In vertebrates, the heart is the first organ to form and function. The basic steps and molecular pathways involved in heart development are highly conserved. Myocardial progenitor-fated cells are among the first cells to migrate during gastrulation away from the primitive streak. These cells move bilaterally to populate the heart-forming region (HFR) in the anterior lateral plate mesoderm (ALPM). Once cells have reached the HFR, they receive the signals necessary to differentiate into myocardial progenitor cells. It is clear that the development of myocardial progenitor cells entails the migration of cells from the lateral embryonic margin to the ALPM. However, it is unclear whether cells are specified for a myocardial progenitor fate early in embryogenesis, a step that may promote their migration specifically to the ALPM, or whether the migration of cells to the ALPM alone is sufficient for differentiation into myocardial progenitor cells. A zebrafish mutant, grinch (grn), was indentified in which there is a defect in the development of myocardial progenitor cells. The mutation resulting in the grn phenotype was mapped to the gene encoding the G protein-coupled receptor Apelin receptor b (Aplnrb). I have used the aplnrb mutant embryo, as well as morpholino-mediated knockdown (morphant embryos) of aplnrb, and its paralog aplnra, to determine the function of Aplnr signaling in myocardial progenitor development. Results demonstrate that Aplnr signaling is necessary for the migration of cells from the lateral embryonic margin of the zebrafish embryo to the heart-forming region. Interestingly, this entails a novel cell-non-autonomous function for Aplnr signaling. Furthermore, both the only identified ligand for the receptor, Apelin, and the canonical mediators of Aplnr signaling, Gαi/o proteins, appear to be dispensable for this process. Loss of Aplnr signaling also appears to affect embryonic patterning of the early embryo through subtle perturbations of Nodal, Wnt, and Bmp signaling and attenuation of Nodal signaling can partially recapitulate the aplnr morphant cardiac phenotype. Taken together, my results suggest that Aplnr signaling plays a role in creating an environment that allows for the migration of cells to the heart-forming region, possibly through the regulation of early embryonic patterning.

In vertebrates, the heart is the first organ to form and function. The basic steps and molecular pathways involved in heart development are highly conserved. Myocardial progenitor-fated cells are among the first cells to migrate during gastrulation away from the primitive streak. These cells move bilaterally to populate the heart-forming region (HFR) in the anterior lateral plate mesoderm (ALPM). Once cells have reached the HFR, they receive the signals necessary to differentiate into myocardial progenitor cells. It is clear that the development of myocardial progenitor cells entails the migration of cells from the lateral embryonic margin to the ALPM. However, it is unclear whether cells are specified for a myocardial progenitor fate early in embryogenesis, a step that may promote their migration specifically to the ALPM, or whether the migration of cells to the ALPM alone is sufficient for differentiation into myocardial progenitor cells. A zebrafish mutant, grinch (grn), was indentified in which there is a defect in the development of myocardial progenitor cells. The mutation resulting in the grn phenotype was mapped to the gene encoding the G protein-coupled receptor Apelin receptor b (Aplnrb). I have used the aplnrb mutant embryo, as well as morpholino-mediated knockdown (morphant embryos) of aplnrb, and its paralog aplnra, to determine the function of Aplnr signaling in myocardial progenitor development. Results demonstrate that Aplnr signaling is necessary for the migration of cells from the lateral embryonic margin of the zebrafish embryo to the heart-forming region. Interestingly, this entails a novel cell-non-autonomous function for Aplnr signaling. Furthermore, both the only identified ligand for the receptor, Apelin, and the canonical mediators of Aplnr signaling, Gαi/o proteins, appear to be dispensable for this process. Loss of Aplnr signaling also appears to affect embryonic patterning of the early embryo through subtle perturbations of Nodal, Wnt, and Bmp signaling and attenuation of Nodal signaling can partially recapitulate the aplnr morphant cardiac phenotype. Taken together, my results suggest that Aplnr signaling plays a role in creating an environment that allows for the migration of cells to the heart-forming region, possibly through the regulation of early embryonic patterning.

The natriuretic peptide system (NPS) is a hormonal system critical to mammalian cardiovascular homeostasis. The purpose of the present study was to investigate the role of ANP during early postnatal cardiac development by i) monitoring the development of cardiac hypertrophy during early postnatal development of the ANP-/- mice, and ii) comparing morphologic, morphometric and molecular differences in ANP+/+ mice compared to ANP-/- mice during this developmental period. Age matched male ANP+/+ and ANP-/- mice, aged day 1 and weeks 1 to 5, were evaluated. Body weight, organ weights and hematocrit were recorded. RNA was isolated and quantitative real-time RT-PCR was used to monitor cardiac gene expression. An additional cohort of animals was used for morphologic and morphometric analysis. Heart weight to body weight ratio (HW/BW) was dramatically higher in ANP-/- animals at all time points, indicating cardiac hypertrophy is established before the advent of adult blood pressure. Molecular analysis of gene expression revealed a compensatory response of the NPS in the ANP-/- mice. Specifically an up-regulation of BNP expression in ANP-/- mice was noted throughout postnatal development. Similarly, NPR-A and NPR-C demonstrated compensatory action for the lack of ANP, as expressional levels also varied throughout development. Morphological analysis of cardiac vasculature revealed striking structural differences between ANP+/+ and ANP-/- mice. Quantitative stereological analysis of LM images indicated a greater vessel volume in ANP-/- compared to ANP+/+ mice. This study demonstrates that alterations in early molecular events, such as changes in NPS expression, may be responsible for the maintenance and progression of cardiac hypertrophy during early postnatal development in the ANP-/- mice. The absence of ANP during this critical period of development has a profound impact on final cardiac structure leading to future pathological states. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2010-02-05 14:15:33.982

Congenital heart defects (CHD) are the largest class of birth defects in humans and are a major cause of infant mortality and morbidity. Deciphering the molecular and genetic etiologies central for heart development and the pathogenesis of congenital heart diseases (CHD) is a challenging puzzle. We have previously demonstrated that the zinc-finger kruppel-like transcription factor KLF13, expressed predominantly in the atria, binds evolutionarily conserved regulatory elements known as CACC-boxes and transcriptionally activates several cardiac promoters. KLF13 loss of function in Xenopus embryos was associated with cardiac developmental defects underscoring its critical role in the heart. In the current study, using in vivo and in vitro approaches, we examined KLF13’s mechanisms of action and its interaction with other cardiac regulators. To test the evolutionary conserved role in the mammalian heart, we deleted the Klf13 gene in transgenic mice using homologous recombination. Mice with homozygote deletion of Klf13 were born at reduced frequency owing to severe heart defects. We also report the existence of a novel isoform of KLF13, referred to here as KLF13b. Furthermore, we report that KLF13 interacts biochemically and genetically with the T-box transcription factor TBX5 which is a key regulator of heart development. Our data provide novel insight into the role of KLF13 in cardiac transcription and suggest that KLF13 maybe a genetic modifier of congenital heart disease. Furthering our knowledge of protein-protein interactions and gene transcription will enhance genotype-phenotype correlation and contribute to better understanding of the etiology of CHD.

I present applications of imaging and spectroscopy to understand mechanical, chemical, and electrical dynamics in biological materials. The first part describes the development and characterization of a protein-based fluorescent calcium and voltage indicator (CaViar). The far-red fluorescence of CaViar faithfully tracks the cardiac action potential in cardiomyocytes. CaViar's green fluorescence reports the resulting calcium transients. I demonstrated the applicability of CaViar in vivo with transgenic zebrafish designed to express CaViar in their hearts. Spinning disk confocal imaging allowed detailed three-dimensional mapping of simultaneous voltage and calcium dynamics throughout the heart of zebrafish embryos, in vivo, as a function of developmental stage. I tested the effect of channel blockers on voltage and calcium dynamics and discovered a chamber-specific transition from a calcium-dependent to a sodium-dependent action potential. I also describe a new measurement technique using a fluorescent voltage indicator to report absolute voltage via the indicator's temporal response. / Physics

Cardiac progenitor cells (CPCs) are abundant in the embryonic heart and have hallmark features which include a rapid rate of cell division and the ability to differentiate into mature heart muscle cells (cardiomyocytes). Based on these features, CPCs are considered an attractive candidate cell type for transplantation therapies which aim to replenish the diseased heart muscle tissue (myocardium) with new muscle forming cells. A better understanding of how pharmacological drugs and endogenous hormones/signalling molecules modulate the balance between proliferation and differentiation of CPCs could be used to develop more effective cell based therapies for myocardial repair. Furthermore, this information could provide valuable new insight into molecular mechanisms regulating normal cardiogenesis during the embryonic period. The specific aims of the present study were to characterize the effects of the Ca2+ channel blocking drug nifedipine and the endogenous hormone/paracrine factor atrial natriuretic peptide (ANP) on CPC proliferation and differentiation. Results showed that primary cultured CPCs, isolated from the ventricles of embryonic day (E) 11.5 mouse embryos, underwent a reduction in cell cycle activity following exposure to nifedipine. Furthermore, systemic administration of nifedipine to adult mice receiving transplanted E11.5 ventricular cells (containing CPCs) was associated with smaller graft sizes compared to control animals that did not receive the drug. Results from the present study also demonstrated that ANP receptor mediated signalling systems are biologically active in E11.5 ventricular cells and have an antiproliferative effect on cultured E11.5 CPCs. Moreover, preliminary data provided evidence that genetic ablation of the ANP high affinity receptor (NPRA) may be associated with impaired development of the ventricular cardiac conduction system. Collectively, work from this thesis provides evidence that interactions between transplanted cells and pharmacological drugs could have a significant impact on the effectiveness of cell based therapies and that ANP signalling systems may play a critical role in cardiac ontogeny by regulating the balance between CPC proliferation and differentiation.

A leading cause of morbidity and mortality in the developed world is cardiovascular disease (CVD). Like many other disease processes the etiology of CVD has origins in both genetic and environmental factors. These factors affect the development of the heart and vasculature and how they respond to physiological and pathological stress. Abnormal heart development can lead to cardiac pathologies that manifest in a shift from normal cardiac geometry and physiology to what is called pathological cardiac remodeling. Often though, pathological remodeling can result from cardiovascular stress even when heart development is normal. Growth factors are essential mediators of cardiac development and physiology and a good number of clinical and experimental studies have implicated growth factors and their signaling effectors as potential therapeutic targets for pathological cardiac remodeling. Of those is Fibroblast Growth Factor 2 (FGF2) which is a potent inducer of fibroblast and cardiomyocyte proliferation in vitro. FGF2 is made in high molecular weight and low molecular weight isoforms (Hi FGF2 and Lo FGF2, respectively). It has already been demonstrated that, in the context of the heart, FGF2 modulates cardiac hypertrophy, cardiac fibrosis and mediates protection against cardiac injury. However, the isoform specific role of FGF2 in cardiac development, physiology and pathological remodeling has not been disclosed, and in this dissertation I address the hypothesis that FGF2 has isoform-specific function in cardiac physiology and remodeling. To test this hypothesis I used mice that are either deficient in Hi FGF2 (Hi KO) or Lo FGF2 (Lo KO) and subjected them to echocardiographic analysis and isoproterenol (Iso) treatment and compared them to wildtype (WT) cohorts. At baseline echocardiographic measurements, female Lo KO hearts are smaller and present with increased peak E-wave velocity, a diminutive A wave, and shortened mitral-flow deceleration time consistent with a restricted filling pattern and myocardial stiffness. Conversely, male Lo KO hearts present with a lower E wave and a higher A-wave velocity and a prolonged isovolumic-relaxation time consistent with impaired left ventricular (LV) relaxation. Female Hi KO hearts display no significant deviation from WT, while male Hi KO hearts exhibit increased systolic function. Hence, a deficiency in Lo FGF2 results in a shift from normal diastolic parameters and geometric measurements which is gender specific. Conversely, a deficiency in Hi FGF2 produces a phenotype in male hearts only. Histological and gravimetric analysis of Lo KO and Hi KO hearts post-Iso treatment reveals that female Lo KO hearts remain smaller even though their cardiomyocytes are hypertrophied while female Hi KO hearts present with a blunted hypertrophic response indicating a hypoplastic myocardium. Male Lo KO hearts present with an exacerbated fibrotic response and increased alpha-smooth muscle actin protein expression while Hi KO hearts exhibit a resistance to the fibrotic response and an induction of atrial natriuretic factor protein expression. Thus, in female hearts Hi FGF2 mediate cardiac hypertrophy while in male hearts Lo FGF2 and Hi FGF2 display an antithetical role in cardiac fibrosis where Lo FGF2 is protective while Hi FGF2 is damaging. Hence, cardiac remodeling following catecholamine overactivation is modulated by FGF2 in isoform- and gender-specific manners. In conclusion, the results presented here provide novel evidence on the interaction of gender and endogenous FGF2 isoforms as modulators of cardiac development, physiology and remodeling. Lo FGF2 signaling is necessary in the male heart for normal myocardial relaxation and for amelioration of the fibrotic response induced by beta-adrenergic stress, while in female hearts Lo FGF2 is necessary for normal cardiac growth and normal myocardial compliance. Hi FGF2 is necessary only in female hearts for mediating the hypertrophic response. Hence, I demonstrate that Lo FGF2 and Hi FGF2 have non-redundant roles in cardiac physiology and remodeling which are gender-specific.

In the heart, the primary role of the L-type calcium channel (LTCC) CaV1.2 is to conduct calcium into cardiomyocytes and initiate contraction. However, part of the CaV1.2 channel itself, the cleaved carboxyl-terminus (CCt) can also localize to the nucleus and regulate gene transcription. Therefore, the goal of this dissertation project was to determine the role and regulation of CCt in the embryonic and adult heart. The global hypothesis of my dissertation project is that CCt localizes to the nucleus in embryonic and adult cardiomyocytes via a calcium-mediated mechanism and regulates transcription. A model of pharmacological LTCC block-induced perturbation of murine embryonic heart development was first utilized to study the role of CCt. Pharmacological block at embryonic day 10 perturbed cardiogenesis and increased CaV1.2 expression. This result was not mimicked by removal of extracellular calcium or inhibition of calcium release from the sarcoplasmic reticulum. Co-currently, pharmacological block decreased CCt nuclear localization in embryonic cardiomyocytes. At the transcriptional level, CCt suppressed the CaV1.2 promoter. This indicated that the observed upregulation of CaV1.2 induced by pharmacological block may be caused by nuclear localization of the transcriptional repressor, CCt. Therefore, the conclusion was made that pharmacological LTCC block perturbed embryonic cardiogenesis by decreasing nuclear localization of the transcription factor CCt; implying a role for CCt in embryonic heart development. Next, CCt regulation was studied in the adult heart. Similar to the embryonic heart, pharmacological LTCC block decreased nuclear localization of CCt. Inhibition of the calcium activated phosphatase calcineurin also decreased CCt nuclear localization. To determine a role for CCt in the adult heart, CCt nuclear localization was measured in response to hypertrophic stimuli. Serum-induced cardiomyocyte hypertrophy significantly increased nuclear localization of CCt. In conclusion, this dissertation supports the hypothesis that CCt localizes to the nucleus in embryonic and adult cardiomyocytes, and that this regulation is mediated by calcium entry into the cardiomyocyte. Furthermore, data from this dissertation suggests that CCt nuclear localization may play an important role in embryonic heart development and adult cardiac hypertrophy.

To understand the role of nonmuscle myosin II in cardiac and skeletal muscle, we used a number of polyclonal antibodies, three detecting nonmuscle myosin heavy chain II-B (NMHC II-B) and two detecting NMHC II-A, to examine the localization of these two proteins in fresh-frozen, acetone-fixed sections of normal human and mouse hearts and human skeletal muscles. Results were similar in both species and were confirmed by examination of fresh- frozen sections of human hearts subjected to no fixation or to treatment with either 4% p-formaldehyde or 50% glycerol. NMHC II-B was diffusely distributed in the cytoplasm of cardiac myocytes during development, but after birth it was localized to the Z-lines and intercalated discs. Dual labeling showed almost complete colocalization of NMHC II-B with α-actinin. Whereas endothelial cells, smooth muscle cells and fibroblasts showed strong immunoreactivity for NMHC II-A and NMHC II-B, cardiac myocytes only showed reactivity for the latter. The Z-lines of human skeletal muscle cells, in contrast to those of cardiac myocytes, gave positive reactions for both NMHC II-A and NMHC II-B. The presence of a motor protein in the Z-lines and intercalated discs raises the possibility that these structures may play a more dynamic role in the contraction/relaxation mechanism of cardiac and skeletal muscle than has been previously suspected.

Indiana University-Purdue University Indianapolis (IUPUI) / MCTP2 (multiple C2 domain transmembrane containing protein 2) encodes a protein with poorly understood roles in lipid metabolism and lipid droplet biogenesis. Genetic studies previously identified variations in MCTP2 in conjunction with left ventricular outflow tract obstructive forms of congenital heart disease (CHD). This dissertation research aimed to delineate the biomedical significance of Mctp2 by investigating its expression and consequences of its genetic deletion in mouse models. Temporal and spatial expression of Mctp2 was investigated by RT-PCR and in-situ hybridization. A novel isoform, designated as isoform 2 in mice, results from alternative pre-mRNA splicing. Similar levels of Mctp2 isoforms 1 and 2 are present in embryonic tissues, whereas isoform 1 is preferentially expressed in adult tissues with high lipid metabolism. During mouse embryonic development, in-situ hybridization suggests expression of Mctp2 at the gut tube, liver bud and near the pharyngeal arches from E8.5 – E10.5. Given association of MCTP2 with CHD, the biological significance of Mctp2 was addressed using gene trap (GT) and conditional mouse models. Survival of Mctp2 GT mice was dependent on the genetic background strain, suggesting a role for strain-specific modifiers. Conditional knockout of Mctp2 in cardiac progenitor cells displayed no effect on survival. The role of Mctp2 in cardiac development remains to be delineated. The role of Mctp2 in cardiac function was addressed in both mouse models. Initial findings suggest Mctp2 allele dosage effects on the development of heart failure. GT mice lacking one, or both, copies of Mctp2 display cardiac systolic dysfunction, with upregulation of heart failure markers at 50 weeks of age in heterozygotes and increases in cardiac fibrosis in homozygotes. Systemic conditional deletion of Mctp2 did not show heart failure phenotypes using the strain protective from lethality. However, cardiac specific deletion of Mctp2 using the Nkx2.5-Cre driver, a line that is sensitized for cardiac dysfunction, led to decreased ejection fraction and fractional shortening in mice with conditional deletion of both copies of Mctp2 as well as Mctp2 dosage dependent penetrance of cardiac dilation. These studies of knockout mice suggest a role for Mctp2 in maintenance of cardiac function and possible genetic interaction with Nkx2.5. / 2022-02-02