Elsevier

Heart Rhythm

Volume 18, Issue 1, January 2021, Pages 109-117
Heart Rhythm

Experimental
Optical capture and defibrillation in rats with monocrotaline-induced myocardial fibrosis 1 year after a single intravenous injection of adeno-associated virus channelrhodopsin-2

https://doi.org/10.1016/j.hrthm.2020.08.002Get rights and content

Background

Optogenetics uses light to regulate cardiac rhythms and terminate malignant arrhythmias.

Objective

The purpose of this study was to investigate the long-term validity of optical capture properties based on virus-transfected channelrhodopsin-2 (ChR2) and evaluate the effects of optogenetic-based defibrillation in an in vivo rat model of myocardial fibrosis enhanced by monocrotaline (MCT).

Methods

Fifteen infant rats received jugular vein injection of adeno-associated virus (AAV). After 8 weeks, 5 rats were randomly selected to verify the effectiveness ChR2 transfection. The remaining rats were administered MCT at 11 months. Four weeks after MCT, the availability of 473-nm blue light to capture heart rhythm in these rats was verified again. Ventricular tachycardia (VT) and ventricular fibrillation (VF) were induced by burst stimulation on the basis of enhanced myocardial fibrosis, and the termination effects of the optical manipulation were tested.

Results

Eight weeks after AAV injection, there was ChR2 expression throughout the ventricular myocardium as reflected by both fluorescence imaging and optical pacing. Four weeks after MCT, significant myocardial fibrosis was achieved. Light could still trigger the corresponding ectopic heart rhythm, and the pulse width and illumination area could affect the light capture rate. VT/VF was induced successfully in 1-year-observation rats, and the rate of termination of VT/VF under light was much higher than that of spontaneous termination.

Conclusion

Viral ChR2 transfection can play a long-term role in the rat heart, and light can successfully regulate heart rhythm and defibrillate after cardiac fibrosis.

Introduction

Optogenetics is a new biomedical technology that combines genetic and photocontrol technologies. The principle is use of virus vector or transgenic animal methods to transfer the photosensitive protein gene into excitable cells such as nerve cells, muscle cells, and glandular cells, and to express the photosensitive protein on the cell membrane.1,2 Because most photosensitive proteins are light-gated ion channels and ion pumps, activation of the protein using light in a specific wavelength range can induce ion flow both inside or outside the cell, which can cause target cells to depolarize or hyperpolarize.3, 4, 5, 6 Cardiovascular studies that have used optogenetic techniques for cardiac pacing, resynchronization, termination of arrhythmias,7, 8, 9 and electrophysiological function detection10, 11, 12 are emerging and have shown the prospects and potential for optogenetic applications in the clinic.

Arrhythmogenic right ventricular cardiomyopathy, pulmonary hypertension, chronic heart failure, and other cardiac diseases can cause myocardial collagen deposition and fibrosis. The viable cardiomyocytes and collagen interlace with each other, and interstitial fibrosis may disrupt the electrical coupling between cardiomyocytes to form discontinuous conduction or ventricular reentry, promoting the development and spread of ventricular arrhythmias.13 There are no specific antifibrosis drugs, and the effective termination of malignant ventricular arrhythmia (MVA) caused by myocardial fibrosis is a problem worth considering. Researchers have used optogenetics to terminate ventricular tachycardia (VT)/ventricular fibrillation (VF) in vitro or in vivo after acute myocardial infarction.14,15 Light defibrillation has the advantages of no contact, easy pacing site (lighting area is the pacing site), and real-time synchronous excitation (spot area is synchronous excitation area) due to the noncontact nature of light propagation, the flexibility of beam movement, the plasticity of spot size and shape, and the nonspreading of the light focus.16, 17, 18, 19 Termination of VT/VF by optogenetics may be another important option to restore the heart’s electrophysiological function in future cardiovascular treatment.

In this study, we clarified the long-term stability of channelrhodopsin-2 (ChR2) transfection and tested the effects of light defibrillation after increasing myocardial fibrosis. We selected and observed rats for 1 year after viral injection during infancy, and we injected monocrotaline (MCT) to increase the degree of myocardial fibrosis and change cardiac morphology at 11 months as subjects for an in vivo study of light pacing and defibrillation. In addition, we selected the most common photosensitive protein ChR2 mutant ChR2(H134R) as the actuator, which can be irradiated by a 473-nm blue laser to realize the ion transient of the photosensitive current and generate the corresponding electrophysiological effect.20,21 ChR2(H134R) was transferred into the intact heart using adeno-associated virus 2/9 (AAV2/9).22

Section snippets

Materials and methods

The expanded materials and methods section can be found in the Supplemental Material and Supplemental Figure 1.

Phase evaluation of light stimulation and histologic results

Eight weeks after virus injection, the results showed that all parts of the rat heart could be captured using 473-nm blue light. This result showed that AAV transfection with ChR2 was effective. We obtained the light intensity thresholds needed to achieve complete rhythm capture at different pulse widths (Figures 1A–D). Immunofluorescence results showed that red immunofluorescence-labeled ChR2 was evenly distributed in the ventricles (Figure 1E). This was consistent with the in vivo light

Discussion

Cardiac optogenetics is a technique that uses light to regulate photocurrent production in cardiomyocytes expressing either excitatory or inhibitory photosensitive proteins. This study observed the long-term efficacy of AAV transfection with ChR2 and the functional validity of ChR2. We showed that the heart can be captured using light to perform light defibrillation for a long time.

In contrast to electrical pacing that raises the membrane potential above a threshold potential by giving an

Conclusion

AAV carrying the ChR2 gene enabled rats to survive and respond to light after 1 year with changes in heart morphology. In addition, light can increase the termination rate of ventricular arrhythmia on the basis of myocardial fibrosis.

Acknowledgment

The authors are grateful for kind support from Hubei Key Laboratory of Cardiology, Wuhan, China.

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    Funding sources: This work was supported by the National Natural Science Foundation of China (No. 81772044), Hainan Provincial Innovative Research Team Project (No. 2016CXTD012), and Hainan Provincial Personnel Support Project (No. 2019RC368).

    Disclosures: The authors have reported that they have no relationships relevant to the contents of this paper to disclose.

    1

    Drs Jianyi Li and Long Wang contributed equally to this study.

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