Increased epicardial nerves and decreased intramyocardial PVAT in acute myocardial infarction

Acute myocardial infarction (AMI) primarily results from thrombus formation following plaque rupture in the epicardial coronary arteries (ECA), leading to a partial or complete occlusion of the coronary artery, with subsequent ischemia and infarction of the associated myocardium. However, dysregulation of coronary vasomotor function and the vascular tone of either ECA and intramyocardial coronary arteries (ICA) can also contribute to AMI induction.1 Important regulators of vascular tone are the perivascular nerve fibres (PVNF)2 and perivascular adipose tissue (PVAT).3 In this study, we analysed PVNF of the ECA, PVNF in the myocardium and PVAT of the ICA in patients that died of an AMI to determine changes that could be present prior to the AMI onset. PVNF activity was shown to increase vascular resistance of canine ECA and ICA following nerve activation,4 while PVAT can induce vasodilation of the ECA and ICA by secreting vasoactive adipokines. PVNF can regulate vascular tone in humans by releasing vasoconstrictive compounds such as Neuropeptide Y (NPY), which is primarily produced in sympathetic nerves and is associated with increased cardiac microvascular resistance in AMI.5 Additionally, structural and functional changes in the neural network have been shown to occur in the heart following AMI in humans.6 For instance, the amount of intramyocardial nerves increased from Day 3 up to 1 month postAMI in the infarcted and noninfarcted ventricular myocardium in nonatherosclerotic dogs,7 while PVAT dysfunction in humans has been described to result in hypertension and endothelial dysfunction.8 It is unknown if PVNF innervation of the ECA or myocardium and PVAT surrounding coronary vessels is already aberrant prior to AMI onset.

Acute myocardial infarction (AMI) primarily results from thrombus formation following plaque rupture in the epicardial coronary arteries (ECA), leading to a partial or complete occlusion of the coronary artery, with subsequent ischemia and infarction of the associated myocardium.However, dysregulation of coronary vasomotor function and the vascular tone of either ECA and intramyocardial coronary arteries (ICA) can also contribute to AMI induction. 1Important regulators of vascular tone are the perivascular nerve fibres (PVNF) 2 and perivascular adipose tissue (PVAT). 3In this study, we analysed PVNF of the ECA, PVNF in the myocardium and PVAT of the ICA in patients that died of an AMI to determine changes that could be present prior to the AMI onset.
PVNF activity was shown to increase vascular resistance of canine ECA and ICA following nerve activation, 4 while PVAT can induce vasodilation of the ECA and ICA by secreting vasoactive adipokines.PVNF can regulate vascular tone in humans by releasing vasoconstrictive compounds such as Neuropeptide Y (NPY), which is primarily produced in sympathetic nerves and is associated with increased cardiac microvascular resistance in AMI. 5 Additionally, structural and functional changes in the neural network have been shown to occur in the heart following AMI in humans. 6For instance, the amount of intramyocardial nerves increased from Day 3 up to 1 month post-AMI in the infarcted and non-infarcted ventricular myocardium in non-atherosclerotic dogs, 7 while PVAT dysfunction in humans has been described to result in hypertension and endothelial dysfunction. 8It is unknown if PVNF innervation of the ECA or myocardium and PVAT surrounding coronary vessels is already aberrant prior to AMI onset.

| Patients
Tissue of autopsied AMI patients (N = 14) and controls without heart disease (N = 7) were included in the study (Table 1).Due to limited material, not all analyses could be performed in all patients.Up to six cross-sectional samples from areas of stenosis were taken per epicardial coronary arteries (ECA) (Left Anterior Descendens [LAD], Left Circumflex [LCX] and Right Coronary Artery [RCA]) from the excised heart at autopsy.Additionally, transmural sections of the left ventricular wall were sampled, taken from the infarcted area in AMI patients.Tissues were fixed overnight in formalin, decalcified (ECA only) and embedded in paraffin.The AMI patients all had an infarction of 3-6 h old without prior infarction: they had macroscopical signs of infarction (decolourization of NBT stained heart slides), but no microscopical changes (inflammation).

| (Immuno)histochemical analysis
Number of (sympathetic) nerve fibres were examined in 4 μm paraffin embedded cross-sections.Following epitope retrieval in TRIS-EDTA for 20 min at 98°C, slides were incubated with primary antibodies (Table 2) for 1 h, incubated with secondary antibody for 30 min, visualized via DAB incubation for 10 min incubation with DAB and counterstained with haematoxylin (1 min).For virtual double staining, the S100-stained slides were visualized with NovaRed, counterstained and scanned.Neurofilament staining was subsequently performed after stripping the primary staining with strip buffer.
PVAT was visualized by incubation in Lawson's solution for 60 min, differentiated in alcohol and incubated in Mayer's haematoxylin solution for 5 min.After a 5 min wash in running tap water, the slides were incubated for 5 min in Van Gieson solution.
Plaque size and plaque stability were determined in haematoxylin-and eosin-stained slides.Slides were incubated in haematoxylin for 5 min and then rinsed in water.The slides were then incubated in eosin for 2 min and rinsed in water with 1% ammonia.

| Immunoscoring
S100+ and NPY+ nerve fibres per mm 2 were quantified using a light microscope in the adventitia and immediately adjacent PVAT.The surface areas of the adventitia and adjacent PVAT (mm 2 ) were measured using a PathScan Enabler IV slide scanner (Meyer Instruments Inc.) and QuickPHOTO MICRO 3.0 software.Stenosis level per coronary artery was determined semi-quantitatively as a percentage of the luminal narrowing on HE-stained slides.Unstable lesions were defined as atherosclerotic plaques with a thin fibrous cap and/or inflammatory cells infiltrating up to the endothelial layer of the intima.

| Statistical analysis
Atherosclerotic plaques, even within one coronary artery branch, are diverse in size and stability, which could affect PVNF density.The cross-sections of the coronary arteries of each plaque rather than patients as whole were therefore used for analysis.Differences between groups were analysed for statistical significance using GraphPad Prism 7.02 software.Appropriate statistical tests were used.A pvalue of <0.05 was considered significantly different.
Reporting of the study conforms to broad EQUATOR guidelines. 9

| Patient characteristics
Patient characteristics (age, gender, STEMI, stenosis, plaque stability, cause of death and co-morbidities) are listed in Table 1.Control patients were 54.8 (±8.0) years old, while AMI patients were 63.6 (±11.7) years old, no significant difference was found between the groups (p = 0.10).AMI patients were primarily STEMI-patients (92.9%), based on transmural LDH staining.The median percentage of stenosis of control and AMI patients was 50%.The median of maximum stenosis in control patients was 50%, while in AMI patients it was 75%.

| Increased epicardial PVNF density in AMI patients
Colocalization of S100 with the pan-neuronal marker neurofilament demonstrated that S100 stains PVNF (Figure S1).PVNF densities (S100+ cells) were analysed in cross-sectional samples in the adventitial adipose tissue of the ECAs with atherosclerotic plaque formation (Figure 1A; Figure S2).The PVNF around the ECAs were present predominantly in the adventitia and the immediate adjacent PVAT.A significant increase in the density of S100+ PVNF (p < 0.01) was found in the ECAs of AMI patients (7.6 PVNF/mm 2 ) compared to control patients (5.2 PVNF/mm 2 ) (Figure 1B).This increase was especially evident in the in the infarct-related ECAs (8.3 PVNF/mm 2 ; p = 0.03) (Figure 1C).
There was no significant difference in maximal stenosis levels between AMI and control patients, but the stenosis level in the infarct-related ECAs was significantly higher than in the non-infarct-related ECAs (Figure 1D).In AMI patients, the PVNF density did not differ between ECAs with low (≤50%) and high (>50%) levels of stenosis (Figure 1E).Furthermore, the PVNF density did not significantly differ based on plaque stability (Figure 1F).As a measure of sympathetic nerves, the densities of PVNF positive for Neuropeptide Y (NPY; Figure 2A) were determined around the ECAs.In AMI patients, the density of NPY+ PVNF (1.0 PVNF/mm 2 ) was significantly higher (p = 0.02) than in controls (0.64 PVNF/ mm 2 ) (Figure 2B).Although the density of NPY+ PVNF of the infarct-related ECAs (1.04 PVNF/mm 2 ) appeared higher compared with the non-infarct-related ECAs (0.91 PVNF/mm 2 ), this was not significant (Figure 2C).Moreover, the NPY+ PVNF density did not relate to the level of ECA stenosis (Figure 2D).Furthermore, there was no significant difference in NPY+ PVNF density around stable and unstable atherosclerotic plaques in control and AMI patients (Figure 2E).Lastly, regarding the epicardial PVAT, only adipose tissue was observed at the adventitial side of ECAs, no fibrosis (not shown).No overt histopathological differences in epicardial PVAT were noticed between the groups.

| PVAT of ICAs is lower in AMI patients compared to control patients
Within the myocardium of both control and AMI patients, S100+ nerve fibres (Figure 3A; Figure S3) and NPY+ nerve fibres (Figure 3B; Figure S3) were predominantly found adjacent to large arterial branches, arterioles and in some cases capillaries.The density of PVNF within the myocardium of controls and AMI patients was both 0.09 PVNF/mm 2 3C).A non-significant (p = 0.53) increase in the density of intramyocardial NPY+ PVNF was observed in AMI patients (0.05 NPY+ nerves/mm 2 ) compared with controls (0.03 NPY+ PVNF/mm 2 ) (Figure 3D).
Both in control and AMI patients, intramyocardial PVAT was found mainly adjacent to large arterial branches.A significantly lower amount of PVAT was found in AMI patients (36.0 mm 2 PVAT per artery) compared to control patients (59.9 mm 2 PVAT per artery; p = 0.02) (Figure 3E; Figure S3).

| DISCUSSION
Perivascular nerve fibres (PVNF) and adipose tissue (PVAT) are important regulators of vascular tone and are thereby co-determinants of myocardial perfusion.This study found a significant increase of nerve fibres around epicardial coronary arteries in patients with very recent (<6 h AMI) that coincided with a significant decrease of PVAT in the myocardium of AMI patients, which may relate to AMI onset.
From a clinical perspective, AMI is roughly divided into ST-elevation myocardial infarction (STEMI) and non-STEMI, depending on the presence of ST segment elevations in the ECG as a marker of transmural ischemia and infarction. 10Although solid evidence is still lacking, some cases of AMI may have aspects of both STEMI and non-STEMI due to a combination of underlying pathophysiological mechanisms, such as ruptured or eroded plaques with an overlying thrombus, or spasm in the ECA.such, it is theorized that the new classification of a Transient STEMI may be due to a combination of mechanisms, such as coronary muscle relaxation combined with thrombus formation, as these mechanisms may trigger each other as well. 10,12n the presence of endothelial dysfunction and flowlimiting coronary stenosis, PVNF activation can constrict the ECAs, reduce coronary flow and result in myocardial ischemia. 13,14The increased S100+ and NPY+ (a known vasoconstrictor 5 ) nerve densities in our AMI patients may reflect increased epicardial PVNF activity.Given that significant atherosclerosis, indicative of endothelial dysfunction and stenosis were present in the ECAs of AMI patients, such increased PVNF activity may possibly have contributed to the onset of AMI.All the more since the PVNF density was especially increased in the infarctrelated ECAs.As AMI patients died within 6 h after onset of AMI, the PVNF densities were likely increased prior to AMI development.
An intricate relation between PVNF in ECAs may relate to atherosclerotic plaque development, as murine atherosclerotic plaque development triggered outgrowth of adventitial axons.This neuronal restructuring contributed to plaque development and instability.Moreover, in human ECAs increased PVNF densities in plaque-burdened segments were observed. 15In our study however, there was no significant difference between the level of stenosis between control and AMI patients and we found no significant associations between stenosis level and PVNF densities or plaque stability and PVNF densities.Therefore, the difference in epicardial PVNF density between AMI patients and controls cannot be explained by a difference in atherosclerotic plaque burden.Given that the structural changes in PVNF density likely occurred prior to AMI, our data may suggest that a higher epicardial PVNF density may predispose towards AMI development, although this remains to be proven.
In contrast to the ECAs, the PVNF density around the intramyocardial blood vessels did not differ between AMI and control patients.In both animal and humans, AMI resulted in structural and functional changes in the neural network in the heart, especially in the peri-infarct area, that occurred from 3 to 4 days post-AMI onwards. 16herefore, the brief period between AMI induction and death in our AMI patients suggests that post-AMI neural remodelling was not a factor in our study.
We found a significantly lower amount of PVAT around the intramyocardial blood vessels of AMI patients.PVAT regulates vascular tone through the release of vasoactive factors, 17 and a decrease in PVAT likely negatively affects cardiac microvascular tone, contributing to cardiac ischemia.Again, these differences in intramyocardial PVAT quantity likely predate AMI onset and could theoretically predispose towards AMI development.The mechanisms behind lowered intramyocardial PVAT amount are at present unknown.Epicardial stenosis was shown in pigs to induce structural changes in the distal microvasculature (e.g.inward remodelling and exaggerated vasoconstriction). 18However, no effects of epicardial stenosis on intramyocardial PVAT amount have been described.Besides, as stenosis levels were similar between AMI patients and controls, such effect would not adequately explain the observed differences.
Lastly, studies have shown sympathetic innervation in PVAT and interaction between PVNF and PVAT for a coordinated effect on vascular tone.Sympathetic stimulation in PVAT triggers the release of the vasodilator adiponectin, while the vasoconstricting noradrenalin (released by PVNF) is stored in PVAT without reaching the vessel. 19ven though we did not observe a difference in intramyocardial NYP+ PVNF densities, a decreased amount of intramyocardial PVAT may jeopardize both the release of adiponectin and noradrenalin storage.
The biggest limitation of this study is the sample size, although the results still indicate differences between control patients and AMI patients.Regarding the baseline characteristics, the mean age of AMI patient group is non-significantly higher (63.6) compared to control patient group (54.8).In addition, most subjects were middle-aged men, and therefore, it can be expected no significant changes occurred due to menopausal effects.As PVNF are decreased with age, 20 one expects less PVNF in our older AMI group.Contrastingly, more PVNF in the AMI group were observed than in control patients, indicating an association between PVNF and AMI.
In conclusion, our study shows a significant increase in epicardial PVNF density and a significant decrease of intramyocardial PVAT in patients with very recent AMI, that may have contributed to AMI development.
U R E 1 (A) Visualization of nerve fibres with the S100 protein (pointed to by arrows), in the adventitia of an ECA (20×).(B) PVNF in ECA of MI patients (N = 125) compared with controls (N = 76).A Mann-Whitney test was used for statistical analysis.(C) PVNF of ECA comparing Infarct-related arteries (N = 64) with Non-Infarct-related arteries (N = 61).A Kruskal-Wallis test was used for statistical analysis.(D) PVNF of ECA in controls (N = 12) and MI patients (N = 33) comparing Infarct related (N = 16) and Non-Infarct related (N = 17) for levels of stenosis.A one-way ANOVA test was used for statistical analysis.(E) PVNF of ECA in controls (N = 44) and MI patients comparing low (N = 67) and high (N = 54) levels of stenosis.A Kruskal-Wallis test was used for statistical analysis.(F) PVNF in ECA of control and MI patients comparing plaque stability (control: stable plaques, N = 27; unstable plaques, N = 27; MI: stable plaques, N = 70; unstable plaques, N = 55).A Kruskal-Wallis test was used for statistical analysis.Bars represent the mean and the SD in all graphs.

F
I G U R E 2 (A) Visualization of NPY-positive PVNF in the adventitia of an ECA (pointed to by arrow).(B) NPYpositive PVNF in ECA comparing MI patients (N = 48) with controls (N = 53).A Mann-Whitney test was used to for statistics.(C) NPY-positive PVNF in ECA comparing infarct-related arteries (N = 20) with non-infarct-related arteries (N = 28) and controls.A Kruskal-Wallis test was used to for statistics.(D) NPY of ECA in controls (N = 44) and MI patients comparing low (N = 15) and high (N = 28) levels of stenosis.A Kruskal-Wallis test was used to for statistics.(E) NPY-positive PVNF in ECA of control and MI patients comparing plaque stability (control: stable plaques, N = 15; unstable plaques, N = 21; MI: stable plaques, N = 22; unstable plaques, N = 18).A Kruskal-Wallis test was used for statistical analysis.Bars represent the mean and the SD in all graphs.

F
I G U R E 3 (A) Visualization of nerve fibres with the S100 protein (pointed to by arrows), present predominantly in the PVAT an ICA.(B) Visualization of NPYpositive PVNF in the adventitia of an ICA (pointed to by arrows).(C) PVNF of the ICA in controls (N = 3) and MI patients (N = 8).A Mann-Whitney test was used for statistical analysis.(D) NPY-positive PVNF of the ICA in controls (N = 7) and MI patients (N = 9).A Mann-Whitney test was used for statistical analysis.(E) PVAT of ICA comparing MI (N = 26) patients with controls (N = 29).A Mann-Whitney test was used for statistical analysis.Bars represent the mean and the SD.
Patient characteristics.