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Vascular smooth muscle cells (VSMCs) play a key role in aortic aneurysm formation. Bone morphogenetic proteins (BMPs) have been implicated as important regulators of VSMC phenotype, and dysregulation of the BMP pathway has been shown to be associated with vascular diseases. The aim of this study was to investigate for the first time the effects of BMP-4 on the VSMC phenotype and to understand its role in the development of thoracic aortic aneurysms (TAAs). Using the angiotensin II (AngII) osmotic pump model in mice, aortas from mice with VSMC-specific BMP-4 deficiency showed changes similar to AngII-infused aortas, characterised by a loss of contractile markers, increased fibrosis, and activation of matrix metalloproteinase 9. When BMP-4 deficiency was combined with AngII infusion, there was a significantly higher rate of apoptosis and aortic dilatation. In vitro, VSMCs with mRNA silencing of BMP-4 displayed a dedifferentiated phenotype with activated canonical BMP signalling. In contrast, BMP-2-deficient VSMCs exhibited the opposite phenotype. The compensatory regulation between BMP-2 and BMP-4, with BMP-4 promoting the contractile phenotype, appeared to be independent of the canonical signalling pathway. Taken together, these results demonstrate the impact of VSMC-specific BMP-4 deficiency on TAA development.

ObjectivesElevated matrix metalloproteinase-2 (MMP-2) tissue levels have been associated with ascending thoracic aortic aneurysm (aTAA). As MMP-2 activation is controlled by interactions among matrix metalloproteinase-14 (MMP-14), a tissue inhibitor of metalloproteinases-2 (TIMP-2) and Pro-MMP-2 in cell culture, this activation process might also play a role in aTAA.MethodsVia gelatin zymography we analyzed tissue levels of MMP-2 isoforms (Pro-MMP-2, active MMP-2, total MMP-2) and via enzyme-linked immunosorbent assay (ELISA,) MMP-14,TIMP-2 and total MMP-2 tissue levels in N = 42 patients with aTAA. As controls, MMP-14 and TIMP-2 aortic tissue levels in N = 9 patients undergoing coronary artery bypass surgery were measured via ELISA, and levels of MMP-2 isoforms in N = 11 patients via gelatin zymography.ResultsActive MMP-2 was significantly higher in aTAA than in controls. Patients with aTAA exhibited significantly lower Pro-MMP-2 and TIMP-2 levels. Total MMP-2 and MMP-14 did not differ significantly between groups. Regression analysis revealed a linear relationship between TIMP-2 and the MMP-14/TIMP-2 ratio, as well as active MMP-2 in aTAA. Aneurysmatic tissue can be accurately distinguished from control aortic tissue (AUC = 1) by analyzing the active MMP-2/Pro-MMP-2 ratio with a cutoff value of 0.11, whereas MMP-14 and TIMP-2 roles are negligible in ROC analysis.ConclusionA larger amount of MMP-2 is activated in aTAA than in control aortic tissue-a factor that seems to be a central process in aneurysm development. When active MMP-2 exceeds 10% compared to Pro-MMP-2, we conclude that it originates from aneurysmatic tissue, which we regard as a starting point for further studies of aTAA biomarkers. The tissue's MMP-14/TIMP-2 ratio may regulate the degree of Pro-MMP-2 activation as a determining factor, while the enzymatic activities of MMP-14 and TIMP-2 do not seem to play a key role in aneurysm development.

Abstract Background Atrial fibrillation has an estimated prevalence of 1.5–2%, making it the most common cardiac arrhythmia. The processes that cause and sustain the disease are still not completely understood. An association between atrial fibrillation and systemic, as well as local, inflammatory processes has been reported. However, the exact mechanisms underlying this association have not been established. While it is understood that inflammatory macrophages can influence cardiac electrophysiology, a direct, causative relationship to atrial fibrillation has not been described. This study investigated the pro-arrhythmic effects of activated M1 macrophages on human induced pluripotent stem cell (hiPSC)-derived atrial cardiomyocytes, to propose a mechanistic link between inflammation and atrial fibrillation. Methods Two hiPSC lines from healthy individuals were differentiated to atrial cardiomyocytes and M1 macrophages and integrated in an isogenic, pacing-free, atrial fibrillation-like coculture model. Electrophysiology characteristics of cocultures were analysed for beat rate irregularity, electrogram amplitude and conduction velocity using multi electrode arrays. Cocultures were additionally treated using glucocorticoids to suppress M1 inflammation. Bulk RNA sequencing was performed on coculture-isolated atrial cardiomyocytes and compared to meta-analyses of atrial fibrillation patient transcriptomes. Results Multi electrode array recordings revealed M1 to cause irregular beating and reduced electrogram amplitude. Conduction analysis further showed significantly lowered conduction homogeneity in M1 cocultures. Transcriptome sequencing revealed reduced expression of key cardiac genes such as SCN5A, KCNA5, ATP1A1, and GJA5 in the atrial cardiomyocytes. Meta-analysis of atrial fibrillation patient transcriptomes showed high correlation to the in vitro model. Treatment of the coculture with glucocorticoids showed reversal of phenotypes, including reduced beat irregularity, improved conduction, and reversed RNA expression profiles. Conclusions This study establishes a causal relationship between M1 activation and the development of subsequent atrial arrhythmia, documented as irregularity in spontaneous electrical activation in atrial cardiomyocytes cocultured with activated macrophages. Further, beat rate irregularity could be alleviated using glucocorticoids. Overall, these results point at macrophage-mediated inflammation as a potential AF induction mechanism and offer new targets for therapeutic development. The findings strongly support the relevance of the proposed hiPSC-derived coculture model and present it as a first of its kind disease model.

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Atrial fibrillation (AF) is regularly accompanied by cardiac fibrosis and concomitant heart failure. Due to the heterogeneous nature and complexity of fibrosis, the knowledge about the underlying mechanisms is limited, which prevents effective pharmacotherapy. A deeper understanding of cardiac fibroblasts is essential to meet this need. We previously described phenotypic and functional differences between atrial fibroblasts from patients in sinus rhythm and with AF. Herein, we established and characterized a novel human atrial fibroblast line, which displays typical fibroblast morphology and function comparable to primary cells but with improved proliferation capacity and low spontaneous myofibroblast differentiation. These traits make our model suitable for the study of fibrosis mechanisms and for drug screening aimed at developing effective antifibrotic pharmacotherapy.

The human heart responds to various diseases with structural, mechanical, and electrical remodelling processes [...]

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.

BackgroundThe ability of heart valve cells to respond to their mechanical environment represents a key mechanism by which the integrity and function of valve cusps is maintained. A number of different mechanotransduction pathways have been implicated in the response of valve cells to mechanical stimulation. In this study, we explore the expression pattern of several mechanosensitive ion channels (MSC) and their potential to mediate mechanosensitive responses of human valve interstitial cells (VIC).MethodsMSC presence and function were probed using the patch clamp technique. Protein abundance of key MSC was evaluated by Western blotting in isolated fibroblastic VIC (VICFB) and in VIC differentiated towards myofibroblastic (VICMB) or osteoblastic (VICOB) phenotypes. Expression was compared in non-calcified and calcified human aortic valves. MSC contributions to stretch-induced collagen gene expression and to VIC migration were assessed by pharmacological inhibition of specific channels.ResultsTwo MSC types were recorded in VICFB: potassium selective and cation non-selective channels. In keeping with functional data, the presence of both TREK-1 and Kir6.1 (potassium selective), as well as TRPM4, TRPV4 and TRPC6 (cationic non-selective) channels was confirmed in VIC at the protein level. Differentiation of VICFB into VICMB or VICOB phenotypes was associated with a lower expression of TREK-1 and Kir6.1, and a higher expression of TRPV4 and TRPC6. Differences in MSC expression were also seen in non-calcified vs calcified aortic valves where TREK-1, TRPM4 and TRPV4 expression were higher in calcified compared to control tissues. Cyclic stretch-induced expression of COL I mRNA in cultured VICFB was blocked by RN-9893, a selective inhibitor of TRPV4 channels while having no effect on the stretch-induced expression of COL III. VICFB migration was blocked with the non-specific MSC blocker streptomycin and by GSK417651A an inhibitor of TRPC6/3.ConclusionAortic VIC express a range of MSC that play a role in functional responses of these cells to mechanical stimulation. MSC expression levels differ in calcified and non-calcified valves in ways that are in part compatible with the change in expression seen between VIC phenotypes. These changes in MSC expression, and associated alterations in the ability of VIC to respond to their mechanical environment, may form novel targets for intervention during aortic valvulopathies.

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.

Freshly isolated primary cardiomyocytes (CM) are indispensable for cardiac research. Experimental CM research is generally incompatible with life of the donor animal, while human heart samples are usually small and scarce. CM isolation from animal hearts, traditionally performed by coronary artery perfusion of enzymes, liberates millions of cells from the heart. However, due to progressive cell remodeling following isolation, freshly isolated primary CM need to be used within 4–8 h post-isolation for most functional assays, meaning that the majority of cells is essentially wasted. In addition, coronary perfusion-based isolation cannot easily be applied to human tissue biopsies, and it does not straightforwardly allow for assessment of regional differences in CM function within the same heart. Here, we provide a method of multi-day CM isolation from one animal heart, yielding calcium-tolerant ventricular and atrial CM. This is based on cell isolation from cardiac tissue slices following repeated (usually overnight) storage of the tissue under conditions that prolong CM viability beyond the day of organ excision by two additional days. The maintenance of cells in their near-native microenvironment slows the otherwise rapid structural and functional decline seen in isolated CM during attempts for prolonged storage or culture. Multi-day slice-based CM isolation increases the amount of useful information gained per animal heart, improving reproducibility and reducing the number of experimental animals required in basic cardiac research. It also opens the doors to novel experimental designs, including exploring same-heart regional differences.

Abstract In a patient with right ventricular outflow tract (RVOT) tachycardia, we identified a heterozygous point mutation in the selectivity filter of the stretch‐activated K2P potassium channel TREK‐1 (KCNK2 or K2P2.1). This mutation introduces abnormal sodium permeability to TREK‐1. In addition, mutant channels exhibit a hypersensitivity to stretch‐activation, suggesting that the selectivity filter is directly involved in stretch‐induced activation and desensitization. Increased sodium permeability and stretch‐sensitivity of mutant TREK‐1 channels may trigger arrhythmias in areas of the heart with high physical strain such as the RVOT. We present a pharmacological strategy to rescue the selectivity defect of the TREK‐1 pore. Our findings provide important insights for future studies of K2P channel stretch‐activation and the role of TREK‐1 in mechano‐electrical feedback in the heart.

The mechanical environment of cardiac cells changes continuously and undergoes major alterations during diseases. Most cardiac diseases, including atrial fibrillation, are accompanied by fibrosis which can impair both electrical and mechanical function of the heart. A key characteristic of fibrotic tissue is excessive accumulation of extracellular matrix, leading to increased tissue stiffness. Cells are known to respond to changes in their mechanical environment, but the molecular mechanisms underlying this ability are incompletely understood. We used cell culture systems and hydrogels with tunable stiffness, combined with advanced biophysical and imaging techniques, to elucidate the roles of the stretch-activated channel Piezo1 in human atrial fibroblast mechano-sensing. Changing the expression level of Piezo1 revealed that this mechano-sensor contributes to the organization of the cytoskeleton, affecting mechanical properties of human embryonic kidney cells and human atrial fibroblasts. Our results suggest that this response is independent of Piezo1-mediated ion conduction at the plasma membrane, and mediated in part by components of the integrin pathway. Further, we show that Piezo1 is instrumental for fibroblast adaptation to changes in matrix stiffness, and that Piezo1-induced cell stiffening is transmitted in a paracrine manner to other cells by a signaling mechanism requiring interleukin-6. Piezo1 may be a new candidate for targeted interference with cardiac fibroblast function.

Mutations in the filamin A (FlnA) gene are frequently associated with severe arterial abnormalities, although the physiological role for this cytoskeletal element remains poorly understood in vascular cells. We used a conditional mouse model to selectively delete FlnA in smooth muscle (sm) cells at the adult stage, thus avoiding the developmental effects of the knockout. Basal blood pressure was significantly reduced in conscious smFlnA knockout mice. Remarkably, pressure-dependent tone of the resistance caudal artery was lost, whereas reactivity to vasoconstrictors was preserved. Impairment of the myogenic behavior was correlated with a lack of calcium influx in arterial myocytes upon an increase in intraluminal pressure. Notably, the stretch activation of CaV1.2 was blunted in the absence of smFlnA. In conclusion, FlnA is a critical upstream element of the signaling cascade underlying the myogenic tone. These findings allow a better understanding of the molecular basis of arterial autoregulation and associated disease states.

The mechanically activated non-selective cation channel Piezo1 is a determinant of vascular architecture during early development. Piezo1-deficient embryos die at midgestation with disorganized blood vessels. However, the role of stretch-activated ion channels (SACs) in arterial smooth muscle cells in the adult remains unknown. Here, we show that Piezo1 is highly expressed in myocytes of small-diameter arteries and that smooth-muscle-specific Piezo1 deletion fully impairs SAC activity. While Piezo1 is dispensable for the arterial myogenic tone, it is involved in the structural remodeling of small arteries. Increased Piezo1 opening has a trophic effect on resistance arteries, influencing both diameter and wall thickness in hypertension. Piezo1 mediates a rise in cytosolic calcium and stimulates activity of transglutaminases, cross-linking enzymes required for the remodeling of small arteries. In conclusion, we have established the connection between an early mechanosensitive process, involving Piezo1 in smooth muscle cells, and a clinically relevant arterial remodeling.

How renal epithelial cells respond to increased pressure and the link with kidney disease states remain poorly understood. Pkd1 knockout or expression of a PC2 pathogenic mutant, mimicking the autosomal dominant polycystic kidney disease, dramatically enhances mechanical stress-induced tubular apoptotic cell death. We show the presence of a stretch-activated K+ channel dependent on the TREK-2 K2P subunit in proximal convoluted tubule epithelial cells. Our findings further demonstrate that polycystins protect renal epithelial cells against apoptosis in response to mechanical stress, and this function is mediated through the opening of stretch-activated K2P channels. Thus, to our knowledge, we establish for the first time, both in vitro and in vivo, a functional relationship between mechanotransduction and mechanoprotection. We propose that this mechanism is at play in other important pathologies associated with apoptosis and in which pressure or flow stimulation is altered, including heart failure or atherosclerosis.

TREK-1 potassium channels are involved in a number of physiopathological processes such as neuroprotection, pain and depression. Molecules able to open or to block these channels can be clinically important. Having a cell model for screening such molecules is of particular interest. Here, we describe the development of the first available cell line that constituvely expresses the TREK-1 channel. The TREK-1 channel expressed by the h-TREK-1/HEK cell line has conserved all its modulation properties. It is opened by stretch, pH, polyunsaturated fatty acids and by the neuroprotective molecule, riluzole and it is blocked by spadin or fluoxetine. We also demonstrate that the h-TREK-1/HEK cell line is protected against ischemia by using the oxygen-glucose deprivation model.