Altered autonomic balance, with increased sympathetic drive and decreased parasympathetic tone, is a hallmark of cardiovascular disease including MI, heart failure, sleep apnea, and diabetes [
22,
31,
110,
115,
118]. Although implantable device-based activation of parasympathetic drive provides potent cardioprotection during disease, there is no non-invasive rapid approach for parasympathetic activation during an acute MI. Our previous work has shown that CVNs receive powerful excitation from a population of hypothalamic PVN-OXT neurons that co-release OXT and GLUT to excite CVNs [
28,
49,
92]. Activation of those PVN-OXT neurons reduces blood pressure and heart rate in conscious unrestrained animals, and those effects are parasympathetically mediated [
34,
97]. In this report, we present new evidence that activation of PVN-OXT neurons soon after an MI is cardioprotective during the 7 days following an MI. We also provide new insight into the molecular basis of the multifaceted aspects underlying that cardioprotection.
Neurotransmission
The present study underscores the profound bidirectional interaction between the central nervous system and the heart after an MI. We found that an MI results in diminished excitatory neurotransmission from PVN-OXT neurons to brainstem parasympathetic neurons, leading to reduced cardioprotection. We also demonstrated that daily activation of PVN-OXT neurons maintains excitatory neurotransmission to brainstem parasympathetic neurons and sustains the expression of muscarinic receptors (Chrm2) within the myocardium, thereby supporting muscarinic-mediated cardioprotective outcomes of the autonomic parasympathetic network.
LV gene expression analysis revealed that activation of PVN-OXT neurons after an MI maintained signaling pathways and transcriptional responses that are protective against myocardial injury. As indicated in Fig.
2C, many of the cardioprotective effects of parasympathetic drive are due to post-ganglionic release of ACh and subsequent activation of inhibitory pathways within myocytes, which was potentially mediated in Treatment animals by increased expression of Chrm2. This result is consistent with others who have reported increased Chrm2 expression in HF rats treated with carvedilol (an α- and β-blocker), indicating that the upregulation of muscarinic receptors is consistent with cardioprotection [
124]. Long-term treatment with carvedilol also restored autonomic tone in patients with moderate HF [
77]. Furthermore, multiple pre-clinical studies have shown cardioprotection following ischemia/reperfusion injury by activating cholinergic muscarinic receptors (mAChRs), as well as nicotinic receptors (nAChRs), either pharmacologically or by direct-current electrical stimulation [
45].
Expression of the muscarinic M2 receptor via Chrm2 transcriptional activity is tightly regulated by the gene silencing transcription factor Rest/Nrsf (RE-1 silencing transcription factor), which may act via epigenetic remodeling to repress neural genes in non-neural cells [
42,
132]. Rest expression was significantly upregulated (2.1-fold) in MI vs. Sham animals (FDR
p = 0.01) but was non-significantly downregulated (1.1-fold) in Treatment vs. Sham animals. This suggests that excitatory signals mediated by PVN-OXT neuron activity may alter Chrm2 abundance through epigenetic actions of Rest/Nrsf. Additionally, Rbm24, an RNA binding protein that drives various post-transcriptional processes and is known to interact with Chrm2 transcript [
69], was reduced after MI yet preserved in Treatment animals. Consistent with altered Chrm2 expression in the heart [
38], we found that HRR was longer in MI animals while Treatment animals had shorter HRR times that were no different than Sham (Fig.
2E). HRR after peak effort is a common assessment of autonomic balance following adverse cardiovascular events, with a longer HRR time associated with increased mortality, sudden cardiac death, and arrhythmic events [
53,
85,
109]. As such, it was not unexpected to find that the untreated MI animals had longer HRR times and higher mortality than animals treated with PVN-OXT neuron activation (Fig.
8B). Consistent with our results, work from others has shown pyridostigmine bromide, a reversible anticholinesterase agent that exerts cholinergic stimulation, improves HRR after exercise [
4], increases heart rate variability, and decreases the density of ventricular arrhythmia in patients with heart failure [
7].
Arrhythmia incidence
In the first 24 h following LAD ligation, both MI and Treatment animals exhibited similar ST segment elevations, indicating a similar level of myocardial injury. In MI animals, there were frequent bursts of arrhythmia, including VF and VT, that often occurred in the first hour after LAD ligation. In Treatment animals, arrhythmias in the 24 h after LAD ligation were significantly less frequent, shorter in duration, and often absent (Fig.
8C). This is reflected in the increased survival of Treatment animals compared to MI (Fig.
8B). The higher mortality of MI animals is consistent with the lower EPSC amplitudes of the parasympathetic DMNX neurons observed for that group (Fig.
2A, B) and reports of reduced vagal drive to the heart being a strong independent risk factor for life-threatening arrhythmias and sudden cardiac death [
9]. The reduced incidence of arrhythmias for Treatment animals is consistent with the cardioprotective effects of cholinergic muscarinic activation, as reported during ischemia/reperfusion injury, where hearts pretreated with choline had significantly decreased ischemia-induced arrhythmia, fewer ventricular premature beats, and a smaller infarct size [
23,
133,
134]. Furthermore, expression of key genes responsible for cardiac excitation and contraction, such as connexin 43 (Gja1), Nav1.5/Scn5a, Cav1.2/Cacna1c, repolarizing K + channels, and the ryanodine receptor (Ryr2), were likewise preserved in Treatment animals compared to MI (Fig.
8F). Significant preservation of ischemic zone Serca2 protein expression in Treatment animals at a level similar to Sham (Fig.
8E) indicates maintenance of the capacity of the sarcoplasmic reticulum to sequester Ca
2+.
Our observations of reduced arrhythmia incidence and the associated preserved expression levels of key genes during PVN-OXT neuron activation in animals with an acute MI are supported by previous studies of electrical vagal nerve stimulation (VNS) during MI. In one study, VNS demonstrated reduced incidence of VF during coronary artery occlusion in canines [
27]. A more recent study found that chronic VNS, applied after permanent MI in Yucatan minipigs, stabilized the LV scar-border zone by reducing heterogeneity in activation and repolarization
in vivo, drastically reducing lethal ventricular arrhythmias [
36]. Although shown to be beneficial in controlled experiments, VNS devices are not selective for cardiac cholinergic fibers and implanting the devices before, or at the onset of, unanticipated episodes of cardiac ischemia, and other triggers of sudden cardiac death, is not clinically feasible [
17]. A recent clinical study demonstrated that low-level tragus stimulation reduced the incidence of reperfusion-related ventricular arrhythmias during the first 24 h after acute MI [
129]. Although highly encouraging, studies of human anatomy found that tragus distribution of the auricular branch of the vagus nerve is present in only 45% of the cases [
89], possibly limiting the efficacy of low-level tragus stimulation in patients with different nerve supplies of the tragus [
40].
Mitochondria
Ischemia causes mitochondrial function and structure alterations that impair ATP production and increase ROS production [
10,
13,
62,
112]. In healthy myocardium, substrate utilization is tightly regulated to meet changes in energy demand and this metabolic regulation is impaired by ischemia and disease [
44]. This involves reduced contribution of fatty acid oxidation to energy production and increased glycolysis, as described for hypertrophied and failing hearts [
3,
120]. Increasing evidence suggests that the loss of metabolic substrate flexibility is a major contributor to the development of cardiac dysfunction and heart failure [
59].
We found that many genes for rate-limiting proteins integral for substrate utilization were reduced in MI and preserved in Treatment animals (Fig.
5A), suggesting that chronic PVN-OXT neuron activation supported the maintenance of metabolic flexibility after an MI. Furthermore, MI animals had reduced expression of crucial enzymes and intermediates involved in glycolysis, the TCA cycle, OXPHOS, and fatty acid beta-oxidation (Fig.
4A), and this was attributed to a decline in mitochondrial quality (Fig.
5B–D) and respiratory capacity rather than decreased mitochondrial content at 7 days post-MI. These results were confirmed by mitochondrial XF assays where we found that complex I and II of the ETC were compromised in response to MI, with complex II impacted more dramatically, and that the function of complex I and II was significantly protected in Treatment animals (Fig.
4B–D). Similar mitoprotection and fuel preference restoration have been observed in response to VNS following MI [
74], isoproterenol-induced ischemia [
125], and ischemia/reperfusion injury, and appears to be mediated through efferent fiber activation which is consistent with our treatment paradigm [
86].
The most differentially regulated upstream pathway between MI and Treatment groups identified by IPA was peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1⍺; Ppargc1a), with activation in Treatment and repression in MI animals (Fig.
5E). PGC-1⍺ is a transcriptional coactivator that regulates metabolic genes and is the master regulator of mitochondrial biogenesis [
26,
27]. The mitochondrial deacetylase, sirtuin 3 (Sirt3), functions as a downstream target gene of PGC-1⍺ and mediates fatty acid metabolism [
41], mitochondrial quality control and dynamics [
102], and is postulated to regulate flux through the TCA cycle [
117]. Evidence suggests that Sirt3 also regulates the mitochondrial unfolded protein response and acts to sort moderately stressed from irreversibly damaged organelles by activating antioxidant machinery or mitophagy [
60,
88]. We found that Sirt3 gene expression was preserved in Treatment animals with a concomitant reduction in mitochondrial unfolded protein response and endoplasmic reticulum stress response (IPA results not shown). This is consistent with the preserved structure and function of our Treatment mitochondria (Figs.
4D,
5B) and prior reports of preserved endothelial cell mitochondria and endoplasmic reticulum after administering ACh following hypoxia/reoxygenation injury[
8,
123].
A mechanism of Sirt3-mediated cardioprotection is the preservation of Opa1 gene expression. Opa1 encodes a protein that is critical for inner mitochondrial membrane fusion and the maintenance of proper cristae structure [
102]. In addition to Opa1, Treatment vs. MI animals had preserved expression of key mitochondrial genes involved in mitochondrial fusion and fission, biogenesis, and mitophagy (Fig.
5E). Preservation of the aforementioned mitochondrial dynamics has been observed in multiple reports using direct ACh application, m2AChR agonist, and VNS, and attributed to preserved cell survival and function [
107,
113]. Consistent with these findings, mitochondrial ultrastructure as well as flow cytometric forward and side scatter analysis suggested that Treatment mitochondria were larger, more granular, and maintained their elongated shape, further supporting improved mitochondrial dynamics and OXPHOS in Treatment animals (Fig.
5C, D). Although the specific mechanisms for the preservation of mitochondrial structure and function conferred by PVN-OXT neuron activation after an MI remain to be rigorously tested, activation of Akt and AMPK (Fig.
2C) are likely candidates because these kinases have been implicated in the prevention of mitochondrial dysfunction by electronic VNS after ischemia/reperfusion injury [
6,
86,
106,
125]. Altogether, our results demonstrate that the outcomes of PVN-OXT neuron activation immediately after an MI may include the preservation of mitochondrial OXPHOS, maintained mitochondrial fusion and biogenesis, reduced mitochondrial ROS, and conserved sirtuin pathway signaling components. These functional outcomes are supported by preserved expression of numerous genes involved in mitochondrial structure and function, and provide new molecular insight into potential cardioprotective pathways.
Inflammation
Although a temporary inflammatory response after MI is required to clear the myocardium of cellular debris and toxic metabolites, excessive chronic inflammation leads to adverse LV remodeling and heart failure. During MI, neutrophils, followed by Ly6C
high (Ly6c) mononuclear cells, are quickly recruited to the infarct [
32]. Circulating monocytes, upon entry into tissues, give rise to dendritic cells and macrophages. Macrophages phenotypically differ from monocytes by increased expression of Cd68 as well as F4/80 (Adgre1). These monocyte-derived macrophages produce both pro-inflammatory and anti-inflammatory mediators (cytokines, chemokines, matrix metalloproteinases, and growth factors), phagocytize dead cells, and promote angiogenesis and scar formation.
Seven days after MI, leukocyte CD45 (Ptprc) marker expression and markers for cardiac macrophages and monocytes were dramatically elevated in MI vs. Sham animals, with a corresponding decrease in Treatment vs. MI animals (Fig.
6A). Increased expression of pro-inflammatory cytokines and chemokines was evident in MI vs. Treatment animals, with increased angiogenic gene expression in Treatment animals (Fig.
6B), suggesting either a timelier resolution of inflammation or reduced injury response in Treatment animals. These results are consistent with differences between MI and Treatment animals in ischemic zone collagen content (Fig.
7B) and 24 h arrhythmia burden (Fig.
8C), aligning with studies that identified a positive correlation between systemic inflammation in the first 5 days after MI with the size of the peri-infarct zone [
94] and the incidence of ventricular arrhythmias [
52].
Cholinergic anti-inflammatory pathways are potently activated by electrical VNS, as demonstrated in previous studies that prevented the release of pro-inflammatory cytokines such as TNF-⍺, IL-1β, IL-6, and IL-18 during endotoxemia [
11], and in patients receiving tragus stimulus following acute MI [
129]. In other studies, activation of cholinergic anti-inflammatory pathways through electrical VNS or muscarinic receptor agonists promoted macrophage M1 to M2 polarization in ischemic heart and lung injury [
20,
66], with AMPK signaling as a central regulator of the response [
101]. Accordingly, in Treatment animals, we found increased M2-type reparative markers (Cd163, IL-10, Ly6c) and a decrease in pro-inflammatory M1 markers, cytokines, and chemokines (Mcp1/Ccl2, Mip-1a/Ccl3, Cd68) (Fig.
6A, B), suggesting that PVN-OXT neuron activation may promote timely inflammatory resolution and promotion of wound healing and tissue repair.
IL-1β protein was elevated in MI vs. Sham animals with no significant elevation in Treatment animals (Fig.
6D). This macrophage-secreted cytokine was shown to induce arrhythmias in metabolically compromised mice [
82] and is a pivotal cytokine in neuroinflammation following MI [
25]. Endothelin-1, and IL-1β contribute to the production of NGF in the rodent heart [
1,
43], the pulmonary bronchi [
33], and the non-neuronal cells of the sciatic [
70]. Increased NGF is one of the immune-stimulated mechanisms responsible for nerve sprouting, sympathetic hyperinnervation, and pathological rise in sympathetic activity following MI [
127]. Regions of denervation and hyperinnervation may lead to heterogeneity of sympathetic nerve distribution and contribute to cardiac arrhythmias [
39]. Interestingly, previously identified underlying mechanisms of VNS-mediated electrical stability include suppressing cardiac neuronal sprouting, inhibiting excessive sympathetic nerve sprouting, and pro-inflammatory response by regulating gene expression [
36,
135]. Accordingly, Ngf transcript was significantly elevated in MI but not in Treatment animals.
Additionally, IL-8 signaling was one of the top ten canonical pathways activated in MI animals but was significantly reduced in Treatment animals. IL-8 pathway activation is clinically associated with larger infarct size, lower LV ejection fraction, larger increase in LV end-diastolic volume, and higher frequency of microvascular obstruction [
104]. Overall, these inflammatory outcomes suggest that during MI, untreated animals, compared to treated animals, may experience more severe tissue injury signals with a corresponding robust and sustained recruitment of immune cells to the injury, which likely increased pathologic structural remodeling and the incidence of arrhythmia.
Structural remodeling
Gene transcripts contributing to LV remodeling were markedly increased following MI, with Treatment animals having significantly fewer DEGs associated with fibrosis and an upregulation of genes that inhibit matrix metalloproteins (Figs.
3B and
7C). Infarct area, measured as collagen area per area of tissue, was also significantly greater in MI vs. Treatment animals. Activation of CVNs has been shown to reduce MI size, not only through heart rate reduction, but through a number of mechanisms including attenuated formation of reactive oxygen species and inflammation, and improved mitochondrial function [
40]. Previous studies indicate that a lack of cardiomyocyte-secreted ACh can cause maladaptive remodeling and cardiac functional decline [
96,
100] and that over-expression of cardiomyocyte vesicular ACh transporter or choline acetyltransferase increases ACh synthesis which then inhibits ventricular remodeling [
99]. Cholinesterase inhibitors, such as donepezil, are also known to improve autonomic balance, and can reduce myocardial infarct size and arrhythmia, and improve LV function following ischemia–reperfusion injury [
56]. Interestingly, in many studies utilizing VNS, the infarct limiting effect is only observed when VNS is applied during ischemia, but not at the onset of reperfusion [
17,
106]. Subsequent studies have shown that activation of cardiac Chrm2 receptors also exerts an infarct limiting effect [
67,
90]. A proposed mechanism of these beneficial outcomes is the inhibition of endoplasmic reticulum stress-induced apoptosis through extracellular signal-regulated kinase (ERK1/2) and the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in combination with the inhibition of adenylyl cyclase activity via Gαi of the m2AChR, thereby reducing cAMP production and further attenuation of ER-stress and apoptosis (Fig.
2C)[
67].
In addition to OXT network activation and VNS, parasympathetic-mediated cardioprotection, particularly following ischemia reperfusion, is likely complex and may involve both vagus-dependent and independent mechanisms. Protection independent of the vagus may be mediated by GLP-1 receptors that act via M3 muscarinic receptor activation [
5]. Consistent with the potential benefit of peripheral muscarinic receptor activation post-ischemia–reperfusion, studies have shown that acetylcholine activation of α7 nicotinic receptors (α7nAChR) on macrophages polarizes the pro-inflammatory into anti-inflammatory subtypes, activating the transcription 3 (STAT3) signaling pathway, inhibiting the secretion of pro-inflammatory cytokines, limiting ischemic injury in the myocardium, and initiating efficient reparative mechanisms [
20]. Furthermore, vagus-mediated ischemic preconditioning and cardioprotection may involve release of humoral factors which subsequently act on many downstream vagal targets and function [
58], including a vago-splenic axis [
68]. Clinical studies suggest remote ischemic conditioning-induced cardioprotection likely involves activation of sensory nerve fibers [
81], while acute caffeine intake can possibly provide a cardioprotective effect through increased vagal tone [
103]. In a mouse model of spared nerve injury (SNI) neuropathic pain, myocardial infarct size and apoptosis were reduced following MI, and this protection was dependent upon activation of the paraventricular thalamus and the autonomic nervous system, as shown by loss of SNI-induced cardioprotection by parasympathetic nerve blockers [
18].