Paul Delgado-Olguín

Traditional histological sectioning has been widely used to examine congenital heart defects (CHDs) in mouse models, providing detailed insights into tissue and cellular architecture. However, this approach is time-consuming, labor-intensive, and can miss subtle defects due to the critical need for precise serial sections. In contrast, three-dimensional imaging of cleared embryonic hearts using light sheet fluorescence microscopy offers a more efficient and comprehensive solution. This technique enables the rapid acquisition of z-stacks, including thousands of sections, without manual sectioning, which can be reconstructed into high-resolution 3D models. As a result, it significantly improves the detection and visualization of CHDs in mouse fetuses.

Cardiomyocyte maturation represents the final stage of functional development in the heart. During this critical process, fetal cardiomyocytes gradually acquire adult phenotypes and become capable of sustaining billions of contraction-relaxation cycles throughout an organism's lifespan. This transition is driven by gene expression changes that remodel cardiomyocyte structure, metabolism, and functions. In mice, cardiomyocyte maturation takes place during embryonic day (E)18.5 to postnatal two weeks, during which the heart loses its regenerative capacity in response to injury. Hence, characterizing the functional and molecular changes during mouse cardiomyocyte maturation and how this process is regulated provides invaluable insights into heart development and disease. Cardiomyocyte-specific analysis requires isolating cardiomyocytes from other cardiac cell populations. Here, we present a standardized protocol for consistent isolation of neonatal and postnatal mouse cardiomyocytes. Leveraging enzymatic and mechanical digestion, coupled with non-cardiomyocyte depletion via pre-plating, our protocol enables high-quality cardiomyocyte isolation in approximately three hours. In addition, we detail methods for cardiomyocyte culture, molecular, and functional characterization.

Genotyping is a valuable tool for identifying organisms that carry genetic variations, including insertions and deletions. This method involves extracting DNA from animal tissue samples and subsequently amplifying genomic regions of interest using the polymerase chain reaction (PCR). The amplified product is analyzed by agarose gel electrophoresis based on DNA size.

Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of Polycomb Repressive Complex 2, catalyses H3K27 trimethylation and directs lineage development programs, yet its function in early multipotent progenitors remains incompletely understood. ISL1 marks cardiopharyngeal and neural crest progenitors that contribute to second heart field derivatives and to craniofacial and limb morphogenesis. Here, Isl1-cre lineage tracing showed that lineage derivatives populated the cardiopharyngeal region and the posteromedial hindlimb bud during mid-gestation, and contributed extensively to the developing right ventricle, outflow tract, and hindlimb. Loss of Ezh2 in Isl1-expressing progenitors resulted in a high incidence of congenital heart defects, dominated by incomplete atrial and ventricular septation and outflow tract malformations, including double outlet right ventricle and persistent truncus arteriosus. These defects were accompanied by ventricular wall thickening at birth. In parallel, Ezh2 mutants exhibited hindlimb skeletal abnormalities, including pelvic and tarsal malformations with impaired ossification. These abnormalities were associated with cyanosis and perinatal demise. Together, these findings identify a requirement for EZH2 in Isl1-lineage contribution to heart and hindlimb development.

Becker muscular dystrophy (BMD) is a rare X-linked recessive neuromuscular disorder, frequently caused by in-frame deletions in the DMD gene that result in the production of a truncated, yet functional, dystrophin protein. The consequences of BMD-causing in-frame deletions on the organism are difficult to predict, especially in regard to long-term prognosis. Here, we used CRISPR-Cas9 to generate a new Dmd Δ52-55 mouse model by deleting exons 52-55 in the Dmd gene, resulting in a BMD-like in-frame deletion. To delineate the long-term effects of this deletion, we studied these mice over 52 weeks by performing histology and echocardiography analyses and assessing motor functions. Our results suggest that truncated dystrophin is sufficient to maintain wildtype-like muscle and heart histology and functions in young mice. However, the truncated protein appeared to be insufficient to maintain normal muscle homeostasis and protect against exercise-induced damage at 52 weeks. To further delineate the effects of this exon 52-55 in-frame deletion, we performed RNA sequencing pre- and post-exercise and identified several differentially expressed pathways that reflect the abnormal muscle phenotype observed at 52 weeks in the BMD model.

Background: Sepsis-induced myocardial dysfunction is a common and critical complication of sepsis. Extracellular vesicles (EVs) from clonally expanded immortalized mesenchymal stromal cells (ciMSCs) contain microRNAs that may be exploited as therapy. Methods: In mouse models of septic cardiomyopathy induced by caecum ligation and puncture, cardiac function was determined by invasive and echocardiographic assessment. Primary cardiomyocytes derived from foetal murine and human adult ventricular tissue, as well as murine hearts were used for mechanistic studies. Studies using post-mortem human hearts or patient plasma, and clinical and echocardiographic measurements were used to establish translational relevance. Results: In preclinical models of sepsis, intravenous administration of either MSCs or ciMSC-EVs, given after the induction of sepsis, prevented a decrease in myocardial ejection fraction, ventricular inflammation, and mortality compared to placebo or platelet-derived control EVs. EV-microRNA sequencing identified enrichment for microRNA-187a-3p (miR-187) in ciMSC-EVs. miR-187 is anti-inflammatory; with interleukin-6 (IL-6) as its major target. Intravenous delivery of lipid nanoparticle (LNP) encapsulated miR-187 improved cardiac function, reduced inflammation, and enhanced survival of septic mice. In cardiomyocytes and in murine hearts, LNP-miR-187 reduces inflammation and expression of myocardial transcription factors linked to fetal gene reactivation in failing septic hearts. In human septic hearts, low circulating miR-187 levels correlate with reduced cardiac function and high sequential organ failure assessment (SOFA) scores. Conclusion: These findings support the development of first-in-class, cell-free, miRNA-based therapy as a novel approach to treat sepsis-induced cardiomyopathy to address a critical gap in sepsis care. One Sentence Summary: miR-based therapy for sepsis The Clinical Perspective: A. What is NEW? Sepsis accounts for 1 in 5 deaths worldwide. Here, we demonstrate that sepsis-induced myocardial dysfunction represents a discrete, targetable sepsis-trait — a distinct biological abnormality characterized by cardiomyocyte inflammation and fetal gene reactivation. This component contributes to the propagation of organ dysfunction and overall mortality and may respond to focused epigenetic-based interventions. B. What are the Clinical implications? Currently, there are no effective treatments to reduce, limit, or reverse the immune dysfunction component of sepsis that contributes to multiorgan failure, such as sepsis-induced cardiomyopathy. We identify miR-187 as a clinically relevant post-transcriptional regulator of cardiac inflammation and cardiomyocyte gene expression. Intravenous delivery of miR-187 encapsulated in a lipid nanoparticle (LNP) represents a fundamentally distinct, effective and pathogen-agnostic approach to correcting sepsis-induced cardiac dysfunction through modulation of cardiomyocyte inflammatory and metabolic pathways.

Introduction: Neonatal necrotizing enterocolitis (NEC) is a severe gastrointestinal disorder with high mortality, characterized by epithelial cell injury and compromised epithelial repair. The mechanisms underlying defective epithelial regeneration remain poorly understood despite advances in single-cell omics. Addressing these challenges is essential for elucidating the pathogenesis of NEC and identifying therapeutic targets to restore epithelial regeneration and replace the damaged epithelial layer. Methods: Multi-omics approaches were employed to investigate molecular and spatial changes in experimental NEC at epigenetic and transcriptomic levels. These included bulk RNA sequencing, single-nucleus RNA sequencing (snRNA-seq), single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq), and multiplexed error-robust fluorescence in situ hybridization (MERFISH) for spatial transcriptomics. Complementary in vitro experiments and in vivo mouse models were utilized to evaluate NEC phenotypes, intestinal tissue morphology, and organoid formation. Results: Changes in cell type composition, transcriptional network remodeling, and chromatin accessibility were observed in the small intestine of neonatal mice with NEC. Chromatin accessibility significantly changed in epithelial cells, highlighting their pivotal roles in NEC. A marked reduction in intestinal stem cells (ISCs) and transit-amplifying cells, along with an increased proportion of enteroendocrine cells, indicates disrupted epithelial regeneration and functional differentiation. These changes correlated with disrupted WNT signaling and stem cell maintenance genes (e.g., Lgr5, Smoc2, Axin2) and activation of inflammatory and hypoxia-related pathways (e.g., Il6, Tnfα). The epigenetic regulator Ezh2 was identified as a critical factor in maintaining LGR5+ ISCs and epithelial homeostasis. Knockdown of Ezh2 reduced stemness and proliferation-related gene expression and exacerbated inflammation. Reactivation of WNT signaling restored Ezh2 and Lgr5 expression, improving intestinal regeneration. Conclusion: This study reveals dynamic transcriptomic, epigenetic, and spatial changes in NEC and highlights Ezh2 as a key regulator of LGR5+ intestinal stem cell function and epithelial regeneration. These findings provide insights into NEC pathogenesis and a basis for therapies targeting Ezh2 and WNT signaling to restore intestinal integrity. Abstract Figure:

Background: As the fetal heart develops, cardiomyocyte proliferation potential decreases while fatty acid oxidative capacity increases in a highly regulated transition known as cardiac maturation. Small noncoding RNAs, such as microRNAs (miRNAs), contribute to the establishment and control of tissue-specific transcriptional programs. However, small RNA expression dynamics and genome-wide miRNA regulatory networks controlling maturation of the human fetal heart remain poorly understood. Results: Transcriptome profiling of small RNAs revealed the temporal expression patterns of miRNA, piRNA, circRNA, snoRNA, snRNA and tRNA in the developing human heart between 8 and 19 weeks of gestation. Our analysis demonstrated that miRNAs were the most dynamically expressed small RNA species throughout mid-gestation. Cross-referencing differentially expressed miRNAs and mRNAs predicted 6200 mRNA targets, 2134 of which were upregulated and 4066 downregulated as gestation progressed. Moreover, we found that downregulated targets of upregulated miRNAs, including hsa-let-7b, miR-1-3p, miR-133a-3p, miR-143-3p, miR-499a-5p, and miR-30a-5p predominantly control cell cycle progression. In contrast, upregulated targets of downregulated miRNAs, including hsa-miR-1276, miR-183-5p, miR-1229-3p, miR-615-3p, miR-421, miR-200b-3p and miR-18a-3p, are linked to energy sensing and oxidative metabolism. Furthermore, integrating miRNA and mRNA profiles with proteomes and reporter metabolites revealed that proteins encoded in mRNA targets and their associated metabolites mediate fatty acid oxidation and are enriched as the heart develops. Conclusions: This study presents the first comprehensive analysis of the small RNAome of the maturing human fetal heart. Our findings suggest that coordinated activation and repression of miRNA expression throughout mid-gestation is essential to establish a dynamic miRNA-mRNA-protein network that decreases cardiomyocyte proliferation potential while increasing the oxidative capacity of the maturing human fetal heart. Our results provide novel insights into the molecular control of metabolic maturation of the human fetal heart.

Becker Muscular Dystrophy (BMD) is a rare X-linked recessive neuromuscular disorder caused by in-frame deletions in the DMD gene that result in the production of a truncated, yet functional, dystrophin protein. BMD is often considered a milder form of Duchenne Muscular Dystrophy, in which mutations typically result in the disruption of the reading frame and the malfunction or loss of dystrophin. The consequences of BMD-causing in-frame deletions on the organism are more difficult to predict, especially in regard to long-term prognosis. Here, we employed CRISPR-Cas9 technology to generate a new Dmd del52-55 mouse model by deleting exons 52-55, resulting in a typical BMD-like in-frame deletion. To delineate the long-term effects of this deletion, we studied these mice over 52 weeks. Our results suggest that a truncated dystrophin is sufficient to maintain wildtype-like muscle and heart functions in young mice. However, the truncated protein appears insufficient to maintain normal muscle homeostasis and protect against exercise-induced damage at 52 weeks. To further delineate the effects of the exons 52-55 in-frame deletion, we performed RNA-Seq pre– and post-exercise and identified several differentially expressed pathways that could explain the abnormal muscle phenotype observed at 52 weeks in the BMD model. Summary Statement: We generated and characterized the long-term effects of a Becker Muscular Dystrophy-like in-frame deletion of exon 52 to 55 in mice.