Liam Brunham

Plasma lipoproteins, such as low-density lipoprotein (LDL), high-density lipoprotein (HDL), and lipoprotein(a) are highly heritable traits and important biomarkers for atherosclerotic cardiovascular disease (ASCVD). LDL and lipoprotein(a) are atherogenic plasma lipoproteins that are often both elevated among individuals with familial hypercholesterolemia; a common, autosomal co-dominant disorder characterized by pathogenic DNA variants in the LDLR, APOB, and PCSK9 genes. These pathogenic variants impair the removal of LDL from the blood and lead to severe hypercholesterolemia and increased risk of ASCVD. Despite the ability to identify individuals with a genetic cause for familial hypercholesterolemia or elevated lipoprotein(a), one of the most challenging aspects in the clinical management of this patient population is the remarkable diversity of ASCVD risk.Alternatively, HDL has been thought to protect against atherosclerosis because low levels of HDL are strongly associated with increased risk of ASCVD. Several recent clinical trials have unsuccessfully attempted to raise HDL cholesterol to reduce the risk of ASCVD. These results have left unanswered questions about the primary function(s) of HDL. Plasma lipoproteins undergo extensive changes in structure, function, and metabolism during severe infections such as sepsis. However, the implications and causality of these changes to clinical outcomes is poorly understood.The central objective was to explore the contribution of genetic variation in plasma lipoprotein traits and metabolism on lipid disorders such as familial hypercholesterolemia, elevated lipoprotein(a), and serious infections such as sepsis. Here, we used genetic epidemiology and mouse models of disease to assess how common and rare germline genetic variation affects the risk of atherosclerotic cardiovascular diseases and infectious diseases. Specifically, 1) can background genetic variation related to LDL and lipoprotein(a) modify the risk of ASCVD for individuals with familial hypercholesterolemia? and 2) are associations between HDL and risk of infectious disease causal? Several broad conclusions can be made. First, background polygenic factors influencing LDL-C and lipoprotein(a) levels modify the penetrance and expressivity of familial hypercholesterolemia. Second, the primordial function of HDL may be related to immunoregulation and resolution of infection. Third, cholesteryl ester protein is an important regulator of HDL levels during sepsis and may be a therapeutic target.

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Doxorubicin is a commonly used chemotherapy drug that treats both adult and childhood cancers, but its clinical usefulness is limited by doxorubicin-induced cardiotoxicity (DIC). The incidence of DIC increases up to 65% at cumulative doses of 550 mg/m², which leads to irreversible heart failure and death. Since some patients suffer from DIC even at low doses, genetic differences may account for some of the inter-individual variability in risk for DIC and several associated genetic variants have been identified. Among these variants, RARG-S427L is one of the top variants that shows strong evidence of association with DIC. Sodium-glucose transport protein 2 inhibitors (SGLT2i) are effective glucose-lowering medications that are indicated for type 2 diabetes mellitus treatment. SGLT2i have also been demonstrated to be cardioprotective for heart failure. We still lack ways to predict and prevent DIC. The goal of this dissertation is to investigate the impacts and mechanism of RARG-S427L variant in DIC and the potential cardioprotective effects of SGLT2i against DIC. I hypothesized that RARG-S427L increases the risk of DIC and the SGLT2i (empagliflozin) protects against DIC. To conduct this work, I developed a patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) model of DIC and used this to show that RARG-S427L increases susceptibility to DIC by orchestrating a DNA repair response to doxorubicin. Furthermore, co-treatment with empagliflozin reduced doxorubicin-induced cell death and up-regulated fatty acid metabolism-related gene expression. In summary, our findings reveal the roles of RARG-S427L in transcriptional response to doxorubicin in cardiomyocytes and identify empagliflozin as a potential cardioprotective agent against DIC, with implications for personalized risk prediction and the potential usage of empagliflozin to prevent this adverse drug reaction.

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Adverse drug reactions (ADRs) constitute the fourth leading cause of death and their incidence is steadily increasing. Cardiovascular toxicity is one of the most common and serious ADR and is the leading cause of drug discontinuation. The overarching goal of this thesis is to investigate cardiac ADRs using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs). Doxorubicin is a chemotherapy drug administered to adult and pediatric patients for the treatment of hematological and solid tumors, however, it can cause doxorubicin induced cardiotoxicity (DIC). We generated induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) from patients who received doxorubicin as part of their chemotherapy regimen. iPSC-CMs from individuals who developed DIC displayed significantly greater sensitivity to doxorubicin compared to iPSC-CMs from patients who did not experience DIC. Several variants associated with DIC have been identified, with RARG –S427L having the strongest genetic evidence. iPSC-CMs together with genome editing, provide a powerful platform to establish causal relationships between genetic variants and ADRs. We used CRISPR/Cas9 to investigate the functional impact of RARG-S427L on DIC in isogenic patient derived iPSC-CMs that differed only at the RARG locus. Genetic correction of RARG-S427L decreased susceptibility to in vitro DIC, whereas introduction of RARG-S427L had the opposite effect. We also used this platform to identify novel cardioprotectants that can be used clinically to prevent DIC. Empagliflozin, a sodium-glucose co-transporter 2 inhibitor, is an FDA-approved medication for the treatment of diabetes that shows cardiovascular benefits. iPSC-CMs treated with empagliflozin exhibited reduced doxorubicin induced cell death. Finally, we showed that enriched cardiomyocyte subtype populations are necessary for accurate drug screening and disease modelling. Ibrutinib is an anticancer drug indicated for the treatment of B cell malignancies; however, it can cause atrial fibrillation. We used atrial and ventricular hPSC-CMs to study ibrutinib induced atrial fibrillation. Ibrutinib had an arrhythmogenic impact on hPSC atrial derived cardiomyocytes, while ventricular hPSC remained unaffected. Collectively, our findings demonstrate that hPSC-CMs represent a powerful platform for disease modelling and drug screening that is amenable to personalized risk prediction for the prevention of adverse drug reactions.

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