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Endothelial Pannexin 1 Regulates Cardiac Response to Myocardial Infarction

Originally published Research. 2021;128:1211–1213

Cardiovascular disease is a leading cause of morbidity and mortality in the world. A recent cohort study identified a significant reduction in cardiovascular disease events, especially myocardial infarction (MI) and stroke, in patients taking probenecid compared with allopurinol.1 Probenecid is a potent Panx1 (pannexin 1) channel blocker. In studies from our group, we have identified endothelial cell (EC) Panx1 as an important regulator of ischemic outcome in multiple organs, specifically playing a role in regulating leukocyte infiltration.2 Here, we examine the functional effects of MI with probenecid pharmacological intervention or genetic deletion of EC Panx1.

Male C57Bl6J mice, 12 to 20 weeks old, underwent coronary ischemia/reperfusion (IR) surgeries (blinded study; approved by the University of Virginia and Tufts Medical Center Animal Care and Use Committees).3 Anesthetized (isoflurane) mice were intubated, and the left anterior descending artery was ligated 1 mm distal to the left atrial appendage, confirmed by myocardium blanching, visual tachycardia, and ST elevation. After 60 minutes, the ligation was removed and saline or 1.1 mg/kg probenecid (Millipore Sigma, St Louis, MO), randomly assigned, was intraperitoneally injected. Hearts were collected 14-day post-IR and fixed (neutral buffered formalin). Probenecid-treated mice had a significantly higher ejection fraction than saline-treated mice 14-day post-IR (Figure [A]). Infarct sizes, quantified using polar plots from stained sections apex to base, and vascular densities, measured using isolectin (data not shown) were not significantly different (Figure [A]).


Figure. Pharmacological inhibition or genetic deletion of endothelial cell (EC) Panx1 (pannexin 1) improves cardiac function postmyocardial infarction (MI). Ejection fraction (A), measured via echocardiogram, in saline (n=9; baseline vs 1-d: P=0.000002; vs 14-d P=0.0000005) or probenecid (n=14; baseline vs 1-d: P=0.00005; vs 14-d P=0.0004; saline vs probenecid 14-d P=0.0002) treated mice. A, Infarct size (Mason trichrome representative images; saline: n=5, probenecid: n=14) 14-d post-IR hearts. B, Ejection fraction and infarct size in Cre (n=9; baseline vs 1-d: P=0.000003; vs 14-d P=0.00003) and Cre+ mice (n=8; baseline vs 1-d: P=0.0003; vs 14-d P=0.0002; Cre vs Cre+ 14-d P=0.009). C, Pressure-volume (PV) loop data from Cre (n=3) and Cre+ (n=5) mice (P=0.03). D, Cytokine release (change from control siRNA) from human aortic EC treated with control or Panx1 siRNA, confirmed with quantitative polymerase chain reaction (qPCR; P=0.007), posthypoxia/reoxygenation. E, Total Ly6C, Ly6CHI+ (P=0.04) and Ly6C low (Ly6CLOW+) expressing monocytes/macrophages infiltration in Cre and Cre+ (n=5) mice. F, Distribution of CCR2+ cells (magenta; % of CCR2+ area within given region; Cre vs Cre+: P=0.03; Cre (n=3): infarct vs border: P=0.02; Cre+ (n=4): noninfarct vs infarct: P=0.003, vs border: P=0.03). Infarct area, reduced autofluorescence area (teal), and infarct border area (≈0.5 mm around the infarct) are delineated by the dashed line. Two-way ANOVA with Sidak multiple comparisons test (Prism 9; A and B): vs baseline: ***P<0.001 and ****P<0.0001; Saline/Cre vs Probenecid/Cre+: ###P<0.001 and ##P<0.01; (F) *P<0.05 and **P<0.01. Student t test (A, B, C, D, and E): *P<0.05. All data passed Shapiro-Wilk normality tests (P>0.05), performed within each group. Scale bar: 1 mm.

To examine if EC Panx1 deletion recapitulated the functional effects seen with probenecid treatment, we used 12- to 20-week inducible EC Panx1 deleted mice (Cdh5-CreERT2+/Panx1fl/fl; Cre+) and control mice (Cdh5-Cre/Panx1fl/fl; Cre) injected with tamoxifen.2 Ejection fractions were significantly increased in Cre+ mice at 14-day post-IR (Figure [B]). Infarct sizes and vascular densities (data not shown) were not significantly different (Figure [B]). Pressure-volume loops at 14-day post-IR showed decreased average rate of pressure decline suggesting improved diastolic function in Cre+ mice. dPdt max, contractility index, and ESPVR were not significantly different, although all trend towards increased contractility (Figure [C]). Thus, acute pharmacological blockade of Panx1 or genetic deletion of EC Panx1 was functionally beneficial in mouse models of MI.

Because Panx1 is a key regulator of inflammatory cell infiltration,2 we examined cytokine release from ECs following in vitro 3-hour hypoxia/24-hour reoxygenation (R&D Systems, Minneapolis, MN). RANTES (Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted), tumor necrosis factor (TNF), and GM-CSF (granulocyte-macrophage colony-stimulating factor), which promote a proinflammatory status, were decreased in Panx1 knockdown cells; and M-CSF (macrophage colony-stimulating factor), an anti-inflammatory macrophage cytokine, was modestly increased (Figure [D]). Perfused and digested hearts, using the Langendorff preparation, 2-day post-IR were examined for infiltration of neutrophils (CD45-FITC: 1:100 and Ly6G-APC-Cy7: 1:200) and macrophages (CD45-FITC and Ly6C-PE: 1:200) using flow cytometry.2,3 Neutrophils were not different (data not shown). In Cre+ mice, we found a significant reduction in the number of Ly6C high (Ly6CHI+) expressing macrophages and no difference in total Ly6C+ macrophages (Figure [E]). The Ly6CHI+ macrophages, identified by CCR2+ (1:100; Abcam, Cambridge United Kingdom), distribution differed between the Cre- and Cre+ mice at 2-day post-IR, with significantly reduced accumulation in the noninfarct region in Cre+ mice (Figure [F]).

Ly6CHI+ macrophages are proinflammatory and predominately found during the initial stages of injury post-MI.4 Our data suggest an early reduction of Ly6CHI+ macrophages. Whether this is due to a greater transition of Ly6CHI+ to Ly6CLOW+ or a change in the infiltration of these monocytes is unclear. This difference in Ly6CHI+ expressing monocytes/macrophages number and localization within the noninfarct region could impact recovery in the heart in numerous ways, including improved tissue remodeling or facilitate cardiac electrical conduction, and remains an active area of study.4,5 Macrophages are regulated by purinergic signaling providing a potential point of crosstalk for Panx1-mediated ATP release and early tissue inflammatory response.

In summary, we found that pharmacological inhibition or genetic deletion of EC Panx1 significantly improved cardiac function following MI possibly through an early shift to an anti-inflammatory status. These data provide strong evidence for the benefit of postreperfusion inhibition of Panx1 using Food and Drug Administration-approved pharmacological drugs and suggest that Panx1 may contribute to the reduced mortality and incidence of cardiovascular disease in patients taking probenecid.1

Nonstandard Abbreviations and Acronyms


endothelial cell




myocardial infarction


pannexin 1


We thank the members of the Pannexin Interest Group, the Flow Cytometry Facility, and the Histology Core at the University of Virginia School of Medicine and the MCRI Mouse Physiology Core Facility at Tufts Medical Center. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Disclosures None.


For Sources of Funding and Disclosures, see page 1212.

Correspondence to: Miranda E. Good, PhD, Molecular Cardiology Research Institute, 800 Tufts Medical Center, Washington St, Box 80, Boston, MA 02111, Email
Matthew J. Wolf, MD, PhD, University of Virginia School of Medicine, Charlottesville, VA, Email
Brant E. Isakson, University of Virginia School of Medicine, Charlottesville, VA, Email


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Jack Rubinstein May. 5, 2021
Multiple roles of probenecid in regulating the cardiac response to injury

We read with interest the research letter by Good and colleagues describing the role of pannexin 1 (Panx1) in regulating the myocardial response to ischemia/reperfusion (I/R) injury [1]. The authors employ a Cre inducible Panx1 knockout mouse (EC Panx1 KO) as well as the drug probenecid as a Panx1 blocker [2]. They report improved cardiac function in the EC Panx1 KO as well as the wild type treated groups in order to reach the conclusion that "pharmacological inhibition or genetic deletion of EC Panx1 significantly improved cardiac function following MI possibly through an early shift to an anti-inflammatory status". 

The data from the genetic deletion of EC Panx1 and the observed inflammatory effects supports this conclusion, though we believe that the improved contractility observed in the WT mice is more easily explained through the other known effect of probenecid as a positive inotrope [3]. 

Our laboratory and others have shown that probenecid is a potent agonist of transient receptor potential vanilloid 2 (TRPV2) channels and have demonstrated that through improved calcium cycling at the myocyte level it increases contractility in murine and porcine models as well as in patients with heart failure (HF) [4][5]. Further, the increased contractility reported by Good in this I/R model is comparable to our data in a very similar ischemic cardiomyopathy model and consistent with studies showing upregulation of TRPV2 in diseased cardiomyocytes [6][7].

We wish to stress that these two hypotheses are not mutually exclusive and are in fact both likely not only true, but also consistent with human data from Kim and colleagues that is referenced in the original paper [8]. Briefly, Kim et al. is the largest clinical report to date that compared cardiovascular outcomes in patients treated for gout with either probenecid or allopurinol. They report in patients treated with probenecid decreased rates of HF exacerbation consistent with the known inotropic effects through TRPV2 channels. In addition, they also document decreased incidence of stroke and myocardial infarction as likely explained through Panx1 mediated anti-inflammatory effects.

In summary, we congratulate the research team on this excellent paper and propose that the observed effects  of probenecid are not only through downregulation of pannexin but also through its agonism of TRPV2 channels and myocyte calcium regulation.


Raymond D. Roberts

Cincinnati VA Medical Center

[email protected]

Jack Rubinstein MD

University of Cincinnati Medical College

Cincinnati VA Medical Center

[email protected]

[1] Good, M. E., et-al. Circulation Research128(8), 1211–1213.

[2] Silverman, W., et-al  American Journal of Physiology-Cell Physiology295(3).

[3]Koch, S. E., et-al. Journal of Molecular and Cellular Cardiology53(1), 134–144. 

[4] Rubinstein, J., et-al. American Journal of Physiology-Heart and Circulatory Physiology306(4).

[5] Rubinstein, J., et-al. Pediatric Cardiology41(8), 1675–1688. 

[6] Aguettaz, E. et-al. Progress in Biophysics and Molecular Biology130, 273–280.

[7] Lorin, C.,et-al.  Cardiovascular Research106(1), 153–162. 

[8]  Kim SC, et-al. J Am Coll Cardiol. 2018;71:994–1004.

Competing Interests