Arrhythmogenesis in a murine model with genetic modifications in the ryanodine receptor.
Lead Research Organisation:
University of Cambridge
Department Name: Physiology
Abstract
Sudden cardiac death (SCD) is common, causing ~70,000 deaths in the UK per annum. Its clinical management has improved but is restricted by the little that is known of its initiation and mechanisms; preventative treatments have consequently proved disappointing. The abnormal heart rhythms (arrhythmias) thought responsible may result from abnormalities in the patterns by which heart beats are generated or conducted. Cellular excitation caused by the abnormal movement of calcium ions is thought to be important both in the initiation and maintenance of arrhythmias. Altered calcium transport has been observed for many through electrical and optical recordings in isolated cells, but not in whole hearts. We shall study this complex pathophysiology using genetically modified mice based on mutations identified in patients with severe arrhythmias. These mice can then be characterised using specialised assays, many of which can also then be applied in patients.
We shall employ one particular disease paradigm causing cardiac arrhythmia and sudden death, called catecholaminergic polymorphic ventricular tachycardia (CPVT). The results should both improve our fundamental understanding of abnormal heart rhythms, as well as the specific pathophysiology of the CPVT condition and help in the design of better drug treatments.
We shall employ one particular disease paradigm causing cardiac arrhythmia and sudden death, called catecholaminergic polymorphic ventricular tachycardia (CPVT). The results should both improve our fundamental understanding of abnormal heart rhythms, as well as the specific pathophysiology of the CPVT condition and help in the design of better drug treatments.
Technical Summary
Medical/scientific opportunity:
Sudden cardiac death (SCD) causes ~70,000 deaths in the UK pa. Improvement of its clinical management requires increased understanding of its mechanisms; the underlying arrhythmias are likely caused by abnormal patterns by which cardiac electrical activity is generated or conducted. These involve not only re-entrant waves of excitation, but also triggered activity. Abnormal Ca2+ homeostasis may be important both in initiating such waves as well as operating as an arrhythmogenic mechanism.
Aims and objectives
This project will study this underlying pathophysiology using GM mice focusing on one particular disease and physiological paradigm: catecholaminergic polymorphic ventricular tachycardia (CPVT). It will utilize the opportunity offered by recent generation of heterozygotic (RyRp/s) and homozygotic (RyRs/s) genetically modified (GM) mice containing P2328S directly recapitulating clinical genetic findings within symptomatic family members with this condition.
Design:
I will test for [1] arrhythmogenic phenotypes in the murine system with anatomical, electrophysiological and pharmacological features translatable to the human condition. [2] the presence and pharmacological properties of alterations in Ca2+ homeostasis, [3] its possible molecular basis. These changes will be related to [4] arrhythmic electrophysiological properties, and [5] the nature of the resulting arrhythmia.
Methodologies:
[1] The presence or absence of arrhythmogenic phenotypes exacerbated by catecholinergic excitation and any accompanying structural change will be tested by: (a) conventional light microscopy of gross cardiac anatomy and 3-dimensional electronmicroscopic organisation of tubular-sarcoplasmic (TSR) junctions and (b) Electrophysiological assessments of spontaneous and provoked arrhythmogenesis on whole Langendorff-perfused wild-type (WT), RyR2p/s, and RyR2s/s murine hearts in the presence and absence of isoproterenol.
[2] Altered Ca2+ homeostasis reflected in evoked Ca2+ signals, and the presence or absence of diastolic Ca2+ peaks and propagated Ca2+ waves will be explored in isolated Fluo-3-AM loaded mouse ventricular myocytes.
[3] Molecular basis for the CPVT phenotype will be examined against a hypothesis implicating altered FKPB12.6 binding using Western blot analyses for detectable FKPB12.6 in heavy sarcoplasmic reticular vesicles from hearts under conditions of arrhythmic activity and catecholaminergic stimulation.
[4] Electrophysiological studies will test a recent hypothesis separating roles for early/delayed afterdepolarizations as initiators of arrhythmogenic activity and repolarization gradient phenomena as substrates for arrhythmogenic activity.
[5] Spatial patterns in observed ventricular tachycardia will be studied in Langendorf perfused hearts to bridge arrhythmogenesis in murine systems to human phenotypes and test a hypothesis implicating Ca2+ driven arrhythmias in bi-directional polymorphic tachycardias, in contrast to the monomorphic torsade de pointe pattern seen with the LQT syndromes.
Sudden cardiac death (SCD) causes ~70,000 deaths in the UK pa. Improvement of its clinical management requires increased understanding of its mechanisms; the underlying arrhythmias are likely caused by abnormal patterns by which cardiac electrical activity is generated or conducted. These involve not only re-entrant waves of excitation, but also triggered activity. Abnormal Ca2+ homeostasis may be important both in initiating such waves as well as operating as an arrhythmogenic mechanism.
Aims and objectives
This project will study this underlying pathophysiology using GM mice focusing on one particular disease and physiological paradigm: catecholaminergic polymorphic ventricular tachycardia (CPVT). It will utilize the opportunity offered by recent generation of heterozygotic (RyRp/s) and homozygotic (RyRs/s) genetically modified (GM) mice containing P2328S directly recapitulating clinical genetic findings within symptomatic family members with this condition.
Design:
I will test for [1] arrhythmogenic phenotypes in the murine system with anatomical, electrophysiological and pharmacological features translatable to the human condition. [2] the presence and pharmacological properties of alterations in Ca2+ homeostasis, [3] its possible molecular basis. These changes will be related to [4] arrhythmic electrophysiological properties, and [5] the nature of the resulting arrhythmia.
Methodologies:
[1] The presence or absence of arrhythmogenic phenotypes exacerbated by catecholinergic excitation and any accompanying structural change will be tested by: (a) conventional light microscopy of gross cardiac anatomy and 3-dimensional electronmicroscopic organisation of tubular-sarcoplasmic (TSR) junctions and (b) Electrophysiological assessments of spontaneous and provoked arrhythmogenesis on whole Langendorff-perfused wild-type (WT), RyR2p/s, and RyR2s/s murine hearts in the presence and absence of isoproterenol.
[2] Altered Ca2+ homeostasis reflected in evoked Ca2+ signals, and the presence or absence of diastolic Ca2+ peaks and propagated Ca2+ waves will be explored in isolated Fluo-3-AM loaded mouse ventricular myocytes.
[3] Molecular basis for the CPVT phenotype will be examined against a hypothesis implicating altered FKPB12.6 binding using Western blot analyses for detectable FKPB12.6 in heavy sarcoplasmic reticular vesicles from hearts under conditions of arrhythmic activity and catecholaminergic stimulation.
[4] Electrophysiological studies will test a recent hypothesis separating roles for early/delayed afterdepolarizations as initiators of arrhythmogenic activity and repolarization gradient phenomena as substrates for arrhythmogenic activity.
[5] Spatial patterns in observed ventricular tachycardia will be studied in Langendorf perfused hearts to bridge arrhythmogenesis in murine systems to human phenotypes and test a hypothesis implicating Ca2+ driven arrhythmias in bi-directional polymorphic tachycardias, in contrast to the monomorphic torsade de pointe pattern seen with the LQT syndromes.