Poster T15 - On-demand, non-invasive creation of ventricular arrythmias in mice through cardiac optogenetics

Introduction: Cardiac arrythmias are a major cause of mortality and morbidity worldwide, causing around 5 million deaths annually (1). Animal models are essential for both understanding mechanisms and testing treatments for arrythmia, and mouse models in particular offer similarity to humans in cardiac action potential shape and electrical conduction (2). Traditional methods of inducing arrythmia require drugs or invasive surgical methods, which are time consuming, have low success rates, and do not offer temporal control over heart rhythm (3). In mice, the small size of the heart often leads to the self-termination of arrythmias. Through cardiac optogenetics, excitatory or inhibitory light sensitive ion channels, called opsins, are introduced into heart tissue through genetic crosses or the use of adeno-associated virus (AAV) injection, but applications in mammalian models are limited due to the attenuation of light in tissue. The previous study of cardiac optogenetics in mice in vivo used the red-shifted excitatory opsin ChRmine to study stress responses to elevated heart rates, but the light power density used through a single LED was up to 240 mW/mm2, above the ANSI safety limit (4). Here, we induce ventricular tachycardia in living transgenic mice through LED array illumination of the excitatory opsin ReaChR, providing tunable arrhythmia on demand, while characterizing pacing effects through both electrocardiogram (ECG) and echocardiogram.

References: (1) Chugh, S. S. et al. Prog. Cardiovasc. Dis. (2008). (2) Zaragoza, C. et al. BioMed Res. Int. (2011). (3) Blackwell, D. J. et al. Circ. Res. (2022). (4) Hsueh, B. et al. Nature (2023).

Materials and

Methods: We crossed a mouse line containing heart-specific Cre recombinase under Nkx2-5 gene control with a line carrying CAG-LSL-ReaChR-mCitrine transgene in the Gt(ROSA)26sor locus, which provides uniform expression of ReaChR throughout the heart tissue. Mice anesthetized with isoflurane were positioned under a 617 nm LED array light source (LCS-0617-03-11, Mightex Inc., Canada), which provided a 10-mm spot size, and were connected to an ECG recording system using needle electrodes. Heart response was characterized using ECG and Echocardiogram in separate experiments. The heart pacing protocol to test the pace-able range consisted of a 15 second rest period, 10 second pacing period, 15 second recovery period. A typical recorded resting heart rate was 8 Hz. Stimulation frequencies ranged from 6 Hz (lower than the resting heart rate of the mouse) to 14 Hz, and the pulse width used was 20 ms. A longer pacing protocol (5-minute resting period, 10-minute pacing period, 10-minute recovery period) was used to analyze arrhythmic conditions caused by pulsing light lower than the resting heart rate. ECG traces were analyzed for heart rhythmicity, where the R-R interval was used to determine the heart rate. Arrhythmicity index was defined as the standard deviation in the heart periods divided by the median heart period. Fractional shortening (FS) and cardiac output (CO) were measured in echocardiogram recordings using the left ventricle and aortic arch views, respectively.

Results, Conclusions, and Discussions: The ECG during pacing, when compared to normal sinus rhythm (Fig 1A), showed both a missing P wave, indicating that the atria are not contracting before the ventricles, and a wider QRS complex, which is indicative of ventricular tachycardia (Fig 1B). Light pacing can be achieved with less than 2 mW/mm2 of irradiance at 617 nm, and rhythmic capture of the light pulses occurs between 8 and 11.5 Hz. Arrhythmic conditions occur when light is pulsed lower than or much higher than the resting heart rate. At 14 Hz, there is initial capture of the light pulses by the heart, and the heart rate reaches 14 Hz. However, it cannot sustain this rate, and a desynchrony between sinus and light-induced beats occurs, causing the heart rate to drop to 7 Hz. Echocardiogram analysis during this period reveals a significant drop in cardiac output during the pacing period (Fig 1C). When the heart is paced at 6 Hz, below its resting heart rate, competition between the sinus rhythm and light induced beats occurs. Here, the arrhythmicity index is elevated for the duration of the pacing period (Fig 1D). Additionally, the cardiac output is significantly decreased when compared to normal sinus rhythm (Fig 1C).
We have created a non-invasive, on-demand arrythmia model through cardiac optogenetics in mice, using transgenic ReaChR expressing mice and LED array illumination. LED array illumination provides deeper penetration of light into tissue while using a safe power density ( < 2 mW/mm2). Arrhythmic beating with a decrease in cardiac output is observed when pacing with frequencies lower than or much greater than the resting heart rate. This model will be extended further to better observe the effects of arrhythmia on blood flow in the brain using optical intrinsic signal imaging (OIS). Additionally, this approach could be used to stress the heart and assess the efficiency of different treatments in preventing arrythmia.

Acknowledgements (Optional): This work was supported by NIH R01-EB025209, R21-EB032684, and R01-HL156265.