The heart is one of the most mechanically active organs in the body. In a mechanism known as the “Bainbridge Reflex”, the heart rate accelerates in response to the mechanical stretch induced by the increase in venous return. The cardiac pacemaker controls heart rate, and while stretch-activated channels have been identified in cardiac tissue, their molecular identity remains unknown. We hypothesize that PIEZO channels are the molecular determinant of the stretch-dependent heart rate acceleration responsible for the Bainbridge reflex. Using quantitative polymerase chain reaction (qPCR), we assessed the presence of Piezo1 and Piezo2 transcripts in the pacemaker, atrium, and ventricle of the mouse heart. Our findings revealed that both Piezo1 and Piezo2 are present in the three regions with significantly higher expression in the pacemaker and atria. Combining immunohystochemistry, tissue clearing, and super-resolution microscopy, we analyzed the distribution of Piezo1 and Piezo2 in mouse pacemaker explants. Our results show that Piezo2 is uniformly expressed in the pacemaker and surrounding atrial tissue, whereas Piezo1 exhibits higher expression levels outside the pacemaker. These results were further confirmed at the single-cell level, with immunostaining of Piezo1 and Piezo2 in isolated pacemaker cells (HCN4+) and transitional cells (HCN4-). We observed similar expression levels of Piezo2 in both cell types and increased Piezo1 expression in transitional cells. In addition, we observed distinct localization patterns for Piezo1 and Piezo2 at the subcellular level. Piezo1 predominantly localizes to the sarcolemma, while Piezo2 exhibits a striated distribution that colocalizes alternately with both the Z- and the M- line of the sarcomere. Given this pattern, half of the Piezo2 bands colocalize with the RyR. These results set the starting point to evaluate the functional role of PIEZO channels in the cardiac pacemaker.

BK channels are potassium channels activated in response to depolarization and elevated intracellular calcium ion levels. It has been observed that BK channels form clusters in cells, but the mechanism for clustering has not been characterized. This project attempts to discover important components that lead to BK channel clustering using super-resolution microscopy, proximity ligation assay, and Fluorescence Recovery After Photobleaching (FRAP) experiments. One possible mechanism relates to denser regions in the plasma membrane of PtdIns(4,5)P₂ as a possible lipid raft, hypothesized to localize proteins. We used tsA-201 cells to express BK channels (α subunit). In FRAP experiments, BK channels were tagged with a green fluorescent protein (GFP). To modify the levels of PtdIns(4,5)P₂, we co-expressed PIP5Kγ, the enzyme that catalyzes the synthesis of PtdIns4P to PtdIns(4,5)P₂. Expression of PIP5Kγ is known to increase PtdIns(4,5)P₂ levels by 30%. In our analysis, we assumed that large, bright fluorescent dots in live cells correspond to BK clusters. We found that co-expression of PIP5Kγ with BK decreases cluster size by 43% in super-resolution experiments and increases the number of puncta (BK clusters) by 41% in PLA experiments. FRAP experiments on a PIP2 biosensor, PH-PLCδ1-GFP, showed reduced fluorescence recovery speed when PIP5Kγ was co-expressed. Future FRAP experiments observing BK channels will allow us to determine if membrane components, such as PtdIns(4,5)P₂, influence the integrity and mobility of BK clusters and if the addition of these lipids is sufficient to induce additional cluster formation.