Cardiovascular diseases (CVDs) are the leading cause of death and disability worldwide. The aging population and the rapidly increasing prevalence of obesity and type 2 diabetes will contribute to a growing epidemic of CVDs globally. Despite the extensive investigations in etiology, the pathogenesis of CVDs still not fully understand, and the treatment and prevention for CVDs are still limited. Significant interest has been raised in gut microbiota-host interaction since increasing evidence revealed that gut microbiomes play an important role in human health and diseases, including CVDs. Among more than two thousand gut microbiota metabolites, a compound named trimethylamine N-oxide (TMAO) was revealed to be closely related to CVDs. However, the impact of TMAO on cardiovascular health is still full of controversy and the direct impact of TMAO on heart tissue and cardiomyocytes has not been fully understood yet.
In the first chapter, we reviewed the literature on TMAO-related atherosclerosis and cardiomyopathy to give us a general aspect of current research progress in the role of TMAO on CVDs. In this context, we provide an overview of the potential mechanisms underlying TMAO-induced cardiovascular diseases at the cellular and molecular levels, with a focus on atherosclerosis and cardiomyopathy. We also address the direct effects of TMAO on cardiomyocytes (a new and under-researched area) and finally propose TMAO as a potential biomarker and/or therapeutic target for the diagnosis and treatment of patients with CVDs.
In the second chapter, the direct impact of TMAO on cardiac function was tested in vivo using wild-type C57B6L mice model. Four experiment groups were enrolled in the feeding protocol, which included 3w (different time points), 6w, and 13w feeding time to reveal the impact of short and longer periods of TMAO consumption on cardiac function. The plasma TMAO was measured by liquid chromatography-tandem mass spectrometry (LC/MS/MS) method at the end of the feeding protocol. Echocardiography and electrocardiography (ECG) were performed to assess the overall heart function. The histopathology staining was used to evaluate the cardiac microstructure change. By the end of the feeding protocol, the plasma TMAO all increased significantly in the TMAO group compared to the control no matter the TMAO feeding period. Echocardiography showed that 6w and 13w TMAO intake could significantly decrease cardiac contractility evidenced by decreased eject fraction (EF) and fraction shortening (FS). The electrocardiography (ECG) showed decreased R wave aptitude in 6w and 13w TMAO feed group with sinus rhythm. However, 3w TMAO intake had no impact on both cardiac contractability and ECG. Moreover, chronic TMAO supplement (13w) showed increased left ventricle (LV) mass on echocardiography and increased LV thickness on the tissue section. Further histology analysis revealed cardiomyocyte hypertrophy in the 13w TMAO-treated male group. Notably, the female mice showed significantly higher TMAO levels both in the control and treated group compared to the male, however, no gender difference was observed as to the ECG and echocardiography. In addition, the plasma inflammation cytokines were also analyzed and the tumor necrosis factor-α (TNF- α), interleukin 10 (IL-10), Fibroblast growth factor 2 (FGF β) and leptin were all increased in the 13w TMAO treated group compared to the control. These results suggest that chronic TMAO exposure led to increased plasma TMAO levels, which contribute to system inflammation and cardiac dysfunction due to cardiac hypertrophy in mice models.
Research in chapter 3 demonstrates the potential underlying mechanisms of TMAO-induced cardiac dysfunction using adult mouse cardiomyocytes. In this study, we examined the direct effect of TMAO on reactive oxidative species (ROS) generation and factors related to cardiomyocyte contractibility, including, microtubule, Connexin43 (Cx43) expression, and gap junction intracellular communication (GJIC), intracellular calcium dynamics and transversal-tubule (T-tubule) both in acute and chronic TMAO challenge. Moreover, we also tested whether TMAO can enter cardiomyocytes directly. The results suggested that TMAO could enter cardiomyocytes through organic cation transporters (OCTs) and promote increased ROS generation via augmentation of NADPH oxidase 4 (Nox4). Moreover, both acute and chronic TMAO exposure could induce microtubule densification, which plays a critical role in intracellular protein transportation and cardiomyocyte morphology maintenance. We also demonstrated chronic TMAO exposure could inhibit the Cx43 expression at both cellular and tissue level, and therefore impact the GJIC for the first time. Besides, we also revealed that TMAO could interrupt intracellular calcium handling both acutely and chronically, especially documented by decreased efficiency in intracellular calcium removal, related to decreased sarcoplasmic reticulum Ca2+-ATPase (Serca2) expression. However, TMAO showed no impact on cardiomyocyte T-tubule network organization. Taken together, we demonstrated a direct destructive role of TMAO on cardiomyocytes' functional properties and provided a novel potential mechanism for TMAO-induced cardiac dysfunction.
Overall, the research in this dissertation demonstrated the direct impact of TMAO on cardiomyocytes and cardiac function both in vivo and in vitro and evaluated the effect of TMAO both acutely and chronically. The TMAO can enter cardiomyocytes and induce Nox4-mediated oxidative stress, which could connect to multiple intracellular pathways, including microtubule densification, decreased Cx43 expression, and GJIC, as well as calcium handling dysfunction. Meanwhile, all these changes were closely related to the cardiomyocyte swelling observed in mice cardiac tissue after chronic TMAO consumption, which could ultimately contribute to cardiac contractile dysfunction and electrophysiology change in mice models. / Doctor of Philosophy / Cardiovascular diseases (CVDs) are a group of diseases related to our heart and blood vessels, such as heart attack and stroke. It is the leading cause of death and disability around the world, more common than diabetes and cancer. According to the reports of the American Heart Association, CVDs cost America 555 billion US dollars in 2016 while by 2035, the cost will reach 1.1 trillion. The individual, population, and economic impact of CVDs are tremendous, making CVD one of the largest public health problems at present. Despite the extensive investigations into the cause of CVDs, the exact underlying reason still not fully understand. The microbiome inside our body has raised much attention recently due to its close relationship with human health, including CVDs. The microbiome from the gut can affect our heart health both by affecting the immune system and its metabolites after we eat daily foods.
Among thousands of metabolites, one named trimethylamine N-oxide (TMAO) has been shown to be related to increased CVDs risks. After we eat choline-rich food such as red meat and eggs, the gut microbiome can use these nutrients and produce TMA as metabolite waste, the TMA then goes into the liver and convert to TMAO via liver enzymes. However, the impact of TMAO on cardiovascular health is not fully understood yet. Our study uses the mice model to test whether TMAO has a direct impact on heat cells and heart function. We fed the mice with water containing 0.12% TMAO for different times including 3w, 6w, and 13w, and then check the mice's heart function through heart ultrasound and ECG. The results showed that TMAO could significantly harm heart function after long-term exposure in mice (13w). Further histology analysis of heart tissue showed increased heart cell size, which may contribute to decreased heart function. Certain blood inflammation cytokines related to CVDs also increased. The experiments using isolated mice heart cells showed that the ROS, which could harm the heart cells and related to lots of other damage processes in human health, were increased after exposure to the TMAO. Several other factors, including cell skeleton, cell channels responsible for cell-to-cell communication, and cell calcium balance were all damaged by TMAO, which could finally induce heart damage and heart diseases.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/113839 |
| Date | 15 February 2023 |
| Creators | Zheng, Youjing |
| Contributors | Biomedical and Veterinary Sciences, He, Jia-Qiang, Luo, Xin, Gourdie, Robert G., Allen, Irving Coy, Li, Liwu |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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