Browsing by Author "Garrity, Deborah, advisor"

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Open Access
Characterization of zebrafish models of filamin C related cardiomyopathy
(Colorado State University. Libraries, 2019) Alnefaie, Rasha M., author; Garrity, Deborah, advisor; Reddy, A. S. N., committee member; Lybourn, Paul, committee member; Muller, Racheal, committee member
Cardiomyopathies are a group of cardiac muscle diseases characterized by abnormal function and/or structure of the myocardium which cause arrhythmia, heart failure or sudden death. In many cases, cardiomyopathy is a genetic disease and the majority of inherited cases are caused by mutations in genes that encode cardiac costameric and sarcomeric proteins. Cardiomyopathies include different types, such as dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy (RCM), and arrythmogenic cardiomyopathy (ARCM). These groups of disorders can have common cellular phenotypes and mechanisms. To date, few studies have described the roles of filamin C in cardiac development or explained how mutations in filamin C cause cardiomyopathic disease. Due to the lack of a suitable animal model, the pathological mechanisms underlying this disease and the role of filamin C in cardiac development remain unclear. Here, we created a zebrafish loss-of-function model for two flnc paralogous genes. We investigated several genetic lines bearing mutations in filamin C that target different sites of the gene. As for humans, zebrafish mutants exhibited variable penetrance and variable expressivity. Double flnca and flncb mutant hearts exhibited more pronounced cardiac morphological defects compared to single mutants, leading to the conclusion that these paralogs play redundant roles in zebrafish heart development. The cardiac morphological phenotype of double flnc mutant embryos is characterized by a decrease in cardiac output and stroke volume as is also observed in patients who suffer from cardiomyopathies. Using a transgenic line expressing GFP in cardiac z-discs, we find that knockdown of flnca and flncb via morpholinos and double flnca and flncb mutant hearts exhibited irregular z-discs. In support of this finding, ultrastructural analysis by transmission electronic microscopy for flncb morphant embryos and double flnca and flncb mutants indicated disorganized myofibrils with fewer consecutive sarcomeres. Particularly, z-discs were irregular or apparently absent, and numerous small vacuoles and potentially autophagous vesicles were observed. Additionally, through double flnca and flncb mutant, we demonstrated that filamin C is required for normal cardiomyocyte morphology and microfilament arrangement. In summary, the zebrafish model demonstrates an essential requirement for filamin C function during heart and skeletal muscle development. Depletion of filamin C impairs both sarcomerogenesis and alters the cytoskeletal architecture of cardiomyocytes.
Open Access
Myocardial afterload regulates atrioventricular valve development in zebrafish
(Colorado State University. Libraries, 2020) Ahuja, Neha, author; Garrity, Deborah, advisor; Sloan, Daniel, committee member; Mykles, Don, committee member; Tjalkens, Ron, committee member
The incidence of congenital heart disease (CHD) is estimated to be 1% of all human births. CHD of the heart valves occurs in over 50% of CHD cases. Despite significant clinical interest, the molecular mechanisms that govern valve development remain poorly elucidated. As the heart develops, blood flow and blood pressure increase rapidly to support the growing demands of the embryo. Our group has previously shown that pressure at the developing atrioventricular valve dramatically increases through development. Consequently, we hypothesized that afterload—defined as the pressure the ventricle must overcome in order to pump blood through the body—may be a cue that cardiac valve cells read and respond to build a valve leaflet. Here, we present a zebrafish model in which afterload has been increased through the use of vasopressin, a vasoconstrictive drug. We first show that application of vasopressin reliably produces an increase in afterload without directly acting on cardiac tissue in zebrafish embryos. To evaluate cardiac function and valve leaflet dynamics, we took a quantitative live-imaging approach. Consistent with pathology seen in adult human patients with clinically high afterload, we see defects in both form and function of the valve leaflets. To identify the cause of this functional defect, we utilized in situ hybridization to evaluate makers of cell differentiation for both valve leaflet cells and the adjacent myocardial cells. Our results suggest that this valve defect is due to changes in atrioventricular myocyte differentiation and signaling, rather than pressure directly acting on the valve leaflet cells. We next took a transcriptomics approach to identify regulators of atrioventricular myocyte differentiation and identified a subset of differentially expressed transcription factors that are putatively responsible for sensing afterload. Together, our results show that afterload regulates the physiological and molecular state of the developing valve.
Open Access
New roles for calcium channel beta subunits in early zebrafish development
(Colorado State University. Libraries, 2008) Ebert, Alicia Marie, author; Garrity, Deborah, advisor
Voltage-gated calcium channels are present on pre-synaptic terminals and at neuromuscular junctions in the adult. In embryos, the channel is primarily expressed in the developing heart. The auxiliary β subunit is responsible for trafficking the pore-forming α subunit to the membrane, and regulating the calcium channel kinetics. In non-canonical roles, the β subunit regulates gene silencing, vesicle docking, and calcium release from pancreatic cells. We report here the cloning and expression of two zebrafish β2 genes and two β4 genes. Morpholino inhibition of the β4 subunit slowed or blocked the morphogenetic movements of gastrulation, causing the blastoderm to retract and the embryos to assume dorsalized phenotypes. The nuclei of the extra-embryonic yolk syncytial layer (YSL) contained extra centrosomes, which led to formation of abnormal mitotic spindle. Microtubule arrays in the yolk were disrupted or absent. In 48 hpf embryos, the axis of the embryo was expanded mediolaterally and shorter anteroposteriorly. Gastrulation defects were present as early as shield formation. These data combined support the hypothesis for a novel role of the β4 subunit in early zebrafish development.
Open Access
Quantifying function in the zebrafish embryonic heart: a study on the role of timed mechanical cues
(Colorado State University. Libraries, 2014) Johnson, Brennan Michael, author; Dasi, Lakshmi, advisor; Garrity, Deborah, advisor; Kisiday, John, committee member; Orton, Christopher, committee member; Venayagamoorthy, Karan, committee member
Congenital heart defects are a relatively common problem, yet the cause is unknown in the large majority of cases. A significant amount of past research has shown that there is a link between altered blood-induced mechanical stress and heart development. However, very little research has been done to examine the effect of altered loading timing. During embryonic development, the heart undergoes a drastic change in morphology from its original valveless tube structure to a complete multi-chambered pump with valves. Blood flow dynamics are consequently altered significantly as well. Given the changes occurring through this period, it is hypothesized that significant and persistent decreases in heart function occur when cardiac loading is altered during certain time windows of early development. The main objectives of this work were to (1) develop a methodology to quantify heart function in the embryonic zebrafish from high-speed bright field images, (2) develop a model for temporary and noninvasive alteration of cardiac loading, and (3) apply the methodology to normal and treated embryos to determine whether certain time windows of altered loading are more impactful than others. Results indicated that altered loading during the tube and early looping stages of development produce persistent changes in heart morphology along with accompanying decreases in cardiac function. Altered loading during late cardiac looping resulted in temporary changes in function which did not persist through the latest time point measured. This work has produced extensive tools for quantifying heart function from high speed images and presents a new model for altered cardiac loading in the zebrafish. Results support the hypothesis that the heart is more sensitive to altered loading during certain windows in development. This provides new insight into how congenital defects may develop and sets the stage for future experiments investigating the effects of altered loading on heart development.
Open Access
Quantitative analysis of the mechanical environment in the embryonic heart with respect to its relationship in cardiac development
(Colorado State University. Libraries, 2015) Bulk, Alexander T., author; Dasi, Lakshmi Prasad, advisor; Garrity, Deborah, advisor; Popat, Ketul, committee member; Orton, Christopher, committee member
In order to understand the causes of congenital heart defects, which afflict at least 4 infants per 1,000 live births, research has implemented the use of animal models to study embryonic heart development. Zebrafish (Danio rerio) have become one of the more prominent of these animal models due to the fact that their heart morphology at the earliest stages of development is remarkably similar to humans, and because embryos lack pigmentation, rendering them transparent. This transparency allows for high-speed images of blood flow to be acquired in the developing heart so that the mechanotransductive relationship between the intracardiac flow environment and myocardial progenitor cell differentiation can be understood. One particular aspect of the flow environment, a cyclic retrograde flow at the junction of the forming atrium and ventricle, has been shown to be necessary for valve formation, though the mechanisms causing it to occur had previously been unknown. By comparing the results of two-dimensional spatiotemporal analysis applied to embryos both with normal retrograde flow and inhibited retrograde flow, this study shows that a particular range of pressures associated with the pumping mechanics of the heart as well as resistance due to systolic contractile closure must exist in order to maintain adequate retrograde flow to induce valve formation. The use of two-dimensional spatiotemporal analysis was sufficient to acquire these results, however when applied to analysis of other aspects of the intracardiac flow environment, this computational method is subject to critical limitations. Therefore, this study includes the development of methodology to integrate the results of spatiotemporal analysis on multiple focal planes bisecting the heart into a more accurate, three-dimensional result. The results of this study not only increase our understanding of the mechanics behind an important factor in embryonic development, but also enable future experiments pertaining to the measurement of embryonic intracardiac blood flow to be performed with increased certainty.
Open Access
The cardiac jelly extracellular matrix contributes to valve development and overall cardiac function
(Colorado State University. Libraries, 2022) Ostwald, Paige, author; Garrity, Deborah, advisor; Bark, David, committee member; Bedinger, Patricia, committee member; Nishimura, Erin, committee member; Peers, Graham, committee member
Nearly 2.6 million infants are born every year with a congenital cardiac anomaly across the entire globe. Congenital heart defects (CHDs) within the valve occur in over 50% of cases. 56% of heart defects have an unknown etiology, illuminating the need for continuous research on heart development and potential causes. Before the valve is a mature structure with established leaflets, the heart forms two endocardial cushions that press together to occlude blood flow between chambers. The cushions are composed of an extracellular matrix called the cardiac jelly (CJ). Previous studies have found evidence of the vital role the cardiac jelly plays within the developing valve for structure, genetic signaling and cell organization. Here, we present a specific role the cardiac jelly plays in valve function and overall cardiac output. To alter the cardiac jelly, we used a morpholino approach in a zebrafish model to increase, decrease and structurally compromise the cardiac jelly. By doing so, we found decreased valve cell differentiation with decreased CJ and increased valve cell differentiation with increased CJ. Using high-speed video technology, we also found decreased valve opening regardless of cardiac jelly alteration, resulting in reduced overall cardiac function. Our results suggest that the function of the endocardial cushions relies on an appropriate presence of CJ. We next investigated just how the cardiac jelly may be altered during development. To do so, we exposed zebrafish embryos to hyperglycemic conditions during the initial and critical heart development period. We found that when embryos absorb over 1.5-fold more D-glucose due to high-glucose conditions, they exhibit significant alterations to CJ width. Altered CJ due to hyperglycemic conditions affected valve differentiation, valve opening, and cardiac function, particularly when embryos have absorbed over a 2-fold increase of glucose. Together, these results show the structural role of the cardiac jelly to support endocardial cushion opening which will supply enough oxygenated blood to the embryo.
Open Access
The role of FLNC in the contractility of the heart and valve development in zebrafish
(Colorado State University. Libraries, 2020) Alshahrani, Areej Ali, author; Garrity, Deborah, advisor; Mueller, Rachel, committee member; Bark, David, committee member
Dilated Cardiomyopathy (DCM) is the most common type of cardiomyopathy disease that causes heart muscle defects. DCM is characterized by a dilated left ventricular chamber and systolic dysfunction that results in congestive heart failure. Although the cause of DCM is not fully understood, evidence supports the hypothesis that costameric proteins contribute to muscle dysfunction linked to cardiomyopathy. In fact, the mutation of FLNC has been linked to many muscle disease including myofibrila myopathies (MFM) and different types of cardiomyopathy. However, the mechanisms underlying the variability between MFM and cardiac disease is a field of interest. Patients with DCM shown to carry truncating variants whereas patients with hypertrophic cardiomyopathy (HCM) carried missense variants. Additionally, patients with other types of cardiomyopathy carried missense or in-frame indel variants (Ader, Groote et al.). This seems to suggest that different mechanisms may be at play regarding the role of FLNC in cardiac developments and they remain unclear. It would be interesting to examine if a similar correlation holds in animal model like zebrafish. Therefore, our group has developed the zebrafish model for study of the FLNC contribution to cardiac phenotypes. Here, we used several FLNC mutant lines to investigate how FLNC directly or indirectly affects development of the atrioventricular (AV) valve. To date, little data indicate whether or not increased RFF is pathologic. This project will test the overarching hypothesis that flnc depletion causes changes in RFF, which lead to aberrant valve development, which in turn affects overall heart function. We find that the cardiac morphological phenotype of most single FLNC alleles showed normal heart parameters such as heart rate, stroke volume, cardiac output and reverse flow fraction. However, flcnbexon14-/- allele exhibited a decreased in stroke volume and cardiac output whereas RFF is intact. Furthermore, using an immunocytochemistry to examine a correlation that may exist between the strength of the cardiac phenotype and the presence of valve defects indicate that valve defects presented in flncb truncation mutants, and suggest that defects in flncbexon35-/- embryos are more severe. In support of this finding, our qPCR study displayed that expression levels of klf2a and klf2b in flncaexon1-/- ; flncbexon14-/- double mutant hearts were significantly decreased. In addition, Prior studies have proposed that massive formation of intracellular protein aggregates imposes toxic impacts that contribute to the skeletal muscle degeneration observed in myofibrillar myopathy (Fichna, Maruszak et al. 2018). We demonstrated that the truncated protein either exerts a direct toxic effect, and/or sequesters wildtype FLNC leading to insufficiency phenotypes.
Open Access
Tugging on the heartstrings: determining the function of Tbx5 in early cardiac development
(Colorado State University. Libraries, 2012) Parrie, Lindsay E., author; Garrity, Deborah, advisor; Bouma, Gerrit, committee member; Mykles, Donald, committee member; Reddy, A. S. N., committee member
During cardiac morphogenesis, the vertebrate heart acquires a characteristic three dimensional shape well-suited for efficient function. The morphology of the developing cardiac organ reflects a series of changes in the cardiomyocytes themselves, which must become specified, migrate, proliferate, grow in size, alter their shape and adhesive properties, and develop ultrastructure, among other differentiated characteristics. Mutation of the T-box transcription factor tbx5 leads to embryonic lethal cardiac phenotypes and forelimb malformations in vertebrate models. Haploinsufficiency of Tbx5 results in Holt-Oram Syndrome (HOS), a human congenital disease characterized by cardiac and forelimb defects. Homozygous mutation of zebrafish tbx5a in heartstrings (hst) embryos also leads to lethal defects in cardiac looping morphogenesis and prevents initiation of pectoral fin formation. Here I describe a new hst mutant allele (tbx5as296) which encodes a premature stop codon within the tbx5a T-box region, a location likely to generate a full loss-of-function allele. Data from comparative genetics and immunoblot analyses indicate that both alleles are null. I find that mutants completely lacking Tbx5a generated normal cardiomyocyte numbers in early chamber morphogenesis stages. Moreover, in situ hybridization data and functional assays support the idea that venous differentiation is not seriously impaired in zebrafish mutants, in contrast to mouse. However, cardiac cell size was significantly smaller in both chambers of tbx5a mutants. Hearts stalled early in the process of cardiac looping, but cell shape changes associated with chamber ballooning surprisingly still occurred. These studies point to a critical role for Tbx5a in growth-related aspects of cardiac differentiation, and suggest that morphologic events of cardiac looping morphogenesis and chamber ballooning are genetically separable. A second zebrafish tbx5 paralog was recently described, termed tbx5b, which showed a lower amount of sequence conservation than is typical for a T-box gene. Based on overlapping expression patterns within the embryonic heart, I hypothesized that functional redundancy between tbx5a and tbx5b might reduce the severity of cardiac phenotypes for tbx5a mutant embryos. I here report that the cardiac phenotypes in tbx5b-depleted fish were similar, but not identical, to those of homozygous tbx5a mutants. In addition, tbx5b-depletion led to defects in the timing and morphogenesis of pectoral fin outgrowth. Somewhat surprisingly, simultaneous depletion of both Tbx5 gene paralogs did not lead to more severe cardiac phenotypes, and injection of wild-type mRNA was not sufficient to cross-rescue the phenotypes of the paralogous gene. In the heart, tbx5a and tbx5b appear to have related essential functions that are nevertheless independently required. In the fin, tbx5a alone was required for fin bud initiation, but both genes are independently required for patterning and morphogenesis. Therefore, this work demonstrates a functional divergence between the two zebrafish tbx5 paralogs.