A Multitubular Kidney-on-Chip to Decipher Pathophysiological Mechanisms in Renal Cystic Diseases.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a major renal pathology provoked by the deletion of PKD1 or PKD2 genes leading to local renal tubule dilation followed by the formation of numerous cysts, ending up with renal failure in adulthood. In vivo, renal tubules are tightly packed, so that dilating tubules and expanding cysts may have mechanical influence on adjacent tubules. To decipher the role of this coupling between adjacent tubules, we developed a kidney-on-chip reproducing parallel networks of tightly packed tubes. This original microdevice is composed of cylindrical hollow tubes of physiological dimensions, parallel and closely packed with 100-200 µm spacing, embedded in a collagen I matrix. These multitubular systems were properly colonized by different types of renal cells with long-term survival, up to 2 months. While no significant tube dilation over time was observed with Madin-Darby Canine Kidney (MDCK) cells, wild-type mouse proximal tubule (PCT) cells, or with PCT Pkd1 +/- cells (with only one functional Pkd1 allele), we observed a typical 1.5-fold increase in tube diameter with isogenic PCT Pkd1 -/- cells, an ADPKD cellular model. This tube dilation was associated with an increased cell proliferation, as well as a decrease in F-actin stress fibers density along the tube axis. With this kidney-on-chip model, we also observed that for larger tube spacing, PCT Pkd1 -/- tube deformations were not spatially correlated with adjacent tubes whereas for shorter spacing, tube deformations were increased between adjacent tubes. Our device reveals the interplay between tightly packed renal tubes, constituting a pioneering tool well-adapted to further study kidney pathophysiology.

TMEM33 regulates intracellular calcium homeostasis in renal tubular epithelial cells.

Mutations in the polycystins cause autosomal dominant polycystic kidney disease (ADPKD). Here we show that transmembrane protein 33 (TMEM33) interacts with the ion channel polycystin-2 (PC2) at the endoplasmic reticulum (ER) membrane, enhancing its opening over the whole physiological calcium range in ER liposomes fused to planar bilayers. Consequently, TMEM33 reduces intracellular calcium content in a PC2-dependent manner, impairs lysosomal calcium refilling, causes cathepsins translocation, inhibition of autophagic flux upon ER stress, as well as sensitization to apoptosis. Invalidation of TMEM33 in the mouse exerts a potent protection against renal ER stress. By contrast, TMEM33 does not influence pkd2-dependent renal cystogenesis in the zebrafish. Together, our results identify a key role for TMEM33 in the regulation of intracellular calcium homeostasis of renal proximal convoluted tubule cells and establish a causal link between TMEM33 and acute kidney injury.

Piezo1-dependent regulation of urinary osmolarity.

The collecting duct (CD) is the final segment of the kidney involved in the fine regulation of osmotic and ionic balance. During dehydration, arginine vasopressin (AVP) stimulates the expression and trafficking of aquaporin 2 (AQP2) to the apical membrane of CD principal cells, thereby allowing water reabsorption from the primary urine. Conversely, when the secretion of AVP is lowered, as for instance upon water ingestion or as a consequence of diabetes insipidus, the CD remains water impermeable leading to enhanced diuresis and urine dilution. In addition, an AVP-independent mechanism of urine dilution is also at play when fasting. Piezo1/2 are recently discovered essential components of the non-selective mechanically activated cationic channels. Using quantitative PCR analysis and taking advantage of a ß-galactosidase reporter mouse, we demonstrate that Piezo1 is preferentially expressed in CD principal cells of the inner medulla at the adult stage, unlike Piezo2. Remarkably, siRNAs knock-down or conditional genetic deletion of Piezo1 specifically in renal cells fully suppresses activity of the stretch-activated non-selective cationic channels (SACs). Piezo1 in CD cells is dispensable for urine concentration upon dehydration. However, urinary dilution and decrease in urea concentration following rehydration are both significantly delayed in the absence of Piezo1. Moreover, decreases in urine osmolarity and urea concentration associated with fasting are fully impaired upon Piezo1 deletion in CD cells. Altogether, these findings indicate that Piezo1 is critically required for SAC activity in CD principal cells and is implicated in urinary osmoregulation.

Smooth muscle filamin A is a major determinant of conduit artery structure and function at the adult stage.

Human mutations in the X-linked FLNA gene are associated with a remarkably diverse phenotype, including severe arterial morphological anomalies. However, the role for filamin A (FlnA) in vascular cells remains partially understood. We used a smooth muscle (sm)-specific conditional mouse model to delete FlnA at the adult stage, thus avoiding the developmental effects of the knock-out. Inactivation of smFlnA in adult mice significantly lowered blood pressure, together with a decrease in pulse pressure. However, both the aorta and carotid arteries showed a major outward hypertrophic remodeling, resistant to losartan, and normally occurring in hypertensive conditions. Notably, arterial compliance was significantly enhanced in the absence of smFlnA. Moreover, reactivity of thoracic aorta rings to a variety of vasoconstrictors was elevated, while basal contractility in response to KCl depolarization was reduced. Enhanced reactivity to the thromboxane A2 receptor agonist U46619 was fully reversed by the ROCK inhibitor Y27632. We discuss the possibility that a reduction in arterial stiffness upon smFlnA inactivation might cause a compensatory increase in conduit artery diameter for normalization of parietal tension, independently of the ROCK pathway. In conclusion, deletion of smFlnA in adult mice recapitulates the vascular phenotype of human bilateral periventricular nodular heterotopia, culminating in aortic dilatation.

Arterial Myogenic Activation through Smooth Muscle Filamin A.

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.

Piezo1 in Smooth Muscle Cells Is Involved in Hypertension-Dependent Arterial Remodeling.

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.

The Piezo Mechanosensitive Ion Channels: May the Force Be with You!

Piezo1 and Piezo2 are critically required for nonselective cationic mechanosensitive channels in mammalian cells. Within the last 5 years, tremendous progress has been made in understanding the function of Piezo1/2 in embryonic development, physiology, and associated disease states. A recent breakthrough was the discovery of a chemical opener for Piezo1, indicating that mechanosensitive ion channels can be opened independently of mechanical stress. We will review these new exciting findings, which might pave the road for the identification of novel therapeutic strategies.

An alternative to force.

Researchers have discovered a synthetic small molecule that activates a mechanosensitive ion channel involved in a blood disorder.

Regional ion channel gene expression heterogeneity and ventricular fibrillation dynamics in human hearts.

RATIONALE: Structural differences between ventricular regions may not be the sole determinant of local ventricular fibrillation (VF) dynamics and molecular remodeling may play a role. OBJECTIVES: To define regional ion channel expression in myopathic hearts compared to normal hearts, and correlate expression to regional VF dynamics. METHODS AND RESULTS: High throughput real-time RT-PCR was used to quantify the expression patterns of 84 ion-channel, calcium cycling, connexin and related gene transcripts from sites in the LV, septum, and RV in 8 patients undergoing transplantation. An additional eight non-diseased donor human hearts served as controls. To relate local ion channel expression change to VF dynamics localized VF mapping was performed on the explanted myopathic hearts right adjacent to sampled regions. Compared to non-diseased ventricles, significant differences (p<0.05) were identified in the expression of 23 genes in the myopathic LV and 32 genes in the myopathic RV. Within the myopathic hearts significant regional (LV vs septum vs RV) expression differences were observed for 13 subunits: Nav1.1, Cx43, Ca3.1, Cavα2δ2, Cavß2, HCN2, Na/K ATPase-1, CASQ1, CASQ2, RYR2, Kir2.3, Kir3.4, SUR2 (p<0.05). In a subset of genes we demonstrated differences in protein expression between control and myopathic hearts, which were concordant with the mRNA expression profiles for these genes. Variability in the expression of Cx43, hERG, Na(+)/K(+) ATPase ß1 and Kir2.1 correlated to variability in local VF dynamics (p<0.001). To better understand the contribution of multiple ion channel changes on VF frequency, simulations of a human myocyte model were conducted. These simulations demonstrated the complex nature by which VF dynamics are regulated when multi-channel changes are occurring simultaneously, compared to known linear relationships. CONCLUSIONS: Ion channel expression profile in myopathic human hearts is significantly altered compared to normal hearts. Multi-channel ion changes influence VF dynamic in a complex manner not predicted by known single channel linear relationships.

Piezo1-dependent stretch-activated channels are inhibited by Polycystin-2 in renal tubular epithelial cells.

Mechanical forces associated with fluid flow and/or circumferential stretch are sensed by renal epithelial cells and contribute to both adaptive or disease states. Non-selective stretch-activated ion channels (SACs), characterized by a lack of inactivation and a remarkably slow deactivation, are active at the basolateral side of renal proximal convoluted tubules. Knockdown of Piezo1 strongly reduces SAC activity in proximal convoluted tubule epithelial cells. Similarly, overexpression of Polycystin-2 (PC2) or, to a greater extent its pathogenic mutant PC2-740X, impairs native SACs. Moreover, PC2 inhibits exogenous Piezo1 SAC activity. PC2 coimmunoprecipitates with Piezo1 and deletion of its N-terminal domain prevents both this interaction and inhibition of SAC activity. These findings indicate that renal SACs depend on Piezo1, but are critically conditioned by PC2.

Circadian rhythms govern cardiac repolarization and arrhythmogenesis.

Sudden cardiac death exhibits diurnal variation in both acquired and hereditary forms of heart disease, but the molecular basis of this variation is unknown. A common mechanism that underlies susceptibility to ventricular arrhythmias is abnormalities in the duration (for example, short or long QT syndromes and heart failure) or pattern (for example, Brugada's syndrome) of myocardial repolarization. Here we provide molecular evidence that links circadian rhythms to vulnerability in ventricular arrhythmias in mice. Specifically, we show that cardiac ion-channel expression and QT-interval duration (an index of myocardial repolarization) exhibit endogenous circadian rhythmicity under the control of a clock-dependent oscillator, krüppel-like factor 15 (Klf15). Klf15 transcriptionally controls rhythmic expression of Kv channel-interacting protein 2 (KChIP2), a critical subunit required for generating the transient outward potassium current. Deficiency or excess of Klf15 causes loss of rhythmic QT variation, abnormal repolarization and enhanced susceptibility to ventricular arrhythmias. These findings identify circadian transcription of ion channels as a mechanism for cardiac arrhythmogenesis.

Early ion-channel remodeling and arrhythmias precede hypertrophy in a mouse model of complete atrioventricular block.

Complete atrioventricular block (CAVB) and related ventricular bradycardia are known to induce ventricular hypertrophy and arrhythmias. Different animal models of CAVB have been established with the most common being the dog model. Related studies were mainly focused on the consequences on the main repolarizing currents in these species, i.e. IKr and IKs, with a limited time point kinetics post-AVB. In order to explore at a genomic scale the electrical remodeling induced by AVB and its chronology, we have developed a novel model of CAVB in the mouse using a radiofrequency-mediated ablation procedure. We investigated transcriptional changes in ion channels and contractile proteins in the left ventricles as a function of time (12h, 1, 2 and 5 days after CAVB), using high-throughput real-time RT-PCR. ECG in conscious and anesthetized mice, left ventricular pressure recordings and patch-clamp were used for characterization of this new mouse model. As expected, CAVB was associated with a lengthening of the QT interval. Moreover, polymorphic ventricular tachycardia was recorded in 6/9 freely-moving mice during the first 24h post-ablation. Remarkably, myocardial hypertrophy was only evident 48 h post-ablation and was associated with increased heart weight and altered expression of contractile proteins. During the first 24 hours post-CAVB, genes encoding ion channel subunits were either up-regulated (such as Nav1.5, +74%) or down-regulated (Kv4.2, -43%; KChIP2, -47%; Navß1, -31%; Cx43, -29%). Consistent with the transient alteration of Kv4.2 expression, I(to) was reduced at day 1, but restored at day 5. In conclusion, CAVB induces two waves of molecular remodeling: an early one (≤24 h) leading to arrhythmias, a later one related to hypertrophy. These results provide new molecular basis for ventricular tachycardia induced by AV block.

Gender-related differences in ion-channel and transporter subunit expression in non-diseased human hearts.

Gender-related differences in ventricular electrophysiology are known to be important determinants of human arrhythmic risk, but the underlying molecular basis is poorly understood. The present work aims to provide the first detailed analysis of gender-related cardiac ion-channel gene-distribution, based on samples from non-diseased human hearts. By using a high-throughput quantitative approach, we investigated at a genome-scale the expression of 79 genes encoding ion-channel and transporter subunits in epicardial and endocardial tissue samples from non-diseased transplant donors (10 males, 10 females). Gender-related expression differences involved key genes implicated in conduction and repolarization. Female hearts showed reduced expression for a variety of K(+)-channel subunits with potentially important roles in cardiac repolarization, including HERG, minK, Kir2.3, Kv1.4, KChIP2, SUR2 and Kir6.2, as well as lower expression of connexin43 and phospholamban. In addition, they demonstrated an isoform switch in Na(+)/K(+)-ATPase, expressing more of the alpha1 and less of the alpha3 subunit than male hearts, along with increased expression of calmodulin-3. Iroquois transcription factors (IRX3, IRX5) were more strongly expressed in female than male epicardium, but the transmural gradient remained. Protein-expression paralleled transcript patterns for all subunits examined: HERG, minK, Kv1.4, KChIP2, IRX5, Nav1.5 and connexin43. Our results indicate that male and female human hearts have significant differences in ion-channel subunit composition, with female hearts showing decreased expression for a number of repolarizing ion-channels. These findings are important for understanding sex-related differences in the susceptibility to ventricular arrhythmias, particularly for conditions associated with repolarization abnormalities like Brugada and Long QT syndrome.

Variable Na(v)1.5 protein expression from the wild-type allele correlates with the penetrance of cardiac conduction disease in the Scn5a(+/-) mouse model.

BACKGROUND: Loss-of-function mutations in SCN5A, the gene encoding Na(v)1.5 Na+ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a(+/-) mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A. METHODOLOGY/PRINCIPAL FINDINGS: Based on ECG, 10-week-old Scn5a(+/-) mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS< or = 18 ms; QRS in wild-type littermates: 10-18 ms). Phenotypic difference persisted with aging. At 10 weeks, the Na+ channel blocker ajmaline prolonged QRS interval similarly in both groups of Scn5a(+/-) mice. In contrast, in old mice (>53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a(+/-) mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A-mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a(+/-) mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a(+/-) mice had similar Na(v)1.5 mRNA but higher Na(v)1.5 protein expression, and moderately larger I(Na) current than severely affected Scn5a(+/-) mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a(+/-) mice than in mildly affected ones. CONCLUSIONS: Scn5a(+/-) mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a(+/-) mice, phenotype severity correlates with wild-type Na(v)1.5 protein expression.

Ion-channel mRNA-expression profiling: Insights into cardiac remodeling and arrhythmic substrates.

Membrane ion channels and transporters are key determinants of cardiac electrical function. Their expression is affected by cardiac region, hemodynamic properties, heart-rate changes, neurohormones and cardiac disease. One of the important determinants of ion-channel function is the level of ion-channel subunit mRNA expression, which governs the production of ion-channel proteins that traffic to the cell-membrane to form functional ion-channels. Ion-channel mRNA-expression profiling can be performed with cDNA microarrays or high-throughput reverse transcription/polymerase chain reaction (PCR) methods. Expression profiling has been applied to evaluate the dependence of ion-channel expression on cardiac region, revealing the molecular basis of regionally-controlled electrical properties as well as the molecular determinants of specialized electrical functions like pacemaking activity. Ion-channel remodeling occurs with cardiac diseases like heart failure, congenital repolarization abnormalities, and atrial fibrillation, and expression profiling has provided insights into the mechanisms by which these conditions affect cardiac electrical stability. Expression profiling has also shown how hormonal changes, antiarrhythmic drugs, cardiac development and altered heart rate affect ion-channel expression patterns to modify cardiac electrical function and sometimes to produce cardiac rhythm disturbances. This article reviews the information obtained to date with the application of cardiac ion-channel expression profiling. With increasing availability and efficiency of high-throughput PCR methods for ion-channel subunit mRNA-expression characterization, it is likely that the application of ion-channel expression profiling will increase and that it will provide important new insights into the determinants of cardiac electrical function in both physiological and pathological situations.

Transfer of rolf S3-S4 linker to HERG eliminates activation gating but spares inactivation.

Studies in Shaker, a voltage-dependent potassium channel, suggest a coupling between activation and inactivation. This coupling is controversial in hERG, a fast-inactivating voltage-dependent potassium channel. To address this question, we transferred to hERG the S3-S4 linker of the voltage-independent channel, rolf, to selectively disrupt the activation process. This chimera shows an intact voltage-dependent inactivation process consistent with a weak coupling, if any, between both processes. Kinetic models suggest that the chimera presents only an open and an inactivated states, with identical transition rates as in hERG. The lower sensitivity of the chimera to BeKm-1, a hERG preferential closed-state inhibitor, also suggests that the chimera presents mainly open and inactivated conformations. This chimera allows determining the mechanism of action of hERG blockers, as exemplified by the test on ketoconazole.

Haplotype-sharing analysis implicates chromosome 7q36 harboring DPP6 in familial idiopathic ventricular fibrillation.

Idiopathic Ventricular Fibrillation (IVF) is defined as spontaneous VF without any known structural or electrical heart disease. A family history is present in up to 20% of probands with the disorder, suggesting that at least a subset of IVF is hereditary. A genome-wide haplotype-sharing analysis was performed for identification of the responsible gene in three distantly related families in which multiple individuals died suddenly or were successfully resuscitated at young age. We identified a haplotype, on chromosome 7q36, that was conserved in these three families and was also shared by 7 of 42 independent IVF patients. The shared chromosomal segment harbors part of the DPP6 gene, which encodes a putative component of the transient outward current in the heart. We demonstrated a 20-fold increase in DPP6 mRNA levels in the myocardium of carriers as compared to controls. Clinical evaluation of 84 risk-haplotype carriers and 71 noncarriers revealed no ECG or structural parameters indicative of cardiac disease. Penetrance of IVF was high; 50% of risk-haplotype carriers experienced (aborted) sudden cardiac death before the age of 58 years. We propose DPP6 as a gene for IVF and increased DPP6 expression as the likely pathogenetic mechanism.

Molecular and functional characterization of a new potassium conductance in mouse ventricular fibroblasts.

The present work is aimed at identifying and characterizing, at a molecular and functional level, new ionic conductances potentially involved in the excitation-secretion coupling and proliferation of cardiac ventricular fibroblasts. Among potassium channel transcripts which were screened by high-throughput real-time PCR, SUR2 and Kir6.1 mRNAs were found to be the most abundant in ventricular fibroblasts. The corresponding proteins were not detected by western blot following 5 days of cell culture, but had appeared at 7 days, increasing with extended cell culture duration as the fibroblasts differentiated into myofibroblasts. Using the inside-out configuration of the patch-clamp technique, single potassium channels could be recorded. These had properties similar to those reported for SUR2/Kir6.1 channels, i.e. activation by pinacidil, inhibition by glibenclamide and activation by intracellular UDP. As already reported for this molecular signature, they were insensitive to intracellular ATP. In the whole-cell configuration, these channels have been shown to be responsible for a glibenclamide-sensitive macroscopic potassium current which can be activated not only by pinacidil, but also by nanomolar concentrations of the sphingolipid sphingosine-1-phosphate (S1P). The activation of this current resulted in an increase in cell proliferation and a decrease in IL-6 secretion, suggesting it has a functional role in situations where S1P increases. Overall, this work demonstrates for the first time that SUR2/Kir6.1 channels represent a significant potassium conductance in ventricular fibroblasts which may be activated in physio-pathological conditions and which may impact on fibroblast proliferation and function.

Transcriptional profiling of ion channel genes in Brugada syndrome and other right ventricular arrhythmogenic diseases.

AIMS: Brugada syndrome is an inherited sudden-death arrhythmia syndrome. Na(+)-current dysfunction is central, but mutations in the SCN5A gene (encoding the cardiac Na(+)-channel Nav1.5) are present in only 20% of probands. This study addressed the possibility that Brugada patients display specific expression patterns for ion-channels regulating cardiac conduction, excitability, and repolarization. METHODS AND RESULTS: Transcriptional profiling was performed on right-ventricular endomyocardial biopsies from 10 unrelated Brugada probands, 11 non-diseased organ-donors, seven heart-transplant recipients, 10 with arrhythmogenic right-ventricular cardiomyopathy, and nine with idiopathic right-ventricular outflow-tract tachycardia. Brugada patients showed distinct clustering differences vs. the two control and two other ventricular-tachyarrhythmia groups, including 14 of 77 genes encoding important ion-channel/ion-transporter subunits. Nav1.5 and K(+)-channels Kv4.3 and Kir3.4 were more weakly expressed, whereas the Na(+)-channel Nav2.1 and the K(+)-channel TWIK1 were more strongly expressed, in Brugada syndrome. Differences were also seen in Ca(2+)-homeostasis transcripts, including stronger expression of RYR2 and NCX1. The molecular profile of Brugada patients with SCN5A mutations did not differ from Brugada patients without SCN5A mutations. CONCLUSION: Brugada patients exhibit a common ion-channel molecular expression signature, irrespective of the culprit gene. This finding has potentially important implications for our understanding of the pathophysiology of Brugada syndrome, with possible therapeutic and diagnostic consequences.