Electroacupuncture Suppresses Premature Ventricular Complexes Occurring Post-myocardial Infarction through corticothalamic circuit

doi:10.21203/rs.3.rs-4473024/v1

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Electroacupuncture Suppresses Premature Ventricular Complexes Occurring Post-myocardial Infarction through corticothalamic circuit

https://doi.org/10.21203/rs.3.rs-4473024/v1

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Electroacupuncture (EA) has been shown to suppress premature ventricular complexes (PVCs) following myocardial infarction (MI) in humans. However, the specific neural circuitry and causal mechanisms underlying this effect remain unclear. Here, we reveal a previously unrecognized connection from the primary motor cortex (M1) to the nucleus rostral ventrolateral medulla (RVLM) circuitry via the layer 5 of the primary motor cortex (M1L5)-zona incerta (ZI) pathway, which selectively suppresses PVCs in post-MI mice. Utilizing viral tracing, fiber photometry recordings, and optogenetic stimulation, we demonstrate that EA inhibits glutamatergic projections from M1L5 to ZI, leading to the activation of local GABAergic neurons and subsequent inhibition of RVLM (M1L5-ZI-RVLM). Furthermore, optogenetic or chemogenetic inhibition of the M1L5-ZI-RVLM circuit replicates the anti-PVC effects observed with EA in MI mice. Artificial activation of M1L5-projecting ZI neurons reverses the suppressive effects of EA on PVCs in MI mice. Overall, our findings highlight the M1L5-ZI-RVLM circuit as a crucial mediator of EA-induced suppression of PVCs following myocardial infarction. Additionally, this newly identified corticothalamic circuit may represent a promising target for mitigating PVCs post-myocardial infarction.

Biological sciences/Neuroscience/Neural circuits

Health sciences/Neurology

Electroacupuncture

myocardial ischemia

Primary motor cortex

premature ventricular complex

CNS

EA suppresses PVCs occurring post-MI

HT7 (Shenmen) acupoint afferent nerves drive a brain-to-heart circuit through M1L5

EA reduces hyperactivity of M1L5 neurons in MI model mice

EA-induced suppression of PVCs occurring post-MI via M1L5-ZI-RVLM circuit

Premature ventricular complexes (PVCs) following myocardial infarction (MI) pose a significant risk factor for subsequent sudden cardiac death (SCD)^1–3. While treatments over the past decade have encompassed antiarrhythmic drug therapy, defibrillation, or ablation, early-stage use of antiarrhythmic drugs such as encainide, flecainide, or moricizine has been associated with increased rates of SCD and overall mortality⁴. Consequently, effectively mitigating PVCs post-MI remains a formidable scientific challenge.

Bioelectronic medicine therapy, which includes nerve electrical stimulation, has emerged as a promising alternative. Acupuncture, a practice with roots dating back over 2000 years in China and now globally recognized, is increasingly utilized as a form of bioelectronic medicine therapy. Electroacupuncture (EA), in particular, has demonstrated efficacy in alleviating PVCs post-MI, ameliorating disorders of the sympathetic system, and reducing the risk of SCD^5–8. The tachycardias precipitating cardiac arrest are primarily driven by abnormal sympathetic excitation^9,10. Human studies have shown that electroacupuncture can regulate abnormal states of autonomic nerves^11,12. Furthermore, expert consensus recommends acupuncture as an effective adjunctive therapy for regulating heartbeat rhythm in arrhythmia patients¹³. Leveraging the somatosensory autonomic nervous system, acupuncture's effects can extend to distant areas^14,15. The inherent characteristics of EA effects have led to the hypothesis that they improve PVCs post-MI via modulation of the autonomic nervous system. However, the precise mechanisms underlying these effects remain to be elucidated.

Shenmen (HT7), an acupoint situated in the anterior carpal region at the ulnar end of the distal metacarpal crease and along the radial margin of the flexor carpi ulnaris tendon (Fig. S1A), has been specifically targeted for the treatment of myocardial ischemia, particularly post-myocardial infarction PVCs ^16,17. Alterations in activities in various brain regions involved in sensation and visceral motor processing have been revealed in functional magnetic resonance imaging (fMRI) studies with individuals receiving EA treatment at HT7¹⁸. The thalamus serves as a relay for multimodal sensory information and reciprocally connects with the sympathetic nervous system¹⁹. Within the thalamus, the zona incerta (ZI) receives diverse inputs from cortical and subcortical regions²⁰, suggesting a potential role for the ZI as a conduit for somatosensory processing during EA. Nevertheless, the specific organization of cell types and the functions of corticothalamic circuits underlying EA-induced suppression of post-myocardial infarction PVCs remain largely unexplored.

In the present study, we investigated the functional organization of the M1L5-ZI-RVLM circuit and elucidated its role in suppressing post-MI PVCs in response to EA stimulation. Artificial manipulation of this circuitry can replicate or abolish the effects of EA-induced suppression of post-MI PVCs. Our findings indicate that inhibition of the M1L5-ZI-RVLM circuit is essential for the manifestation of EA's suppressive effects on post-MI PVCs.

EA suppresses PVCs depends on balance sympathetic nervous system in mice

We investigated whether stimulation of the HT7 acupoint by EA suppresses post-myocardial infarction PVCs in mice. Mouse models of premature ventricular complexes were induced by ligation of the left anterior descending coronary artery (MI) (Fig. 1A). Male mice were the focus of our study unless otherwise specified. At 3 hours post-ligation, mice exhibited a significant increase in PVCs and ST-segment elevation compared to sham-operated mice (Fig. S1B, S1C). To assess the effect of different EA intensities on post-MI PVC suppression in mice, we evaluated the impact of EA treatment at two intensities (0.5mA or 3mA). We found that only an EA intensity of 0.5mA elicited PVC suppression in MI mice (Fig. 1B-D). Additionally, the 0.5mA EA intensity reduced the QRS width in MI mice (Fig. 1E). It is noteworthy that QRS width reflects ventricular electrical activity and exhibits abnormal prolongation during ventricular arrhythmias²¹.

We investigated whether EA suppresses PVCs in MI mice by potentially reducing sympathetic nerve activation. The autonomic nervous system significantly contributes to the development of PVCs^22,23. In human studies, inhibiting abnormal sympathetic excitation after myocardial ischemia effectively reduces PVC occurrence²⁴. We conducted heart rate variability (HRV) analysis and detected cardiac norepinephrine (NE) content to assess the potential effects of EA (0.5mA, 2Hz, pulse width of 50 μs) on the cardiac sympathetic component of PVCs occurring post-MI in mice. Our findings indicate that compared to the MI group, EA treatment significantly attenuated cardiac sympathetic excitability (Fig. 1F, 1G) (p = 0.0045, Sham: n = 7; MI: n = 7; EA: n = 6), reduced cardiac NE content (Fig. 1H) (p = 0.0053, n = 6), improved survival rate in MI mice (Fig. 1I) (p = 0.006, Sham: n = 8; MI: n = 11; EA: n = 13), and decreased myocardial damage in MI mice (Fig. 1J) (AAR/LV: p = 0.0027; n = 13). We also observed that EA 0.5mA reduced QRS width (p = 0.026; n = 6) and ST deviation (p = 0.0319; n = 6) in MI mice during electrocardiogram (ECG) analysis. However, it did not affect RR interval, heart rate, PR interval, QTc interval, and R amplitude (Fig. S1H, S1I). Increased cardiac sympathetic activity can induce severe PVCs during MI^25,26, which worsens damage to myocardial cells and fibrosis. Furthermore, activated myocardial fibrosis perpetuates PVC occurrence^27,28. Several studies have shown that reducing excessive cardiac sympathetic nerve activation is crucial for improving cardiac function and reducing PVCs^9,29.

We then examined ventricular infarction and fibrosis levels through histological analysis in MI mice following EA treatment. We observed reductions in cell atrophy and nuclear shrinkage proportions, cross-section of cardiomyocytes, interstitial congestion, and inflammatory cell infiltration (Fig. S1D), and a decrease in cell fibrosis in the MI+EA 0.5mA group compared to the MI group (Fig. S1E, S1F) (p = 0.0076, n = 6). Furthermore, considering the unique structure and restricted expression of cardiac troponin T (cTnT) and creatine kinase (CK)-MB by heart myocytes, they are utilized as biomarkers for acute myocardial injury. Hence, we assessed the expression of cTnT and CK-MB in myocardial tissue. Our results indicated significantly lower expression of cTnT (Fig. 1K) (p = 0.0088, n = 6) and CK-MB (Fig. S1G) (p = 0.0083, n = 6) in the MI+EA 0.5mA group compared to the MI group. These findings suggest that EA at 0.5mA effectively reduces PVCs and substantially mitigates risk of sudden cardiac death in MI mice.

HT7 (Shenmen) acupoint afferent nerves drive a brain-to-heart circuitthrough the Layer 5 neurons in motor cortex (M1L5)

We then investigated the neural circuitry underlying the observed suppression of PVCs following post-MI induced by EA. Given the pivotal role of the primary cortex in processing and integrating afferent somatosensory inputs^30,31, we examined the neurons receiving somatosensory input from EA (HT7 acupoint) sites to the primary cortex. We employed herpes simplex virus (HSV) as a tracer, as it selectively targets somatosensory neurons and can traverse the somatosensory circuit. Considering the widespread distribution of somatosensory neurons at the HT7 acupoint in the dorsal root ganglion (DRG) of the T3 spinal cord segment (Fig. S2C, S2D, S2E, S2F), we performed microinjections to directly administer the virus into the T3 DRG, ensuring a high concentration around the soma (Fig. 2A, 2B). Additionally, to mitigate immune elimination by the host, we combined DRG microinjection with bortezomib to enhance HSV infection in T3 DRG neurons³² (Fig. S3A). Our observations revealed that HSV requires a minimum of 48 hours to transport from the soma of DRG neurons to the central terminals (Fig. S3B). Secondary spinal cord neurons can be labeled approximately 72 hours after viral injection (Fig. S3C), after which the virus spreads along the ascending pathway and gradually labels the downstream neurons (Fig. S3D). We found that S1 and M1 neurons of the cortical area were labeled by HSV (green) (Fig. 2C, 2D) (p = 0.9383, n = 6).

The M1 serves as a crucial command center in somatosensory signal processing. Recent studies have suggested the possibility that dorsal root ganglion neurons directly transmit sensory signals to the motor cortex through ascending transmission of spinal cord projection neurons^33,34. To verify whether afferent somatosensory nerves from the HT7 acupoint project to the M1L5 via a direct spino-cortical circuit, we injected the EnvA-pseudotyped glycoprotein (G)-deleted rabies virus (EnvA-RV-ΔG-eGFP)21 after expressing AAV2/9-DIO-RVG-TVA-mCherry for 3 weeks in the M1 of mice, with fluorogold (FG) injected at the HT7 acupoint site (Fig. 2E). We observed a cluster of eGFP-labeled neurons located in the IV to VI layers of the T3 thoracic spinal cord. Remarkably, in the same animals, FG-positive fibers were also identified in the IV to VI layers of the T3 thoracic spinal cord (Fig. 2F, 2G). Conversely, no labeled neurons were observed in the dorsal horn of the T3 spinal cord after injecting AAV2/9-DIO-TVA-mCherry alone into the layer 5 neurons in M1 (Fig. 2H, 2I). These findings suggest that afferent somatosensory nerves from the HT7 acupoint relay information to the M1L5. Furthermore, the direct spinal cortical pathway may be implicated in the transmission of HT7 acupoint afferent somatosensory nerves.

Although the HT7 acupoint's afferent somatosensory nerves sending projections to the M1L5 in mice are well established, it remains unknown whether the M1L5 can modulate sympathetic outflow to the heart. To identify cortical neurons involved in heart control and sympathetic nervous regulation, we initiated experiments by injecting pseudorabies virus (PRV) 531 encoding EGFP into the left ventricular wall of C57BL/6J mice to label upstream neurons retrogradely and trans-synaptically (Fig. 2J). Validating this method, we began observations 80-90 hours post PRV injection, revealing the emergence of viral labeling in subcortical areas previously implicated in cardiac function (Fig. S2A).

Critically, at 110-120 hours post PRV injection, A small cluster of EGFP-positive neurons was consistently observed in the bilateral primary motor cortex (M1; bregma: 0.20 to –0.85 mm, lateral from midline: ±0.78 to ±1.36 mm) (Fig. 2K). Cell counting indicated that the labeled cortical region was situated in M1 rather than the primary sensory cortex (S1) (Fig. 2L, 2M) (p < 0.0001, n = 8). In contrast, control animals receiving the same PRV injection into the chest or other viscera showed no labeled neurons in these regions (Fig. 2N, 2O, and Fig. S2B). Moreover, the overwhelming majority of EGFP+ neurons in M1 (~95%) were localized in layer 5 and exhibited the characteristic morphology of projecting pyramidal neurons. Through immunofluorescence identification, we found that the upstream M1L5 pyramidal neurons of the heart primarily co-labeled with glutamatergic antibodies rather than GABAergic antibodies (Fig. 2P, 2Q) (p < 0.0001, n = 6). Based on these findings, the M1L5 glutamatergic neuron (M1L5^Glu) links HT7 acupoint afferent somatosensory nerves to the mice heart sympathetic outflow center.

The necessity of cardiac sympathetic regulation by M1L5 ^Glu

For a long time, the ventrolateral region of the rostral medulla (RVLM), housing cardiac sympathetic premotor neurons, has been considered pivotal in cardiovascular disease³⁵. Subsequently, we investigated c-Fos expression in the RVLM, observing an increase in c-Fos levels in the RVLM of post-MI mice compared to sham mice, with this phenomenon reversed by EA treatment (Fig. 3A, 3B) (p = 0.0439, n = 6). To further analyze whether M1L5 activation can induce RVLM activity and augment sympathetic outflow, we chemogenetically activated glutamatergic (Glu) neurons in M1L5, significantly increasing c-Fos expression in RVLM neurons (Fig. 3C, 3D) (p = 0.0019, n = 4).

Next, we investigated whether stimulation of M1L5^Glu input to the heart alters cardiac sympathetic activity. Specifically, we conducted optogenetic manipulations that were temporally restricted using the CaMKIIα promoter-driven expression of Channelrhodopsin (ChR2). Optogenetic stimulation of Glu neurons in M1L5 in WT mice injected with AAV1-CaMKIIα-ChR2-mCherry (20Hz, 5ms, 3-5mw/mm³) significantly increased. We also verified ChR2 cells co-expression and anti-glutamate positive cells (Fig. S4A, S4B), heart rate (Fig. 3E, 3F), and the HRV LF/HF ratio (an indication of cardiac sympathetic nerve excitation) (Fig. 3G, S4C) in male mice. Additionally, the score of cardiac sympathetic nerve activity (Fig. S4E) and the content of NE in cardiac tissue increased after optogenetic stimulation (Fig. 3H) (p = 0.0034, n = 7). Conversely, there was no change in heart NE levels in mice injected with the control virus at the end of 20 minutes of optogenetic stimulation. These data suggest that M1L5^Glu represents a potential target for EA to suppress ventricular extrasystole after myocardial infarction.

The effect of electroacupuncture on PVCs occurring post-MI mice depends on the M1L5^Glu neurons

Based on our aforementioned findings, we sought to examine changes in M1L5^Glu activity in MI mice induced by EA stimulation. We conducted c-Fos staining, a marker for neuronal activation, on M1L5 neurons upstream of the heart (Fig. 4A). We observed increased c-Fos expression in M1L5 pyramidal neurons from MI mice. Interestingly, c-Fos expression was reduced in MI+EA mice compared with MI mice (Fig. 4B, 4C) (p = 0.0010, n = 18). To further investigate M1L5 neuron activity in MI+EA mouse models, we performed in vivo electrophysiological recordings (Fig. 4D). Recorded M1L5 neurons were classified as putative excitatory pyramidal neurons (Eps, trough to peak duration 406.4 ± 3.312 μs, n = 154) and putative inhibitory interneurons (INs, trough to peak duration 593.3 ± 9.399 μs, n = 113) using unsupervised clustering techniques (Fig. 4E) and cross-correlogram analyses (Fig. S5A, S5B) based on the area under peak, trough to peak duration, and firing rate³⁶. Interestingly, we observed an increase in the spike action potential firing rate of M1L5^Eps neurons in MI mice compared to sham mice, and this effect was reversed by EA treatment (Fig. 4F) (p = 0.0044, Sham: n = 53; MI: n = 27; MI+EA: n = 37).

To delve deeper into the in vivo neuronal activity of M1L5 glutamatergic neurons following EA treatment, we conducted fiber photometry recordings in MI+EA mice by infusing an adeno-associated virus (AAV) expressing the fluorescent Ca²⁺ indicator GCaMP6m (AAV-CaMKIIα-GCaMP6m) into M1L5 (Fig. 4G, 4H). We observed that under EA treatment (0.5mA, 2Hz, pulse width of 50 μs), the fluorescence intensity of GCaMP6m-expressing neurons in MI mice was lower than before treatment (mean ΔF/F(%), p = 0.0107; calcium events, p = 0.0062, n = 7) (Fig. 4I, 4J, 4K), but this effect was not observed under 3mA EA intensity (Fig. S5D, S5E, S5F). Additionally, to mitigate the effects of isoflurane, we conducted fiber photometry recordings in freely moving MI mice (Fig. S5G). We found that the average ΔF/F(%) and calcium events of M1L5 neurons in MI+EA mice were significantly lower than those in MI model mice (mean ΔF/F(%), p = 0.0021; calcium events, p = 0.0024, n = 7) (Fig. S5H, S5I, S5J, S5K). Microendoscopic calcium imaging (Fig. 4L, 4M) revealed that the fluorescence intensity (p = 0.0221, Sham: n = 47; MI: n = 122; EA+MI: n = 95) (Fig. 4N, 4O) and spontaneous calcium event rates (p = 0.0434) (Fig. 4P) from M1L5 neurons were significantly enhanced in MI mice compared with those in sham mice, and this enhancement was reversed by EA treatment.

The aforementioned studies consistently demonstrate a heightened firing rate in M1L5 Glu neurons during PVCs, which was effectively reversed by EA treatment. To ascertain the necessity of M1L5 neurons in EA-mediated suppression of PVCs in MI mice, we employed a pharmacogenetic approach to selectively activate bilateral M1L5^Glu neurons (Fig. 4Q). Specifically, we utilized an AAV vector to deliver hM3Dq, a designer receptor exclusively activated by the inert agonist clozapine-n-oxide (CNO) into the M1L5 region of mice (AAV-CaMKIIα-hM3Dq-mCherry)³⁷.

Subsequently, we assessed the therapeutic efficacy of EA in MI mice following the pharmacogenetic activation of M1L5^Glu neurons by analyzing PVC counts (refer to Fig. 4R), LF/HF ratio (Fig. 4S), QRS width (Fig. 4T), and NE levels (Fig. 4U). Intriguingly, our findings indicate that the EA-induced reduction in PVCs and attenuation of cardiac sympathetic excitability in MI mice were significantly impeded upon activation of M1L5^Glu excitatory neurons.

The ZI receives direct inputs from M1L5^Glu neurons

The primary motor cortex (M1) has been extensively studied for its role in regulating the autonomic nervous system and its involvement in motor initiation processes^38,39. Notably, the cerebral cortex appears to be crucial in activating somatic-visceral reflexes induced by electroacupuncture. Studies utilizing intracortical recordings and transcranial magnetic stimulation have revealed that the perception of cardiac activity triggers responses in the human primary motor cortex⁴⁰. One of the most distinctive features of the cerebral cortex is its reciprocal connections with the thalamus and midbrain^41,42. In order to elucidate the acupoint (HT7)-heart pathway between these regions, we investigated the functional connections of various cortical circuits. Initially, an AAV expressing channelrhodopsin-2 (AAV-CAMKIIα–ChR2–mCherry) was administered into the M1L5 of wild-type mice (Fig. 5A). Subsequently, three weeks later, we observed mCherry⁺ fibers in the ZI (Fig. 5B) and in the ventrolateral periaqueductal gray (vlPAG) of the midbrain (Fig. S6A). Notably, anterograde viral tracing experiments revealed that the RVLM does not receive direct fiber projections from M1L5^Glu (Fig. S6B), indicating indirect downstream modulation.

To further elucidate this pathway, we initially expressed ChR2 in all Glu neurons of the M1L5 and optogenetically stimulated M1 projection fibers to the ZI (Fig. S6C) or the vlPAG (Fig. S6D). Remarkably, we observed that optogenetic stimulation of M1L5^Glu-ZI, but not M1L5^Glu-vlPAG, increased the expression of c-Fos (a neuronal activity marker) in the RVLM (Fig. S6E). Additionally, optogenetic stimulation of M1 projection fibers to the ZI (Fig. S6F) elevated the HRV LF/HF ratio in male mice (Fig. S6G, S6H), and increased the content of NE in cardiac tissue (Fig. S6I, S6J).

To elucidate the organization of the M1L5-ZI connection, we utilized a retrograde trans-monosynaptic tracing system employing a modified rabies virus (EnvA-pseudotyped RV-△G-eGFP) and Cre-dependent helper viruses (AAV-Ef1a-DIO-TVA-GFP and AAV-Ef1a-DIO-RVG) in mice (Fig.5C). Incorporation of these helper viruses facilitated the monosynaptic retrograde spread of the RV. Neurons intensely labeled with eGFP were identified in several brain regions, including the anterior cingulate cortex (ACC), primary somatosensory cortex (S1L4), primary visual cortex (V1L5), and bilateral primary motor cortex (M1L5) (Fig.S7A-C). Notably, the eGFP+ signal was predominantly observed in layer 5 of M1, co-localizing with the glutamate-specific antibody signal, while being absent in layers 1, 2, 3, 4, and 6 (Fig.5D,E).

Despite these observations, there exists a paucity of knowledge regarding the cellular circuitry underlying the modulation of the ZI by M1, a field which remains unexplored. The ZI, a subthalamic structure conserved across mammals, primarily comprises GABAergic neurons exerting widespread inhibitory influence throughout the brain⁴³. ZI neurons display functional heterogeneity, exhibiting specificity in modulating various behaviors such as defensive behaviors^44,45, binge eating⁴⁶, hunting⁴⁷, and sleep^48,49, contingent upon their neurochemical properties and subdivisions across different anatomical domains. Considering potential tracer biases and limitations associated with viral tropism, we conducted retrograde tracing using retro-AAV-YFP virus injected into the ZI. Subsequently, we observed labeled neurons in layer 5 of the M1 (Fig. 5N), while layer 6 of the M1 remained devoid of labeled neurons.

Several observations collectively suggest that M1 activation primarily enhances output from the ZI to non-GABAergic neurons. Firstly, employing a viral strategy for fluorescent labeling of synaptic projection targets and GABAergic neurons, we identified that GABA neurons in the ZI predominantly reside in ZIv. Most ZI neurons receiving monosynaptic inputs from the M1 mainly consist of non-GABAergic neurons from ZId and are not co-labeled with GABA neurons in ZIv (Fig. 5F, 5G). Notably, employing an anterograde trans-monosynaptic tracing system to track downstream neurons of M1L5^Glu-ZId^non-GABAcircuit revealed that ZIv^GABAneurons received fiber projections from M1L5^Glu-ZId^non-GABA. Secondly, optogenetic stimulation of the M1 in mice with MI led to increased c-Fos expression in Zid but not in Ziv, which was non-GABAergic neuron-specific (Fig. 5H, 5I) (Fig. S7D, S7E). In summary, GABAergic neurons exhibited a decrease in c-Fos expression following M1 stimulation, suggesting modulation within the local ZI microcircuit.

To elucidate the reciprocal modulation of neuronal microcircuits in ZI (Fig. 5J), we administered AAV carrying eNpHR4.0 (AAV-CAMKIIα-eNpHR-mCherry) into the M1L5 of MI mice, enabling optical inhibition of M1L5^Glu terminals in ZI (Fig. 5K). A significant presence of mCherry nerve fibers in ZI was observed. Immunostaining revealed that c-Fos primarily colocalized with the GABA antibody (Fig. 5L). Furthermore, we selectively monitored the responses of ZI^GABA neurons in freely behaving mice following optogenetic activation or inhibition of the M1L5^Glu-ZI circuit. To achieve this, we delivered Retro-AAV1-CAMKIIα-NpHR4.0-mCherry/Retro-AAV1-CAMKIIα-ChR2-mCherry and AAV-GAD2-GCaMP6m virus into the ZI of mice. Fiber photometry recordings demonstrated a notable decrease in fluorescence intensity observed in GCaMP6m-expressing ZI^GABA neurons after optogenetic stimulation of ChR2-expressing M1L5^Glu neurons (Fig. 5M). Concurrently, the activity of ZI^GABA neurons increased after optogenetic inhibition of M1L5^Glu-ZI (Fig. 5O). These findings unveil modulation within local ZId-ZIv microcircuits (Fig. 5P).

M1L5-ZI-RVLM circuit regulates cardiac sympathetic and mediates EA to reduce PVCs occurring post-MI in mice

We investigated the impact of EA on ZI^GABA neurons in mice with MI (Fig. 6A). AAV-GAD2-GCaMP6m virus was introduced into the ZI, and optic fibers were implanted above these regions in MI mice (Fig. 6B). By examining the fluorescence intensity of GCaMP6m-expressing ZI^GABA neurons, we established a direct correlation between their activity and EA stimulation. Notably, EA significantly increased the fluorescence intensity of these neurons (mean △F/F (%), p = 0.0002; calcium events, p = 0.0048; n = 6) (Fig. 6C, 6D, 6E). Subsequently, we bilaterally administered AAV2/9-CAMKIIα-hM3dq-mCherry into the M1L5 region, activating M1L5^Glu neurons chemogenetically with CNO via intraperitoneal injection prior to EA stimulation (Fig. S9A, S9B). Interestingly, we observed that the EA-induced increase in ZI^GABA neuron activity in MI mice was inhibited (mean △F/F (%), p = 0.0004; calcium events, p = 0.0028; n = 7) (Fig. S9C, S9D, S9E, S9F). Concurrently, the EA-induced reduction in c-Fos expression in RVLM neurons was abolished (Fig. S9G). Given previous findings suggesting the presence of non-GABA neurons in the ZI, we deemed it crucial to verify that ZIGABA neuron activation resulted from EA stimulation rather than technical artifacts or isoflurane effects.

To address this, we utilized Fos-targeted recombination in active populations (Fos-TRAP) labeling technology to selectively identify EA-activated neurons in a time-dependent manner. Recombinant adeno-associated viruses rAAV-TRE-tight-mCherry and rAAV-c-Fos-tTA were injected into the ZI (Fig. 6F). To validate Fos-TRAP labeling system reliability, we compared mCherry expression in neurons across different conditions (Fig. 6G). Following a 30-day period of drinking non-Dox water, approximately 57.27±4.09% of ZI neurons exhibited mCherry expression. Conversely, Dox treatment resulted in only about 1.87±0.15% of ZI neurons expressing mCherry, confirming effective TRE promoter suppression by Dox. Furthermore, 12.72±2.09% of ZI neurons expressed mCherry three days after EA stimulation at the HT7 acupoint in MI mice (Fig. 6I, 6J). Notably, there was no significant difference in water consumption between Dox water-drinking mice and non-Dox water-drinking mice(Fig. 6H). Subsequent immunofluorescence staining revealed that approximately 60.27±9.64% of the mCherry signal colocalized with a specific GABA antibody (Fig. 6K), confirming the activation of ZI^GABA neurons following EA.

We then proceeded to examine the intricate circuits connecting the ZI to the medulla. Employing sparse neuronal type-specific labeling, we introduced rAAV-EF1a-DIO-Ypet-2A-mGFP-WPRE-pA and AAV2/9-Vgat-Cre virus into the ZI, facilitating selective labeling of ZI^GABA dendritic spines. Subsequently, mGFP⁺ fibers were observed in various brain regions (Fig. 6M and Fig. S8A). Given our previous investigation demonstrating the reversal of cardiac sympathetic nerve hyperexcitability in MI mice through EA, we correlated this with the well-documented projections of the M1, particularly emphasizing the RVLM (Fig. 6L). This discovery is intriguing, given the ZI's role as an integrative hub for modulating sensory integration, behavioral control, and visceral activity regulation^43,50,51.

To verify the ZI-RVLM projection, we infused AAV-DIO–eGFP virus into the ZI and AAV-hsyn-Cre virus into the RVLM of C57BL/6J mice, leading to the identification of eGFP-expressing neurons in the RVLM (Fig. 6N). However, the specific neuronal types in the ZI projecting to the RVLM remained unknown. Therefore, we conducted anterograde trans-monosynaptic tracing by administering AAV1-Vgat-cre/AAV-DIO-TK-mRFP helper virus and HSV-△TK-eGFP into the ZI of mice. Remarkably, we found that RVLM primarily receives fiber projections from ZI^GABA neurons (Fig. 6O and fig. S8B). Subsequently, we employed a retrograde trans-monosynaptic tracing system, introducing modified rabies virus (EnvA-pseudotyped RV-△G-eGFP) into the RVLM along with Cre-dependent helper viruses (AAV-Ef1a-DIO-TVA-mCherry and AAV-Ef1a-DIO-RVG) in mice. Our results revealed relatively abundant eGFP signals in the ventral part of the Zona Incerta (ZIv) but scarcity in the dorsal part (ZId) (Fig. 6P). Notably, no eGFP signal was detected in M1L5.

To explore the functional connections within the M1L5^Glu-ZI^GABA-RVLM circuit, we administered Retro-AAV-CaMKIIα-ChR2 virus and AAV-Vgat-hM3dq-mCherry virus into the ZI of C57 mice (Fig. 7A). Using fiber photometry recordings, we stimulated ChR2-containing upstream ZI cells in M1L5 and observed an increase in △F/F and calcium events in the RVLM. Notably, these responses were attenuated by chemogenetic activation of ZI^GABA neurons (Fig. 7B-D). These observations elucidate the microcircuitry wherein ZI^GABA neurons receive innervation from local ZI non^-GABA interneurons, both of which directly receive inputs from M1L5^Glu neurons. Given that premotor neurons for cardiac sympathetic activity are located in the RVLM, and M1L5 mediates the somatosensory effects of acupuncture at the HT7 point, we investigated whether the functional connection of the M1L5^Glu-ZI^GABA-RVLM circuit contributes to the anti-PVCs effects induced by 0.5mA EA in MI mice.

To this end, we infused AAV2/9-Vgat-hM3Dq-mCherry & Retro-AAV-CaMKIIα-ChR2 virus into the bilateral ZI and implanted optic fibers above the ipsilateral M1L5 regions in MI mice (Fig. 7E). Upon optogenetic activation of the M1L5Glu-ZI pathway, we observed a blockade in the EA-induced anti-PVCs effect in MI mice (Fig. 7F), along with a blockage in the reduction of cardiac sympathetic excitability induced by EA. However, simultaneous activation of both M1L5Glu and ZIGABA neurons reduced PVCs in MI mice subjected to EA (Fig. 7G-I). Subsequently, we investigated the potential physiological characteristics of the M1L5^Glu-ZI^GABA-RVLM pathway in anti-PVCs in MI mice (Fig. S9H, S9I). The 0.5mA EA-induced reduction in PVC counts in MI mice was replicated upon optogenetic inhibition of the M1L5Glu-ZI pathway and was blocked upon neural activation of the RVLM (Fig. S9J-S9O).

The neural pathway underlying the regulation of cardiac function via acupuncture at the HT7 point has long been enigmatic, with an intricate interplay between somatosensory systems and internal organs. Our investigation has delineated a complex polysynaptic circuit comprising M1L5-ZI-RVLM neurons, pivotal in conveying HT7 afferent signals to modulate sympathetic outflow and, consequently, heart function. Specifically, this M1L5-ZI-RVLM pathway we've elucidated suppresses post-myocardial infarction PVCs in response to EA stimulation, underscoring its role in the cardioprotective effects of HT7 activation in MI-afflicted mice.

Employing HSV for polysynaptic tracing of HT7 acupoint somatosensory neural networks has proven highly effective. HSV's broad host range, particularly its robust amplification and propagation efficiency in mice⁵², establishes it as the optimal tracer for such investigations. Nonetheless, variations in host immunity necessitated the use of the immunosuppressant bortezomib⁵³ to enhance HSV infection rates consistently. While our focus primarily centered on HSV-labeled cases within the S1 and M1 regions, intriguingly, our observations revealed the involvement of not just the S1 but also the M1 in the reception and processing of sensory input from the HT7 acupoint.

For tracking autonomic motor networks, PRV emerged as the preferred tracer. Our investigation identified a distinct cluster of M1L5 pyramidal neurons, primarily located upstream of the heart in the cortex, emphasizing the sensory-cardiac autonomic motor connection specifically within the M1L5 region. Notably, recent human imaging studies have corroborated a parallel pattern of sensory-motor integration within the M1L5 cortex, further substantiating our findings. In essence, the intricate intertwining⁵⁴ of sensory and motor pathways, forged through evolutionary processes, emerges as a fundamental mechanism underpinning life's development.

Recent research has shed light on the influence of transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS) on the modulation of sympathetic outflow to the heart through motor cortex activity⁵⁵. While the corticothalamic circuit appears to be a key player in PVCs⁵⁶, the specific organization of corticothalamic connections by cell types and their roles in PVCs remain largely unexplored.

This study aims to investigate the involvement of M1 neurons in PVCs using a mouse model of myocardial ischemia-induced left ventricular PVCs. Our findings reveal heightened activity in the RVLM and M1L5^Glu neurons, along with reduced activity in ZIGABA neurons in MI mice. Notably, inhibition of either M1L5 or ZI^GABA neuronal activity leads to the suppression of PVCs induced by MI, underscoring their respective roles. Our data provide compelling evidence for the modulation of PVCs by the M1L5-ZI circuit via the RVLM.

Support for this hypothesis comes from human imaging studies suggesting distinct roles for M1 and ZI neurons in regulating sympathetic outflow to the heart^57,58. Compared to extensively studied autonomic nervous pathways activated by acupoints^59–61, the specific central pathway transmitting afferent nerve signals from HT7 acupoints to sympathetic outflow towards the heart remains uncharted. Employing virus tracing techniques, we identified neurons that were innervated by spinal afferents from HT7 acupoints, predominantly congregating in M1L5. These glutamatergic neurons within M1L5 project to the ZI, and subsequently to the RVLM, serving as a pivotal conduit for regulating sympathetic outflow to the heart. While the somatosensory cortex is conventionally recognized as the primary pathway for receiving somatosensory signals, relying on thalamocortical relay pathways in the thalamus^62,63, our findings reveal a novel route. We discovered that layer 5 of the somatic motor cortex (M1L5) directly receives somatosensory signals, bypassing the thalamus, thus providing morphological evidence for the regulation of visceral function, particularly the heart, through bioelectrical stimulation at HT7.

It is noteworthy that the neural mechanism underlying the regulation of cardiac function by human acupuncture at HT7 is undoubtedly more intricate than observed in mice. Human studies have indicated the involvement of multiple brain regions in autonomic regulation, including the hypothalamus, cerebellum, prefrontal cortex, and amygdala, in response to acupuncture^64–66. Consequently, further exploration is necessary to clarify how other downstream brain regions receiving projections from M1L5 (excluding ZI and PAG), or transmitting afferent nerve signals from HT7 acupoints, may elucidate the acupuncture treatment for MI. While our findings underscore the significant role of the brain in processing acupuncture signals, we acknowledge the potential for acupuncture to directly modulate the autonomic nervous system of the heart through sensory transmission in the spinal cord.

Overall, our study has identified a neural pathway linking the brain and the heart, implicating the role of HT7 in the processing of heart function, which could catalyze research into the connection between the body surface and internal organs. In the future, these discoveries hold promise for inspiring the development of alternative interventions for myocardial diseases.

Limitations of the study

In this investigation, we primarily observed cortical involvement in the inhibitory effect of EA at HT7 on PVCs. However, our findings also indicate widespread distribution of somatosensory neurons at HT7 within the dorsal root ganglion of the T3 spinal cord. Given that the cardiac sympathetic nerve is distributed in the T3-T4 spinal cord, further investigation is warranted to ascertain whether a direct connection exists between HT7 and the cardiac sympathetic nerve at the spinal cord level, thereby contributing to the effect of EA at HT7. Simultaneously, somatosensory neural projections from the HT7 acupoint extend to multiple brain regions, including the periaqueductal gray, lateral hypothalamic area, and parvocellular medial vestibular nucleus. However, whether these projections are implicated in the suppression of PVCs in MI mice by EA remains unknown.

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (82074536) awarded to RLC, Natural Science Foundation of Anhui Province (2108085Y30) awarded to RLC, Distinguished Young Youth Scientific Research Project in Universities of Anhui Province (2022AH020043) awarded to RLC, Research Funds of Center for Xin'an Medicine and Modernization of Traditional Chinese Medicine of IHM (2023CXMMTCM019) awarded to RLC, The Plans for Major Provincial Science & Technology Projects (202303a07020002) to ZZ, National Natural Science Foundation of China (82104999) awarded to QY, Natural Science Foundation of Anhui Province (2108085QH364) awarded to QY, Excellent Young Youth Scientific Research Project in Universities of Anhui Province (2022AH030062) awarded to QY, Higher Education Teaching Quality and Teaching Reform Project of Anhui Provincial (2023xscx092) awarded to FZ, National Natural Science Foundation of China (81973757) awarded to LH, College Natural Science Project of Anhui Provincial(2022AH050514).

AUTHOR CONTRIBUTIONS

Conceptualization: Fan ZHANG, Rong-lin CAI, Qing YU, Zhi ZHANG, Xia ZHU. Data curation: Fan ZHANG, Qian-yi WANG, Li-bin WU, Jie ZHOU, Liu YANG, Wen-xiu DUAN. Formal analysis: Fan ZHANG, Qian-yi WANG, Xiang ZHOU, Bin ZHANG. Funding acquisition: Ling HU, Qing YU, Rong-lin CAI, Zhi ZHANG. Investigation: Fan ZHANG, Li-bin WU, Qi SHU. Methodology: Fan ZHANG, Xia WEI, Qing YU, Hui-min CHANG. Project administration: Zhi ZHANG, Ling HU, Rong-lin CAI, Qing YU. Resources: Rong-lin CAI, Qing YU, Xia ZHU. Software: Fan ZHANG, Yan WU, Qian-yi WANG, Wen-jing SHAO, Zheng-jie LUO. Supervision: Fan ZHANG, Ling HU, Qing YU, Rong-lin CAI, Zhi ZHANG. Validation: Qing YU, Rong-lin CAI. Visualization: Fan ZHANG, Rong-lin CAI. Writing–original draft: Fan ZHANG, Qian-yi WANG, Xia WEI. Writing – review & editing: Fan ZHANG, Rong-lin CAI. All authors have read and agreed to the published version of the manuscript.

DECLARATION OF INTERESTS

The authors declare that they have no conflicts of interest.

Data availability statement

The author will unreservedly provide the raw data to support the conclusions of this article. All data generated in this study can be found at: https://data.mendeley.com/datasets/vs34h73gr3/1, or through contact of the lead author, Rong-lin CAI ([email protected]).

Ethics statement

We obtained six-week-old male C57BL/6J mice from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd., for inclusion in this study. The production license for experimental animals is SCXK (ZHE) 2019-0004. The mice were housed in cages with ad libitum access to food and water, and the ambient temperature was maintained at a relative range of 23–25 °C. All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and received approval from the Animal Nursing and Utilization Committee of Anhui University of Chinese Medicine (AHUCM-mouse-2022083).

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Electroacupuncture Suppresses Premature Ventricular Complexes Occurring Post-myocardial Infarction through corticothalamic circuit

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