Extension of Endocardium-Derived Vessels Generate Coronary Arteries in Neonates

Background: Unraveling how new coronary arteries develop may provide critical information for establishing novel therapeutic approaches to treating ischemic cardiac diseases. There are two distinct coronary vascular populations derived from different origins in the developing heart. Understanding the formation of coronary arteries may provide insights into new ways of promoting coronary artery formation after myocardial infarction. Methods: To understand how intramyocardial coronary arteries are generated to connect these two coronary vascular populations, we combined genetic lineage tracing, light-sheet microscopy, fluorescence micro-optical sectioning tomography, and tissue-specific gene knockout approaches to understand their cellular and molecular mechanisms. Results: We show that a subset of intramyocardial coronary arteries form by angiogenic extension of endocardium-derived vascular tunnels in the neonatal heart. Three-dimensional whole-mount fluorescence imaging showed that these endocardium-derived vascular tunnels or tubes adopt an arterial fate in neonates. Mechanistically, we implicate Mettl3 and Notch signaling in regulating endocardium-derived intramyocardial coronary artery formation. Functionally, these intramyocardial arteries persist into adulthood and play a protective role after myocardial infarction. Conclusions: A subset of intramyocardial coronary arteries form by extension of endocardium-derived vascular tunnels in the neonatal heart.

Characterizing Neonatal Heart Maturation, Regeneration, and Scar Resolution Using Spatial Transcriptomics

The neonatal mammalian heart exhibits a remarkable regenerative potential, which includes fibrotic scar resolution and the generation of new cardiomyocytes. To investigate the mechanisms facilitating heart repair after apical resection in neonatal mice, we conducted bulk and spatial transcriptomic analyses at regenerative and non-regenerative timepoints. Importantly, spatial transcriptomics provided near single-cell resolution, revealing distinct domains of atrial and ventricular myocardium that exhibit dynamic phenotypic alterations during postnatal heart maturation. Spatial transcriptomics also defined the cardiac scar, which transitions from a proliferative to secretory phenotype as the heart loses regenerative potential. The resolving scar is characterized by spatially and temporally restricted programs of inflammation, epicardium expansion and extracellular matrix production, metabolic reprogramming, lipogenic scar extrusion, and cardiomyocyte restoration. Finally, this study revealed the emergence of a regenerative border zone defined by immature cardiomyocyte markers and the robust expression of Sprr1a. Taken together, our study defines the spatially and temporally restricted gene programs that underlie neonatal heart regeneration and provides insight into cardio-restorative mechanisms supporting scar resolution.

Neonatal heart rate variability: a contemporary scoping review of analysis methods and clinical applications

BackgroundNeonatal heart rate variability (HRV) is widely used as a research tool. However, HRV calculation methods are highly variable making it difficult for comparisons between studies.ObjectivesTo describe the different types of investigations where neonatal HRV was used, study characteristics, and types of analyses performed.Eligibility criteriaHuman neonates ≤1 month of corrected age.Sources of evidenceA protocol and search strategy of the literature was developed in collaboration with the McGill University Health Center’s librarians and articles were obtained from searches in the Biosis, Cochrane, Embase, Medline and Web of Science databases published between 1 January 2000 and 1 July 2020.Charting methodsA single reviewer screened for eligibility and data were extracted from the included articles. Information collected included the study characteristics and population, type of HRV analysis used (time domain, frequency domain, non-linear, heart rate characteristics (HRC) parameters) and clinical applications (physiological and pathological conditions, responses to various stimuli and outcome prediction).ResultsOf the 286 articles included, 171 (60%) were small single centre studies (sample size <50) performed on term infants (n=136). There were 138 different types of investigations reported: physiological investigations (n=162), responses to various stimuli (n=136), pathological conditions (n=109) and outcome predictor (n=30). Frequency domain analyses were used in 210 articles (73%), followed by time domain (n=139), non-linear methods (n=74) or HRC analyses (n=25). Additionally, over 60 different measures of HRV were reported; in the frequency domain analyses alone there were 29 different ranges used for the low frequency band and 46 for the high frequency band.ConclusionsNeonatal HRV has been used in diverse types of investigations with significant lack of consistency in analysis methods applied. Specific guidelines for HRV analyses in neonates are needed to allow for comparisons between studies.

Targeting cardiomyocyte proliferation as a key approach of promoting heart repair after injury

Cardiovascular diseases such as myocardial infarction (MI) is a major contributor to human mortality and morbidity. The mammalian adult heart almost loses its plasticity to appreciably regenerate new cardiomyocytes after injuries, such as MI and heart failure. The neonatal heart exhibits robust proliferative capacity when exposed to varying forms of myocardial damage. The ability of the neonatal heart to repair the injury and prevent pathological left ventricular remodeling leads to preserved or improved cardiac function. Therefore, promoting cardiomyocyte proliferation after injuries to reinitiate the process of cardiomyocyte regeneration, and suppress heart failure and other serious cardiovascular problems have become the primary goal of many researchers. Here, we review recent studies in this field and summarize the factors that act upon the proliferation of cardiomyocytes and cardiac repair after injury and discuss the new possibilities for potential clinical treatment strategies for cardiovascular diseases.

Yap Promotes Noncanonical Wnt Signals From Cardiomyocytes for Heart Regeneration

Rationale: During neonatal heart regeneration, the fibrotic response, which is required to prevent cardiac rupture, resolves via poorly understood mechanisms. Deletion of the Hippo pathway gene Sav in adult cardiomyocytes increases Yap activity and promotes cardiac regeneration, partly by inducing fibrosis resolution. Deletion of Yap in neonatal cardiomyocytes leads to increased fibrosis and loss of neonatal heart regeneration, suggesting that Yap inhibits fibrosis by regulating intercellular signaling from cardiomyocytes to cardiac fibroblasts (CFs). Objective: We investigated the role of Wntless ( Wls ), which is a direct target gene of Yap, in communication between cardiomyocytes and CFs during neonatal heart regeneration. Methods and Results: We generated 2 mouse models to delete Wls specifically in cardiomyocytes ( Myh6-Cas9 combined with AAV9-Wls-g RNAs, and Myh6 cre-ERT2/+ ; Wls flox/flox ). Reanalysis of single-cell RNA sequencing data revealed that Wnt ligands are expressed in cardiomyocytes, whereas Wnt receptors are expressed in CFs, suggesting that Wnt signaling is directional from cardiomyocytes to CFs during neonatal heart regeneration. Wls deletion in neonatal cardiomyocytes disrupted Wnt signaling, revealed by reduced noncanonical Wnt signaling in non-cardiomyocytes. Four weeks after neonatal heart myocardial infarction, heart function was measured by echocardiography. Wls deletion in neonatal cardiomyocytes after myocardial infarction impairs neonatal heart regeneration, marked by decreased contractile function and increased fibrosis. Wls mutant hearts display CF activation, characterized by increased extracellular matrix secretion, inflammation, and CF proliferation. Conclusions: These data indicate that during neonatal heart regeneration, intercellular signaling from cardiomyocytes to CFs occurs via noncanonical Wnt signaling to rebuild cardiac architecture after myocardial infarction.

Nrf1 promotes heart regeneration and repair by regulating proteostasis and redox balance

Following injury, cells in regenerative tissues have the ability to regrow. The mechanisms whereby regenerating cells adapt to injury-induced stress conditions and activate the regenerative program remain to be defined. Here, using the mammalian neonatal heart regeneration model, we show that Nrf1, a stress-responsive transcription factor encoded by the Nuclear Factor Erythroid 2 Like 1 (Nfe2l1) gene, is activated in regenerating cardiomyocytes. Genetic deletion of Nrf1 prevented regenerating cardiomyocytes from activating a transcriptional program required for heart regeneration. Conversely, Nrf1 overexpression protected the adult mouse heart from ischemia/reperfusion (I/R) injury. Nrf1 also protected human induced pluripotent stem cell-derived cardiomyocytes from doxorubicin-induced cardiotoxicity and other cardiotoxins. The protective function of Nrf1 is mediated by a dual stress response mechanism involving activation of the proteasome and redox balance. Our findings reveal that the adaptive stress response mechanism mediated by Nrf1 is required for neonatal heart regeneration and confers cardioprotection in the adult heart.

Abstract P446: Cyclophilin D Inhibition Rescues Cardiac Function And Electron Transport Chain Assembly And Activity In Hypoxia Exposed Neonatal Mice

Introduction: Mitochondria play a critical role in cardiac myocyte physiology and differentiation. Hypoxia decreases cardiac function. Changes in embryonic heart metabolism at the level of the electron transport chain (ETC) are regulated by a chaperone protein known as cyclophilin D (CypD). Inhibition of CypD with chemicals such as cyclosporin A (CsA) and N-methyl-4-isoleucine cyclosporin (NIM811) leads to more complex mitochondrial structure and effective oxidative phosphorylation. Hypothesis: Inhibition of CypD with CsA or NIM811 will rescue the detrimental effects of hypoxia on cardiac function and on ETC assembly and activity. Methods: Mice were exposed to continuous hypoxia (12% oxygen) immediately before birth (gestational age E19.5) to postnatal day 7 (P7). Hypoxic mice received no treatment (No Tx) or intraperitoneal injections 10mg/kg of vehicle (VEH), CsA, or NIM811 from P1 to P6. Litters of mice born into room air served as controls. On P7, mice were anesthetized and underwent echocardiography and/or hearts were harvested for mitochondrial isolation. Enzymatic activity of ETC complexes was quantified using spectrophotometry and normalized to total protein. To measure physical assembly of complex I & V of the ETC, high resolution clear native polyacrylamide gel electrophoresis (HCRN PAGE) followed by in-gel assays were utilized using appropriate protein loading controls. Results: Cardiac ejection fraction was decreased in hypoxic No Tx (P<0.0001) and VEH (P<0.0001), but was rescued by CsA or NIM811 when compared to room air controls (P>0.05). Heart weight to body weight ratio was increased in No Tx and VEH groups (P< 0.0001) and rescued in the CsA and NIM811 groups when compared to room air controls (P>0.05). Complex I enzymatic activity was rescued with treatment with CsA and NIM811. HCRN PAGE followed by in-gel ETC complex assay demonstrated assembly of complexes I and V into dimers and tetramer in the room air, CsA, and NIM811 groups that was not seen in the No Tx and VEH groups. Conclusion: Pharmacologic inhibition of CypD reversed the effects of hypoxia on cardiac function and ETC activity and assembly in the neonatal heart. Our studies may help develop therapies to treat neonatal cardiomyopathies and the effects of hypoxia on the neonatal heart.