Impairment of TRPC1-BK Complex in Diabetic Rat Coronary Artery
Diabetes mellitus (DM) is a chronic disease that poses significant risks to human health and can lead to various complications. Among these, coronary artery disease is one of the most severe complications in diabetic patients. Recent evidence suggests that the pathophysiology of diabetic coronary diseases is closely related to the dysfunction of ion channels in coronary artery smooth muscle cells (SMCs). Understanding the ionic mechanisms underlying diabetic coronary artery dysfunction is therefore of critical clinical importance.
Two key ion channels in coronary artery SMCs are the transient receptor potential C (TRPC) channels and the large-conductance calcium-activated potassium (BK) channels. The TRPC1 channel, a member of the classical TRP cation channels family, is widely distributed in vascular SMCs. Calcium depletion in the sarcoplasmic reticulum induces the opening of TRPC1 channels, leading to calcium influx, cell depolarization, and vasoconstriction. The BK channel, also widely distributed in vascular SMCs, responds to cell membrane depolarization or increased cytosolic calcium concentrations by opening, which leads to membrane hyperpolarization in vascular SMCs and subsequent blood vessel relaxation. It has been reported that TRPC1 channels are physically associated with BK channels in rat aorta SMCs. In this study, we confirmed that the TRPC1 channel and BK channel form a complex in normal rat coronary artery SMCs. This complex plays a role in increased calcium entry and decreased BK channel activity, leading to changes in vascular reactivity in coronary arteries. These findings suggest that dysfunction of the TRPC1-BK complex could be involved in the development of coronary diseases and cardiac adverse events in diabetes.
To investigate this, streptozotocin-induced diabetic rats were sacrificed 8 weeks after the development of hyperglycemia. First, the co-immunolocalization of TRPC1 and BK channels in coronary arteries was evaluated using co-immunoprecipitation. Immunoblot experiments confirmed a single BK-a protein expression band in the anti-TRPC1 antibody pull-down from coronary artery lysates. Similarly, TRPC1 proteins were detected in anti-BK-a antibody precipitates. Immunoprecipitation fraction intensity quantifications were similar in control and diabetic groups. Double-labeling immunofluorescence experiments further suggested that TRPC1 and BK channels are co-immunolocalized on diabetic coronary artery SMCs. These results indicate a physical association between TRPC1 and BK channels in both control and diabetic coronary artery SMCs.
Next, the expressions of TRPC1 and BK channels in diabetic coronary arteries were examined. Protein and mRNA expressions of TRPC1 channels were significantly increased in the diabetic group. The protein and mRNA expressions of the BK-a subunit were similar between control and diabetic rats, while the BK-b1 subunit expressions were significantly downregulated in diabetes.
Cytosolic calcium concentrations before and after incubation with SKF96365, a relatively specific TRPC1 blocker, were measured using Fura-2/AM, a cytoplasmic calcium indicator. Coronary artery SMCs were pre-treated with thapsigargin to deplete endoplasmic reticulum calcium stores in calcium-free D-Hanks buffer, causing a rise in intracellular calcium concentration. Extracellular calcium was then applied to activate TRPC1 channels, leading to another increase in intracellular calcium. The Dratios were higher in diabetic coronary artery SMCs compared to controls and significantly decreased in both control and diabetic groups after incubation with SKF96365.
Total potassium currents at baseline and after exposure to 100 nmol/L iberiotoxin (IBTX), a BK channel blocker, were recorded in control and diabetic rat coronary SMCs. Current densities of IBTX-sensitive BK channels decreased in diabetic rats compared to controls. Additionally, total potassium currents at baseline and after exposure to 30 mmol/L NS1619, a BK channel agonist, were recorded. Current densities of NS1619-activated BK channels in diabetic rats were significantly higher compared to controls.
To investigate the effects of TRPC1-BK complex imbalance on diabetic coronary artery function, vascular tension measurements were performed. Vascular tensions in the presence of the vasoconstrictor endothelin-1 (ET-1) did not show significant differences between control and diabetic rats. The BK channel inhibitor IBTX induced coronary vessel contraction, which was increased in diabetic rats compared to controls. In the presence of the BK channel agonist NS1619 and the TRPC1 channel blocker SKF96365, coronary vessels pre-contracted with ET-1 showed vasodilation. The induced coronary vasodilation percentages decreased with NS1619 and increased with SKF96365 in diabetic rats compared to controls. These data confirm that diabetic coronary artery vasorelaxation and vasoconstriction dysfunction can be related to the TRPC1-BK complex imbalance.
In summary, this study presents novel findings on the ionic mechanisms underlying diabetic coronary dysfunction. First, the TRPC1 channel is associated with the BK channel, forming a complex on diabetic coronary artery SMCs, similar to normal ones. Second, the expression of the TRPC1-BK complex is imbalanced in diabetic coronary arteries, with increased TRPC1 channel expression and decreased BK channel expression. Third, dysregulation of calcium signaling and BK channel activation by the TRPC1-BK complex results in coronary artery dysfunction in diabetic rats. These findings indicate that the TRPC1-BK complex is crucial for the regulation of coronary artery function. Furthermore, the imbalance of the TRPC1-BK complex in diabetic coronary arteries may lead to serious cardiovascular complications and cardiac adverse events.
doi.org/10.1097/CM9.0000000000001904
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