Cardiac Biology Research Articles

Heart Organoids and Engineered Heart Tissues: Novel Tools for Modeling Human Cardiac Biology and Disease.

Organoids are three-dimensional in vitro cell constructs that recapitulate organ properties and structure to a significant extent. They constitute particularly useful models to study unapproachable states in humans, such as embryonic and fetal development, or early disease progression in adults. In recent years organoids have been implemented to model a wide range of different organs and disease conditions. However, the technology for their fabrication and application to cardiovascular studies has been lagging significantly when compared to other organoid types (e.g., brain, pancreas, kidney, intestine). This is a surprising fact since cardiovascular disease (CVD) and congenital heart disease (CHD) constitute the leading cause of mortality and morbidity in the developed world, and the most common birth defect in humans, respectively, and collectively constitute one of the largest unmet medical needs in the modern world. There is a critical need to establish in vitro models of the human heart that faithfully recapitulate its biology and function, thus enabling basic and translational studies to develop new therapeutics. Generating heart organoids that truly resemble the heart has proven difficult due to its complexity, but significant progress has been made recently to overcome this obstacle. In this review, we will discuss progress in novel heart organoid generation methods, the advantages and disadvantages of each approach, and their translational applications for advancing cardiovascular studies and the treatment of heart disorders.

Glutamate 73 Promotes Anti-arrhythmic Effects of Voltage-Dependent Anion Channel Through Regulation of Mitochondrial Ca2+ Uptake.

Mitochondria critically regulate a range of cellular processes including bioenergetics, cellular metabolism, apoptosis, and cellular Ca2+ signaling. The voltage-dependent anion channel (VDAC) functions as a passageway for the exchange of ions, including Ca2+, across the outer mitochondrial membrane. In cardiomyocytes, genetic or pharmacological activation of isoform 2 of VDAC (VDAC2) effectively potentiates mitochondrial Ca2+ uptake and suppresses Ca2+ overload-induced arrhythmogenic events. However, molecular mechanisms by which VDAC2 controls mitochondrial Ca2+ transport and thereby influences cardiac rhythmicity remain elusive. Vertebrates express three highly homologous VDAC isoforms. Here, we used the zebrafish tremblor/ncx1h mutant to dissect the isoform-specific roles of VDAC proteins in Ca2+ handling. We found that overexpression of VDAC1 or VDAC2, but not VDAC3, suppresses the fibrillation-like phenotype in zebrafish tremblor/ncx1h mutants. A chimeric approach showed that moieties in the N-terminal half of VDAC are responsible for their divergent functions in cardiac biology. Phylogenetic analysis further revealed that a glutamate at position 73, which was previously described to be an important regulator of VDAC function, is sevolutionarily conserved in VDAC1 and VDAC2, whereas a glutamine occupies position 73 (Q73) of VDAC3. To investigate whether E73/Q73 determines VDAC isoform-specific anti-arrhythmic effect, we mutated E73 to Q in VDAC2 (VDAC2E73Q) and Q73 to E in VDAC3 (VDAC3Q73E). Interestingly, VDAC2E73Q failed to restore rhythmic cardiac contractions in ncx1 deficient hearts, while the Q73E conversion induced a gain of function in VDAC3. In HL-1 cardiomyocytes, VDAC2 knockdown diminished the transfer of Ca2+ from the SR into mitochondria and overexpression of VDAC2 or VDAC3Q73E restored SR-mitochondrial Ca2+ transfer in VDAC2 deficient HL-1 cells, whereas this rescue effect was absent for VDAC3 and drastically compromised for VDAC2E73Q. Collectively, our findings demonstrate a critical role for the evolutionary conserved E73 in determining the anti-arrhythmic effect of VDAC isoforms through modulating Ca2+ cross-talk between the SR and mitochondria in cardiomyocytes.

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

Exercise Training Preserves Myocardial Strain and Improves Exercise Tolerance in Doxorubicin-Induced Cardiotoxicity.

Doxorubicin causes cardiotoxicity and exercise intolerance. Pre-conditioning exercise training seems to prevent doxorubicin-induced cardiac damage. However, the effectiveness of the cardioprotective effects of exercise training concomitantly with doxorubicin treatment remains largely unknown. To determine whether low-to-moderate intensity aerobic exercise training during doxorubicin treatment would prevent cardiotoxicity and exercise intolerance, we performed exercise training concomitantly with chronic doxorubicin treatment in mice. Ventricular structure and function were accessed by echocardiography, exercise tolerance by maximal exercise test, and cardiac biology by histological and molecular techniques. Doxorubicin-induced cardiotoxicity, evidenced by impaired ventricular function, cardiac atrophy, and fibrosis. Exercise training did not preserve left ventricular ejection fraction or reduced fibrosis. However, exercise training preserved myocardial circumferential strain alleviated cardiac atrophy and restored cardiomyocyte cross-sectional area. On the other hand, exercise training exacerbated doxorubicin-induced body wasting without affecting survival. Finally, exercise training blunted doxorubicin-induced exercise intolerance. Exercise training performed during doxorubicin-based chemotherapy can be a valuable approach to attenuate cardiotoxicity.

Role of GSK-3 in Cardiac Health: Focusing on Cardiac Remodeling and Heart Failure.

Glycogen synthase kinase 3 (GSK-3) is a ubiquitously expressed serine/threonine kinase and was first identified as a regulator of glycogen synthase enzyme and glucose homeostasis. It regulates cellular processes like cell proliferation, metabolism, apoptosis and development. Recent findings suggest that GSK-3 is required to maintain the normal cardiac homeostasis that regulates cardiac development, proliferation, hypertrophy and fibrosis. GSK-3 is expressed as two isoforms, α and β. The role of GSK-3α and GSK-3β in cardiac biology is well documented. Both isoforms have common as well as isoform-specific functions. Human data also suggests that GSK-3β is downregulated in hypertrophy and heart failure and acts as a negative regulator. Pharmacological inhibition of GSK-3α and GSK-3β leads to endogenous cardiomyocyte proliferation and cardiac regeneration via the upregulation of cell cycle regulators, which results in cell cycle re-entry and DNA synthesis. It was found that cardiac-specific knockout (KO) of GSK-3α retained cardiac function, inhibited cardiovascular remodelling and restricted scar expansion during ischemia. Further, knockout of GSK-3α decreases cardiomyocyte apoptosis and enhances its proliferation. However, GSK-3β KO also results in hypertrophic myopathy due to cardiomyocyte hyper-proliferation. Thus GSK-3 inhibitors are named as a double-edged sword because of their beneficial and off-target effects. This review focuses on the isoform-specific functions of GSK-3 that will help in better understanding the role of GSK-3α and GSK-3β in cardiac biology and pave the way for the development of new isoform-specific GSK-3 modulator for the treatment of ischemic heart disease, cardiac regeneration and heart failure.

Iron-Deficiency Anemia Results in Transcriptional and Metabolic Remodeling in the Heart Toward a Glycolytic Phenotype.

Iron deficiency is the most prevalent micronutrient disorder globally. When severe, iron deficiency leads to anemia, which can be deleterious to cardiac function. Given the central role of iron and oxygen in cardiac biology, multiple pathways are expected to be altered in iron-deficiency anemia, and identifying these requires an unbiased approach. To investigate these changes, gene expression and metabolism were studied in mice weaned onto an iron-deficient diet for 6 weeks. Whole-exome transcriptomics (RNAseq) identified over 1,500 differentially expressed genes (DEGs), of which 22% were upregulated and 78% were downregulated in the iron-deficient group, relative to control animals on an iron-adjusted diet. The major biological pathways affected were oxidative phosphorylation and pyruvate metabolism, as well as cardiac contraction and responses related to environmental stress. Cardiac metabolism was studied functionally using in vitro and in vivo methodologies. Spectrometric measurement of the activity of the four electron transport chain complexes in total cardiac lysates showed that the activities of Complexes I and IV were reduced in the hearts of iron-deficient animals. Pyruvate metabolism was assessed in vivo using hyperpolarized 13C magnetic resonance spectroscopy (MRS) of hyperpolarized pyruvate. Hearts from iron-deficient and anemic animals showed significantly decreased flux through pyruvate dehydrogenase and increased lactic acid production, consistent with tissue hypoxia and induction of genes coding for glycolytic enzymes and H+-monocarboxylate transport-4. Our results show that iron-deficiency anemia results in a metabolic remodeling toward a glycolytic, lactic acid-producing phenotype, a hallmark of hypoxia.

Monoclonal Antibody-Based Immunotherapy and Its Role in the Development of Cardiac Toxicity.

Simple SummaryThe application of immunotherapies to treat cancer patients has significantly improved over the last two decades and extended many patients’ life spans. Monoclonal antibodies are synthetic proteins employed as immunotherapies to treat and manage cancers that require a complete understanding of cancer biology and the host’s immune system. However, activated immune responses, by monoclonal antibodies, can target nonspecific cancer cells, causing frequent immune-related adverse events that can lead to permanent disorders among cancer patients. The immune-related adverse events pose a risk of cardiac toxicity that includes hypertension, heart failure, arrhythmias, and left ventricular dysfunction during and after monoclonal antibody immunotherapy. Moreover, with the mortality rate of 47% attributed to heart disease and cancer, it is imperative to employ reliable, sensitive, and clinically relevant models for efficacy and safety assessment of immune drugs for cancer and the prevention of cardiotoxicities.Immunotherapy is one of the most effective therapeutic options for cancer patients. Five specific classes of immunotherapies, which includes cell-based chimeric antigenic receptor T-cells, checkpoint inhibitors, cancer vaccines, antibody-based targeted therapies, and oncolytic viruses. Immunotherapies can improve survival rates among cancer patients. At the same time, however, they can cause inflammation and promote adverse cardiac immune modulation and cardiac failure among some cancer patients as late as five to ten years following immunotherapy. In this review, we discuss cardiotoxicity associated with immunotherapy. We also propose using human-induced pluripotent stem cell-derived cardiomyocytes/ cardiac-stromal progenitor cells and cardiac organoid cultures as innovative experimental model systems to (1) mimic clinical treatment, resulting in reproducible data, and (2) promote the identification of immunotherapy-induced biomarkers of both early and late cardiotoxicity. Finally, we introduce the integration of omics-derived high-volume data and cardiac biology as a pathway toward the discovery of new and efficient non-toxic immunotherapy.

Mitochondrial Calcium Uniporter Deficiency in Zebrafish Causes Cardiomyopathy With Arrhythmia

Mitochondrial Ca2 + uptake influences energy production, cell survival, and Ca2 + signaling. The mitochondrial calcium uniporter, MCU, is the primary route for uptake of Ca2 + into the mitochondrial matrix. We have generated a zebrafish MCU mutant that survives to adulthood and exhibits dramatic cardiac phenotypes resembling cardiomyopathy and sinus arrest. MCU hearts contract weakly and have a smaller ventricle with a thin compact layer and reduced trabecular density. Damaged myofibrils and swollen mitochondria were present in the ventricles of MCU mutants, along with gene expression changes indicative of cell stress and altered cardiac structure and function. Using electrocardiography, we found that MCU hearts display conduction system defects and abnormal rhythm, with extended pauses resembling episodes of sinus arrest. Together, our findings suggest that proper mitochondrial Ca2 + homeostasis is crucial for maintaining a healthy adult heart, and establish the MCU mutant as a useful model for understanding the role of mitochondrial Ca2 + handling in adult cardiac biology.

Deficit of glucocorticoid-induced leucine zipper amplifies angiotensin-induced cardiomyocyte hypertrophy and diastolic dysfunction.

Poor prognosis in heart failure and the lack of real breakthrough strategies validate targeting myocardial remodelling and the intracellular signalling involved in this process. So far, there are no effective strategies to counteract hypertrophy, an independent predictor of heart failure progression and death. Glucocorticoid‐induced leucine zipper (GILZ) is involved in inflammatory signalling, but its role in cardiac biology is unknown. Using GILZ‐knockout (KO) mice and an experimental model of hypertrophy and diastolic dysfunction, we addressed the role of GILZ in adverse myocardial remodelling. Infusion of angiotensin II (Ang II) resulted in myocardial dysfunction, inflammation, apoptosis, fibrosis, capillary rarefaction and hypertrophy. Interestingly, GILZ‐KO showed more evident diastolic dysfunction and aggravated hypertrophic response compared with WT after Ang II administration. Both cardiomyocyte and left ventricular hypertrophy were more pronounced in GILZ‐KO mice. On the other hand, Ang II–induced inflammatory and fibrotic phenomena, cell death and reduction in microvascular density, remained invariant between the WT and KO groups. The analysis of regulators of hypertrophic response, GATA4 and FoxP3, demonstrated an up‐regulation in WT mice infused with Ang II; conversely, such an increase did not occur in GILZ‐KO hearts. These data on myocardial response to Ang II in mice lacking GILZ indicate that this protein is a new element that can be mechanistically involved in cardiovascular pathology.

Influence of perfusable microvasculature on excitation-contraction coupling in IPSC-derived myocardium

Abstract The myocardium is one of the most densely vascularised tissues in the body, with dynamic metabolic demand from beating cardiomyocytes (CM) necessitating an intimate relationship with microvasculature. Endothelial cells (EC) produce a diverse array of cardio-active factors which acutely and chronically modulate myocardial phenotype. Disruption of CM-EC signalling results in pathological remodelling, and ultimately organ failure. However, as physiologically relevant recapitulation of CM-EC interaction has been difficult to achieve in vitro, many molecular mechanisms governing their interaction remain poorly understood. To induce cardiac vasculogenesis in vitro, we have developed microfluidic chips which subject 3D hydrogel cultures to precisely controlled flow. We then co-cultured human cardiac microvascular ECs, human left ventricular fibroblasts (FB), and human induced pluripotent stem cell-derived cardiomyocytes for 5 days under a pro-vasculogenic protocol (0.5 ul/min flow rate, 50ng/ml VEGF, 100ng/ml Ang-1). Via live and fixed immunofluorescence microscopy, we observed spontaneous formation of a microvasculature network with a continuously open lumen embedded within beating myocardium. Simultaneous quantification of iPSC-CM contractility and perfused red blood cell velocity reveals biomimetic pulsatile flow profile within the microvasculature. To evaluate the influence of microvasculature on CM function, we incorporated CMs differentiated from stem cells with the genetically encoded calcium biosensor GCaMP6F. Compared to CM only control, vascularised preparations demonstrate significantly faster calcium transient time to peak (−11.5%, p=0.007) and time to 50% relaxation (−15%, p=0.01). Under static conditions and 1Hz electrical stimulation, presence of EC was associated with reduced iPSC-CM arrhythmia at baseline (p<0.0001) and during 1uM isoprenaline treatment (p=0.0003), while maintaining isoprenaline induced Ca2+ handling quickening. To the best of our knowledge, this work represents the first fully perfusable model of the myocardial microvasculature, and highlights the importance of EC regulatory influence on CM function. Further work aims to investigate underlying molecular mechanisms to provide therapeutically relevant insight into cardiac biology. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): British Heart Foundation

Heart Development and Regeneration in Non-mammalian Model Organisms.

Cardiovascular disease is a serious threat to human health and a leading cause of mortality worldwide. Recent years have witnessed exciting progress in the understanding of heart formation and development, enabling cardiac biologists to make significant advance in the field of therapeutic heart regeneration. Most of our understanding of heart development and regeneration, including the genes and signaling pathways, are driven by pioneering works in non-mammalian model organisms, such as fruit fly, fish, frog, and chicken. Compared to mammalian animal models, non-mammalian model organisms have special advantages in high-throughput applications such as disease modeling, drug discovery, and cardiotoxicity screening. Genetically engineered animals of cardiovascular diseases provide valuable tools to investigate the molecular and cellular mechanisms of pathogenesis and to evaluate therapeutic strategies. A large number of congenital heart diseases (CHDs) non-mammalian models have been established and tested for the genes and signaling pathways involved in the diseases. Here, we reviewed the mechanisms of heart development and regeneration revealed by these models, highlighting the advantages of non-mammalian models as tools for cardiac research. The knowledge from these animal models will facilitate therapeutic discoveries and ultimately serve to accelerate translational medicine.

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes.

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression. The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

The Genetic Makeup of the Electrocardiogram.

The electrocardiogram (ECG) is one of the most useful non-invasive diagnostic tests for a wide array of cardiac disorders. Traditional approaches to analyzing ECGs focus on individual segments. Here, we performed comprehensive deep phenotyping of 77,190 ECGs in the UK Biobank across the complete cycle of cardiac conduction, resulting in 500 spatial-temporal datapoints, across 10 million genetic variants. In addition to characterizing polygenic risk scores for the traditional ECG segments, we identified over 300 genetic loci that are statistically associated with the high-dimensional representation of the ECG. We established the genetic ECG signature for dilated cardiomyopathy, associated the BAG3, HSPB7/CLCNKA, PRKCA, TMEM43, and OBSCN loci with disease risk and confirmed this association in an independent cohort. In total, our work demonstrates that a high-dimensional analysis of the entire ECG provides unique opportunities for studying cardiac biology and disease and furthering drug development. A record of this paper's transparent peer review process is included in the Supplemental Information.

Differentiation of Human Induced Pluripotent Stem Cells into Epicardial-Like Cells.

The epicardium is a multipotent cell layer that is vital to myocardial development and regeneration. Epicardial cells contribute to cardiac fibroblast and smooth muscle populations of the heart and secrete paracrine factors that promote cardiomyocyte proliferation and angiogenesis. Despite a central role in cardiac biology, the mechanisms by which epicardial cells influence cardiac growth are largely unknown, and robust models of the epicardium are needed. Here, we review our protocol for differentiating induced pluripotent stem cells (iPSCs) into epicardial-like cells through temporal modulation of canonical Wnt signaling. iPSC-derived epicardial cells (iECs) resemble in vivo epicardial cells morphologically and display markers characteristic of the developing epicardium. We also review our protocol for differentiating iECs into fibroblasts and smooth muscle cells through treatment with bFGF and TGF-β1, respectively. iECs provide a platform for studying fundamental epicardial biology and can inform strategies for therapeutic heart regeneration.

Novel cardiac cell subpopulations: Pnmt-derived cardiomyocytes.

Diversity among highly specialized cells underlies the fundamental biology of complex multi-cellular organisms. One of the essential scientific questions in cardiac biology has been to define subpopulations within the heart. The heart parenchyma comprises specialized cardiomyocytes (CMs). CMs have been canonically classified into a few phenotypically diverse subpopulations largely based on their function and anatomic localization. However, there is growing evidence that CM subpopulations are in fact numerous, with a diversity of genetic origin and putatively different roles in physiology and pathophysiology. In this chapter, we introduce a recently discovered CM subpopulation: phenylethanolamine-N-methyl transferase (Pnmt)-derived cardiomyocytes (PdCMs). We discuss: (i) canonical classifications of CM subpopulations; (ii) discovery of PdCMs; (iii) Pnmt and the role of catecholamines in the heart; similarities and dissimilarities of PdCMs and canonical CMs; and (iv) putative functions of PdCMs in both physiological and pathological states and future directions, such as in intra-cardiac adrenergic signalling.

Abstract MP145: Potential Functions of the Nuclear Basket Associated With Dilated Cardiomyopathy

Objectives: NUP153 overexpression is clinically correlated with cardiomyopathies, specifically dilated cardiomyopathy (DCM). In combination with the nuclear pore complex (NPC) proteins TPR and NUP50, all three form the basket structure of the NPC that sits within the nuclear envelope. The nuclear basket regulates chromatin dynamics and nuclear export, yet its potential role in cardiopathological development and progression remains unknown. This study was thus carried out to elucidate possible functional roles for nuclear basket nucleoporins in cardiac biology. Methods: Human induced pluripotent stem cells (hiPSCs) with endogenous mEGFP tagged NUP153 were differentiated into cardiomyocytes (hiPSC-CMs) via direct differentiation. Beating started ~ Day 10 and contractile areas were analyzed for contraction velocity and magnitude. NUP153 mobility in hiPSCs and hiPSC-CMs was determined by iFRAP experiments. Bioinformatic analysis of a clinical dilated cardiomyopathy dataset (GSE29819) returned a focused ‘nuclear envelope’ gene list that was uploaded to Ingenuity Pathways Analysis for network analysis. Results: Within hiPSC-CM beating foci (A), NUP153 mobility decreased and went from a biphasic to a monophasic decay curve (B). Bioinformatic analysis of a DCM dataset (C) revealed expression changes in TPR and NUP50 , with no significant changes in NUP153 (D). Conclusions: A switch to monophasic decay kinetics of NUP153 in hiPSC-CMs suggests prioritization of stable slow pools that may reflect changes in global transcriptome regulation. In DCM, differential nucleoporin gene expression changes implicate discrete functional disruptions within the nuclear basket.

Abstract MP112: Optogenetic Manipulation of Macrophage Electrical Excitability Modulates Cardiac Rhythm in Developing Zebrafish

Despite the unremitting research surrounding heart development and disease, congenitalheart defects (CHD) remain the most common birth defect worldwide. Arrhythmias are aleading cause of morbidity and mortality in CHD patients, though the etiologies ofarrhythmia are not well understood. Notably, there have been considerable advances inour understanding of cardiac macrophage biology in both regulating developmentalprocesses and modulating adult cardiac conduction. However, it is not known 1) whenmacrophage-derived signals influence heart electrical function, nor 2) if macrophages areintegral to the development and function of the embryonic or juvenile conduction system.Using transparent zebrafish, a genetically tractable and well-established developmentalmodel, we employ advanced microscopy techniques to visualize cardiac macrophages inreal-time and in vivo throughout heart development. We demonstrate here that embryoniccardiac macrophages display synchronous calcium activity in-time with heartbeat. Usingoptogenetics to stimulate macrophage electrical activity via light-gated ion channels, weshow that macrophage-specific expression of channelrhodopsin, a cation channel, orhalorhodopsin, a chloride channel, can directly influence heartrate. Thus, embryonicmacrophages can functionally manipulate cardiac rhythm in developing zebrafish.Further, loss of embryonic macrophages, either through drug-inducible ablation orembryonic macrophage genetic mutation, disrupts organization of both the embryonic andadult ventricular myocardium. We therefore hypothesize cardiac macrophages arefunctionally important in embryonic conduction and fetal heart development, such thatembryonic macrophage loss negatively impacts long-term cardiac health. This work willprovide novel insight into potential contributing factors of cardiac developmentaldysregulation and macrophages as targets of intervention for arrhythmia.

Heart Regeneration by Endogenous Stem Cells and Cardiomyocyte Proliferation

Ischemic heart disease is the leading cause of death worldwide. Myocardial infarction results in an irreversible loss of cardiomyocytes with subsequent adverse remodeling and heart failure. Identifying new sources for cardiomyocytes and promoting their formation represents a goal of cardiac biology and regenerative medicine. Within the past decade, many types of putative cardiac stem cells (CSCs) have been reported to regenerate the injured myocardium by differentiating into new cardiomyocytes. Some of these CSCs have been translated from bench to bed with reported therapeutic effectiveness. However, recent basic research studies on stem cell tracing have begun to question their fundamental biology and mechanisms of action, raising serious concerns over the myogenic potential of CSCs. We review the history of different types of CSCs within the past decade and provide an update of recent cell tracing studies that have challenged the origin and existence of CSCs. In addition to the potential role of CSCs in heart regeneration, proliferation of preexisting cardiomyocytes has recently gained more attention. This review will also evaluate the methodologic and technical aspects of past and current studies on CSCs and cardiomyocyte proliferation, with emphasis on technical strengths, advantages, and potential limitations of research approaches. While our understanding of cardiomyocyte generation and regeneration continues to evolve, it is important to address the shortcomings and inaccuracies in this field. This is best achieved by embracing technological advancements and improved methods to label single cardiomyocytes/progenitors and accurately investigate their developmental potential and fate/lineage commitment.

Metabolic Regulation of Human Pluripotent Stem Cell-Derived Cardiomyocyte Maturation.

This review summarizes the important role that metabolism plays in driving maturation of human pluripotent stem cell-derived cardiomyocytes. Human pluripotent stem cell-derived cardiomyocytes provide a model system for human cardiac biology. However, these models have been unable to fully recapitulate the maturity observed in the adult heart. By simulating the glucose to fatty acid transition observed in neonatal mammals, human pluripotent stem cell-derived cardiomyocytes undergo structural and functional maturation also accompanied by transcriptional changes and cell cycle arrest. The role of metabolism in energy production, signaling, and epigenetic modifications illustrates that metabolism and cellular phenotype are intimately linked. Further understanding of key metabolic factors driving cardiac maturation will facilitate the generation of more mature human pluripotent stem cell-derived cardiomyocyte models. This will increase our understanding of cardiac biology and potentially lead to novel therapeutics to enhance heart function.