Background: Neonatal mouse cardiomyocytes undergo a metabolic switch from glycolysis to oxidative phosphorylation, which results in a significant increase in reactive oxygen species (ROS) production that induces DNA damage. These cellular changes contribute to cardiomyocyte cell cycle exit and loss of the capacity for cardiac regeneration. The mechanisms that regulate this metabolic switch and the increase in ROS production have been relatively unexplored. Current evidence suggests that elevated ROS production in ischemic tissues occurs due to accumulation of the mitochondrial metabolite succinate during ischemia via succinate dehydrogenase (SDH), and this succinate is rapidly oxidized at reperfusion. Interestingly, mutations in SDH in familial cancer syndromes have been demonstrated to promote a metabolic shift into glycolytic metabolism, suggesting a potential role for SDH in regulating cellular metabolism. Whether succinate and SDH regulate cardiomyocyte cell cycle activity and the cardiac metabolic state remains unclear. Methods: Here, we investigated the role of succinate and succinate dehydrogenase (SDH) inhibition in regulation of postnatal cardiomyocyte cell cycle activity and heart regeneration. Results: Our results demonstrate that injection of succinate in neonatal mice results in inhibition of cardiomyocyte proliferation and regeneration. Our evidence also shows that inhibition of SDH by malonate treatment after birth extends the window of cardiomyocyte proliferation and regeneration in juvenile mice. Remarkably, extending malonate treatment to the adult mouse heart following myocardial infarction injury results in a robust regenerative response within 4 weeks following injury via promoting adult cardiomyocyte proliferation and revascularization. Our metabolite analysis following SDH inhibition by malonate induces dynamic changes in adult cardiac metabolism. Conclusions: Inhibition of SDH by malonate promotes adult cardiomyocyte proliferation, revascularization, and heart regeneration via metabolic reprogramming. These findings support a potentially important new therapeutic approach for human heart failure.
Cardiac nerves regulate neonatal mouse heart regeneration and are susceptible to pathological remodeling following adult injury. Understanding cardiac nerve remodeling can lead to new strategies to promote cardiac repair. Our current understanding of cardiac nerve architecture has been limited to two-dimensional analysis. Here, we use genetic models, whole-mount imaging, and three-dimensional modeling tools to define cardiac nerve architecture and neurovascular association during development, disease, and regeneration. Our results demonstrate that cardiac nerves sequentially associate with coronary veins and arteries during development. Remarkably, our results reveal that parasympathetic nerves densely innervate the ventricles. Furthermore, parasympathetic and sympathetic nerves develop synchronously and are intertwined throughout the ventricles. Importantly, the regenerating myocardium reestablishes physiological innervation, in stark contrast to the non-regenerating heart. Mechanistically, reinnervation during regeneration is dependent on collateral artery formation. Our results reveal how defining cardiac nerve remodeling during homeostasis, disease, and regeneration can identify new therapies for cardiac disease.
The human T‐lymphotropic virus type 1 (HTLV‐1) RNA genome includes two programmed ‐1 ribosomal frameshift (‐1 PRF) sites. These sites allow ribosomes access to alternate reading frames encoding critical viral enzymes. The gag‐proframeshift site includes a slippery sequence, spacer, and stem‐loop structure. How the stem‐loop acts to promote frameshifting is unclear. Previous HTLV‐2 research showed that changes to the gag‐pro frameshift site stem‐loop thermodynamic stability influenced its frameshift efficiency to a modest degree. There is substantial conservation between the HTLV‐1 and HTLV‐2 gag‐pro frameshift site sequences (86%) and structures. We hypothesized that the HTLV‐1 gag‐pro frameshift efficiency would be similarly influenced by its stem‐loop thermodynamic stability. To test this hypothesis, we designed 15 stem‐loop mutants (SLMs) with varied base‐pair composition. These mutations decoupled changes in overall thermodynamic stability from those localized to the stem‐loop base. The SLM thermodynamic stabilities were calculated using nearest neighbor parameters and the in vitro frameshift efficiencies were measured with a dual‐luciferase assay. Correlations between frameshift efficiency and thermodynamic stability were subsequently assessed. Preliminarily results reveal a moderate correlation between the SLM stem‐loop overall thermodynamic stability and frameshifting efficiency. No correlation was observed between the thermodynamic stability of the stem‐loop base and frameshifting efficiency. While the overall thermodynamic stability does impact the frameshift efficiency, it cannot be used exclusively to predict it. This reflects a complex interplay between the frameshift site elements. Overall, our preliminary results suggest a conserved function for the gag‐pro frameshift site stem‐loop between the HTLV‐1 and HTLV‐2 retroviruses.
Insights regarding the regulated secretion of insulin from pancreatic β‐cells has implications for understanding diabetes and regulation of glucose metabolism. Piccolo is a high‐molecular weight protein and part of a multi‐molecular complex involved in insulin secretion. Interestingly, genetic variants of the Piccolo gene (PCLO) have been modestly associated with increased risk of early‐onset type II diabetes among Pima Indians. Piccolo may regulate insulin secretion through a novel mechanism by serving as a Ca2+ sensor that interacts with L‐type voltage‐dependent Ca2+ channels and other molecules associated with insulin secretory vesicles. The purpose of this study is to examine the role of Piccolo and its splice variants in insulin secretion. Piccolo was originally identified as a protein involved in regulation of neurotransmitter secretion at presynaptic active zones. In neurons, PCLO undergoes extensive alternative splicing. Two major splice variants have been found in both neurons and pancreatic cells: isoform‐1 (I‐1) and isoform‐2 (I‐2). I‐1 has two Ca2+‐binding C2 domains, C2A and C2B, while I‐2 includes only C2A. Alternative splicing also leads to additional minor splice variants with altered Ca2+‐binding properties. Specifically, an exon‐skipping event results in a splice variant that lacks exon 16, a 27‐nucleotide (nt) exon within the C2A domain. Exclusion of this exon (i.e. short variant) enhances the Ca2+‐ and phospholipid binding affinity of C2A motif of rat Piccolo. In this study, we sought to examine the role of Piccolo and its splice variants in Ca2+‐mediated insulin secretion in rat INS‐832/13 pancreatic β‐cells. We demonstrate that INS‐832/13 cells express both I‐1 and I‐2 and also the long and short variant of the C2A domain. In addition, we used CRISPR‐CAS gene editing to selectively target exon 1 of PCLO to examine how loss of function of Piccolo may affect glucose‐stimulated insulin secretion. Finally, we examined the effect of overexpression of the short or long C2A‐domain on insulin secretion.Support or Funding InformationResearch in this study was funded by NIH‐Maximizing Access to Research Careers Undergraduate Student Training in Academic Research (MARC U*STAR) award number T34GM092711, start‐up funds provided to Dr. Steven Fenster (Fort Lewis College), and funds received from SSNAP (Science Scholars the Native American Path) by SACNAS (Dr. Les Sommerville, Fort Lewis College). We would also like to thank Dr. Christopher Newgaard at Duke University for supplying the pancreatic cells and culturing procedure.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Worldwide, congenital anomalies of the kidney and urinary tract (CAKUT) are the leading cause of chronic renal disease in children and play a significant causative factor in pediatric end‐stage renal disease. Current research being conducted is reliant upon animal model systems to monitor embryonic kidney development; however, a system modeling the human kidney organogenesis is a necessity in order to elucidate the intertwined genetic and molecular networks related to CAKUT pathogenesis.One known genetic correlation to CAKUT is the mutation within the gene encoding for the RET receptor tyrosine kinase, which plays a crucial role in kidney development. Mutations within the gene encoding for RET affect cellular pathways correlated to congenital anomalies such as Hirschsprung's disease and renal agenesis, which are due to the maldevelopment of neural crest cells and kidney progenitor cells, respectively. These defects indicate that RET plays a unique role in cellular mechanisms related to the kidney and ureter maturation, allowing for RET to be utilized as a renal biomarker. Monitoring RET protein expression allows for the efficiency of differentiation protocols to generate cells of kidney and neural crest lineages to be validated. Furthermore, utilizing RET as a renal biomarker could elucidate signaling molecules needed for multipotent stem cell development into a complex urinary system.There is a necessity to characterize the proficiency of protocols for Human Induced Pluripotent Stem Cells (hiPSCs) differentiation into kidney organoid and Neural Crest Stem Cells (NCSCs), in order to advance the methods for obtaining functioning kidney lineages. A novel hiPSC RET reporter cell line was created via CRISPR‐Cas9 technology to determine the efficacy of current differentiation protocols. The hiPSC RET reporter cell line was an invaluable asset to the protocol characterization process due to the role of RET in collecting duct and enteric nervous system lineages. With the ability to detect RET utilizing the fluorescent marker, as well as via immunohistochemistry, the ability to produce ureteric bud progenitor cells and NCSCs was able to be reliably verified. The RNA and protein product was analyzed via qPCR and immunofluorescence microscopy, respectively, for the presence of several renal precursor and NCSC biomarkers.Analyzation of the RNA throughout differentiation provided strong evidence for the generation of kidney progenitor cells and NCSCs. Moreover, antibody staining of the kidney organoids and neural crest cells revealed the presence of protein biomarkers produced by ureteric bud and neuronal precursor cells, respectively. Kidney organoids and neural crest cells are a promising paradigm to monitor the pathogenesis of CAKUT. Currently, a model system of the human embryonic kidney development relies on discovery of additional biomarkers in order to verify cell identity. Ultimately, the potential of having hiPSCs as a renewable resource to produce NCSCs and organoids has clinical applications in drug screening, disease modeling, and stem cell therapies – all which may lead to a novel cure for CAKUT and neurocristopathies.Support or Funding InformationAmgen Scholars Program at Washington University in St. LouisThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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