Integrative proteomic and metabolomic elucidation of cardiomyopathy with in vivo and in vitro models and clinical samples

Abstract

Cardiomyopathy is a prevalent cardiovascular disease that affects individuals of all ages and can lead to life-threatening heart failure. Despite its variety in types, each with distinct characteristics and causes, our understanding of cardiomyopathy at a systematic biology level remains incomplete. Mass spectrometry-based techniques have emerged as powerful tools, providing a comprehensive view of the molecular landscape and aiding in the discovery of biomarkers and elucidation of mechanisms. This review highlights the significant potential of integrating proteomic and metabolomic approaches with specialized databases to identify biomarkers and therapeutic targets across different types of cardiomyopathies. In vivo and in vitro models, such as genetically modified mice, patient-derived or induced pluripotent stem cells, and organ chips, are invaluable in exploring the pathophysiological complexities of this disease. By integrating omics approaches with these sophisticated modeling systems, our comprehension of the molecular underpinnings of cardiomyopathy can be greatly enhanced, facilitating the development of diagnostic markers and therapeutic strategies. Among the promising therapeutic targets are those involved in extracellular matrix remodeling, sarcomere damage, and metabolic remodeling. These targets hold the potential to advance precision therapy in cardiomyopathy, offering hope for more effective treatments tailored to the specific molecular profiles of patients.

Keywords: cardiomyopathy; diagnostic marker; metabolomics; proteomics; therapeutic target.

Graphical abstract

Figure 1

Schematic illustration of mass spectrometry-based proteomics workflows Firstly, samples of human heart tissue and serum/plasma were collected for analysis. A procedure involving the removal of high-abundance proteins or the enrichment of low-abundance proteins in serum/plasma samples was conducted to mitigate the inhibitory impact of high-abundance proteins on low-abundance proteins. For tissue samples, the DISCO-MS (three-dimensional imaging of solvent-cleared organs profiled by mass spectrometry) method can be employed for highly efficient protein extraction. Subsequently, samples were subjected to a series of pretreatment steps, including protein reductive alkylation, digestion of proteins into peptides, and peptide desalting. The peptides were then analyzed using LC-MS/MS. The most recent developments in quantitative proteomics are characterized by the use of cutting-edge techniques, including untargeted methods, such as DDA, DIA, and TMT, and targeted approaches, such as MRM and PRM. Ultimately, the data are integrated with machine learning for comprehensive database searching, statistical analysis, and bioinformatic analysis.

Figure 2

Schematic representation of metabolomics methods based on MS After serum/plasma and tissue sample collection and preparation, such as tissue homogenization, deproteinization, and lyophilization, metabolites extracted from samples were reconstituted in appropriate solvents for subsequent analysis by LC-MS. In GC-MS, derivatization is a common procedure to enhance the detection sensitivity of specific molecules. Following the data acquisition by LC-MS or GC-MS, the data undergo processing steps, including noise filtering, peak picking, feature alignment, etc. For untargeted metabolomics, metabolites are identified via database searching. Pathway analysis and cluster analysis facilitate a more comprehensive examination of metabolomics data, enabling the linkage of specific metabolites to the biological context under investigation. In the context of marker validation in targeted metabolomics, MRM and PRM assays are frequently employed as strategies for the simultaneous quantitation of multiple metabolites. Furthermore, metabolic flux analysis employing stable isotope tracer methods to monitor the dynamic metabolic activities of biological systems can be applied.

Figure 3

Common animal models in cardiomyopathy research Rodent models are a prevalent choice of animal model in the field of cardiovascular disease research. Surgical models and genetically engineered models are two particularly common model types. In the case of surgical models, the most commonly used ones are the TAC model and the MI model. The TAC model, which is constructed by narrowing the transverse aorta, is most frequently employed for the study of pressure-overload-induced left ventricular hypertrophy and its progression to HF. MI models are induced by the permanent or transient occlusion of the LAD coronary artery and widely used to simulate myocardial infarction and ischemia-reperfusion injury. For genetically engineered models, inherited mutations previously identified in humans with cardiomyopathy are modeled in mice to reflect human phenotype. Genes associated with DCM include Lmna and TNNT2, genes associated with HCM include TNNT2 and OBSCN, and genes associated with RCM include BAG3 and DES.

Figure 4

Schematic representation of human iPSCs as models for studying cardiomyopathy iPSCs are derived from the skin or blood cells of donors to capture their genetic backgrounds. By introducing reprogramming factors, such as OCT4, SOX2, KLF4, and c-MYC, into a specialized patient’s cells (e.g., fibroblast), the somatic cells were reprogramed to iPSCs. The patient-derived iPSCs can subsequently be differentiated into specialized cells (e.g., cardiomyocytes). To generate isogenic patient-derived iPSC lines, certain genetic alterations can be performed utilizing genome editing tools, such as CRISPR-Cas9. The iPSC-derived cells can be characterized to identify disease features and investigate underlying causes. The iPSC-CMs are donor specific, enabling the establishment of genotype-phenotype connections and providing a tailored drug-screening platform for personalized patient therapy. iPSCs, induced pluripotent stem cells; iPSC-CMs, induced pluripotent stem cell-derived cardiomyocytes.

Figure 5

Potential therapeutic targets of cardiomyopathy TGF-β is a critical profibrotic molecule. ECM remodeling and fibrosis represent crucial pathological features of HF, with the cardiac ECM offering a unique opportunity for therapeutic intervention. Alterations were observed in Clip1, a downstream target of TGF-β, in the context of cardiac ECM remodeling. Proteoglycans play a pivotal role in cardiac matrix remodeling and fibrosis and may represent a promising avenue for therapeutic intervention. ADAMTS proteases have been identified as pivotal for versican degradation and ECM-mediated intercellular communication in the heart. In addition, the sarcomeric structure is essential for heart function. HDAC6-mediated deacetylation reduces myofibril stiffness, and microtubule inhibitors may possess cardioprotective properties. Furthermore, metabolic remodeling contributes to HF development under hemodynamic stress. It has been proposed that enhancing PKM1-mediated glucose metabolism, pharmacologically activating PKM2, and the myocardial bioenergetics downstream of Rev-erb may represent potential therapeutic targets for HF treatment. Reproduced from Kessler et al. by permission of the publisher Oxford University Press. Reproduced from Lin et al., Schuldt et al., Li et al., and Tang et al. by permission of the publisher Wolters Kluwer Health.