MECHANICAL CONTROL OF HEART RATE: INVESTIGATING DETERMINANTS OF THE CHRONOTROPIC RESPONSE TO SINOATRIAL NODE STRETCH
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The sinoatrial node (SAN) is heavily regulated by multiple factors, including stretch, enabling adaptation of heart rate to changes in physiological demand. Right atrial distention, caused by an increase in venous return to the heart, increases heart rate. Thus, changes in mechanical load result in beat-by-beat adaptation of SAN function. Chronotropic responses to stretch have been demonstrated in transplanted and isolated hearts, in isolated SAN tissue, and even in single SAN cells, indicating that the stretch response is intrinsic to the SAN itself. However, mechanisms and determinants responsible for the SAN stretch response are incompletely understood. The zebrafish represents an attractive alternative experimental model for the study of cardiac electrophysiological mechanisms, due to its many physiological similarities with human and the relative ease of its genetic manipulation. Thus, my first objective was to determine the response of the zebrafish isolated SAN to varying degrees of controlled stretch. Consistent with humans and most mammals studied, stretch of the zebrafish SAN results in increased beating rate (BR) in a magnitude-dependent manner. These studies also indicated that baseline force and the direction of stretch may play an important role in determining the magnitude of the chronotropic response. Further, these data demonstrate the viability of zebrafish as a model species for studying SAN stretch responses. It is thought that the chronotropic response to SAN stretch is due to activation of cation non-selective stretch-activated channels (SACNS). As the reversal potential of SACNS is between the maximum diastolic and systolic potential of the SAN action potential (~ -10 mV), we predicted that SAN BR could be modulated through acute SACNS activation, resulting in an increase or decrease in BR depending on the timing of activation within the cardiac cycle. My second objective was to investigate the importance of activation timing and action potential morphology in the chronotropic response to SAN stretch. To test this, I utilised transgenic zebrafish and mice expressing a light-activated ion channel (channelrhodopsin-2), which passes a non-specific cation current with similar characteristics to SACNS, including reversal potential properties. In both species, and with all light intensities, an increase or a decrease in BR could be elicited, depending on the timing of light application within the SAN cycle. This was confirmed, and further explored, using rabbit and mouse SAN cell computational models. Finally, using a pharmacological intervention to manipulate the action potential duration of mouse SAN, we caused a shift in the change in BR, supporting the importance of the interaction between the SACNS reversal potential and action potential morphology for the SAN stretch response. We also hypothesised that the magnitude of the response to SAN stretch is influenced by tissue mechanics. In my final objective I compared how the response of the isolated rabbit and mouse SAN relates to tissue stiffness and whether structural differences may account for observed responses. We measured applied force and tissue stiffness and compared this to chronotropic responsiveness and utilised second- harmonic generation imaging of stretched and unstretched SAN tissue to visualise changes in collagen crimp and alignment. We found differences in the relationship between SAN mechanical properties and the electrophysiological response to stretch between rabbit and mouse, as well as species-differences in SAN structure both at baseline and changes with stretch. Overall, my thesis provides further insight into the electrophysiological and mechanical determinants of the control of SAN function by stretch, which is critical for a fundamental understanding of mechanically induced changes in cardiac rhythm.