Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2019.
Myotonic dystrophy type 1 (DM1) is a toxic RNA-mediated disease; nuclear retained mRNAs containing expanded CUG repeats exhibit gain-of-function toxicity which causes a progressive and debilitating multi-systemic disorder. DM1 patients exhibit one of the largest risks for sudden cardiac death of all patient populations and abnormalities in the electrocardiogram (ECG), including prolongations in PR-, QRS- and QT-interval durations, are observed in up to 80% of all DM1 patients. However, little is known about the specific biophysical and molecular mechanisms that underlie cardiac conduction defects in DM1. Using a novel mouse model of cardiac RNA toxicity (referred to as LC15 mice), the primary objective of this thesis was to fully characterize the effect of expanded CUG-repeat RNA expression on cardiac conduction and to identify the underlying biophysical and molecular mechanisms. LC15 mice possess a transgene that results in global expression of an mRNA
containing expanded CUG repeats, with nearly 4-fold higher levels of expanded CUG-repeat expression in the heart than in skeletal muscle. Continuous ECG data collected from conscious LC15 mice, using surgically implanted radiotelemeters, demonstrated significant prolongation of QRS- and corrected QT (QTc)-intervals compared to those of age-matched wildtype littermate mice, which is consistent in part with the cardiac phenotype of DM1 patients. Single cell current clamp recordings using isolated cardiac myocytes showed that a significant reduction in maximal phase-0 action potential upstroke velocity developed in ventricular myocytes from LC15 hearts when stimulation frequency increased to 9 Hz. Subsequent voltage clamp recordings demonstrated that the inward peak sodium current density was not altered in these myocytes, suggesting that voltage-gated sodium channels in LC15 hearts may exhibit slower recovery from inactivation. Additionally, current clamp recordings also revealed that the action potential duration (APD) in ventricular myocytes isolated from LC15 mice was significantly prolonged compared to that of wildtype mice. Complimentary voltage clamp
recordings revealed a decrease in the peak total outward potassium current density in LC15 ventricular myocytes compared to wildtype. Importantly, a 4-Aminopyridine (4-AP) sensitive current was found to be significantly reduced in ventricular myocytes from LC15 mice. This current may be attributed to at least two different outward potassium current components; Ito,slow and IK,slow. Results from exponential curve fitting of the outward potassium current decay (from peak to steady state) suggest that both of these outward potassium current kinetic
components are reduced in LC15 myocytes. Together, these results indicate that expanded CUG-repeat RNA toxicity in the heart leads to defects in ventricular muscle action potential upstroke and repolarization that contribute to prolongations of the QRS- and QTc-intervals, respectively. We hope this work will guide future
endeavors to identify specific molecular mechanisms that underlie conduction defects in DM1 patients and lead to efforts to identify therapeutic interventions that will help to mitigate the heightened risk for sudden cardiac death.
