Ventricular conduction and long-term heart failure outcomes and mortality in African Americans: insights from the Jackson Heart Study.

BACKGROUND: QRS prolongation is associated with adverse outcomes in mostly white populations, but its clinical significance is not well established for other groups. We investigated the association between QRS duration and mortality in African Americans. METHODS AND RESULTS: We analyzed data from 5146 African Americans in the Jackson Heart Study stratified by QRS duration on baseline 12-lead ECG. We defined QRS prolongation as QRS≥100 ms. We assessed the association between QRS duration and all-cause mortality using Cox proportional hazards models and reported the cumulative incidence of heart failure hospitalization. We identified factors associated with the development of QRS prolongation in patients with normal baseline QRS. At baseline, 30% (n=1528) of participants had QRS prolongation. The cumulative incidences of mortality and heart failure hospitalization were greater with versus without baseline QRS prolongation: 12.6% (95% confidence interval [CI], 11.0-14.4) versus 7.1% (95% CI, 6.3-8.0) and 8.2% (95% CI, 6.9-9.7) versus 4.4% (95% CI, 3.7-5.1), respectively. After risk adjustment, QRS prolongation was associated with increased mortality (hazard ratio, 1.27; 95% CI, 1.03-1.56; P=0.02). There was a linear relationship between QRS duration and mortality (hazard ratio per 10 ms increase, 1.06; 95% CI, 1.01-1.12). Older age, male sex, prior myocardial infarction, lower ejection fraction, left ventricular hypertrophy, and left ventricular dilatation were associated with the development of QRS prolongation. CONCLUSIONS: QRS prolongation in African Americans was associated with increased mortality and heart failure hospitalization. Factors associated with developing QRS prolongation included age, male sex, prior myocardial infarction, and left ventricular structural abnormalities.

Later Life Consequences of Subteratogenic Exposure to a Complex PAH Mixture in the Atlantic Killifish (Fundulus heteroclitus)

Subteratogenic and other low-level chronic exposures to toxicant mixtures are an understudied threat to environmental and human health. It is especially important to understand the effects of these exposures for contaminants, such as polycyclic aromatic hydrocarbons (PAHs) a large group of more than 100 individual compounds, which are important environmental (including aquatic) contaminants. Aquatic sediments constitute a major sink for hydrophobic pollutants, and studies show PAHs can persist in sediments over time. Furthermore, estuarine systems (namely breeding grounds) are of particular concern, as they are highly impacted by a wide variety of pollutants, and estuarine fishes are often exposed to some of the highest levels of contaminants of any vertebrate taxon. Acute embryonic exposure to PAHs results in cardiac teratogenesis in fish, and early life exposure to certain individual PAHs and PAH mixtures cause heart alterations with decreased swimming capacity in adult fish. Consequently, the heart and cardiorespiratory system are thought to be targets of PAH mixture exposure. While many studies have investigated acute, teratogenic PAH exposures, few studies have longitudinally examined the impacts of subtle, subteratogenic PAH mixture exposures, which are arguably more broadly applicable to environmental contamination scenarios. The goal of this dissertation was to highlight the later-life consequences of early-life exposure to subteratogenic concentrations of a complex, environmentally relevant PAH mixture.

A unique population of Fundulus heteroclitus (the Atlantic killifish or mummichog, hereafter referred to as killifish), has adapted to creosote-based polycyclic aromatic hydrocarbons (PAHs) found at the Atlantic Wood Industries (AW) Superfund site in the southern branch of the Elizabeth River, VA, USA. This killifish population survives in a site heavily contaminated with a mixture of PAHs from former creosote operations. They have developed resistance to the acute toxicity and teratogenic effects caused by the mixture of PAHs in sediment from the site. The primary goal of this dissertation was to compare and contrast later-life outcomes of early-life, subteratogenic PAH mixture exposure in both the Atlantic Wood killifish (AW) and a naïve reference population of killifish from King’s Creek (KC; a relatively uncontaminated tributary of the Severn River, VA). Killifish from both populations were exposed to subteratogenic concentrations of a complex PAH-sediment extract, Elizabeth River Sediment Extract (ERSE), made by collecting sediment from the AW site. Fish were reared over a 5-month period in the laboratory, during which they were examined for a variety of molecular, physiological and behavioral responses.

The central aims of my dissertation were to determine alterations to embryonic gene expression, larval swimming activity, adult behavior, heart structure, enzyme activity, and swimming/cardiorespiratory performance following subteratogenic exposure to ERSE. I hypothesized that subteratogenic exposure to ERSE would impair cardiac ontogenic processes in a way that would be detectable via gene expression in embryos, and that the misregulation of cardiac genes would help to explain activity changes, behavioral deficits, and later-life swimming deficiencies. I also hypothesized that fish heart structure would be altered. In addition, I hypothesized that the AW killifish population would be resistant to developmental exposures and perform normally in later life challenges. To investigate these hypotheses, a series of experiments were carried out in PAH-adapted killifish from Elizabeth River and in reference killifish. As an ancillary project to the primary aims of the dissertation, I examined the toxicity of weaker aryl hydrocarbon receptor (AHR) agonists in combination with fluoranthene (FL), an inhibitor of cytochrome P4501A1 (CYP1A1). This side project was conducted in both Danio rerio (zebrafish) and the KC and AW killifish.

Embryonic gene expression was measured in both killifish populations over an ERSE dose response with multiple time points (12, 24, 48, and 144 hours post exposure). Genes known to play critical roles in cardiac structure/development, cardiac function, and angiogenesis were elevated, indicating cardiac damage and activation of cardiovascular repair mechanisms. These data helped to inform later-life swimming performance and cardiac histology studies. Behavior was assessed during light and dark cycles in larvae of both populations following developmental exposure to ERSE. While KC killifish showed activity differences following exposure, AW killifish showed no significant changes even at concentrations that would cause overt cardiac toxicity in KC killifish. Juvenile behavior experiments demonstrated hyperactivity following ERSE exposure in KC killifish, but no significant behavioral changes in AW killifish. Adult swimming performance via prolonged critical swimming capacity (Ucrit) demonstrated performance costs in the AW killifish. Furthermore, swimming performance decline was observed in KC killifish following exposure to increasing dilutions of ERSE. Lastly, cardiac histology suggested that early-life exposure to ERSE could result in cardiac structural alteration and extravasation of blood into the pericardial cavity.

Responses to AHR agonists resulted in a ranking of relative potency for agonists, and determined which agonists, when combined with FL, caused cardiac teratogenesis. These experiments showed interesting species differences for zebrafish and killifish. To probe mechanisms responsible for cardiotoxicity, a CYP1A-morpholino and a AHR2-morpholino were used to mimic FL effects or attempt to rescue cardiac deformities respectively. Findings suggested that the cardiac toxicity elicited by weak agonist + FL exposure was likely driven by AHR-independent mechanisms. These studies stand in contrast to previous research from our lab showing that moderate AHR agonist + FL caused cardiac toxicity that can be partially rescued by AHR-morpholino knockdown.

My findings will form better characterization of mechanisms of PAH toxicity, and advance our understanding of how subteratogenic mixtures of PAHs exert their toxic action in naïve killifish. Furthermore, these studies will provide a framework for investigating how subteratogenic exposures to PAH mixtures can impact aquatic organismal health and performance. Most importantly, these experiments have the potential to help inform risk assessment in fish, mammals, and potentially humans. Ultimately, this research will help protect populations exposed to subtle PAH-contamination.

Arterial pole progenitors interpret opposing FGF/BMP signals to proliferate or differentiate.

During heart development, a subpopulation of cells in the heart field maintains cardiac potential over several days of development and forms the myocardium and smooth muscle of the arterial pole. Using clonal and explant culture experiments, we show that these cells are a stem cell population that can differentiate into myocardium, smooth muscle and endothelial cells. The multipotent stem cells proliferate or differentiate into different cardiovascular cell fates through activation or inhibition of FGF and BMP signaling pathways. BMP promoted myocardial differentiation but not proliferation. FGF signaling promoted proliferation and induced smooth muscle differentiation, but inhibited myocardial differentiation. Blocking the Ras/Erk intracellular pathway promoted myocardial differentiation, while the PLCgamma and PI3K pathways regulated proliferation. In vivo, inhibition of both pathways resulted in predictable arterial pole defects. These studies suggest that myocardial differentiation of arterial pole progenitors requires BMP signaling combined with downregulation of the FGF/Ras/Erk pathway. The FGF pathway maintains the pool of proliferating stem cells and later promotes smooth muscle differentiation.

Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration.

Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, beta-arrestin1(-/-), and beta-arrestin2(-/-) mice. Neointimal hyperplasia was enhanced in beta-arrestin1(-/-) mice, and diminished in beta-arrestin2(-/-) mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in beta-arrestin2(-/-) mice was not altered by transplantation with either wild-type or beta-arrestin2(-/-) bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in beta-arrestin1(-/-) and decreased in beta-arrestin2(-/-) mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in beta-arrestin1(-/-) SMCs and less in beta-arrestin2(-/-) SMCs. Proliferation was less than wild type in beta-arrestin2(-/-) SMCs but not in beta-arrestin2(-/-) endothelial cells. We conclude that beta-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by beta-arrestin2 and beta-arrestin1. These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.

Nrg1 is an injury-induced cardiomyocyte mitogen for the endogenous heart regeneration program in zebrafish.

Heart regeneration is limited in adult mammals but occurs naturally in adult zebrafish through the activation of cardiomyocyte division. Several components of the cardiac injury microenvironment have been identified, yet no factor on its own is known to stimulate overt myocardial hyperplasia in a mature, uninjured animal. In this study, we find evidence that Neuregulin1 (Nrg1), previously shown to have mitogenic effects on mammalian cardiomyocytes, is sharply induced in perivascular cells after injury to the adult zebrafish heart. Inhibition of Erbb2, an Nrg1 co-receptor, disrupts cardiomyocyte proliferation in response to injury, whereas myocardial Nrg1 overexpression enhances this proliferation. In uninjured zebrafish, the reactivation of Nrg1 expression induces cardiomyocyte dedifferentiation, overt muscle hyperplasia, epicardial activation, increased vascularization, and causes cardiomegaly through persistent addition of wall myocardium. Our findings identify Nrg1 as a potent, induced mitogen for the endogenous adult heart regeneration program.

Magnetic resonance imaging of graded skeletal muscle injury in live rats.

INTRODUCTION: Increasing number of stretch-shortening contractions (SSCs) results in increased muscle injury. METHODS: Fischer Hybrid rats were acutely exposed to an increasing number of SSCs in vivo using a custom-designed dynamometer. Magnetic resonance imaging (MRI) imaging was conducted 72 hours after exposure when rats were infused with Prohance and imaged using a 7T rodent MRI system (GE Epic 12.0). Images were acquired in the transverse plane with typically 60 total slices acquired covering the entire length of the hind legs. Rats were euthanized after MRI, the lower limbs removed, and tibialis anterior muscles were prepared for histology and quantified stereology. RESULTS: Stereological analyses showed myofiber degeneration, and cellular infiltrates significantly increased following 70 and 150 SSC exposure compared to controls. MRI images revealed that the percent affected area significantly increased with exposure in all SSC groups in a graded fashion. Signal intensity also significantly increased with increasing SSC repetitions. DISCUSSION: These results suggest that contrast-enhanced MRI has the sensitivity to differentiate specific degrees of skeletal muscle strain injury, and imaging data are specifically representative of cellular histopathology quantified via stereological analyses.

A Tissue-Engineered Microvascular System to Evaluate Vascular Progenitor Cells for Angiogenic Therapies

The ability of tissue engineered constructs to replace diseased or damaged organs is limited without the incorporation of a functional vascular system. To design microvasculature that recapitulates the vascular niche functions for each tissue in the body, we investigated the following hypotheses: (1) cocultures of human umbilical cord blood-derived endothelial progenitor cells (hCB-EPCs) with mural cells can produce the microenvironmental cues necessary to support physiological microvessel formation in vitro; (2) poly(ethylene glycol) (PEG) hydrogel systems can support 3D microvessel formation by hCB-EPCs in coculture with mural cells; (3) mesenchymal cells, derived from either umbilical cord blood (MPCs) or bone marrow (MSCs), can serve as mural cells upon coculture with hCB-EPCs. Coculture ratios between 0.2 (16,000 cells/cm2) and 0.6 (48,000 cells/cm2) of hCB-EPCs plated upon 3.3 µg/ml of fibronectin-coated tissue culture plastic with (80,000 cells/cm2) of human aortic smooth muscle cells (SMCs), results in robust microvessel structures observable for several weeks in vitro. Endothelial basal media (EBM-2, Lonza) with 9% v/v fetal bovine serum (FBS) could support viability of both hCB-EPCs and SMCs. Coculture spatial arrangement of hCB-EPCs and SMCs significantly affected network formation with mixed systems showing greater connectivity and increased solution levels of angiogenic cytokines than lamellar systems. We extended this model into a 3D system by encapsulation of a 1 to 1 ratio of hCB-EPC and SMCs (30,000 cells/µl) within hydrogels of PEG-conjugated RGDS adhesive peptide (3.5 mM) and PEG-conjugated protease sensitive peptide (6 mM). Robust hCB-EPC microvessels formed within the gel with invasion up to 150 µm depths and parameters of total tubule length (12 mm/mm2), branch points (127/mm2), and average tubule thickness (27 µm). 3D hCB-EPC microvessels showed quiescence of hCB-EPCs (<1% proliferating cells), lumen formation, expression of EC proteins connexin 32 and VE-cadherin, eNOS, basement membrane formation by collagen IV and laminin, and perivascular investment of PDGFR-β+/α-SMA+ cells. MPCs present in <15% of isolations displayed >98% expression for mural markers PDGFR-β, α-SMA, NG2 and supported hCB-EPC by day 14 of coculture with total tubule lengths near 12 mm/mm2. hCB-EPCs cocultured with MSCs underwent cell loss by day 10 with a 4-fold reduction in CD31/PECAM+ cells, in comparison to controls of hCB-EPCs in SMC coculture. Changing the coculture media to endothelial growth media (EBM-2 + 2% v/v FBS + EGM-2 supplement containing VEGF, FGF-2, EGF, hydrocortisone, IGF-1, ascorbic acid, and heparin), promoted stable hCB-EPC network formation in MSC cocultures over 2 weeks in vitro, with total segment length per image area of 9 mm/mm2. Taken together, these findings demonstrate a tissue engineered system that can be utilized to evaluate vascular progenitor cells for angiogenic therapies.

Muscle Logic: New Knowledge Resource for Anatomy Enables Comprehensive Searches of the Literature on the Feeding Muscles of Mammals.

BACKGROUND: In recent years large bibliographic databases have made much of the published literature of biology available for searches. However, the capabilities of the search engines integrated into these databases for text-based bibliographic searches are limited. To enable searches that deliver the results expected by comparative anatomists, an underlying logical structure known as an ontology is required. DEVELOPMENT AND TESTING OF THE ONTOLOGY: Here we present the Mammalian Feeding Muscle Ontology (MFMO), a multi-species ontology focused on anatomical structures that participate in feeding and other oral/pharyngeal behaviors. A unique feature of the MFMO is that a simple, computable, definition of each muscle, which includes its attachments and innervation, is true across mammals. This construction mirrors the logical foundation of comparative anatomy and permits searches using language familiar to biologists. Further, it provides a template for muscles that will be useful in extending any anatomy ontology. The MFMO is developed to support the Feeding Experiments End-User Database Project (FEED, https://feedexp.org/), a publicly-available, online repository for physiological data collected from in vivo studies of feeding (e.g., mastication, biting, swallowing) in mammals. Currently the MFMO is integrated into FEED and also into two literature-specific implementations of Textpresso, a text-mining system that facilitates powerful searches of a corpus of scientific publications. We evaluate the MFMO by asking questions that test the ability of the ontology to return appropriate answers (competency questions). We compare the results of queries of the MFMO to results from similar searches in PubMed and Google Scholar. RESULTS AND SIGNIFICANCE: Our tests demonstrate that the MFMO is competent to answer queries formed in the common language of comparative anatomy, but PubMed and Google Scholar are not. Overall, our results show that by incorporating anatomical ontologies into searches, an expanded and anatomically comprehensive set of results can be obtained. The broader scientific and publishing communities should consider taking up the challenge of semantically enabled search capabilities.

Preservation of myocardial beta-adrenergic receptor signaling delays the development of heart failure after myocardial infarction.

When the heart fails, there is often a constellation of biochemical alterations of the beta-adrenergic receptor (betaAR) signaling system, leading to the loss of cardiac inotropic reserve. betaAR down-regulation and functional uncoupling are mediated through enhanced activity of the betaAR kinase (betaARK1), the expression of which is increased in ischemic and failing myocardium. These changes are widely viewed as representing an adaptive mechanism, which protects the heart against chronic activation. In this study, we demonstrate, using in vivo intracoronary adenoviral-mediated gene delivery of a peptide inhibitor of betaARK1 (betaARKct), that the desensitization and down-regulation of betaARs seen in the failing heart may actually be maladaptive. In a rabbit model of heart failure induced by myocardial infarction, which recapitulates the biochemical betaAR abnormalities seen in human heart failure, delivery of the betaARKct transgene at the time of myocardial infarction prevents the rise in betaARK1 activity and expression and thereby maintains betaAR density and signaling at normal levels. Rather than leading to deleterious effects, cardiac function is improved, and the development of heart failure is delayed. These results appear to challenge the notion that dampening of betaAR signaling in the failing heart is protective, and they may lead to novel therapeutic strategies to treat heart disease via inhibition of betaARK1 and preservation of myocardial betaAR function.

Engineering Highly-functional, Self-regenerative Skeletal Muscle Tissues with Enhanced Vascularization and Survival in Vivo

Tissue engineering of biomimetic skeletal muscle may lead to development of new therapies for myogenic repair and generation of improved in vitro models for studies of muscle function, regeneration, and disease. For the optimal therapeutic and in vitro results, engineered muscle should recreate the force-generating and regenerative capacities of native muscle, enabled respectively by its two main cellular constituents, the mature myofibers and satellite cells (SCs). Still, after 20 years of research, engineered muscle tissues fall short of mimicking contractile function and self-repair capacity of native skeletal muscle. To overcome this limitation, we set the thesis goals to: 1) generate a highly functional, self-regenerative engineered skeletal muscle and 2) explore mechanisms governing its formation and regeneration in vitro and survival and vascularization in vivo.

By studying myogenic progenitors isolated from neonatal rats, we first discovered advantages of using an adherent cell fraction for engineering of skeletal muscles with robust structure and function and the formation of a SC pool. Specifically, when synergized with dynamic culture conditions, the use of adherent cells yielded muscle constructs capable of replicating the contractile output of native neonatal muscle, generating >40 mN/mm2 of specific force. Moreover, tissue structure and cellular heterogeneity of engineered muscle constructs closely resembled those of native muscle, consisting of aligned, striated myofibers embedded in a matrix of basal lamina proteins and SCs that resided in native-like niches. Importantly, we identified rapid formation of myofibers early during engineered muscle culture as a critical condition leading to SC homing and conversion to a quiescent, non-proliferative state. The SCs retained natural regenerative capacity and activated, proliferated, and differentiated to rebuild damaged myofibers and recover contractile function within 10 days after the muscle was injured by cardiotoxin (CTX). The resulting regenerative response was directly dependent on the abundance of SCs in the engineered muscle that we varied by expanding starting cell population under different levels of basic fibroblast growth factor (bFGF), an inhibitor of myogenic differentiation. Using a dorsal skinfold window chamber model in nude mice, we further demonstrated that within 2 weeks after implantation, initially avascular engineered muscle underwent robust vascularization and perfusion and exhibited improved structure and contractile function beyond what was achievable in vitro.

To enhance translational value of our approach, we transitioned to use of adult rat myogenic cells, but found that despite similar function to that of neonatal constructs, adult-derived muscle lacked regenerative capacity. Using a novel platform for live monitoring of calcium transients during construct culture, we rapidly screened for potential enhancers of regeneration to establish that many known pro-regenerative soluble factors were ineffective in stimulating in vitro engineered muscle recovery from CTX injury. This led us to introduce bone marrow-derived macrophages (BMDMs), an established non-myogenic contributor to muscle repair, to the adult-derived constructs and to demonstrate remarkable recovery of force generation (>80%) and muscle mass (>70%) following CTX injury. Mechanistically, while similar patterns of early SC activation and proliferation upon injury were observed in engineered muscles with and without BMDMs, a significant decrease in injury-induced apoptosis occurred only in the presence of BMDMs. The importance of preventing apoptosis was further demonstrated by showing that application of caspase inhibitor (Q-VD-OPh) yielded myofiber regrowth and functional recovery post-injury. Gene expression analysis suggested muscle-secreted tumor necrosis factor-α (TNFα) as a potential inducer of apoptosis as common for muscle degeneration in diseases and aging in vivo. Finally, we showed that BMDM incorporation in engineered muscle enhanced its growth, angiogenesis, and function following implantation in the dorsal window chambers in nude mice.

In summary, this thesis describes novel strategies to engineer highly contractile and regenerative skeletal muscle tissues starting from neonatal or adult rat myogenic cells. We find that age-dependent differences of myogenic cells distinctly affect the self-repair capacity but not contractile function of engineered muscle. Adult, but not neonatal, myogenic progenitors appear to require co-culture with other cells, such as bone marrow-derived macrophages, to allow robust muscle regeneration in vitro and rapid vascularization in vivo. Regarding the established roles of immune system cells in the repair of various muscle and non-muscle tissues, we expect that our work will stimulate the future applications of immune cells as pro-regenerative or anti-inflammatory constituents of engineered tissue grafts. Furthermore, we expect that rodent studies in this thesis will inspire successful engineering of biomimetic human muscle tissues for use in regenerative therapy and drug discovery applications.