Association of coronary microvascular dysfunction and cardiac bridge integrator 1, a cardiomyocyte dysfunction biomarker

Abstract Background Coronary microvascular dysfunction (CMD) is associated with heart failure with preserved ejection fraction (HFpEF); however, pathophysiology is not well described. Hypothesis We hypothesized that CMD in women with suspected ischemia with no obstructive coronary artery disease (INOCA) is associated with cardiomyocyte dysfunction reflected by plasma levels of a cardiomyocyte calcium handling protein, cardiac bridge integrator 1 (cBIN1). Methods Women with suspected INOCA undergoing coronary function testing were included. Coronary flow reserve, vasodilation to nitroglycerin, change in coronary blood flow (ΔCBF), and vasodilation to acetylcholine (ΔAch) were evaluated. cBIN1 score (CS) levels in these women (n = 39) were compared to women with HFpEF (n = 20), heart failure with reduced ejection fraction (HFrEF) (n = 36), and reference controls (RC) (n = 50). Higher CS indicates cardiomyocyte tubule dysfunction. Results INOCA, HFpEF, and HFrEF women were older than RC (p < .05). Higher CS was associated with vasoconstriction to acetylcholine (r = −0.43, p = .011) with a trend towards lower ΔCBF (r = 0.30, p = .086). Higher CS was specific for ΔAch and ΔCBF but had limited sensitivity. INOCA women had higher CS than RC, but lower CS than HFpEF/HFrEF groups (p < .001). Conclusions CS, a plasma biomarker indicating poor cardiomyocyte health, was higher in women with suspected INOCA as compared to RC, but lower than in women with HFpEF. Elevated CS in suspected INOCA patients represents an intermediate group between health and disease, supporting the hypothesis that CMD may progress to HFpEF. Larger prospective cohort studies are needed to confirm the pathophysiological relationship between cBIN1, CMD, and HFpEF.

The most frequent of these events is heart failure 2 with preserved ejection fraction (HFpEF); 3,4 however, underlying mechanisms remain poorly understood. CMD, linked with traditional cardiovascular risk factors 5 and stressors, through microvascular rarefication and decreased cardiomyocyte energy availability, also has the potential to disrupt mechanistic functioning of cardiomyocytes, including transverse tubules, contributing to increased ventricular remodeling and stiffness. 6 This may be associated with dysfunctional LV mechanics prior to the onset of overt myocardial dysfunction, fibrosis, and symptoms of heart failure and a circulating biomarker for transverse tubule disruption would be useful for identifying patients with CMD at risk of developing HFpEF.
Cardiac bridge integrator 1 (cBIN1) is a protein distributed along cardiomyocyte t-tubules organizing calcium releasing dyads responsible for calcium influx and calcium-induced calcium release, essential for efficient excitation-contraction coupling and normal contractility. [7][8][9] Elevated cBIN1 score (CS), a reciprocal dimensionless score derived from plasma cBIN1 levels, is associated with adverse cardiac remodeling, 10 and has been observed HFpEF 11 and heart failure with reduced ejection fraction (HFrEF), 12  Coronary function testing was performed in women with suspected INOCA as previously described. 5,13 Coronary flow reserve (CFR) in response to intracoronary (IC) adenosine (Normal > 2.5) was assessed.
CFR < 2.32, previously shown to be of prognostic significance, 1 was also considered as a significant threshold in statistical analyses below.
Coronary artery diameter response to IC nitroglycerin (ΔNTG) (Normal > 20%), coronary artery diameter response to IC acetylcholine (ΔAch) (Normal > 0%), and coronary blood flow increase in to IC T A B L E 1 Comparison of cBIN1 score and baseline characteristics across different groups of women acetylcholine (ΔCBF) (Normal > 50%) were measured. 13 The presence of CMD was defined as a limitation in ≥1 of these tests.

| cBIN1 assay
cBIN1 is a membrane scaffolding protein that localizes to t-tubules and facilitates microtubule-dependent forward delivery of calcium channels. 9 It is a plasma biomarker associated with cardiomyocyte health, 15,16 lower levels are associated with cardiomyopathy, overt heart failure and increased arrhythmias in both animal 17,18 and human studies. 15

| Statistical analysis
Descriptive analysis was conducted using means ± SD or counts and  and area under the curve (AUC) are presented in Figure 2. CS > 1.0 discriminated between women with ΔAch < 0% and ΔCBF < 50% (AUC 0.63 and 0.72, respectively), but was a poor discriminator for CFR < 2.5

| CS across groups
Comparison of mean CS in the women with suspected INOCA to RC, HFpEF and HFrEF women is shown in Figure 3. p < .01). Age was also independently associated to CS after adjustment for covariates (p = .03), and there was an interaction between age and study group, reflecting that as age increased, smaller differences in CS across groups were expected. Functional capacity was not related to CS levels within and across groups (data not shown).

| DISCUSSION
CS, a plasma biomarker indicative of poor cardiomyocyte health and adverse myocardial remodeling, is associated to certain CMD pathways and may potentially be a discriminator of certain mechanisms of CMD in women with suspected INOCA. We found that CS > 1.0 demonstrated high specificity for ΔAch and CBF but limited sensitivity. CS was higher in women with suspected INOCA as compared to RC but lower than CS measured in women with either HFpEF or HFrEF.
Despite the absence of obstructive coronary disease, higher CS levels in 56% of our suspected INOCA women suggests that CMD may be associated with alteration in the intracellular mechanisms responsible for appropriate myocyte function.
CS levels were independently associated to different disease groups, as was age, which may modulate differences in CS levels observed between groups. This finding contrasts with previous reports concerning the association between CS and age, 19 may be due to the inclusion of women with a different clinical phenotype in this study, and potentially suggests that levels of CS may potentially better discriminate myocyte dysfunction in younger women, although larger studies are needed to confirm this observed interaction.
Our prior work in women with CMD demonstrates HF hospitalization is the most frequently adverse cardiovascular event at 5-7 year F I G U R E 3 Comparison of CS levels across groups. CS, cBIN1 score; HFpEF, cohort of women with heart failure with preserved ejection fraction (n = 20); HFrEF, cohort of women with heart failure with reduced ejection fraction (n = 36); RC, reference controls with no previous history of heart disease (n = 50); suspected INOCA, invasive coronary function testing cohort (n = 39) follow-up, 2 the vast majority confirmed to be HFpEF. 3 We have hypothesized that CMD may progress to HFpEF via risk factor condi- CMD and HFpEF share similar risk factors, with both being more common in women, and in subjects with diabetes and hypertension. 21,22 Myocytes of subjects with HFpEF demonstrate lower cyclic guanisine monophosphate (cGMP) and reduced nitric oxide activity, 23 a characteristic, which has also been previously associated with abnormal coronary endothelial function. 21,24 Previous studies have suggested a link between coronary endothelial dysfunction, reduced NO production and the release of reactive oxygen species, which promote myocyte dysfunction via increased collagen deposition. 24 Higher CS levels, reflecting low-serum cBIN1, appear to be have high specificity for abnormalities in the assessment of predominantly endothelial-dependent pathways of microvascular dysfunction, which are often assessed through evaluation of coronary artery diameter and CBF in response to acetylcholine. 13

| LIMITATIONS
Limitations of our study include relatively small sample sizes; these findings are therefore hypothesis generating and subsequent evaluation in larger and prospective cohorts is needed to confirm and extend these initial findings. Our exclusive study of women undergoing clini-

ACKNOWLEDGMENTS
We thank Sarcotein Diagnostics (www.sarcotein.com) for providing the ELISA-based CS assay.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.