Doctor of Philosophy

dissertationAndersen-Tawil syndrome Type 1 (ATS1) is a disorder linked to a loss of function of the inward rectifier current IK1. Such a reduction in repolarization reserve has an established link with heterogeneous action potential duration (APD) prolongation. This in turn can serve as a substrate for reentrant arrhythmias. While APD prolongation and increased dispersion have been reported in pharmacological models of ATS1 they have not been linked with arrhythmogenesis. APD prolongation secondary to reduced IK1 can increase Ca2+ entry into myocytes. The resultant accumulation of cytosolic Ca2+ has been linked with ventricular ectopies which can trigger arrhythmias. Indeed this mechanism of arrhythmogeneis has been proposed in ATS1 based on previous in silico and ex vivo studies. However, ATS1- associated cytosolic Ca2+ overload and increased arrhythmia propensity has not been demonstrated in tissue preparations. The overall goal of this research was to characterize the factors that underlie arrhythmia propensity in a pharmacological model of ATS1. To this end we performed two studies. The first study analyzed APD gradients to determine whether they were sufficient for induction of reentrant arrhythmias. The results indicated they were not. However, this study revealed increased arrhythmia propensity which correlated with cytosolic Ca2+ overload. Therefore, the second study focused on Ca2+ handling and showed that ectopic activity originated from regions of higher Na+/Ca2+ exchanger iv (NCX) functional expression relative to sarcoplasmic reticulum Ca2+ ATPase (SERCA2a), which we term "NCX dominant" regions of the heart. In support of the idea that NCX is an important determinant of ectopy, we were able to modulate both the timing as well as the frequency of ectopy by pharmacologically modulating NCX dominance. The data presented in this dissertation provide an insight into the factors by which Ca2+ handling contributes to ectopy under conditions of partial loss of IK1 function. This concept may aid in the identification of novel targets for antiarrhythmic therapy when IK1 is reduced in ATS1, as well as more prevalent disorders such as heart failure

Optogenetics in striated muscle: defibrillation of the heart and direct stimulation of skeletal muscles with light

Optogenetic depolarization of cells using the non-selective cation channel Channelrhodopsin-2 (ChR2) enables precise control over the membrane potential of cells within a specific area of intact organs. Furthermore, the selective overexpression of light-gated proteins allows cell type-specific and pain-free stimulation which could be of great benefit for future scientific and therapeutic approaches. In my thesis, I explored two potential applications of optogenetic methods in striated muscle: optogenetic defibrillation to terminate ventricular arrhythmia in intact mouse hearts and direct optogenetic stimulation of skeletal muscles. These new approaches could lead in the future to the development of optogenetic defibrillators and laryngeal pacemakers. Most experiments were performed with explanted hearts, isolated skeletal fibers and muscles or larynges from transgenic ChR2 expressing mice. To add translational perspectives, we also explored optogenetic defibrillation and intralaryngeal muscles stimulation after ChR2 gene transfer to wild type mice using adeno-associated virus (AAV). Optogenetic defibrillation by epicardial illumination was highly efficient in terminating ventricular arrhythmia in transgenic hearts and the success rate of optogenetic defibrillation was depending on the pulse duration, the size of illumination and the light intensity. Importantly, we were also able to terminate ventricular arrhythmia in non-transgenic hearts even one year after AAV mediated gene transfer. The potential applicability of optogenetic defibrillation in the human heart was assessed in experimentally-calibrated computer simulations of a patient’s heart with infarct-related ventricular tachycardia. Because optogenetic stimulation would be in principle pain-free in patients, the proof for its feasibility could lay the foundation for the development of a new treatment option for patients at high risk for ventricular arrhythmia. Direct optogenetic stimulation of skeletal muscle was first proven in isolated Flexor digitorum brevis fibers and in intact soleus muscles, which could both be stimulated using brief light pulses. The force of light-induced single twitches could be precisely controlled by varying the pulse duration and light intensity. Optogenetic stimulation was most efficient with 10 ms long pulses at a repetition rate of 40 Hz reaching ~84% of the maximum force generated by electrical stimulation with 100 Hz. Recurrent nerve paralysis is a severe complication of neck surgery, malignant processes or central neurological diseases and results in a fixed paramedian position of the vocal cords as well as life-threatening dyspnea in the case of bilateral paralysis. Current treatment options consist only of destructive surgery. Unfortunately the use of local electrical stimulation to restore laryngeal function faces severe technical limitations. Therefore I sought to explore direct optogenetic stimulation of intralaryngeal muscles in explanted larynges from ChR2 transgenic mice. Specific illumination of the individual intralaryngeal muscle groups led to an opening or closing of the vocal cords depending on the site of illumination. This proves the sufficient spatial resolution of light for selective stimulation of the intralaryngeal muscles groups. In addition, we were able to induce opening of the vocal cords in wild type mice after AAV-based gene transfer of ChR2 with light. Thus optogenetic stimulation could become a new treatment option for patients suffering from bilateral laryngeal paralysis. In conclusion, optogenetic stimulation can overcome the severe limitations of electrical stimulation of the heart and skeletal muscles. The new technologies, I have developed and characterized in this thesis, allow for the design of completely new stimulation patterns to address open questions in muscle physiology. Furthermore, optogenetic stimulation of striated muscles could become a new treatment option for patients enabling selective and pain-free stimulation with few side effects

Electromechanical reciprocity and arrhythmogenesis in long-QT syndrome and beyond.

An abundance of literature describes physiological and pathological determinants of cardiac performance, building on the principles of excitation-contraction coupling. However, the mutual influencing of excitation-contraction and mechano-electrical feedback in the beating heart, here designated 'electromechanical reciprocity', remains poorly recognized clinically, despite the awareness that external and cardiac-internal mechanical stimuli can trigger electrical responses and arrhythmia. This review focuses on electromechanical reciprocity in the long-QT syndrome (LQTS), historically considered a purely electrical disease, but now appreciated as paradigmatic for the understanding of mechano-electrical contributions to arrhythmogenesis in this and other cardiac conditions. Electromechanical dispersion in LQTS is characterized by heterogeneously prolonged ventricular repolarization, besides altered contraction duration and relaxation. Mechanical alterations may deviate from what would be expected from global and regional repolarization abnormalities. Pathological repolarization prolongation outlasts mechanical systole in patients with LQTS, yielding a negative electromechanical window (EMW), which is most pronounced in symptomatic patients. The electromechanical window is a superior and independent arrhythmia-risk predictor compared with the heart rate-corrected QT. A negative EMW implies that the ventricle is deformed-by volume loading during the rapid filling phase-when repolarization is still ongoing. This creates a 'sensitized' electromechanical substrate, in which inadvertent electrical or mechanical stimuli such as local after-depolarizations, after-contractions, or dyssynchrony can trigger abnormal impulses. Increased sympathetic-nerve activity and pause-dependent potentiation further exaggerate electromechanical heterogeneities, promoting arrhythmogenesis. Unraveling electromechanical reciprocity advances the understanding of arrhythmia formation in various conditions. Real-time image integration of cardiac electrophysiology and mechanics offers new opportunities to address challenges in arrhythmia management

INVESTIGATION OF CARDIAC ELECTROPHYSIOLOGY IN HUMAN VENTRICULAR TISSUE

Individuals with cardiomyopathy are at higher risk to die from sudden cardiac arrest than those with non-failing (NF) hearts. This study examined the differences in electrical properties of failing and NF human hearts in terms of cardiac memory through explicit control of diastolic intervals in a sinusoidal fashion, restitution of action potential duration (APD) through standard and dynamic pacing protocols, maximum rate of depolarization and APD alternans. Recordings of transmembrane potentials were made in tissues extracted from patients with heart failure and one donor NF heart. Computational simulations were performed using the O’Hara Rudy model for generating surrogates of control data. Significant differences were seen between left ventricular (LV) tissue and NF LV tissue in tilt, and measures of memory in terms of area and thickness during the sinusoidal 400ms protocol. Minimum delay was also significantly higher in the failing LV during the sinusoidal 150ms protocol. Failing tissues showed a higher restitution slope and prolonged AP which is consistent with previous studies and is hypothesized to contribute to the increased susceptibility to unstable alternans. This study further explored how disease alters the electrical functioning of the heart and why these patients are at a higher risk of ventricular arrhythmia

Multiple targets for flecainide action: implications for cardiac arrhythmogenesis

Flecainide suppresses cardiac tachyarrhythmias including paroxysmal atrial fibrillation, supraventricular tachycardia and arrhythmic long QT syndromes (LQTS), as well as the Ca(2+) -mediated, catecholaminergic polymorphic ventricular tachycardia (CPVT). However, flecainide can also exert pro-arrhythmic effects most notably following myocardial infarction and when used to diagnose Brugada syndrome (BrS). These divergent actions result from its physiological and pharmacological actions at multiple, interacting levels of cellular organization. These were studied in murine genetic models with modified Nav channel or intracellular ryanodine receptor (RyR2)-Ca(2+) channel function. Flecainide accesses its transmembrane Nav 1.5 channel binding site during activated, open, states producing a use-dependent antagonism. Closing either activation or inactivation gates traps flecainide within the pore. An early peak INa related to activation of Nav channels followed by rapid de-activation, drives action potential (AP) upstrokes and their propagation. This is diminished in pro-arrhythmic conditions reflecting loss of function of Nav 1.5 channels, such as BrS, accordingly exacerbated by flecainide challenge. Contrastingly, pro-arrhythmic effects attributed to prolonged AP recovery by abnormal late INaL following gain-of-function modifications of Nav 1.5 channels in LQTS3 are reduced by flecainide. Anti-arrhythmic effects of flecainide that reduce triggering in CPVT models mediated by sarcoplasmic reticular Ca(2+) release could arise from its primary actions on Nav channels indirectly decreasing [Ca(2+) ]i through a reduced [Na(+) ]i and/or direct open-state RyR2-Ca(2+) channel antagonism. The consequent [Ca(2+) ]i alterations could also modify AP propagation velocity and therefore arrhythmic substrate through its actions on Nav 1.5 channel function. This is consistent with the paradoxical differences between flecainide actions upon Na(+) currents, AP conduction and arrhythmogenesis under circumstances of normal and increased RyR2 function.S.C.S. and A.P.J. are funded by the British Heart Foundation (PG/14/79/31102). K.J. is funded by the Fundamental Research Grant Scheme (FRGS/2/2014/SKK01/PERDANA/ 02/1), Ministry of Education, Malaysia and the Research Support Fund, Faculty of Health and Medical Science, University of Surrey. A.F.D. is funded by the National Health and Medical Research Council (NHMRC) of Australia (APP1126201). A.J.T. is funded by the British Heart Foundation (PG/13/39/3029). C.L.H.H. is funded by the Medical Research Council (MR/M001288/1), Wellcome Trust (105727/Z/14/Z), British Heart Foundation (PG/14/79/ 31102), the McVeigh Benefaction and SADS UK