Adult male rats (Sprague Dawley, weight 180–210 g) were randomized into the ChR2 group (n = 10) and control group (n = 10) who received adeno-associated virus (AAV) or empty control. All experimental procedures were approved by the Animal Care and Use Ethics Committee of Yijishan Hospital and performed in accordance with the Guideline for the Care and Use of Laboratory Animals of the National Institutes of Health's guiding principles.

All rats were placed in a spinal stereotaxic frame with forceps clamped to spinal processes C2 and T2 after anesthetizing with 10% chloral hydrate (0.3 g/kg body weight, i.p.). Spinal surgery was performed as previously described (21). AAV9-containing Ca(2+)/calmodulin-activated protein kinase II-α (CaMKIIα) prompter with or without -hChR2(H134R) (PackGene Biotech, 10¹³ vg/ml) was drawn up into a 10 μl Hamilton syringe with a glass pipette and pulled to a 20 μm tip (Hamilton, Shanghai, Ch). Then AAVs (3 μl) were injected into the dorsal horn (0.5 mm depth) under control by a stereotaxic micromanipulator through previously reported methods (11). The injection and needle retention were kept for 10 min to let the virus penetrate evenly into the dorsal horn (Figure 1A).

Needle electrodes were inserted into the left upper limb 4 weeks after virus injection to record electromyography (EMG) (100–30,000 Hz filters) offline (RM6240, Chengdu, China). After EMG implantation, the muscles were carefully dissected from C2 to T2. The animals were then clamped to the dorsal C2 and T1 processes of the spinal cord in the spinal stereotactic frame to prevent spinal cord movement during the whole process of optical stimulation (11) (Details were shown in Supplementary Materials). Optical stimulation was performed by a laser stimulator with blue light (473 nm, Newton Co., Ltd., Hangzhou, China) through a 200 μm diameter optical fiber. Before ischemia and reperfusion, the rats received optogenetic activation for 30 min (1 min laser on and 4 min laser off, repeated 6 times). Different stimulation parameters (0, 2, 5, 10, 20, 50, and 50 mW, 10 ms pulse at frequency of 1 Hz) were applied to each side of the dorsal horn. As shown in Figures 1D,E, EMG gradually increased with enhanced stimulation intensity. Stimulation parameters with a pulse width of 10 ms and intensity of 20 mW/m² were selected as the final study. According to previous reports, SCS with 90% of the motor threshold can protect the ischemic heart (22). In this study, we selected proper optical stimulation parameters which induce EMG but no limb movement (11).

According to our previous report, we created a rat model of myocardial ischemia-reperfusion (23). In brief, rats were subjected to occlusion of the coronary artery to induce myocardial ischemia for 15 min and then released for 30 min for reperfusion. Successful myocardial ischemia was judged by the ST-segment elevation on ECG and pallor changes in the heart immediately after ligation. Blue light stimulation was administered 30 min prior to ischemia.

Monophasic action potentials (MAPs) were recorded and analyzed using a Biosignal analysis software according to our previously developed method (RM6240, Chengdu, China) (24). In brief, a pair of electrodes were placed on the base and free wall of the heart, and the control electrode was connected to the skin. The MAP data were recorded and stored in a computer for further analysis (24, 25) (Figure 3A). APD90 was defined as the duration of 90% repolarization (APD90). APD dispersion (APDd) was calculated by subtracting the minimum APD from the maximum APD.

Electrocardiography (ECG) recording and arrhythmia identification complied with Lambeth Convention criteria (26). Ischemia-reperfusion induced arrhythmia analysis was used to calculate the duration of ventricular arrhythmia (VA), ventricular fibrillation (VF), and ventricular arrhythmia score, shown as follows: 0: < 10 ventricular premature contractions (VPCs); 1: ≥ 10 VPCs; 2: 1–5 episodes of ventricular tachycardia (VT); 3: > 5 VTs or 1 ventricular fibrillation (VF), 4: 2–5 episodes of VF; 5: > 5 episodes of VF; 6: VT, VF, or both for a duration ≤ 300 s; 7: VT, VF, or both for a duration > 300 s.

Cervical sympathetic nerve activity (CSNA) was recorded as previously described (2, 20). In brief, CSNA was recorded by a biological data acquisition system (RM6240, Chengdu, China) using two pairs of hook electrodes (2 mm interval) for offline further analysis. The CSNA amplitude was continuously recorded during baseline, light stimulation, myocardial ischemia, and reperfusion phases.

The fluorescent protein mCherry, the glutamatergic neuron marker VGLUT2, the GABAergic neuron marker GAD65, and the cholinergic neuron marker ChAT (VGLUT2, 1:200; GAD65, 1:300; ChAT, 1:100; Abcam) were used for colocalization analysis of the ratio of different neurons. Myocardial tissue sections were processed for immunofluorescence as before (24).

All data were expressed as means ± SD. Student t-test was used to evaluate the difference between the ChR2 and Control groups. A paired t-test was used to compare variables with or without optical stimulation. Significant differences among baseline, ischemia, and reperfusion were evaluated using one-way ANOVA, followed by a post-hoc Newman–Keuls multiple comparison test. Statistical analyses were performed using SPSS 16.0 (IBM, New York, USA), and P < 0.05 was considered significant.

As shown in Figure 1B, red signals of mCherry can be seen at the ipsilateral dorsal horn. However, there were only a few mCherry positive signals in the contralateral dorsal horn (Figures 1B,C). As shown in Figures 1D,E, an increased EMG was recorded at the left upper limb of rats. When the stimulation intensity was above 50 mW/mm, forelimb contraction could be observed in rats. Thus, stimulation parameters (20.3 ± 2.8 mW/mm, pulse width: 10 ms) were selected as subsequent spinal cord stimulation. As shown in Figures 1D,F, immunofluorescence double staining showed that most merged neurons were co-stained with the glutamatergic neuron marker VGLUT2 and mCherry(+). Only a few neurons were co-stained with GABAergic neuron marker GAD65. Almost no neurons co-expressed the cholinergic neuron marker ChAT with mCherry (Figure 1G).

As shown in Figure 2A, both myocardial ischemia and reperfusion-induced ventricular arrhythmias like VPCs, VT, and VF. Optical stimulation significantly reduced the VT durations during ischemia (12.6 ± 5.2 s vs. 79.2 ± 14.3 s, P < 0.05) and reperfusion (9.6 ± 3.6 s vs. 22.8 ± 4.9 s, P < 0.05; Figure 2B). Optical stimulation significantly reduces the arrhythmia score of VA during the ischemia (2.8 ± 0.8 vs. 5.7 ± 0.7, P < 0.01) and reperfusion (1.7 ± 0.7 vs. 3.9 ± 1.1, P < 0.05; Figure 2C).

During the myocardial ischemia and reperfusion, APD was significantly prolonged in the non-infarct zone (NZ) but shortened in the infarct zone (NZ) (Figures 3A–C). As a result, APD dispersion in the control group increased significantly during myocardial ischemia (47.7 ± 5.7 ms vs. 7.8 ± 1.5 ms, P < 0.01) and reperfusion (39.9 ± 6.2 ms vs. 7.8 ± 1.5 ms, P < 0.01). Compared with the control group, optical stimulation significantly reduced APD dispersion both in ischemia (21.2 ± 7.9 ms vs. 47.7 ± 5.7 ms, P < 0.01) and reperfusion phrase (24.2 ± 7.0 vs. 39.9 ± 6.2 ms, P < 0.01; Figure 3D).

Optical stimulation of the dorsal horn neurons significantly increased CSNA compared with the control group (55.3 ± 4.9 vs. 38.9 ± 6.6 μV, P < 0.05). CSNA significantly increased during myocardial ischemia and reperfusion in the control group (202.9 ± 47.7 vs. 33.6 ± 4.7 μV, P < 0.01; Figures 4A,B). However, optical stimulation of dorsal horn glutamatergic neurons significantly prevented CSNA during ischemia (82.6 ± 8.7 vs. 202.9 ± 47.7 μV, P < 0.01) and reperfusion (88.7 ± 8.7 vs. 235.3 ± 50.2 μV, P < 0.01; Figure 4B).