Role of calcium mobilising messengers in cardiac arrhythmias

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent calcium ion (Ca2+) activating second messenger due to its ability to release Ca2+ from intracellular acidic stores at nanomolar concentrations. NAADP has been known to play an important role in controlling excitation-contraction coupling in the heart. The ADP-ribosyl cyclase (ARC) CD38, is believed to be responsible for the production of NAADP in the heart.
The first part of the thesis assesses the role of NAADP pathway in calcium handling of cardiac atrial tissues by genetically (CD38-/- mice) and pharmacologically (bafilomycin A1) impairing NAADP pathway. Optical mapping techniques were used to observe the calcium transients and action potentials of mouse atrial tissues. At baseline conditions (without the activation of β-adrenoceptors), no contribution of the NAADP signalling pathway was observed in cardiac atria with respect to its calcium handling, action potential characteristics and resting heart rate. However, under the effect of the β- adrenoceptor agonist isoprenaline, CD38-/- mouse atria and bafilomycin A1 treated wildtype mouse atria showed a blunted increase in calcium transient amplitudes and heart rate as compared to control wildtype atria.
The second part of the thesis focuses on understanding the association of NAADP signalling pathway with the sarcoplasmic reticulum (SR), using a Ca2+ indicator to directly observe the SR Ca2+ content. NAADP was observed to increase the Ca2+ content of the SR in wildtype and CD38-/- mice ventricular cardiomyocytes in a bafilomycin A1 sensitive manner. Moreover, stimulating β-adrenergic response using cyclic adenosine monophosphate (cAMP) showed a reduced effect on SR Ca2+ increase in CD38-/- myocytes as compared to wildtype myocytes and disruption of lysosomes using bafilomycin A1 in wildtype myocytes also reduced the SR Ca2+ increase in response to cAMP. These observations provide further evidence that the NAADP signalling pathway has a prominent role in β-adrenergic response in the heart.
Although CD38 has been suggested as an enzyme that can produce both NAADP and cyclic adenosine diphosphate ribose (cADPR), several studies have attributed a non- CD38 ARC to catalyse the production of cADPR in the heart. The third part of the thesis, therefore, attempted to characterise the ARC present in sheep cardiac SR membrane preparations (SRMP). Functional and molecular biology experiments in this thesis strongly suggests that the ARC found in sheep SRMP is CD38.
Finally, in the last section of this thesis, a protocol for combining optical mapping with optogenetic pacing is discussed to study intracellular Ca2+ transients in atrial tissues of mice genetically modified to express channelrhodopsin 2 (ChR2) on phenylethanolamine N-methyltransferase (Pnmt)-derived cardiomyocytes (PdCMs). On optogenetic pacing, a shift in primary pacemaker sites could be seen towards the left atrium where the PdCMs were localised with minimal disturbance on the Ca2+ handling properties of the atrial tissue. Although this cell type-specific optogenetic pacing strategy targeting PdCMs as candidates for biopacemaking is still at an early stage of development, it could potentially lay foundation for an exciting prospect of clinical applications in treating heart rhythm disorders in the future.

Advisor: Terrar, D
Lei, M