Non-Invasive Electrocardiographic Mapping of Arrhythmia and Arrhythmogenic substrate in the Human Ventricle.

PhD Theses.The ablation of ventricular tachycardia often involves mapping when the arrhythmia is ongoing. This is often limited by haemodynamic instability. Non-invasive electrocardiographic mapping (ECGI) may aid in the mapping process by allowing expedient localisation. However, insufficient testing of this technology against ground truth data has been conducted. Furthermore, the system could have utility in detection of arrhythmogenic substrate. Current clinical practice uses echocardiography to risk stratify patients for implantation of intracardiac defibrillators (ICDs). Invasive epicardial electrogram data was collected in 8 patients. Activation and repolarisation times were compared to ECGI derived data showing modest correlation. A detailed analysis of ventricular tachycardia sites of origin in the heart was elucidated using validated electrophysiological techniques. These were compared to ECGI derived data in 18 patients, showing better accuracy than the 12 lead ECG with a resolution of ~2.2cm suggesting it may be a useful adjunctive tool in mapping unstable VT. ECGI derived data collected during sinus rhythm was compared to invasive electrogram maps in 16 patients. The capacity of ECGI to localise scar showed modest accuracy. ECGI and Cardiac MRI scans were performed in 21 patients with cardiac amyloidosis. ECGI showed cardiac amyloidosis to be associated with both ventricular conduction and repolarization abnormalities, supporting the hypothesis that arrhythmic mechanisms may be linked to mortality in this condition

Investigation of arrhythmogenesis in the desmoplakin knockout mouse: A model of arrhythmogenic cardiomyopathy

Arrhythmogenic cardiomyopathy (ACM), in contrast with other cardiomyopathies, often presents with lethal ventricular arrhythmias with athletes affected more severely. It has the characteristic feature of fibrofatty replacement of the right ventricular myocardium, although left ventricular variants have been reported. It has been associated with desmosomal protein mutations – structural proteins involved in cell-cell adhesion at the intercalated disk between cardiomyocytes. Arrhythmias are often noted to occur in a ‘concealed phase’ with minimal or no evidence of structural change. There is a need for better understanding of the mechanisms promoting arrhythmogenesis in order to improve arrhythmic risk prediction. A cardiac restricted heterozygous desmoplakin (DSP) knockout mouse was developed using the Cre-lox system with the cardiac restricted αMHC promoter (αMHC-Cre DSP flox/+) as a model of ‘concealed phase’ ACM and was studied with ECG, electrophysiology study and histology. This model recapitulated the ventricular arrhythmias seen in patients with evidence of conduction delay at electrophysiology study. This was no evidence of fibrofatty replacement of the myocardium on histology. Immunohistochemistry, however, revealed connexin 43 (Cx43) mislocalization away from the intercalated disk and a reduction in mRNA expression. Cx43 is a protein that makes up gap junctions which are involved in allowing rapid electrical conduction in the heart. The sodium channel is also located at the intercalated disk, but no change in its distribution, mRNA expression or change in the sodium current was noted. This suggests that interactions between Cx43 and desmosomal proteins are a key driver of arrhythmogenesis in ACM. In order to assess the effect of exercise on the arrhythmic phenotype, the mice were allowed to exercise freely before electrophysiology study. One group had slow release β blocker pellets implanted prior to exercise. Exercise made the mice more prone to arrhythmia, consistent with human studies. β blockers significantly reduced the numbers of mice developing ventricular arrhythmia as well as reducing the conduction delay observed at electrophysiology study. Cx43 showed less mislocalization in the β blocker treated mice, suggesting a role in slowing disease progression. Using the CreER promoter, which knocks DSP out in the adult mouse, the effect of DSP loss in adulthood was investigated. Mice with a complete knockout of DSP in adulthood became rapidly unwell and died, with bradycardia the only notable arrhythmia. However, heterozygous CreER knockout mice did not develop arrhythmia. The αMHC promoter is maximally expressed in early postnatal life. This suggests that this period, when desmosomes and adherens junctions are forming the mixed cell-cell junctions called the area composita, is significant in forming functional gap junctions to allow normal conduction. This mechanism may be relevant to arrhythmogenesis in other inherited cardiomyopathies. HL-1 cells were used as a cellular cardiomyocyte model to express DSP mutations identified in our ACM patient cohort. These two mutations (R1113X and T586fsX594) were both nonsense mutations at the N terminus. Cx43 was also found to be mislocalised in this model and shows similarity between the heterozygous knockout murine model and a cellular m0del of disease causing mutations. Sodium channel localisation was variable and showed less membrane localisation with the R1113X mutation. This may account for differences in the arrhythmia burden amongst ACM patients and shows the complex nature of the interactions at the intercalated disk. Plakoglobin was found to be localised at the nucleus with mutant DSP. This shows it is a key binding partner for desmoplakin at the intercalated disk and may also promote arrhythmogenesis by alterations in nuclear signalling. In conclusion, this work has established the heterozygous DSP knockout mouse and HL-1 cells as useful models for investigating the mechanisms of arrhythmogenesis in ACM. The key mechanism is interaction of desmoplakin and Cx43 at the intercalated disk. Restriction on exercise and treatment with β blockers for ACM patients is supported by this model. Further investigation of the mechanisms of interaction of DSP with other desmosomal proteins, the sodium channel and Cx43 may allow better prediction of arrhythmic risk and targeted therapies for ACM patients

Electrophysiological and cellular analysis of filamin-C mutations causing cardiomyopathy using human iPSC-derived cardiomyocytes

Background: Arrhythmogenic Cardiomyopathy (AC) is a genetic cardiac disease resulting from different mutations within proteins constituting the intercalated disc, including desmosomal and nondesmosomal proteins. Recent studies have revealed that mutations in filamin-C (FLNC) may lead to AC. The arrhythmogenesis and electrophysiological effects of FLNC-related AC are incompletely understood. Therefore, the aim of this study is to assess the potential electrophysiological consequences of FLNC loss as occurs in AC in human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs). Specifically, I aimed to characterise abnormal electrical activity and the expression and function of key proteins in cardiac electrical activity such as gap junction protein connexin 43 (Cx43).// Methods: hiPSC-CMs were differentiated and observed by immunofluorescence microscopy. Small interfering RNA (siRNA) transfection was utilised to knockdown the expression of FLNC in hiPSC-CMs. Protein analysis was performed using western blotting to confirm the knockdown efficiency. Electrophysiological properties were recorded using a multielectrode array and manual patch clamping. Optical recording of membrane potential and calcium activity from hiPSC-CMs were also carried out using parameter sensitive dyes.// Results: Silencing of FLNC led to markedly decreased immunofluorescence signals of FLNC, Cx43, desmoplakin, and junctional plakoglobin. No significant reductions were noted in the immunofluorescence signals of voltage-gated sodium channel (Nav1.5) and plakophilin-2 compared with control hiPSC-CMs. Western blotting showed the reduction of FLNC and Cx43 expression following silencing of FLNC. Knockdown of FLNC resulted in disturbances to the recorded action and field potential signals of hiPSC-CMs and arrhythmic likeevents. Transfected hiPSC-CMs with siRNA-FLNC were associated with prolongation of calcium transient durations, optical action potential duration, and action potentials measured with patch clamping.// Conclusion: The current findings indicated that loss of FLNC resulted in a complex arrhythmogenic phenotype in hiPSC-CM

Methods for Arrhythmogenic Substrate Identification and Procedural Improvements for Ventricular Arrhythmias.

Ventricular arrhythmias (VA) are a frequent precursor to sudden cardiac death (SCD) in patients with structural heart disease (SHD). Patients with SHD are at risk of recurrent ventricular tachycardia (VT), which generally occurs due to re-entry within and around the presence of an arrhythmogenic scar. Therefore, scarred myocardium forms the necessary substrate for arrhythmogenesis to occur. A scar may occur due to obstructive coronary artery disease, causing ischaemic cardiomyopathy (ICM), or from cardiac injury due to several other causes, including inflammatory, infiltrative, toxin-mediated, or genetic heart disease, termed non-ischaemic cardiomyopathy (NICM). An implantable cardioverting defibrillator (ICD) can abort SCD from recurrent VAs. However, they do not stop VAs from occurring in the first place. Anti-arrhythmic drugs (AADs) may reduce the frequency and burden of VAs but have limited efficacy. Some have a narrow therapeutic window or the potential for multiorgan toxicity and can be poorly tolerated. Catheter ablation (CA) is a class I indication for treating sustained monomorphic VT refractory to AADs. CA reduces VT burden, the number of defibrillator therapies, greater freedom from recurrent ventricular arrhythmia, and improves quality of life. However, recurrences can be experienced in up to 50% of patients with SHD-related VT. Some reasons for the failure of CA include reliable identification of critical components of substrate that can harbour VAs both in sinus rhythm and during ongoing VT using electroanatomic mapping (EAM) and imaging techniques, as well as limitations in assessing intraprocedural endpoints. Further refinement of electroanatomic mapping techniques is required to improve the efficacy of CA. This thesis aims to expand on current techniques for substrate identification and methods to improve the efficacy of VA ablation procedures