Impacts of Phosphofructokinase-2 on Cardiac Electrophysiology and Function in Diabetes - Cardiovascular disease is the leading cause of death in individuals with type 1 diabetes (T1D) and arrhythmia serves as a prominent contributor to this mortality. Prolongation of the QT interval represents vulnerability to fatal ventricular tachyarrhythmia and is strongly associated with T1D. However, the cause of QT prolongation in this population is incompletely understood. A decrease in PFKFB2, the cardiac isoform of the critical glycolytic regulator phosphofructokinase-2 (PFK-2), may contribute. Despite degradation of PFKFB2 in diabetes, little is known regarding consequences of its loss on cardiac function and electrophysiology. A lack of suitable models manipulating PFK-2-mediated glycolytic regulation using a cardiac-specific approach has limited investigation into these relationships. However, we are now poised to address this using a novel cardiomyocyte-specific Pfkfb2 knockout mouse (cKO) as well as a model that overexpresses constitutively active PFK-2 in the heart (GlycoHi). Interestingly, we have recently shown that cKO animals exhibit QT prolongation, dilation, and decreased cardiac function. This occurs in concordance with activation of the hexosamine biosynthesis pathway (HBP), a metabolic pathway which branches from glycolysis directly upstream of phosphofructokinase and drives pathologic functional and electrophysiological remodeling in the diabetic heart. The overarching hypothesis of this proposal is that glycolytic dysregulation due to loss of cardiac PFK-2 is responsible for impaired cardiac function and arrhythmogenic predisposition observed in T1D. Aim 1 will investigate mechanistic underpinnings of the relationship between glycolytic regulation in the heart and cardiac mechanical and electrical function. I will utilize an AAV9-mediated knockdown of the rate-limiting HBP enzyme in cKO and wildtype mice and measure cardiac functional and electrophysiological parameters. I will use collected hearts to assess posttranslational modification of known HBP-targeted transcriptional and electrophysiological regulators. In further mechanistic studies utilizing isolated cardiomyocytes, I will pharmacologically target the HBP and an immediate downstream effector. We will then investigate impacts on ion channel expression, action potential duration, and relevant K+ currents. Aim 2 will determine whether upregulation of PFK-2 preserves metabolic substrate preference and rescues the pathologic changes to electrical and mechanical function observed in T1D. Using a streptozotocin-induced T1D stress in the GlycoHi model, I will interrogate mitochondrial respiration, metabolic enzyme abundance, glycolytic flux, cardiac function, and electrophysiological parameters. These experiments will illuminate an unexplored relationship between PFK-2, a critical glycolytic regulator, and electrocardiographic and functional changes commonly observed in a disease marked by its degradation. Further, a protective effect of PFK-2 activity in T1D would provide a new therapeutic direction for diabetic cardiomyopathy while advancing our understanding of how glucose metabolism interacts with electrophysiology in the heart. Lastly, these studies will provide me with key training opportunities in electrophysiology, echocardiography, biochemistry, and molecular physiology.
