase, catalase (CAT) and glutathione peroxidase were evaluated. The results from in vitro showed that curcumin-loaded NPs showed better DPPH and NO radical scavenging activity than native curcumin in a concentrations range of 2.5-20 µM. It was also observed that the nanoparticulate curcumin was comparatively more effective than native curcumin in protecting against ROS-induced membrane damage by reducing LPx and LDH leakage at low concentrations of 5-10 µM. Further, curcumin NPs performed better in facilitating the activities of antioxidant enzymes under in vitro and in vivo conditions with respect to time and concentrations, resulting in reduced cellular ROS levels. In this scenario, we anticipate that curcumin-loaded NPs can serve as a better antioxidant than its native counterpart in protecting the heart from oxidative stress-related diseases.

Growth/differentiation factor 15 (GDF15) expression in the heart after myocardial infarction and cardioprotective effect of pre-ischemic rGDF15 administration.

Growth/differentiation factor-15 (GDF15) is considered an unfavourable prognostic biomarker for cardiovascular disease in clinical data, while experimental studies suggest it has cardioprotective potential. This study focuses on the direct cardiac effects of GDF15 during ischemia-reperfusion injury in Wistar male rats, employing concentrations relevant to patients at high cardiovascular risk. Initially, we examined circulating levels and heart tissue expression of GDF15 in rats subjected to ischemia-reperfusion and sham operations in vivo. We then evaluated the cardiac effects of GDF15 both in vivo and ex vivo, administering recombinant GDF15 either before 30 min of ischemia (preconditioning) or at the onset of reperfusion (postconditioning). We compared infarct size and cardiac contractile recovery between control and rGDF15-treated rats. Contrary to our expectations, ischemia-reperfusion did not increase GDF15 plasma levels compared to sham-operated rats. However, cardiac protein and mRNA expression increased in the infarcted zone of the ischemic heart after 24 h of reperfusion. Notably, preconditioning with rGDF15 had a cardioprotective effect, reducing infarct size both in vivo (65 ± 5% in control vs. 42 ± 6% in rGDF15 groups) and ex vivo (60 ± 4% in control vs. 45 ± 4% in rGDF15 groups), while enhancing cardiac contractile recovery ex vivo. However, postconditioning with rGDF15 did not alter infarct size or the recovery of contractile parameters in vivo or ex vivo. These novel findings reveal that the short-term exogenous administration of rGDF15 before ischemia, at physiologically relevant levels, protects the heart against ischemia-reperfusion injury in both in vivo and ex vivo settings. The ex vivo results indicate that rGDF15 operates independently of the inflammatory, endocrine and nervous systems, suggesting direct and potent cardioprotective properties against ischemia-reperfusion injury.

MARK4 aggravates cardiac dysfunction in mice with STZ-induced diabetic cardiomyopathy by regulating ACSL4-mediated myocardial lipid metabolism.

Diabetic cardiomyopathy is a specific type of cardiomyopathy. In DCM, glucose uptake and utilization are impaired due to insulin deficiency or resistance, and the heart relies more heavily on fatty acid oxidation for energy, resulting in myocardial lipid toxicity-related injury. MARK4 is a member of the AMPK-related kinase family, and improves ischaemic heart failure through microtubule detyrosination. However, the role of MARK4 in cardiac regulation of metabolism is unclear. In this study, after successful establishment of a diabetic cardiomyopathy model induced by streptozotocin and a high-fat diet, MARK4 expression was found to be significantly increased in STZ-induced DCM mice. After AAV9-shMARK4 was administered through the tail vein, decreased expression of MARK4 alleviated diabetic myocardial damage, reduced oxidative stress and apoptosis, and facilitated cardiomyocyte mitochondrial fusion, and promoted myocardial lipid oxidation metabolism. In addition, through the RNA-seq analysis of differentially expressed genes, we found that MARK4 deficiency promoted lipid decomposition and oxidative metabolism by downregulating the expression of ACSL4, thus reducing myocardial lipid accumulation in the STZ-induced DCM model.

Ondansetron or beta-sitosterol antagonizes inflammatory responses in liver, kidney, lung and heart tissues of irradiated arthritic rats model.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder mainly affecting joints, yet the systemic inflammation can influence other organs and tissues. The objective of this study was to unravel the ameliorative capability of Ondansetron (O) or ß-sitosterol (BS) against inflammatory reactions and oxidative stress that complicates Extra-articular manifestations (EAM) in liver, kidney, lung, and heart of arthritic and arthritic irradiated rats. METHODS: This was accomplished by exposing adjuvant-induced arthritis (AIA) rats to successive weekly fractions of total body γ-irradiation (2 Gray (Gy)/fraction once per week for four weeks, up to a total dose of 8 Gy). Arthritic and/or arthritic irradiated rats were either treated with BS (40 mg/kg b.wt. /day, orally) or O (2 mg/kg) was given ip) or were kept untreated as model groups. RESULTS: Body weight changes, paw circumference, oxidative stress indices, inflammatory response biomarkers, expression of Janus kinase-2 (JAK-2), Signal transducer and activator of transcription 3 (STAT3), high mobility group box1 (HMGB1), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), as well as pro- and anti-inflammatory mediators in the target organs, besides histopathological examination of ankle joints and extra-articular tissues. Treatment of arthritic and/or arthritic irradiated rats with BS or O powerfully alleviated changes in body weight gain, paw swelling, oxidative stress, inflammatory reactions, and histopathological degenerative alterations in articular and non-articular tissues. CONCLUSION: The obtained data imply that BS or O improved the articular and EAM by regulating oxidative and inflammatory indices in arthritic and arthritic irradiated rats.

Semaglutide ameliorates cardiac remodeling in male mice by optimizing energy substrate utilization through the Creb5/NR4a1 axis.

Semaglutide, a glucagon-like peptide-1 receptor agonist, is clinically used as a glucose-lowering and weight loss medication due to its effects on energy metabolism. In heart failure, energy production is impaired due to altered mitochondrial function and increased glycolysis. However, the impact of semaglutide on cardiomyocyte metabolism under pressure overload remains unclear. Here we demonstrate that semaglutide improves cardiac function and reduces hypertrophy and fibrosis in a mouse model of pressure overload-induced heart failure. Semaglutide preserves mitochondrial structure and function under chronic stress. Metabolomics reveals that semaglutide reduces mitochondrial damage, lipid accumulation, and ATP deficiency by promoting pyruvate entry into the tricarboxylic acid cycle and increasing fatty acid oxidation. Transcriptional analysis shows that semaglutide regulates myocardial energy metabolism through the Creb5/NR4a1 axis in the PI3K/AKT pathway, reducing NR4a1 expression and its translocation to mitochondria. NR4a1 knockdown ameliorates mitochondrial dysfunction and abnormal glucose and lipid metabolism in the heart. These findings suggest that semaglutide may be a therapeutic agent for improving cardiac remodeling by modulating energy metabolism.

Biomarkers Screening and Mechanisms Analysis of the Restraint Stress-Induced Myocardial Injury in Hyperlipidemia ApoE-/- Mice.

OBJECTIVES: To explore the biomarkers and potential mechanisms of chronic restraint stress-induced myocardial injury in hyperlipidemia ApoE-/- mice. METHODS: The hyperlipidemia combined with the chronic stress model was established by restraining the ApoE-/- mice. Proteomics and bioinformatics techniques were used to describe the characteristic molecular changes and related regulatory mechanisms of chronic stress-induced myocardial injury in hyperlipidemia mice and to explore potential diagnostic biomarkers. RESULTS: Proteomic analysis showed that there were 43 significantly up-regulated and 58 significantly down-regulated differentially expressed proteins in hyperlipidemia combined with the restraint stress group compared with the hyperlipidemia group. Among them, GBP2, TAOK3, TFR1 and UCP1 were biomarkers with great diagnostic potential. KEGG pathway enrichment analysis indicated that ferroptosis was a significant pathway that accelerated the myocardial injury in hyperlipidemia combined with restraint stress-induced model. The mmu_circ_0001567/miR-7a/Tfr-1 and mmu_circ_0001042/miR-7a/Tfr-1 might be important circRNA-miRNA-mRNA regulatory networks related to ferroptosis in this model. CONCLUSIONS: Chronic restraint stress may aggravate myocardial injury in hyperlipidemia mice via ferroptosis. Four potential biomarkers are selected for myocardial injury diagnosis, providing a new direction for sudden cardiac death (SCD) caused by hyperlipidemia combined with the restraint stress.

Postmortem Diffusion of Aconitum Alkaloids and Their Metabolites in Rabbits.

OBJECTIVES: To explore the postmortem diffusion rule of Aconitum alkaloids and their metabolites in poisoned rabbits, and to provide a reference for identifying the antemortem poisoning or postmortem poisoning of Aconitum alkaloids. METHODS: Twenty-four rabbits were sacrificed by tracheal clamps. After 1 hour, the rabbits were administered with aconitine LD50 in decocting aconite root powder by intragastric administration. Then, they were placed supine and stored at 25 ℃. The biological samples from 3 randomly selected rabbits were collected including heart blood, peripheral blood, urine, heart, liver, spleen, lung and kidney tissues at 0 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h and 96 h after intragastric administration, respectively. Aconitum alkaloids and their metabolites in the biological samples were analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). RESULTS: At 4 h after intragastric administration, Aconitum alkaloids and their metabolites could be detected in heart blood, peripheral blood and major organs, and the contents of them changed dynamically with the preservation time. The contents of Aconitum alkaloids and their metabolites were higher in the spleen, liver and lung, especially in the spleen which was closer to the stomach. The average mass fraction of benzoylmesaconine metabolized in rabbit spleen was the highest at 48 h after intragastric administration. In contrast, the contents of Aconitum alkaloids and their metabolites in kidney were all lower. Aconitum alkaloids and their metabolites were not detected in urine. CONCLUSIONS: Aconitum alkaloids and their metabolites have postmortem diffusion in poisoned rabbits, diffusing from high-content organs (stomach) to other major organs and tissues as well as the heart blood. The main mechanism is the dispersion along the concentration gradient, while urine is not affected by postmortem diffusion, which can be used as the basis for the identification of antemortem and postmortem Aconitum alkaloids poisoning.

Effects of hibernation on two important contractile tissues in tibetan frogs, Nanorana parkeri: a perspective from transcriptomics and metabolomics approaches.

BACKGROUND: In response to seasonal cold and food shortage, the Xizang plateau frogs, Nanorana parkeri (Anura: Dicroglossidae), enter a reversible hypometabolic state where heart rate and oxygen consumption in skeletal muscle are strongly suppressed. However, the effect of winter hibernation on gene expression and metabolic profiling in these two tissues remains unknown. In the present study, we conducted transcriptomic and metabolomic analyses of heart and skeletal muscle from summer- and winter-collected N. parkeri to explore mechanisms involved in seasonal hibernation. RESULTS: We identified 2407 differentially expressed genes (DEGs) in heart and 2938 DEGs in skeletal muscle. Enrichment analysis showed that shared DEGs in both tissues were enriched mainly in translation and metabolic processes. Of these, the expression of genes functionally categorized as "response to stress", "defense mechanisms", or "muscle contraction" were particularly associated with hibernation. Metabolomic analysis identified 24 and 22 differentially expressed metabolites (DEMs) in myocardium and skeletal muscle, respectively. In particular, pathway analysis showed that DEMs in myocardium were involved in the pentose phosphate pathway, glycerolipid metabolism, pyruvate metabolism, citrate cycle (TCA cycle), and glycolysis/gluconeogenesis. By contrast, DEMs in skeletal muscle were mainly involved in amino acid metabolism. CONCLUSIONS: In summary, natural adaptations of myocardium and skeletal muscle in hibernating N. parkeri involved transcriptional alterations in translation, stress response, protective mechanisms, and muscle contraction processes as well as metabolic remodeling. This study provides new insights into the transcriptional and metabolic adjustments that aid winter survival of high-altitude frogs N. parkeri.

Single-Cell RNA Sequencing Reveals Cardiac Fibroblast-Specific Transcriptomic Changes in Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is the most common cause of heart failure, with a complex aetiology involving multiple cell types. We aimed to detect cell-specific transcriptomic alterations in DCM through analysis that leveraged recent advancements in single-cell analytical tools. Single-cell RNA sequencing (scRNA-seq) data from human DCM cardiac tissue were subjected to an updated bioinformatic workflow in which unsupervised clustering was paired with reference label transfer to more comprehensively annotate the dataset. Differential gene expression was detected primarily in the cardiac fibroblast population. Bulk RNA sequencing was performed on an independent cohort of human cardiac tissue and compared with scRNA-seq gene alterations to generate a stratified list of higher-confidence, fibroblast-specific expression candidates for further validation. Concordant gene dysregulation was confirmed in TGFß-induced fibroblasts. Functional assessment of gene candidates showed that AEBP1 may play a significant role in fibroblast activation. This unbiased approach enabled improved resolution of cardiac cell-type-specific transcriptomic alterations in DCM.