This prospective study included men and women with ischemic-sounding chest pain and non-obstructive coronary arteries (defined by epicardial stenosis <50%), who underwent invasive coronary physiology studies between Oct 2019 and April 2021. Every patient had been referred by cardiologists or other cardiovascular specialists to a subspeciality clinic (Cardiovascular Integrated Physiology Program) for the assessment and management of ongoing symptoms presumed to be of cardiac origin.

This study was conducted in accordance with the principles of the Declaration of Helsinki, and all patients provided written informed consent prior to enrollmen (SRHC protocol S-021-2021).

The baseline characteristics are presented in Table 1. Patients with Ach-induced chest pain were compared with those who denied chest pain. Patients with Ach-induced chest pain were more likely to have a history of hyperlipidemia and experience chest pain during graded exercise testing. With respect to anginal burden and physical quality of life, patients with Ach-induced chest pain had numerical trends toward worse clinical status that did not reach statistical significance.

Patients with Ach-induced chest pain had significantly increased baseline coronary blood flow (1/T_mn = 1.6 ± 0.7 vs. 1.2 ± 0.4, p = 0.004) (Table 2) reflecting decreased baseline transit times (T_mn 0.75 ± 0.33 vs. 0.94 ± 0.33, p = 0.009). Baseline cardiac efficiency was lower in patients who subsequently experienced Ach-induced chest pain (7,613 ± 3,250 vs. 9,143 ± 3,610, p = 0.04).

During adenosine infusion, there were no significant differences in Ad-IMR: (21.1 ± 10.7 vs. 21.8 ± 8.2 p = 0.76) (Table 2). Coronary flow reserve was significantly lower in patients with Ach-induced chest pain due to the elevated baseline coronary blood flow (Ad-CFR 3.1 ± 1.3 vs. 3.7 ± 1.4, p = 0.04). Patients with Ach-induced chest pain were more likely to experience chest pain during intravenous adenosine infusion (40/52 = 77% vs. 7/35 = 20%, p < 0.00001). No differences were seen in Ad-induced ECG changes which were infrequent.

During Ach infusion: patients with Ach-induced chest pain had significantly higher coronary blood flow 1/T_mn (3.6 ± 1.7 vs. 2.7 ± 1.3, p = 0.01), and decreased microvascular resistance Ach-IMR (29.7 ± 16.3 vs. 40.4 ± 17.1, p = 0.004). Ach-induced chest pain was associated with a higher rate of Ach-induced ECG changes (58% vs. 41%, p = 0.013).

Endothelial dysfunction was more prevalent in patients with Ach-induced chest pain (63% vs. 40%, p = 0.03, see Table 2). Patients with endothelial dysfunction had significantly lower Ach-IMR (29.5 ± 16.2 vs. 39.3 ± 17.4 p = 0.008) and significantly higher Ach-CFR (2.7 ± 1.2 vs. 2.2 ± 0.8, p = 0.04). There were no differences in the microvascular response to adenosine.

Patients with endothelial dysfunction demonstrated a trend toward increased baseline coronary blood flow (1.57 ± 0.7 vs. 1.33 ± 0.57, p = 0.08), and significantly reduced cardiac efficiency (7,568 ± 2,926 vs. 9,124 ± 3,767, p = 0.03).

The interaction between epicardial endothelial dysfunction and Ach-induced chest pain was explored. Patients with both (n = 33) or neither (n = 21) were compared. There were no significant differences in the microvascular response to adenosine. Those with both chest pain and endothelial dysfunction demonstrated significantly lower Ach-IMR (26.7 ± 14.8 vs. 43.4 ± 16.3, p = 0.0003) and higher Ach-CFR (2.7 ± 1.3 vs. 2 ± 0.5, p = 0.04). The concurrent endothelial dysfunction and Ach-induced chest pain identified patients with the highest baseline coronary blood flow and the highest blood flow in response to intracoronary Ach (Figure 2). This same group also demonstrated the lowest cardiac efficiency at baseline; 7,060 ± 3,086 vs. 9,526 ± 4,148 p = 0.02 (Figure 3).

Multivariable logistic regression analysis is presented in Table 3. Significant predictors of Ach-induced chest pain included Adenosine-induced chest pain, chest pain during exercise testing, lower Ach-IMR and a history of hyperlipidemia.

Approximately 30% of patients with chest pain syndromes have non-obstructive coronary angiograms. Crea and Lanza (19) have noted that this syndrome has multiple potential mechanisms, and there is an opportunity cost to assuming the wrong mechanism in individual patients. Ach-induced chest pain is currently an important part of the diagnostic guidelines. The combination with electrocardiographic changes is assumed to be the result of acute Ach-induced microvascular spasm and to represent an “inappropriate susceptibility to microvascular constriction” (20). The current data does not support this hypothesis. The significantly decreased Ach-IMR is not consistent with widespread microvascular spasm though it does not rule out maldistribution and subendocardial steal due to altered autoregulation (21).

This observation is clinically relevant because vasodilators are routinely recommended for the management of coronary microvascular spasm (18). In the absence of objective measures of increased microvascular resistance (acute or chronic), it is not clear that this strategy has a sound mechanistic underpinning. This may help to explain the failure of diltiazem to improve symptoms in patients with angina but no obstructed coronary arteries (22). Further, the assumption that Ach-induced chest pain represents microvascular spasm distracts from other potential mechanisms that require ongoing investigation.

While Ach-induced chest pain was associated increased overall coronary blood flow, this does not eliminate the possibility of ischemia due to regional perfusion heterogeneity (23). On univariate analysis, Ach-induced chest pain was associated with slightly higher incidence of Ach-induced ECG changes. However, this occurred only 17% more frequently in those with versus without chest pain. In a post-hoc analysis, concurrent chest pain and ECG changes were not associated with decreased CBF or increased IMR (data not shown). Independent of chest pain, Ach-induced ECG changes were not associated with significant differences in baseline coronary blood flow, cardiac efficiency, Ad-IMR, Ad-CFR, Ach-IMR or Ach-CFR (data not shown). In multivariable analysis, Ach-induced chest pain was not associated with Ach-induced ECG changes. Ach augments ECG changes via vagal influence in patient with Brugada syndrome (24, 25), and in a canine model induces a variety of ST segment changes (26). Indeed, chest pain and ECG changes are relatively common during coronary angiography without any clear mechanistic explanation (27). Thus ECG changes alone should not be considered diagnostic of ischemia. Transcardiac lactate production might be helpful, but it is important to note that Ach may induce lactate production via mechanisms that are independent of ischemia (28–31). This may explain why a previous subset of patients with both Ach-induced chest pain and ECG changes had transcardiac lactate production despite increased coronary blood flow (6). These data mirror our own. While it is likely that some Ach-induced chest pain is secondary to true myocardial ischemia, the current data suggest that Ach-induced chest pain, with or without ECG changes, may not be sufficiently specific to be used as a criterion for this diagnosis.

Altered pain sensation is well-documented in patients with chest pain and normal coronary arteries (32). Chronic pain may be associated with abnormal nicotinic Ach receptor activity (33). Sixty percent of our patients complained of chest pain during Ach infusion. Seventy-five percent of these patients also complained of chest pain during adenosine infusion despite normal microvascular responses. They were also more likely to have exercise-induced chest pain without other signs of ischemia. Previous studies have suggested that adenosine induces chest pain “by mechanisms other than myocardial ischemia” (34). In multivariable analysis, adenosine-induced chest pain was the strongest predictor of Ach-induced chest pain. These findings raise the hypothesis that Ach-induced chest pain is a marker for abnormal nociception. Future studies could examine the correlation between Ach- and adenosine-induced chest pain with peripheral pain thresholds (35).

Ach-induced chest pain was associated with an increased prevalence of endothelial dysfunction, which in turn was associated with both increased baseline coronary blood flow, and reduced Ach-IMR.

These findings are initially counterintuitive but may be partially explained by the differential effects of Ach on large versus smaller coronary vessels. L-N monomethylargnine (L-NMMA) abolishes the vasodilatation of the epicardial arteries (36) and larger arterioles (37) but only partially inhibits the vasodilatation of the smaller microvasculature. At least 60% of the microvascular vasodilatory effect of Ach is via mechanisms other than nitric oxide (38). Thus, while epicardial vasodilatation (and endothelial dysfunction) is essentially completely dependent upon Ach-induced nitric oxide production, this effect plays a secondary role in the microvasculature. Our intuitively paradoxical findings mirror a previous study in which 48% of hypertensive patients had constriction of the epicardial conduit arteries but vasodilatation of the microvasculature in response to intracoronary Ach (39).

Endothelial dysfunction has also been associated with chronic pain syndromes. Deep somatic sympathetic afferents contribute to pain syndromes, raising the possibility of a direct effect of Ach on nociception in the heart (40). The association between endothelial dysfunction and Ach-induced chest pain may reflect the complex mechanisms underlying chronic pain syndromes.

We have previously reported that patients with abnormal CFR but normal IMR have decreased cardiac efficiency (9). Our current data identifies both Ach-induced chest pain and endothelial dysfunction as synergist markers of a similar endotype. Looking specifically at patients with or without chest pain the underlying mechanisms are unclear but it is worth noting that the prevalence of hyperlipidemia was 66% in patients with both chest pain and endothelial dysfunction compared with 29% with neither (p = 0.008). We did not measure free fatty acid concentrations, but increased levels are associated with decreased metabolic efficiency and increased coronary blood flow (41) in addition to endothelial dysfunction and the activation of specific pain receptors (42). The potential role of free fatty acids, endothelial dysfunction and myocardial metabolic efficiency should be considered in future studies.

These data should not be interpreted as a repudiation of Ach-induced chest pain as a relevant clinical variable. The correlation with decreased cardiac efficiency raises the possibility of impaired myocardial energetics (9). Ach-induced chest pain was associated with epicardial endothelial dysfunction which itself is associated with adverse clinical outcomes (10). The potential association with chronic pain opens new avenues for research (33). These findings suggest that Ach-induced chest pain is not benign and argue for further investigations into the underlying mechanisms. As stated previously, the current data suggest an association with increased coronary blood flow but do not exclude the possibility of regional hypoperfusion. Specific transcardiac markers of ischemia may be required to differentiate these competing mechanisms.

The current study includes only patients with a chest pain syndrome and no obstructive coronary arteries. Thus, the measures of microvascular resistance in the “control” group may not reflect the population at large.

The utilization of thermodilution techniques during Ach provocation for measurement of IMR and CFR requires further validation. The microvascular-specific metric of Ach-IMR is associated with relevant risk factors and non-invasive measures of vascular dysfunction (7, 8), but it's prognostic significance has not yet been demonstrated.

Epicardial vasomotion was determined by visual inspection. Quantitative coronary angiography would add precision to this measurement. This study did not include free fatty acid measurements. Thus, any commentary on free fatty acids are purely speculative.