Recent Developments in Clinical Plasma Proteomics—Applied to Cardiovascular Research

The human plasma proteome mirrors the physiological state of the cardiovascular system, a fact that has been used to analyze plasma biomarkers in routine analysis for the diagnosis and monitoring of cardiovascular diseases for decades. These biomarkers address, however, only a very limited subset of cardiovascular diseases, such as acute myocardial infarct or acute deep vein thrombosis, and clinical plasma biomarkers for the diagnosis and stratification cardiovascular diseases that are growing in incidence, such as heart failure and abdominal aortic aneurysm, do not exist and are urgently needed. The discovery of novel biomarkers in plasma has been hindered by the complexity of the human plasma proteome that again transforms into an extreme analytical complexity when it comes to the discovery of novel plasma biomarkers. This complexity is, however, addressed by recent achievements in technologies for analyzing the human plasma proteome, thereby facilitating the possibility for novel biomarker discoveries. The aims of this article is to provide an overview of the recent achievements in technologies for proteomic analysis of the human plasma proteome and their applications in cardiovascular medicine.

Is 'Not Different' Enough to Conclude Similar Cardiovascular Responses Across Sexes?

The number of research studies investigating whether similar or different cardiovascular responses or adaptations exist between males and females are increasing. Traditionally, difference-based statistical methods (e.g., t-test, ANOVA, etc.) have been implemented to compare cardiovascular function between males and females, with a P-value >0.05 used to denote similarity between sexes. However, an absence of evidence (i.e., large P-value) is not evidence of absence (i.e., no sex differences). Equivalence testing determines whether two measures or groups provide statistically equivalent outcomes, in that they differ by less than an 'ideally prespecified' smallest effect size of interest. Our perspective discusses the applicability and utility of integrating equivalence testing when conducting sex comparisons in cardiovascular research. An emphasis is placed on how cardiovascular researchers may conduct equivalence testing across multiple study designs (e.g., cross-sectional comparisons, repeated measures intervention, etc.). The strengths and weaknesses of this statistical tool are discussed. Equivalence analyses are relatively simple to conduct, may be used in conjunction with traditional hypothesis testing to interpret findings, and permits the determination of statistically equivalent responses between sexes. We recommend that cardiovascular researchers consider implementing equivalence testing to better our understanding of similar and different cardiovascular processes between sexes.

Recent Progress in Cardiovascular Research Involving Single-Cell Omics Approaches

Cardiovascular diseases are among the leading causes of morbidity and mortality worldwide. Although the spectrum of the heart from development to disease has long been studied, it remains largely enigmatic. The emergence of single-cell omics technologies has provided a powerful toolbox for defining cell heterogeneity, unraveling previously unknown pathways, and revealing intercellular communications, thereby boosting biomedical research and obtaining numerous novel findings over the last 7 years. Not only cell atlases of normal and developing hearts that provided substantial research resources, but also some important findings regarding cell-type-specific disease gene program, could never have been established without single-cell omics technologies. Herein, we briefly describe the latest technological advances in single-cell omics and summarize the major findings achieved by such approaches, with a focus on development and homeostasis of the heart, myocardial infarction, and heart failure.

From Spheroids to Organoids: The Next Generation of Model Systems of Human Cardiac Regeneration in a Dish

Organoids are tiny, self-organized, three-dimensional tissue cultures that are derived from the differentiation of stem cells. The growing interest in the use of organoids arises from their ability to mimic the biology and physiology of specific tissue structures in vitro. Organoids indeed represent promising systems for the in vitro modeling of tissue morphogenesis and organogenesis, regenerative medicine and tissue engineering, drug therapy testing, toxicology screening, and disease modeling. Although 2D cell cultures have been used for more than 50 years, even for their simplicity and low-cost maintenance, recent years have witnessed a steep rise in the availability of organoid model systems. Exploiting the ability of cells to re-aggregate and reconstruct the original architecture of an organ makes it possible to overcome many limitations of 2D cell culture systems. In vitro replication of the cellular micro-environment of a specific tissue leads to reproducing the molecular, biochemical, and biomechanical mechanisms that directly influence cell behavior and fate within that specific tissue. Lineage-specific self-organizing organoids have now been generated for many organs. Currently, growing cardiac organoid (cardioids) from pluripotent stem cells and cardiac stem/progenitor cells remains an open challenge due to the complexity of the spreading, differentiation, and migration of cardiac muscle and vascular layers. Here, we summarize the evolution of biological model systems from the generation of 2D spheroids to 3D organoids by focusing on the generation of cardioids based on the currently available laboratory technologies and outline their high potential for cardiovascular research.

Pathology and Advanced Imaging—Characterization of a Congenital Cardiac Defect and Complex Hemodynamics in a Pig: A Case Report

Domestic pigs are widely used in cardiovascular research as the porcine circulatory system bears a remarkable resemblance to that of humans. In order to reduce variability, only clinically healthy animals enter the study as their health status is assessed in entry examination. Like humans, pigs can also suffer from congenital heart disease, such as an atrial septal defect (ASD), which often remains undetected. Due to the malformation of the endocardial cushion during organ development, mitral valve defects (e.g., mitral clefts) are sometimes associated with ASDs, further contributing to hemodynamic instability. In this work, we report an incidental finding of a hemodynamically highly relevant ASD in the presence of incompetent mitral and tricuspid valves, in an asymptomatic, otherwise healthy juvenile pig. In-depth characterization of the cardiac blood flow by four-dimensional (4D) flow magnetic resonance imaging (MRI) revealed a prominent diastolic left-to-right and discrete systolic right-to-left shunt, resulting in a pulmonary-to-systemic flow ratio of 1.8. Severe mitral (15 mL/stroke) and tricuspid (22 mL/stroke) regurgitation further reduced cardiac output. Pathological examination confirmed the presence of an ostium primum ASD and found a serous cyst of lymphatic origin that was filled with clear fluid partially occluding the ASD. A large mitral cleft was identified as the most likely cause of severe regurgitation, and histology showed mild to moderate endocardiosis in the coaptation area of both atrio-ventricular valves. In summary, although not common, congenital heart defects could play a role as a cause of experimental variability or even intra-experimental mortality when working with apparently heathy, juvenile pigs.

Single-Cell RNA Sequencing of Mouse Left Ventricle Reveals Cellular Diversity and Intercommunication

Previous studies have revealed the diversity of the whole cardiac cellulome but not refined the left ventricle, which was essential for finding therapeutic targets. Here, we characterized single-cell transcriptional profiles of the mouse left ventricular cellular landscape using single-cell RNA sequencing (10×Genomics). Detailed t-Distributed Stochastic Neighbor Embedding (tSNE) analysis revealed the cell types of left ventricle with gene markers. Left ventricular cellulome contained cardiomyocytes highly expressed Trdn, endothelial cells highly expressed Pcdh17, fibroblast highly expressed Lama2 and macrophages highly expressed Hpgds, also proved by in situ hybridization. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analysis (ListHits>2, p<0.05) were employed with the DAVID database to investigate subtypes of each cell type with the underlying functions of differentially expressed genes (DEGs). Endothelial cells included five subtypes, fibroblasts comprised of seven subtypes and macrophages contained eleven subtypes. The key representative DEGs (p<0.001) were Gja4 and Gja5 in cluster 3 of endothelial cells, Aqp2 and Thbs4 in cluster 2 of fibroblasts, as well as Clec4e and Trem-1 in in cluster 3 of marcophages perhaps involved in the occur of atherosclerosis, heart failure and acute myocardial infarction proved by literature review. We also revealed extensive networks of intercellular communication in left ventricle. We suggested possible therapeutic targets for cardiovascular disease and autocrine and paracrine signaling underpins left ventricular homeostasis. This study provided new insights into the structure and function of the mammalian left ventricular cellulome and offers an important resource that will stimulate studies in cardiovascular research.