Asparagine Synthetase Marks a Distinct Dependency Threshold for Cardiomyocyte Dedifferentiation.

BACKGROUND: Adult mammalian cardiomyocytes have limited proliferative capacity, but in specifically induced contexts they traverse through cell-cycle reentry, offering the potential for heart regeneration. Endogenous cardiomyocyte proliferation is preceded by cardiomyocyte dedifferentiation (CMDD), wherein adult cardiomyocytes revert to a less matured state that is distinct from the classical myocardial fetal stress gene response associated with heart failure. However, very little is known about CMDD as a defined cardiomyocyte cell state in transition. METHODS: Here, we leveraged 2 models of in vitro cultured adult mouse cardiomyocytes and in vivo adeno-associated virus serotype 9 cardiomyocyte-targeted delivery of reprogramming factors (Oct4, Sox2, Klf4, and Myc) in adult mice to study CMDD. We profiled their transcriptomes using RNA sequencing, in combination with multiple published data sets, with the aim of identifying a common denominator for tracking CMDD. RESULTS: RNA sequencing and integrated analysis identified Asparagine Synthetase (Asns) as a unique molecular marker gene well correlated with CMDD, required for increased asparagine and also for distinct fluxes in other amino acids. Although Asns overexpression in Oct4, Sox2, Klf4, and Myc cardiomyocytes augmented hallmarks of CMDD, Asns deficiency led to defective regeneration in the neonatal mouse myocardial infarction model, increased cell death of cultured adult cardiomyocytes, and reduced cell cycle in Oct4, Sox2, Klf4, and Myc cardiomyocytes, at least in part through disrupting the mammalian target of rapamycin complex 1 pathway. CONCLUSIONS: We discovered a novel gene Asns as both a molecular marker and an essential mediator, marking a distinct threshold that appears in common for at least 4 models of CMDD, and revealing an Asns/mammalian target of rapamycin complex 1 axis dependency for dedifferentiating cardiomyocytes. Further study will be needed to extrapolate and assess its relevance to other cell state transitions as well as in heart regeneration.

mTORC1 regulates the metabolic switch of postnatal cardiomyocytes during regeneration.

The metabolic switch from glycolysis to fatty acid oxidation in postnatal cardiomyocytes contributes to the loss of the cardiac regenerative potential of the mammalian heart. However, the mechanisms that regulate this metabolic switch remain unclear. The protein kinase complex mechanistic target of rapamycin complex 1 (mTORC1) is a central signaling hub that regulates cellular metabolism and protein synthesis, yet its role during mammalian heart regeneration and postnatal metabolic maturation is undefined. Here, we use immunoblotting, rapamycin treatment, myocardial infarction, and global proteomics to define the role of mTORC1 in postnatal heart development and regeneration. Our results demonstrate that the activity of mTORC1 is dynamically regulated between the regenerating and the non-regenerating hearts. Acute inhibition of mTORC1 by rapamycin or everolimus reduces cardiomyocyte proliferation and inhibits neonatal heart regeneration following injury. Our quantitative proteomic analysis demonstrates that transient inhibition of mTORC1 during neonatal heart injury did not reduce protein synthesis, but rather shifts the cardiac proteome of the neonatal injured heart from glycolysis towards fatty acid oxidation. This indicates that mTORC1 inhibition following injury accelerates the postnatal metabolic switch, which promotes metabolic maturation and impedes cardiomyocyte proliferation and heart regeneration. Taken together, our results define an important role for mTORC1 in regulating postnatal cardiac metabolism and may represent a novel target to modulate cardiac metabolism and promote heart regeneration.

Integrated Proteomics Identifies Troponin I Isoform Switch as a Regulator of a Sarcomere-Metabolism Axis During Cardiac Regeneration.

Adult mammalian cardiomyocytes have limited proliferative potential, and after myocardial infarction (MI), injured cardiac tissue is replaced with fibrotic scar rather than with functioning myocardium. In contrast, the neonatal mouse heart possesses a regenerative capacity governed by cardiomyocyte proliferation; however, a metabolic switch from glycolysis to fatty acid oxidation during postnatal development results in loss of this regenerative capacity. Interestingly, a sarcomere isoform switch also takes place during postnatal development where slow skeletal troponin I (ssTnI) is replaced with cardiac troponin I (cTnI). In this study, we first employ integrated quantitative bottom-up and top-down proteomics to comprehensively define the proteomic and sarcomeric landscape during postnatal heart maturation. Utilizing a cardiomyocyte-specific ssTnI transgenic mouse model, we found that ssTnI overexpression increased cardiomyocyte proliferation and the cardiac regenerative capacity of the postnatal heart following MI compared to control mice by histological analysis. Our global proteomic analysis of ssTnI transgenic mice following MI reveals that ssTnI overexpression induces a significant shift in the cardiac proteomic landscape. This shift is characterized by an upregulation of key proteins involved in glycolytic metabolism. Collectively, our data suggest that the postnatal TnI isoform switch may play a role in the metabolic shift from glycolysis to fatty acid oxidation during postnatal maturation. This underscores the significance of a sarcomere-metabolism axis during cardiomyocyte proliferation and heart regeneration.

Formation evaluation of Abu Madi reservoir in Baltim gas field, Nile Delta, using well logs, core analysis and pressure data.

Baltim Eastern and Northern gas fields in the offshore Nile Delta have very high gas condensate accumulations. Therefore, the present research evaluates Abu Madi and Qawasim Formations and defines the petrophysical parameters for them using various data from five wells composed of wireline logs (gamma-ray, density, neutron, sonic, resistivity), core data, pressure data, and cross-plots. In the current study, the formations of the main reservoirs were evaluated qualitatively and quantitatively based on the petrophysical analysis to assess the production potential. Based on the lithological identification, the two main reservoirs (Abu Madi and Qawasim Formations) are composed of sandstone, calcareous shale, and siltstone. The main petrophysical parameters (Shale volume, effective porosity, net thickness, and fluid saturations) were mapped to track the areal petrophysical variations in the field. The results of the petrophysical analysis reveal that the main reservoirs are promising for the hydrocarbon potential with effective porosity of 18%, low shale content with an average value of about 21%, higher gas saturation of average value of nearly 58%, net reservoir thickness ranges from 25.5 to 131.5 m, net pay thickness (effective thickness) ranges from 6 to 61 m. Also, the conventional core analysis affirms that the main reservoirs are of good effective porosity with high horizontal and vertical permeability values. There is a good match between the well-log results and the pressure data with the production data (DST "perforation tests"). Baltim East (BE3) well has the most desired petrophysical characteristics in the Baltim East gas field, while, the Baltim North-1 (BN1) well showed the most favorable petrophysical parameters in the Baltim North gas field. Different fluid contacts (gas water contact GWC) were detected by integrating all reservoir pressures. The integration of different data in our present work (well logs, core measurements, and pressure data) could reduce the drilling risks and help to determine the best locations for future exploration and development, which is considered a big challenge in the petroleum industry.

Metabolic switches during development and regeneration.

Metabolic switches are a crucial hallmark of cellular development and regeneration. In response to changes in their environment or physiological state, cells undergo coordinated metabolic switching that is necessary to execute biosynthetic demands of growth and repair. In this Review, we discuss how metabolic switches represent an evolutionarily conserved mechanism that orchestrates tissue development and regeneration, allowing cells to adapt rapidly to changing conditions during development and postnatally. We further explore the dynamic interplay between metabolism and how it is not only an output, but also a driver of cellular functions, such as cell proliferation and maturation. Finally, we underscore the epigenetic and cellular mechanisms by which metabolic switches mediate biosynthetic needs during development and regeneration, and how understanding these mechanisms is important for advancing our knowledge of tissue development and devising new strategies to promote tissue regeneration.

mTORC1 Regulates the Metabolic Switch of Postnatal Cardiomyocytes During Regeneration.

The metabolic switch from glycolysis to fatty acid oxidation in postnatal cardiomyocytes contributes to the loss of the cardiac regenerative potential of the mammalian heart. However, the mechanisms that regulate this metabolic switch remain unclear. The protein kinase complex mechanistic target of rapamycin complex 1 (mTORC1) is a central signaling hub that regulates cellular metabolism and protein synthesis, yet its role during mammalian heart regeneration and postnatal metabolic maturation is undefined. Here, we use immunoblotting, rapamycin treatment, myocardial infarction, and global proteomics to define the role of mTORC1 in postnatal heart development and regeneration. Our results demonstrate that the activity of mTORC1 is dynamically regulated between the regenerating and the non-regenerating hearts. Acute inhibition of mTORC1 by rapamycin or everolimus reduces cardiomyocyte proliferation and inhibits neonatal heart regeneration following injury. Our quantitative proteomic analysis demonstrates that transient inhibition of mTORC1 during neonatal heart injury did not reduce protein synthesis, but rather shifts the cardiac proteome of the neonatal injured heart from glycolysis towards fatty acid oxidation. This indicates that mTORC1 inhibition following injury accelerates the postnatal metabolic switch, which promotes metabolic maturation and impedes cardiomyocyte proliferation and heart regeneration. Taken together, our results define an important role for mTORC1 in regulating postnatal cardiac metabolism and may represent a novel target to modulate cardiac metabolism and promote heart regeneration.

Parasympathetic and sympathetic axons are bundled in the cardiac ventricles and undergo physiological reinnervation during heart regeneration.

Sympathetic innervation influences homeostasis, repair, and pathology in the cardiac ventricles; in contrast, parasympathetic innervation is considered to have minimal contribution and influence in the ventricles. Here, we use genetic models, whole-mount imaging, and three-dimensional modeling to define cardiac nerve architecture during development, disease, and regeneration. Our approach reveals that parasympathetic nerves extensively innervate the cardiac ventricles. Furthermore, we identify that parasympathetic and sympathetic axons develop synchronously and are bundled throughout the ventricles. We further investigate cardiac nerve remodeling in the regenerative neonatal and the non-regenerative postnatal mouse heart. Our results show that the regenerating myocardium undergoes a unique process of physiological reinnervation, where proper nerve distribution and architecture is reestablished, in stark contrast to the non-regenerating heart. Mechanistically, we demonstrate that physiological reinnervation during regeneration is dependent on collateral artery formation. Our results reveal clinically significant insights into cardiac nerve plasticity which can identify new therapies for cardiac disease.

LRRC10 regulates mammalian cardiomyocyte cell cycle during heart regeneration.

Leucine-rich repeat containing 10 (LRRC10) is a cardiomyocyte-specific protein, but its role in cardiac biology is little understood. Recently Lrrc10 was identified as required for endogenous cardiac regeneration in zebrafish; however, whether LRRC10 plays a role in mammalian heart regeneration remains unclear. In this study, we demonstrate that Lrrc10-/- knockout mice exhibit a loss of the neonatal mouse regenerative response, marked by reduced cardiomyocyte cytokinesis and increased cardiomyocyte binucleation. Interestingly, LRRC10 deletion disrupts the regenerative transcriptional landscape of the regenerating neonatal mouse heart. Remarkably, cardiac overexpression of LRRC10 restores cardiomyocyte cytokinesis, increases cardiomyocyte mononucleation, and the cardiac regenerative capacity of Lrrc10-/- mice. Our results are consistent with a model in which LRRC10 is required for cardiomyocyte cytokinesis as well as regulation of the transcriptional landscape during mammalian heart regeneration.

Integrated proteomics reveals alterations in sarcomere composition and developmental processes during postnatal swine heart development.

The neonatal swine heart possesses an endogenous ability to regenerate injured myocardium through the proliferation of pre-existing cardiomyocyte (CM) populations. However, this regenerative capacity is lost shortly after birth. Normal postnatal developmental processes and the regenerative capacity of mammalian hearts are tightly linked, but not much is known about how the swine cardiac proteome changes throughout postnatal development. Herein, we integrated robust and quantitative targeted "top-down" and global "bottom-up" proteomic workflows to comprehensively define the dynamic landscape of the swine cardiac proteome throughout postnatal maturation. Using targeted top-down proteomics, we were able to identify significant alterations in sarcomere composition, providing new insight into the proteoform landscape of sarcomeres that can disassemble, a process necessary for productive CM proliferation. Furthermore, we quantified global changes in protein abundance using bottom-up proteomics, identified over 700 differentially expressed proteins throughout postnatal development, and mapped these proteins to changes in developmental and metabolic processes. We envision these results will help guide future investigations to comprehensively understand endogenous cardiac regeneration toward the development of novel therapeutic strategies for heart failure.

Defining Cardiac Nerve Architecture During Development, Disease, and Regeneration.

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.

Quantifying Cardiomyocyte Proliferation and Nucleation to Assess Mammalian Cardiac Regeneration.

Neonatal mice display a remarkable ability to regenerate their heart following an injury during the first week of life. A key facet of successful cardiac regeneration is the proliferation of cardiomyocytes to replace the lost cells. Stimulating cardiomyocyte proliferation in the adult heart is a very promising approach to restore cardiac structure and function following injury. Here, we outline our approach to assess cardiomyocyte proliferation following a myocardial injury via the cell cycle markers phospho-histone H3 and Aurora B. We additionally discuss how we assess successful regeneration using wheat germ agglutinin to measure cardiomyocyte size, nuclear staining to quantify cardiomyocyte nucleation, and Trichrome staining to identify myocardial regeneration and scarring in the myocardium.

The Role of Metabolism in Heart Failure and Regeneration.

Heart failure is the leading cause of death worldwide. The inability of the adult mammalian heart to regenerate following injury results in the development of systolic heart failure. Thus, identifying novel approaches toward regenerating the adult heart has enormous therapeutic potential for adult heart failure. Mitochondrial metabolism is an essential homeostatic process for maintaining growth and survival. The emerging role of mitochondrial metabolism in controlling cell fate and function is beginning to be appreciated. Recent evidence suggests that metabolism controls biological processes including cell proliferation and differentiation, which has profound implications during development and regeneration. The regenerative potential of the mammalian heart is lost by the first week of postnatal development when cardiomyocytes exit the cell cycle and become terminally differentiated. This inability to regenerate following injury is correlated with the metabolic shift from glycolysis to fatty acid oxidation that occurs during heart maturation in the postnatal heart. Thus, understanding the mechanisms that regulate cardiac metabolism is key to unlocking metabolic interventions during development, disease, and regeneration. In this review, we will focus on the emerging role of metabolism in cardiac development and regeneration and discuss the potential of targeting metabolism for treatment of heart failure.

The role of hormones and neurons in cardiomyocyte maturation.

The heart undergoes profound morphological and functional changes as it continues to mature postnatally. However, this phase of cardiac development remains understudied. More recently, cardiac maturation research has attracted a lot of interest due to the need for more mature stem cell-derived cardiomyocytes for disease modeling, drug screening and heart regeneration. Additionally, neonatal heart injury models have been utilized to study heart regeneration, and factors regulating postnatal heart development have been associated with adult cardiac disease. Critical components of cardiac maturation are systemic and local biochemical cues. Specifically, cardiac innervation and the concentration of various metabolic hormones appear to increase perinatally and they have striking effects on cardiomyocytes. Here, we first report some of the key parameters of mature cardiomyocytes and then discuss the specific effects of neurons and hormonal cues on cardiomyocyte maturation. We focus primarily on the structural, electrophysiologic, metabolic, hypertrophic and hyperplastic effects of each factor. This review highlights the significance of underappreciated regulators of cardiac maturation and underscores the need for further research in this exciting field.

Malonate Promotes Adult Cardiomyocyte Proliferation and Heart Regeneration.

BACKGROUND: Neonatal mouse cardiomyocytes undergo a metabolic switch from glycolysis to oxidative phosphorylation, which results in a significant increase in reactive oxygen species 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 reactive oxygen species production have been relatively unexplored. Current evidence suggests that elevated reactive oxygen species production in ischemic tissues occurs as a result of accumulation of the mitochondrial metabolite succinate during ischemia via succinate dehydrogenase (SDH), and this succinate is rapidly oxidized at reperfusion. 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 SDH inhibition in regulation of postnatal cardiomyocyte cell cycle activity and heart regeneration. RESULTS: Our results demonstrate that injection of succinate into 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 after myocardial infarction injury results in a robust regenerative response within 4 weeks after injury via promoting adult cardiomyocyte proliferation and revascularization. Our metabolite analysis after 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.

Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging.

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in cardiac injury, primarily in the form of an acute myocardial infarction. With little resilience following injury, the once healthy cardiac tissue will be replaced by fibrous, non-contractile scar tissue and often be a prelude to heart failure. To identify novel treatment options in regenerative medicine, research has focused on vertebrates with innate regenerative capabilities. One such model organism is the neonatal mouse, which responds to cardiac injury with robust myocardial regeneration. In order to induce an injury in the neonatal mouse that is clinically relevant, we have developed a surgery to occlude the left anterior descending artery (LAD), mirroring a myocardial infarction triggered by atherosclerosis in the human heart. When matched with the technology to track changes both within cardiomyocytes and non-myocyte populations, this model provides us with a platform to identify the mechanisms that guide heart regeneration. Gaining insight into changes in cardiac cell populations following injury once relied heavily on methods such as tissue sectioning and histological examination, which are limited to two-dimensional analysis and often damage the tissue in the process. Moreover, these methods lack the ability to trace changes in cell lineages, instead providing merely a snapshot of the injury response. Here, we describe how technologically advanced methods in lineage tracing models, whole organ clearing, and three-dimensional (3D) whole-mount microscopy can be used to elucidate mechanisms of cardiac repair. With our protocol for neonatal mouse myocardial infarction surgery, tissue clearing, and 3D whole organ imaging, the complex pathways that induce cardiomyocyte proliferation can be unraveled, revealing novel therapeutic targets for cardiac regeneration.

Stimulating ideas for heart regeneration: the future of nerve-directed heart therapy.

Ischemic heart disease is the leading cause of death worldwide. The blockade of coronary arteries limits oxygen-rich blood to the heart and consequently there is cardiomyocyte (CM) cell death, inflammation, fibrotic scarring, and myocardial remodeling. Unfortunately, current therapeutics fail to effectively replace the lost cardiomyocytes or prevent fibrotic scarring, which results in reduced cardiac function and the development of heart failure (HF) in the adult mammalian heart. In contrast, neonatal mice are capable of regenerating their hearts following injury. However, this regenerative response is restricted to the first week of post-natal development. Recently, we identified that cholinergic nerve signaling is necessary for the neonatal mouse cardiac regenerative response. This demonstrates that cholinergic nerve stimulation holds significant potential as a bioelectronic therapeutic tool for heart disease. However, the mechanisms of nerve directed regeneration in the heart remain undetermined. In this review, we will describe the historical evidence of nerve function during regeneration across species. Specifically, we will focus on the emerging role of cholinergic innervation in modulating cardiomyocyte proliferation and inflammation during heart regeneration. Understanding the role of nerves in mammalian heart regeneration and adult cardiac remodeling can provide us with innovative bioelectronic-based therapeutic approaches for treatment of human heart disease.

Apolipoprotein E is a pancreatic extracellular factor that maintains mature ß-cell gene expression.

The in vivo microenvironment of tissues provides myriad unique signals to cells. Thus, following isolation, many cell types change in culture, often preserving some but not all of their in vivo characteristics in culture. At least some of the in vivo microenvironment may be mimicked by providing specific cues to cultured cells. Here, we show that after isolation and during maintenance in culture, adherent rat islets reduce expression of key ß-cell transcription factors necessary for ß-cell function and that soluble pancreatic decellularized matrix (DCM) can enhance ß-cell gene expression. Following chromatographic fractionation of pancreatic DCM, we performed proteomics to identify soluble factors that can maintain ß-cell stability and function. We identified Apolipoprotein E (ApoE) as an extracellular protein that significantly increased the expression of key ß-cell genes. The ApoE effect on beta cells was mediated at least in part through the JAK/STAT signaling pathway. Together, these results reveal a role for ApoE as an extracellular factor that can maintain the mature ß-cell gene expression profile.

A Breakdown in Cooperativity Leads to Cardiac Identity Crisis.

Using induced pluripotent stem cells, Ang et al. elucidate how a mutation in the transcription factor GATA4 causes congenital heart disease. They find that, although the recruitment of GATA4 to cardiac super-enhancers is retained, it no longer functions in partnership with another key transcription factor, leading to misexpression of non-cardiomyocyte genes.