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Hereditary Xerocytosis, a rare hemolytic anemia, is due to gain of function mutations in PIEZO1, a non-selective cation channel activated by mechanical stress. How these PIEZO1 mutations impair channel function and alter red blood cell (RBC) physiology, is not completely understood. Here, we report the characterization of mutations in the N-terminal part of the protein (V598M, F681S and the double mutation G782S/R808Q), a part of the channel that was subject of many investigations to decipher its role in channel gating. Our data show that the electrophysiological features of these PIEZO1 mutants expressed in HEK293T cells are different from previously characterized PIEZO1 mutations that are located in the pore or at the C-terminal extracellular domain of the protein. Although RBC with PIEZO1 mutations showed a dehydrated phenotype, the activity of V598M, F681S or R808Q in response to stretch was not significantly different from the WT channels. In contrast, the G782S mutant showed larger currents compared to the WT PIEZO1. Interestingly, basal activity of all the mutated channels was not significantly altered at the opposite of what was expected according to the decreased water and cation contents of resting RBC. In addition, the features of mutant PIEZO1 expressed in HEK293 cells do not always correlate with the observation in RBC where PIEZO1 mutations induced a cation leak associated with an increased conductance. Our work emphasizes the role of the membrane environment in PIEZO1 activity and the need to characterize RBC permeability to assess pathogenicity to PIEZO1 mutants associated with erythrocyte diseases.

Cardiac fibroblasts express multiple voltage-dependent ion channels. Even though fibroblasts do not generate action potentials, they may influence cardiac electrophysiology by electrical coupling via gap junctions with cardiomyocytes, and through fibrosis. Here, we investigate the electrophysiological phenotype of cultured fibroblasts from right atrial appendage tissue of patients with sinus rhythm (SR) or atrial fibrillation (AF). Using the patch-clamp technique in whole-cell mode, we observed steady-state outward currents exhibiting either no rectification or inward and/or outward rectification. The distributions of current patterns between fibroblasts from SR and AF patients were not significantly different. In response to depolarizing voltage pulses, we measured transient outward currents with fast and slow activation kinetics, an outward background current, and an inward current with a potential-dependence resembling that of L-type Ca2+ channels. In cell-attached patch-clamp mode, large amplitude, paxilline-sensitive single channel openings were found in ≈65% of SR and ∼38% of AF fibroblasts, suggesting the presence of “big conductance Ca2+-activated K+ (BKCa)” channels. The open probability of BKCa was significantly lower in AF than in SR fibroblasts. When cultured in the presence of paxilline, the shape of fibroblasts became wider and less spindle-like. Our data confirm previous findings on cardiac fibroblast electrophysiology and extend them by illustrating differential channel expression in human atrial fibroblasts from SR and AF tissue.

In search of more efficacious and safe pharmacological treatments for atrial fibrillation (AF), atria-selective antiarrhythmic agents have been promoted that target ion channels principally expressed in the atria. This concept allows one to engage antiarrhythmic effects in atria, but spares the ventricles from potentially proarrhythmic side effects. It has been suggested that cardiac small conductance Ca2+-activated K+ (SK) channels may represent an atria-selective target in mammals including humans. However, there are conflicting data concerning the expression of SK channels in different stages of AF, and recent findings suggest that SK channels are upregulated in ventricular myocardium when patients develop heart failure. To address this issue, RNA-sequencing was performed to compare expression levels of three SK channels (KCNN1, KCNN2, and KCNN3) in human atrial and ventricular tissue samples from transplant donor hearts (no cardiac disease), and patients with cardiac disease in sinus rhythm or with AF. In addition, for control purposes expression levels of several genes known to be either chamber-selective or differentially expressed in AF and heart failure were determined. In atria, as compared to ventricle from transplant donor hearts, we confirmed higher expression of KCNN1 and KCNA5, and lower expression of KCNJ2, whereas KCNN2 and KCNN3 were statistically not differentially expressed. Overall expression of KCNN1 was low compared to KCNN2 and KCNN3. Comparing atrial tissue from patients with AF to sinus rhythm samples we saw downregulation of KCNN2 in AF, as previously reported. When comparing ventricular tissue from heart failure patients to non-diseased samples, we found significantly increased ventricular expression of KCNN3 in heart failure, as previously published. The other channels showed no significant difference in expression in either disease. Our results add weight to the view that SK channels are not likely to be an atria-selective target, especially in failing human hearts, and modulators of these channels may prove to have less utility in treating AF than hoped. Whether targeting SK1 holds potential remains to be elucidated.

The healthy heart adapts continuously to a complex set of dynamically changing mechanical conditions. The mechanical environment is altered by, and contributes to, multiple cardiac diseases. Mechanical stimuli are detected and transduced by cellular mechano-sensors, including stretch-activated ion channels (SAC). The precise role of SAC in the heart is unclear, in part because there are few SAC-specific pharmacological modulators. That said, most SAC can be activated by inducers of membrane curvature. The lectin LecA is a virulence factor of Pseudomonas aeruginosa and essential for P. aeruginosa-induced membrane curvature, resulting in formation of endocytic structures and bacterial cell invasion. We investigate whether LecA modulates SAC activity. TREK-1 and Piezo1 have been selected, as they are widely expressed in the body, including cardiac tissue, and they are “canonical representatives” for the potassium selective and the cation non-selective SAC families, respectively. Live cell confocal microscopy and electron tomographic imaging were used to follow binding dynamics of LecA, and to track changes in cell morphology and membrane topology in human embryonic kidney (HEK) cells and in giant unilamellar vesicles (GUV). HEK cells were further transfected with human TREK-1 or Piezo1 constructs, and ion channel activity was recorded using the patch-clamp technique. Finally, freshly isolated cardiac cells were used for studies into cell type dependency of LecA binding. LecA (500 nM) binds within seconds to the surface of HEK cells, with highest concentration at cell-cell contact sites. Local membrane invaginations are detected in the presence of LecA, both in the plasma membrane of cells (by 17 min of LecA exposure) as well as in GUV. In HEK cells, LecA sensitizes TREK-1, but not Piezo1, to voltage and mechanical stimulation. In freshly isolated cardiac cells, LecA binds to non-myocytes, but not to ventricular or atrial cardiomyocytes. This cell type specific lack of binding is observed across cardiomyocytes from mouse, rabbit, pig, and human. Our results suggest that LecA may serve as a pharmacological tool to study SAC in a cell type-preferential manner. This could aid tissue-based research into the roles of SAC in cardiac non-myocytes.

Fibrosis is associated with aging and many cardiac pathologies. It is characterized both by myofibroblast differentiation and by excessive accumulation of extracellular matrix proteins. Fibrosis-related tissue remodeling results in significant changes in tissue structure and function, including passive mechanical properties. This research area has gained significant momentum with the recent development of new tools and approaches to better characterize and understand the ability of cells to sense and respond to their biophysical environment. We use a novel hydrogel, termed CyPhyGel, to provide an advanced in vitro model of remodeling-related changes in tissue stiffness. Based on light-controlled dimerization of a Cyanobacterial Phytochrome, it enables contactless and reversible tuning of hydrogel mechanical properties with high spatial and temporal resolution. Human primary atrial fibroblasts were cultured on CyPhyGels. After 4 days of culturing on stiff (~4.6 kPa) or soft (~2.7 kPa) CyPhyGels, we analyzed fibroblast cell area and stiffness. Cells grown on the softer substrate were smaller and softer, compared to cells grown on the stiffer substrate. This difference was absent when both soft and stiff growth substrates were combined in a single CyPhyGel, with the resulting cell areas being similar to those on homogeneously stiff gels and cell stiffnesses being similar to those on homogeneously soft substrates. Using CyPhyGels to mimic tissue stiffness heterogeneities in vitro, our results confirm the ability of cardiac fibroblasts to adapt to their mechanical environment, and suggest the presence of a paracrine mechanism that tunes fibroblast structural and functional properties associated with mechanically induced phenotype conversion toward myofibroblasts. In the context of regionally increased tissue stiffness, such as upon scarring or in diffuse fibrosis, such a mechanism could help to prevent abrupt changes in cell properties at the border zone between normal and diseased tissue. The light-tunable mechanical properties of CyPhyGels and their suitability for studying human primary cardiac cells make them an attractive model system for cardiac mechanobiology research. Further investigations will explore the interactions between biophysical and soluble factors in the response of cardiac fibroblasts to spatially and temporally heterogeneous mechanical cues.