Project 01: Roles of Nox4 in the regulation of cardiac metabolism in human iPSC-CMs

PI London: A.M. Shah; PI Göttingen: K. Streckfuß-Bömeke; PhD: G. Emanuelli - finished PhD, Double degree

Heart failure is one of the leading causes of death in the Western World today. Heart failure and many of the conditions that predispose to heart failure are associated with oxidative stress. This is considered to be important in the pathophysiology of the condition but clinical trials of antioxidant approaches to prevent cardiovascular morbidity and mortality have been unsuccessful. Indeed, it is now recognised that reactive oxygen species (ROS)-dependent signalling may exert beneficial as well as detrimental roles, depending upon the ROS source and the physiological or disease context. In the heart, ROS may be generated by multiple sources, including NADPH oxidases of the Nox family (1). During cardiac stresses, the expression level of Nox4 increases and, unexpectedly, we previously found a protective role of Nox4 during chronic haemodynamic overload in a mouse model (2). Our recently published data indicate that Nox4 exerts significant effects on cardiac metabolism both in mouse models in vivo and cultured rat cardiomyocytes (NRC) in vitro, which may contribute to these protective effects. Redox state and metabolic reprogramming are crucial in cell growth, differentiation, adaptation and survival. Recent work from our lab shows that Nox4 is involved in cardiomyocyte proliferation and differentiation (3). However, it is still unclear whether Nox4-dependent regulation of cardiac metabolism occurs in the human system. This project aims to investigate the influence of Nox4 on redox regulation and metabolism in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs). Protocols to generate and characterize hiPSCs-derived cardiomyocytes from healthy donors or patients are well established in the laboratory of the co-supervisor, Dr. Katrin Streckfuß-Bömeke (Prof. Gerd Hasenfuß, Cardiology and Pneumology, University of Göttingen) (4). These cells were shown to be a good source for analysis of the pathophysiology of monogenic and complex cardiac diseases. The PhD candidate will be involved in the establishment of hiPSC-CMs as a model system to study redox regulation of metabolic reprogramming in cardiomyocytes. Maturing cells will be characterise with a focus on metabolism. Nox4 levels will be manipulated by lentiviral-mediated knock-down or overexpression, respectively, during cardiac hiPSC-differentiation. Bioenergetics will be studied using extracellular flux (XF) analysis, and correlated with cell differentiation and function. Experiments will include cellular (cell culture of undifferentiated hiPSCs and differentiation into cardiomyocytes) and molecular biology (qRT-PCR), immunofluorescence, western blotting and bioenergetics measurements using XF analysis (Seahorse biosciences); metabolites utilisation will be assessed using labelled substrates for flux experiments, in combination with mass spec. Readouts to study iPSC-CMs differentiation and function will include the expression of cardiac-specific and metabolic markers, Ca2+-handling genes, measurements of calcium transients, cell size and sarcomeric structure (α-actinin, cardiac troponinT, MLC2v, MLC2a and titin). The eventual perspective is to study patient-specific iPSC-CMs with genetic differences in regulators of redox state.

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