ABSTRACT

Background: Diabetic hyperglycemia promotes vasoconstriction by activating an ATP-dependent P2Y_11L (P2Y₁₁-like receptor)/AC5 (adenylyl cyclase 5)/AKAP5 (A-kinase anchoring protein 5)/PKA (protein kinase A)/Ca_V1.2 (L-type voltage-dependent calcium channel 1.2) nanocomplex in arterial myocytes, but upstream mechanisms are unclear. We hypothesized that Panx1 (pannexin 1) channels, which facilitate ATP release, are associated with the complex in arterial myocytes and mediate its activation to induce vasoconstriction during diabetic hyperglycemia.

Methods: Multidisciplinary approach using extracellular ATP and cAMP biosensors, patch-clamp electrophysiology, super-resolution nanoscopy, proximity ligation assay, pressure myography, and laser speckle imaging to test premises in arterial myocytes and vessels from wild-type and genetically modified mice, including an inducible smooth muscle-specific Panx1 knockout and a global AKAP5 knockout.

Results: We found that elevating extracellular glucose (eg, high glucose, hyperglycemia) triggered an increase in extracellular ATP levels, and this was reduced in the presence of the Panx1 inhibitor spironolactone, in inducible smooth muscle-specific Panx1 knockout cells, and by inhibiting glucose metabolism. Panx1 was found in complex with P2Y_11L, AC5, AKAP5, PKA, and Ca_V1.2 in arterial myocytes. The protein complex was strengthened in response to hyperglycemia, which required Panx1 and AKAP5. Hyperglycemia-induced cAMP production, Ca_V1.2 potentiation, sustained vasoconstriction, and in vivo changes in cerebral artery myogenic tone and blood flow were ameliorated by spironolactone and in inducible smooth muscle-specific Panx1 knockout samples. Panx1 expression was elevated in arterial lysates from a mouse model of type 1 diabetes (eg, streptozotocin), and increased Ca_V1.2 activity and enhanced ex vivo and in vivo myogenic tone were prevented in arterial myocytes and arteries from inducible smooth muscle-specific Panx1 knockout mice.

Conclusions: These results suggest a key role for Panx1 in controlling ATP signaling through the P2Y_11L/AC5/AKAP5/PKA/Ca_V1.2 axis in arterial myocytes. This Panx1-led complex modulates cAMP levels, Ca_V1.2 activity, and vascular reactivity in response to diabetic hyperglycemia. Thus, Panx1 could be a new therapeutic target to mitigate vascular complications during diabetes.

# README – Dataset for Hong et al. (DOI: 10.1161/CIRCRESAHA.125.326260)

## Overview
This repository contains the datasets and statistical analyses associated with the publication:

**Hong et al. “Arterial Myocyte Pannexin 1 Channel Controls Vascular Reactivity in Diabetic Hyperglycemia”**
*Circulation Research (2025) 137(5):646-663.* DOI: 10.1161/CIRCRESAHA.125.326260

The data have been organized to ensure transparency and reproducibility. Each figure has an independent folder containing an Excel file with the data points, the summary statistics, and the GraphPad Prism (`.prism`) files used for statistical analysis.

## Contents

- **`Hong et al Excel files (e.g. Fig 1 Data+Statistics.xlsx)`**  
  Contains numerical data underlying each figure panel. Sheets are organized by figure and/or panel (e.g., `Fig1A`, `Fig1B`, …). Each sheet includes individual biological and technical replicates.  

  In addition, there is a tab containing the results of statistical analyses. Each sheet corresponds to the same figure/panel naming convention as above. Statistical tests (e.g., unpaired t-tests, ANOVA with post-hoc corrections, non-parametric tests) are reported as used in the manuscript.  

- **Figure Folder**
  Each figure has its own folder (e.g., `Figure 1`, …). Inside each folder:
  - **Data+Statistic File (`.xlsx`)** – Values plotted and statistics as described in the manuscript figure.
  - **GraphPad Prism File (`.prism`)** – File used to run analyses and generate plots for publication for each panel, plus additional comparisons as necessary.

This structure allows users to directly trace values from replicates through statistical analysis to the final figure.  

## File Naming and Structure
- **Folder Naming Convention**: `Figure#` (e.g., `Figure 1`).  
- **Excel Sheet Naming**: Matches figure/panel labeling in the manuscript (e.g., `Fig 1 Data+Statistics.xlsx`).  
- **Prism File Naming**: Matches figure/panel labeling, with suffix `_analysis` where applicable (e.g., `Fig 1`).  

## How to Use
- **Excel Files (`.xlsx`)** – Open in Microsoft Excel, LibreOffice, or similar software.  
- **Prism Files (`.prism`)** – Open with *GraphPad Prism* to review statistical models, replicate grouping, and analysis steps.  
- **Cross-Referencing** – Each folder contains the dataset, analysis results, and Prism file for direct cross-checking.  

## Metadata
- **Biological Replicates**: where applicable, each data point corresponds to an independent biological replicate, unless otherwise noted.  
- **Technical Replicates**: Averaged or presented as individual points when explicitly stated. Nested grouping was done and is reflected in the PRISM files.  
- **Statistics**: Statistical test, N, p-values, and post-hoc corrections are indicated in the `statistics.xlsx` file and in the Prism project file.  
- **Transparency**: Providing the Prism files allows users to reproduce all plots and analyses exactly as performed for the publication.  

## Citation
When using this dataset, please cite both the original publication and this Zenodo record:  

Hong et al. *Circulation Research*, (2025) 137(5):646-663.* DOI: 10.1161/CIRCRESAHA.125.326260  
Zenodo Repository: [DOI: 10.5281/zenodo.17259523]