Signal-Dependent Transcriptional Regulation of Vascular Smooth Muscle Cell Differentiation

Vascular smooth muscle cells (VSMCs) play a key role in development as they are the major source of extracellular matrix components of vessel walls. During development, VSMCs will both proliferate and differentiate to form components of the vasculature. Differentiated VSMCs (contractile phenotype) line vessel walls to regulate blood flow. The proliferative phenotype of VSMCs (synthetic phenotype) refers to migration and proliferation of these cells to specific sites to contribute to the formation of the vasculature. Interestingly, VSMCs maintain the ability to proliferate post-natally in response to vascular injury. Therefore, the purpose of this body of work was to investigate the signalling pathways that regulate transcriptional control in VSMCs. Calcium sensitivity in VSMCs is regulated by RhoA/ROCK-mediated inhibition of the myosin light chain phosphatase complex, and alterations in smooth muscle gene expression. We found that calcium signalling stimulates ROCK-mediated phosphorylation of the PP1α inhibitor CPI-17 at threonine 38, leading to derepression of MEF2C by PP1α and increased myocardin expression, which lies upstream of smooth muscle-specific structural genes. Furthermore, TGF-β also potently induces VSMC marker genes at the transcriptional and protein levels in 10T1/2 mouse embryonic fibroblast cells. We found that the potent transcriptional regulator and nuclear retention factor, TAZ, is required for TGF-β induction of smooth muscle genes and is required in the maintenance of the differentiated VSMC phenotype. A synergistic interaction between TAZ and SRF in regulating smooth muscle gene activation and differentiation has also been observed, and TAZ expression enhances SRF binding to the smooth muscle α-actin promoter. This work addresses several important aspects of signalling pathways involved in the regulation of the vascular smooth muscle phenotype and provides a further understanding of the role of SRF in vascular development and vascular disease

Non-Coding RNAs in Cell-to-Cell Communication: Exploiting Physiological Mechanisms as Therapeutic Targets in Cardiovascular Pathologies

Cardiovascular disease, the leading cause of death worldwide, has been characterized at the molecular level by alterations in gene expression that contribute to the etiology of the disease. Such alterations have been shown to play a critical role in the development of atherosclerosis, cardiac remodeling, and age-related heart failure. Although much is now known about the cellular and molecular mechanisms in this context, the role of epigenetics in the onset of cardiovascular disease remains unclear. Epigenetics, a complex network of mechanisms that regulate gene expression independently of changes to the DNA sequence, has been highly implicated in the loss of homeostasis and the aberrant activation of a myriad of cellular pathways. More specifically, non-coding RNAs have been gaining much attention as epigenetic regulators of various pathologies. In this review, we will provide an overview of the ncRNAs involved in cell-to-cell communication in cardiovascular disease, namely atherosclerosis, cardiac remodeling, and cardiac ageing, and the potential use of epigenetic drugs as novel therapeutic targets

Epigenetics of aging and disease: a brief overview

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Therapeutic Potential of EVs: targeting cardiovascular diseases

Due to their different biological functions, extracellular vesicles (EVs) have great potential from a therapeutic point of view. They are released by all cell types, carrying and delivering different kinds of biologically functional cargo. Under pathological events, cells can increase their secretion of EVs and can release different amounts of cargo, thus making EVs great biomarkers as indicators of pathological progression. Moreover, EVs are also known to be able to transport and deliver cargo to different recipient cells, having an important role in cellular communication. Interestingly, EVs have recently been explored as biological alternatives for the delivery of therapeutics, being considered natural drug delivery carriers. Because cardiovascular disorders (CVDs) are the leading cause of death worldwide, in this review, we will discuss the up-to-date knowledge regarding the biophysical properties and biological components of EVs, focusing on myocardial infarction, diabetic cardiomyopathy, and sepsis-induced cardiomyopathy, three very different types of CVDs

A chromEM-staining protocol optimized for cardiac tissue

Three-dimensional (3D) chromatin organization has a key role in defining the transcription program of cells during development. Its alteration is the cause of gene expression changes responsible for several diseases. Thus, we need new tools to study this aspect of gene expression regulation. To this end, ChromEM was recently developed: this is an electron-microscopy staining technique that selectively marks nuclear DNA without altering its structure and, thus, allows better visualization of 3D chromatin conformation. However, despite increasingly frequent application of this staining technique on cells, it has not yet been applied to visualize chromatin ultrastructure in tissues. Here, we provide a protocol to carry out ChromEM on myocardial tissue harvested from the left ventricles of C57BL/6J mice and use this in combination with transmission electron microscopy (TEM) to measure some morphological parameters of peripheral heterochromatin in cardiomyocytes. This protocol could also be used, in combination with electron tomography, to study 3D chromatin organization in cardiomyocytes in different aspects of heart pathobiology (e.g., heart development, cardiac aging, and heart failure) as well as help to set-up ChromEM in other tissues

Unlocking cardiac motion: assessing software and machine learning for single-cell and cardioid kinematic insights

The heart coordinates its functional parameters for optimal beat-to-beat mechanical activity. Reliable detection and quantification of these parameters still represent a hot topic in cardiovascular research. Nowadays, computer vision allows the development of open-source algorithms to measure cellular kinematics. However, the analysis software can vary based on analyzed specimens. In this study, we compared different software performances in in-silico model, in-vitro mouse adult ventricular cardiomyocytes and cardioids. We acquired in-vitro high-resolution videos during suprathreshold stimulation at 0.5-1-2 Hz, adapting the protocol for the cardioids. Moreover, we exposed the samples to inotropic and depolarizing substances. We analyzed in-silico and in-vitro videos by (i) MUSCLEMOTION, the gold standard among open-source software; (ii) CONTRACTIONWAVE, a recently developed tracking software; and (iii) ViKiE, an in-house customized video kinematic evaluation software. We enriched the study with three machine-learning algorithms to test the robustness of the motion-tracking approaches. Our results revealed that all software produced comparable estimations of cardiac mechanical parameters. For instance, in cardioids, beat duration measurements at 0.5 Hz were 1053.58 ms (MUSCLEMOTION), 1043.59 ms (CONTRACTIONWAVE), and 937.11 ms (ViKiE). ViKiE exhibited higher sensitivity in exposed samples due to its localized kinematic analysis, while MUSCLEMOTION and CONTRACTIONWAVE offered temporal correlation, combining global assessment with time-efficient analysis. Finally, machine learning reveals greater accuracy when trained with MUSCLEMOTION dataset in comparison with the other software (accuracy > 83%). In conclusion, our findings provide valuable insights for the accurate selection and integration of software tools into the kinematic analysis pipeline, tailored to the experimental protocol

The Emerging Role of Epigenetics in Therapeutic Targeting of Cardiomyopathies

Cardiomyopathies (CMPs) are a heterogeneous group of myocardial diseases accountable for the majority of cases of heart failure (HF) and/or sudden cardiac death (SCD) worldwide. With the recent advances in genomics, the original classification of CMPs on the basis of morphological and functional criteria (dilated (DCM), hypertrophic (HCM), restrictive (RCM), and arrhythmogenic ventricular cardiomyopathy (AVC)) was further refined into genetic (inherited or familial) and acquired (non-inherited or secondary) forms. Despite substantial progress in the identification of novel CMP-associated genetic variations, as well as improved clinical recognition diagnoses, the functional consequences of these mutations and the exact details of the signaling pathways leading to hypertrophy, dilation, and/or contractile impairment remain elusive. To date, global research has mainly focused on the genetic factors underlying CMP pathogenesis. However, growing evidence shows that alterations in molecular mediators associated with the diagnosis of CMPs are not always correlated with genetic mutations, suggesting that additional mechanisms, such as epigenetics, may play a role in the onset or progression of CMPs. This review summarizes published findings of inherited CMPs with a specific focus on the potential role of epigenetic mechanisms in regulating these cardiac disorders

Using Epigenetics as a Pharmacological Tool in Heart Regeneration

Heart failure (HF) is the ultimate outcome of many cardiovascular pathologies and the leading cause of morbidity and mortality worldwide. An exciting therapeutic strategy for HF is cardiac regeneration, whose feasibility is supported by the ability of the neonatal heart to regenerate following cardiac injury. However, the major limitation for the application of this strategy is the inability of adult cardiomyocytes to proliferate. This is a consequence of the gene expression program underlying cardiomyocyte differentiation that promotes silencing of proliferative genes. One mechanism that plays a key role in defining this gene expression program is epigenetics. Recent reports have shown that epigenetic mechanisms similar to those that govern gene expression in heart development govern the gene expression changes occurring in HF. This chapter provides an overview of the epigenetic mechanisms involved in cardiac development and disease and how they can influence cardiac reprogramming and regeneration

Epigenetics of aging and disease: a brief overview

Aging is an important risk factor for several human diseases such as cancer, cardiovascular disease and neurodegenerative disorders, resulting from a combination of genetic and environmental factors (e.g., diet, smoking, obesity and stress), which, at molecular level, cause changes in gene expression underlying the decline of physiological function. Epigenetics, which include mechanisms regulating gene expression independently of changes to DNA sequence, regulate gene expression by modulating the structure of chromatin or by regulating the binding of transcriptional machinery to DNA. Several studies showed that an impairment of epigenetic mechanisms promotes alteration of gene expression underlying several aging-related diseases. Alteration of these mechanisms is also linked with changes of gene expression that occurs during aging processes of different tissues. In this review, we will outline the potential role of epigenetics in the onset of two age-related pathologies, cancer and cardiovascular diseases