Carlos García-Padilla

Cardiovascular disease displays marked sex-related heterogeneity across the life course, and the rise in cardiometabolic and vascular risk during the menopausal transition has focused attention on estrogen signaling. Healthy female hearts are not simply scaled-down male hearts; population averages differ in cardiac mass, chamber geometry, myocardial deformation, hemodynamics, and repolarization. This narrative review integrates experimental, epidemiological, genetic, and clinical evidence on estrogen action in the cardiovascular system. Estrogens signal through ERalpha and ERbeta, their membrane-associated pools, and GPER, whereas estrogen-related receptors coordinate overlapping metabolic programs without binding estrogen. These pathways regulate endothelial nitric oxide production, redox balance, inflammation, mitochondrial energetics, autophagy, calcium handling, electrophysiology, extracellular-matrix remodeling, and non-coding RNA networks. Their effects depend on cell type, age, receptor abundance, vascular substrate, comorbidities, and genetic or epigenetic variation. This context dependence helps explain why endogenous estrogen exposure cannot be equated with menopausal hormone therapy. Current evidence does not support hormone therapy for primary or secondary cardiovascular prevention; treatment of menopausal symptoms requires individualized assessment of indication, timing, formulation, route, thrombotic risk, and baseline cardiovascular health. Future studies should combine sex-aware human models, longitudinal multi-omics, and validated biomarkers to link estrogen-pathway variation with clinically meaningful cardiovascular outcomes.

Cardiogenesis begins during gastrulation with the bilateral specification of precardiac mesoderm on either side of the embryo. These fields fuse at the embryonic midline to form the linear heart tube, which subsequently undergoes rightward looping and chamber formation. Expansion and septation of the primitive atrial and ventricular chambers establish separate systemic and pulmonary circuits with correctly aligned inflow and outflow tracts. These morphogenetic events depend on the coordinated activation and repression of cell type-specific cardiac gene programmes controlled at transcriptional and post-transcriptional levels by cardiac transcription factors and paracrine/autocrine signals. Non-coding RNAs, particularly microRNAs and long non-coding RNAs, add an essential regulatory layer. MicroRNAs are endogenous ≈22-nt RNAs that modulate gene expression predominantly at the post-transcriptional level. The muscle-enriched miR-1/miR-133 cluster is essential for normal cardiogenesis, whereas dysregulation of miR-1 and miR-133 contributes to heart failure and its associated pathological processes. Here, we provide a state-of-the-art review of the genomic regulation, developmental functions, context-dependent interactions, and translational potential of the miR-1/miR-133 cluster in cardiogenesis and heart failure.

Cardiogenesis is initiated soon during gastrulation as bilateral precardiac mesoderm, progressive and symmetrically determined at both sides of the developing embryo. The precardiac mesoderm subsequently fuse at the embryonic midline constituting an embryonic linear heart tube. As development progress, the embryonic tube heart suffers an invariably rightward looping generating a prospective embryonic ventricular and atrial chambers. As cardiac development progresses, the primitive atrial and ventricular chambers enlarged and distinct left and right compartments emerge by formation of the interatrial and interventricular septa, respectively. The last steps of cardiac morphogenesis are represented by the completion of atrial and ventricular septation, resulting in the configuration of a double circuitry with distinct systemic and pulmonary chambers, each of them with distinct inlets and outlets connections. The heart development is resulted of the activation and repression of several cardiac specific gene programs in different cell types. These cardiac specific gene pathways are modulated at transcriptional and post-transcriptional levels by key cardiac transcription factors and different paracrine and autocrine signals. Over the last decade, a novel layer of complexity has emerged with the discovery of non-coding RNAs, particularly microRNAs and lncRNAs. MicroRNAs are endogenous ≈22-nt non-coding RNAs that participate in the regulation of gene expression at post-transcriptional level on most cellular processes. In this line, miR-1/miR-133 cluster is one of the muscle-specific cluster microRNAs. Proper expression of members of this cluster is essential for cardiogenesis. Furthermore, dysregulation expression of miR-1 and miR-133 expression exerts an important role in heart failure and related pathological processes. Herein, we provide a state-of-the-art review of the contribution of miR-1/miR-133 cluster in cardiogenesis and heart failure development.

The epicardium is an embryonic tissue layer essential for heart morphogenesis, providing progenitor cells and regulatory signals that support myocardial growth and coronary vessel formation. Epicardial cells arise from the proepicardium (PE) and spread over the myocardium to form the embryonic epicardium (EE), a transition that requires tight coordination between proliferation, migration, and lineage priming. However, the molecular mechanisms controlling this developmental timing remain incompletely understood. Here, we identify Trim71 as a key regulator of epicardial cell behaviour during the PE-to-EE transition. Trim71 is enriched in the PE and subsequently downregulated as cells acquire migratory competence. Functional analyses show that loss of Trim71 function decreases proliferation while promoting migration, as well as inducing the expression of epicardial commitment markers, suggesting that Trim71 is a controller of a progenitor-like state. We further demonstrate that Trim71 is necessary for these processes through a reciprocal feedback loop with the microRNAs let-7c and miR-30c. Our findings establish Trim71 as a temporal gatekeeper that coordinates the balance between progenitor maintenance and migration during early epicardial development. This Trim71-miRNAs axis constitutes a novel post-transcriptional layer of regulation that ensures the correct timing of epicardium development during cardiogenesis.

Background: Methylene blue (MB), a versatile redox agent, is emerging as a promising therapeutic in diseases associated with mitochondrial dysfunction. Its ability to optimize the electron transport chain increases ATP synthesis (30–40%) and reduces oxidative stress, protecting cellular components such as mitochondrial DNA. The protective role of this compound has been described in several neurodegenerative disease such as Alzheimer’s and Parkinson’s diseases. However, its role in cardiovascular disease has been poorly explored. Methods: In this study, we explored the impact of MB on murine (HL1) and human (AC16) cardiomyocyte redox signaling and cellular survival using RT-Qpcr analysis and immunochemistry assays. Results: Our results revealed that MB increased functional mitochondria, reversed H₂O₂-induced oxidative damage, and modulated antioxidant gene expression. Furthermore, it regulated the microRNA16–UPR signaling axis, reducing CHOP expression and promoting cell survival. Conclusions: These findings underscore its potential in cardioprotective therapy; however, its putative use as a drug requires in vivo validation in preclinical animal models.

Cardiac development is a complex developmental process. The early cardiac straight tube is composed of an external myocardial layer and an internal endocardial lining. Soon after rightward looping, the embryonic heart becomes externally covered by a new epithelial lining, the embryonic epicardium. A subset of these embryonic epicardial cells migrate and colonize the embryonic myocardium, contributing to the formation of distinct cell types. In recent years, our understanding of the molecular mechanisms that govern proepicardium and embryonic epicardium formation has greatly increased. We have recently witnessed the discovery of a novel layer of complexity governing gene regulation with the discovery of non-coding RNAs. Our laboratory recently identified three distinct lncRNAs, adjacent to the Wt1, Bmp4 and Fgf8 chicken gene loci, with enhanced expression in the proepicardium that are distinctly regulated by Bmp, Fgf and thymosin β4, providing support for their plausible implication in epicardial formation. The expression of lncRNAs was analyzed in different chicken and mouse tissues as well as their subcellular distribution in chicken proepicardial, epicardial, ventricle explants and in different murine cardiac cell types. lncRNA transcriptional regulation was analyzed by using siRNAs and expression vectors of different transcription factors in chicken and mouse models, whereas antisense oligonucleotides were used to inhibit Gm14014 expression. Furthermore, RT-qPCR, immunocytochemistry, RNA pulldown, Western blot, viability and cell migration assays were conducted to investigate the biological functions of Wt1_76127 and Gm14014. We demonstrated that Wt1_76127 in chicken and its putative conserved homologue Gm14014 in mice are widely distributed in different embryonic and adult tissues and distinctly regulated by cardiac-enriched transcription factors, particularly Mef2c and Nkx2.5. Furthermore, silencing assays demonstrated that mouse Gm14014, but not chicken Wt1_76127, is essential for epicardial, but not endocardial or myocardial, cell migration. Such processes are governed by partnering with Myl9, promoting cytoskeletal remodeling. Our data show that Gm14014 plays a pivotal role in epicardial cell migration essential for heart regeneration under these experimental conditions.

Excess lipid accumulation in the heart is associated with lipotoxicity and cardiac dysfunction due to excessive fatty acid oxidation. Peroxisome proliferator-activated receptor gamma (PPARγ) modulates the expression of key molecules involved in the FA metabolic pathway. Cardiomyocyte-specific overexpression of PPARγ causes dilated cardiomyopathy associated with lipotoxicity in mice. miR-130b-3p has been shown to be downregulated in the plasma of idiopathic dilated cardiomyopathy patients, but its role in modulating cardiomyocyte lipotoxicity via PPARγ remains unclear. Our objective was to investigate the protective role of miR-130b-3p against palmitate-induced lipotoxicity in cardiomyocytes through the modulation of the PPARγ signaling pathway. Human cardiomyoblasts were treated with palmitate. Intracellular lipid accumulation and expression of PPARγ and its downstream targets (CD36, FABP3, CAV1, VLDLR) were analyzed. Mitochondrial oxidative stress was assessed via MitoTracker Green and Redox Sensor Red staining and expression of CPT1B and SOD2. Endoplasmic reticulum stress and apoptosis were determined by examining GRP78, ATF6, XBP1s, CHOP, and caspase-3 expression. miR-130b-3p overexpression was achieved using transfection methods, and its effect on these parameters was evaluated. Luciferase assays were used to confirm PPARγ as a direct target of miR-130b-3p. Palmitate treatment led to increased lipid accumulation and upregulation of PPARγ and its downstream targets in human cardiomyoblasts. Palmitate also increased mitochondrial oxidative stress, endoplasmic reticulum stress and apoptosis. miR-130b-3p overexpression reduced PPARγ expression and its downstream signaling, alleviated mitochondrial oxidative stress and decreased endoplasmic reticulum stress and apoptosis in palmitate-stimulated cardiomyoblasts. Luciferase assays confirmed PPARγ as a direct target of miR-130b-3p. Our findings suggest that miR-130b-3p plays a protective role against palmitate-induced lipotoxicity in cardiomyocytes by modulating the PPARγ signaling pathway.

Background: Cardiac development is a complex developmental process. The early cardiac straight tube is composed of an external myocardial layer and an internal endocardial lining. Soon after rightward looping, the embryonic heart becomes externally covered by a new epithelial lining, the embryonic epicardium. A subset of these embryonic epicardial cells migrate and colonize the embryonic myocardium, contributing to distinct cell types. In recent years, our understanding of the molecular mechanisms that govern proepicardium and embryonic epicardium formation has greatly increased. We have recently witnessed a novel layer of complexity governing gene regulation with the discovery of non-coding RNAs. Our laboratory recently identified three distinct lncRNAs, adjacent to the Wt1, Bmp4 and Fgf8 chicken gene loci, with enhanced expression in the proepicardium that are distinctly regulated by Bmp, Fgf and thymosin 4, providing support of their plausible implication in epicardial formation. Methods: Expression of lncRNAs was analyzed in different chicken and mouse tissues as well as their subcellular distribution in chicken proepicardial, epicardial, ventricle explants and in different murine cardiac cell types. lncRNA transcriptional regulation was analyzed by using siRNAs and expression vectors of different transcription factors in chicken and mouse. Antisense oligonucleotides were used to inhibit Gm14014 expression. Cardiac injury was induced ex vivo in mouse ventricle explants by cryoinjury. Furthermore, RT-qPCR, immunocytochemistry, RNA pulldown, Western blot, viability and cell migration assays were conducted to investigate the biological function of Wt1_76127 and Gm14014. Results: We demonstrated that Wt1_76127 in chicken and its evolutionarily conserved homologue Gm14014 in mice are widely distributed in different embryonic and adult tissues and distinctly regulated by cardiac enriched transcription factors, particularly Mef2c and Nkx2.5. Gm14014 is distinctly regulated in mouse ventricular cryoinjury ex vivo models, displaying a negative correlation with epicardial and epithelial to mesenchymal transition markers. Furthermore, silencing assays demonstrated that mouse Gm14014, but not chicken Wt1_76127, is essential for epicardial, but not endocardial or myocardial cell migration. Such process is governed by partnering with Myl9, promoting cytoskeletal remodeling. Conclusion: Our data evidence that Gm14014 plays a pivotal role in epicardial cell migration essential for heart regeneration.

A large diversity of epigenetic factors, such as microRNAs and histones modifications, are known to be capable of regulating gene expression without altering DNA sequence itself. In particular, miR-1 is considered the first essential microRNA in cardiac development. In this study, miR-1 potential role in early cardiac chamber differentiation was analyzed through specific signaling pathways. For this, we performed in chick embryos functional experiments by means of miR-1 microinjections into the posterior cardiac precursors—of both primitive endocardial tubes—committed to sinoatrial region fates. Subsequently, embryos were subjected to whole mount in situ hybridization, immunohistochemistry and RT-qPCR analysis. As a relevant novelty, our results revealed that miR-1 increased Amhc1, Tbx5 and Gata4, while this microRNA diminished Mef2c and Cripto expressions during early differentiation of the cardiac sinoatrial region. Furthermore, we observed in this developmental context that miR-1 upregulated CrabpII and Rarß and downregulated CrabpI, which are three crucial factors in the retinoic acid signaling pathway. Interestingly, we also noticed that miR-1 directly interacted with Hdac4 and Calm1/Calmodulin, as well as with Erk2/Mapk1, which are three key factors actively involved in Mef2c regulation. Our study shows, for the first time, a key role of miR-1 as an epigenetic regulator in the early differentiation of the cardiac sinoatrial region through orchestrating opposite actions between retinoic acid and Mef2c, fundamental to properly assign cardiac cells to their respective heart chambers. A better understanding of those molecular mechanisms modulated by miR-1 will definitely help in fields applied to therapy and cardiac regeneration and repair.

Transcriptional regulation constitutes a key step in gene expression regulation. Myocyte enhancer factor 2C (MEF2C) is a transcription factor of the MADS box family involved in the early development of several cell types, including muscle cells. Over the last decade, a novel layer of complexity modulating gene regulation has emerged as non-coding RNAs have been identified, impacting both transcriptional and post-transcriptional regulation. microRNAs represent the most studied and abundantly expressed subtype of small non-coding RNAs, and their functional roles have been widely documented. On the other hand, our knowledge of the transcriptional and post-transcriptional regulatory mechanisms that drive microRNA expression is still incipient. We recently demonstrated that MEF2C is able to transactivate the long, but not short, regulatory element upstream of the miR-23a-miR-27a-miR-24-2 transcriptional start site. However, MEF2C over-expression and silencing, respectively, displayed distinct effects on each of the miR-23a-miR-27a-miR-24-2 mature cluster members without affecting pri-miRNA expression levels, thus supporting additional MEF2C-driven regulatory mechanisms. Within this study, we demonstrated a complex post-transcriptional regulatory mechanism directed by MEF2C in the regulation of miR-23a-miR-27a-miR-24-2 cluster members, distinctly involving different domains of the MEF2C transcription factor and the physical interaction with pre-miRNAs and Ksrp, HnRNPa3 and Ddx17 transcripts.