SGLT2 Inhibitor Empagliflozin Modulates Ion Channels in Adult Zebrafish Heart.

Empagliflozin, an inhibitor of sodium-glucose co-transporter 2 (iSGLT2), improves cardiovascular outcomes in patients with and without diabetes and possesses an antiarrhythmic activity. However, the mechanisms of these protective effects have not been fully elucidated. This study aimed to explore the impact of empagliflozin on ion channel activity and electrophysiological characteristics in the ventricular myocardium. The main cardiac ionic currents (INa, ICaL, ICaT, IKr, IKs) and action potentials (APs) were studied in zebrafish. Whole-cell currents were measured using the patch clamp method in the isolated ventricular cardiomyocytes. The conventional sharp glass microelectrode technique was applied for the recording of APs from the ventricular myocardium of the excised heart. Empagliflozin pretreatment compared to the control group enhanced potassium IKr step current density in the range of testing potentials from 0 to +30 mV, IKr tail current density in the range of testing potentials from +10 to +70 mV, and IKs current density in the range of testing potentials from -10 to +20 mV. Moreover, in the ventricular myocardium, empagliflozin pretreatment shortened AP duration APD as shown by reduced APD50 and APD90. Empagliflozin had no influence on sodium (INa) and L- and T-type calcium currents (ICaL and ICaT) in zebrafish ventricular cardiomyocytes. Thus, we conclude that empagliflozin increases the rapid and slow components of delayed rectifier K+ current (IKr and IKs). This mechanism could be favorable for cardiac protection.

Functional Analysis of SCN5A Genetic Variants Associated with Brugada Syndrome.

BACKGROUND: Brugada syndrome (BrS) is a rare inherited cardiac arrhythmia with increased risk of sudden cardiac death. Mutations in gene SCN5A, which encodes the α-subunit of cardiac voltage-gated sodium channel NaV1.5, have been identified in over 20% of patients with BrS. However, only a small fraction of NaV1.5 variants, which are associated with BrS, are characterized in electrophysiological experiments. RESULTS: Here we explored variants V281A and L1582P, which were found in our patients with BrS, and variants F543L and K1419E, which are reportedly associated with BrS. Heterologous expression of the variants in CHO-K1 cells and the Western blot analysis demonstrated that each variant appeared at the cell surface. We further measured sodium current in the whole-cell voltage clamp configuration. Variant F543L produced robust sodium current with a hyperpolarizing shift in the voltage dependence of steady-state fast inactivation. Other variants did not produce detectable sodium currents, indicating a complete loss of function. In a recent cryoEM structure of the hNaV1.5 channel, residues V281, K1419, and L1582 are in close contacts with residues whose mutations are reportedly associated with BrS, indicating functional importance of respective contacts. CONCLUSIONS: Our results support the notion that loss of function of NaV1.5 or decrease of the channel activity is involved in the pathogenesis of BrS.

Small G-protein RhoA is a potential inhibitor of cardiac fast sodium current.

Small G-proteins of Rho family modulate the activity of several classes of ion channels, including K+ channels Kv1.2, Kir2.1, and ERG; Ca2+ channels; and epithelial Na+ channels. The present study was aimed to check the RhoA potential regulatory effects on Na+ current (INa) transferred by Na+ channel cardiac isoform NaV1.5 in heterologous expression system and in native rat cardiomyocytes. Whole-cell patch-clamp experiments showed that coexpression of NaV1.5 with the wild-type RhoA in CHO-K1 cell line caused 2.7-fold decrease of INa density with minimal influence on steady-state activation and inactivation. This effect was reproduced by the coexpression with a constitutively active RhoA, but not with a dominant negative RhoA. In isolated ventricular rat cardiomyocytes, a 5-h incubation with the RhoA activator narciclasine (5 × 10-6 M) reduced the maximal INa density by 38.8%. The RhoA-selective inhibitor rhosin (10-5 M) increased the maximal INa density by 25.3%. Experiments with sharp microelectrode recordings in isolated right ventricular wall preparations showed that 5 × 10-6 M narciclasine induced a significant reduction of action potential upstroke velocity after 2 h of incubation. Thus, RhoA might be considered as a potential negative regulator of sodium channels cardiac isoform NaV1.5.

Characterization of a novel SCN5A genetic variant A1294G associated with mixed clinical phenotype.

Mutations in gene SCN5A, which encodes cardiac voltage-gated sodium channel Nav1.5, are associated with multiple clinical phenotypes. Here we describe a novel A1294G genetic variant detected in a male patient with combined clinical phenotype including atrioventricular II block, Brugada-like ECG, septal fibrosis, right ventricular dilatation and decreased left ventricular contractility. Residue A1294 is located in the IIIS3-S4 extracellular loop, in proximity to several residues whose mutations are associated with sodium channelopathies. The wild-type channel Nav1.5 and mutant Nav1.5-A1294G were expressed in the CHO-K1 and HEK293T cells and whole-cell sodium currents were recorded using the patch-clamp method. The A1294G channels demonstrated a negative shift of steady-state inactivation, accelerated fast and slow inactivation and decelerated recovery from intermediate inactivation. Our study reveals biophysical mechanism of the Nav1.5-A1294G dysfunction, which may underlie the combined phenotypic manifestation observed in the patient.

Novel role of Rac1/WAVE signaling mechanism in regulation of the epithelial Na+ channel.

The epithelial Na(+) channel (ENaC) is an essential channel responsible for Na(+) reabsorption in the aldosterone-sensitive distal nephron. Consequently, ENaC is a major effector impacting systemic blood volume and pressure. We have shown recently that Rac1 increases ENaC activity, whereas Cdc42 fails to change channel activity. Here we tested whether Rac1 signaling plays a physiological role in modulating ENaC in native tissue and polarized epithelial cells. We found that Rac1 inhibitor NSC23766 markedly decreased ENaC activity in freshly isolated collecting ducts. Knockdown of Rac1 in native principal cells decreased ENaC-mediated sodium reabsorption and the number of channels at the apical plasma membrane. Members of the Wiskott-Aldrich syndrome protein (WASP) family play a central role in the control of the actin cytoskeleton. N-WASP functions downstream of Cdc42, whereas WAVEs are effectors of Rac1 activity. N-WASP and all 3 isoforms of WAVE significantly increased ENaC activity when coexpressed in Chinese hamster ovary cells. However, wiskostatin, an inhibitor of N-WASP, had no effect on ENaC activity. Immunoblotting demonstrated the presence of WAVE1 and WAVE2 and absence of N-WASP and WAVE3 in mpkCCD(c14) and M-1 principal cells. Immunohistochemistry analysis also revealed localization of WAVE1 and WAVE2 but not N-WASP in the cortical collecting duct of Sprague-Dawley rat kidneys. Moreover, patch clamp analysis revealed that Rac1 and WAVE1/2 are parts of the same signaling pathway with respect to activation of ENaC. Thus, our findings suggest that Rac1 is essential for ENaC activity and regulates the channel via WAVE proteins.

The actin cytoskeleton and small G protein RhoA are not involved in flow-dependent activation of ENaC.

BACKGROUND: Epithelial cells are exposed to a variety of mechanical stimuli. Epithelial Na+ channels (ENaC) mediate sodium transport across apical membranes of epithelial cells that line the distal nephron, airway and alveoli, and distal colon. Early investigations into stretch sensitivity of ENaC were controversial. However, recent studies are supportive of ENaC's mechanosensitivity. This work studied whether flow-dependent activation of ENaC is modulated by changes in the state of the actin cytoskeleton and whether small GTPase RhoA is involved in flow-mediated increase of ENaC activity. FINDINGS: Pretreatment with Cytochalasin D and Latrunculin B for 20 min and 1-2 hrs to disassemble F-actin had no effect on flow-mediated increase of amiloride-sensitive current. Overexpression of ENaC with constitutively active (G14V) or dominant negative (T19N) RhoA similarly had no effect on flow-dependent activation of ENaC activity. In addition, we did not observe changes when we inhibited Rho-kinase with Y27632. CONCLUSIONS: Our results suggest that the flow-dependent activation of ENaC is not influenced by small GTPase RhoA and modifications in the actin cytoskeleton.

Intact cytoskeleton is required for small G protein dependent activation of the epithelial Na+ channel.

BACKGROUND: The Epithelial Na(+) Channel (ENaC) plays a central role in control of epithelial surface hydration and vascular volume. Similar to other ion channels, ENaC activity is regulated, in part, by cortical cytoskeleton. Besides, the cytoskeleton is an established target for small G proteins signaling. Here we studied whether ENaC activity is modulated by changes in the state of the cytoskeleton and whether cytoskeletal elements are involved in small G protein mediated increase of ENaC activity. METHODS AND FINDINGS: First, the functional importance of the cytoskeleton was established with whole-cell patch clamp experiments recording ENaC reconstituted in CHO cells. Pretreatment with Cytochalasin D (CytD; 10 microg/ml; 1-2 h) or colchicine (500 microM; 1-3 h) to disassembly F-actin and destroy microtubules, respectively, significantly decreased amiloride sensitive current. However, acute application of CytD induced rapid increase in macroscopic current. Single channel measurements under cell-attached conditions revealed similar observations. CytD rapidly increased ENaC activity in freshly isolated rat collecting duct, polarized epithelial mouse mpkCCD(c14) cells and HEK293 cells transiently transfected with ENaC subunits. In contrast, colchicine did not have an acute effect on ENaC activity. Small G proteins RhoA, Rac1 and Rab11a markedly increase ENaC activity. 1-2 h treatment with colchicine or CytD abolished effects of these GTPases. Interestingly, when cells were coexpressed with ENaC and RhoA, short-term treatment with CytD decreased ENaC activity. CONCLUSIONS: We conclude that cytoskeleton is involved in regulation of ENaC and is necessary for small G protein mediated increase of ENaC activity.

Peroxisome proliferator-activated receptor gamma antagonists decrease Na+ transport via the epithelial Na+ channel.

The epithelial sodium channel (ENaC) is believed to represent the rate-limiting step for sodium absorption in the renal collecting duct. Consequently, ENaC is a central effector affecting systemic blood volume and pressure. Sodium and water transport are dysregulated in diabetes mellitus. Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists are currently used in the treatment of type 2 diabetes, although their use remains limited by fluid retention. The effects of PPARgamma agonists on ENaC activity remain controversial. Although PPARgamma agonists were shown to stimulate ENaC-mediated renal salt absorption, probably via the serum- and glucocorticoid-regulated kinase 1, other studies reported that the PPARgamma agonist-induced fluid retention is independent of ENaC activity. Here we confirmed that four chemically distinct PPARgamma agonists [pioglitazone, rosiglitazone, troglitazone, and 15-deoxy-Delta12,14-prostaglandin J2 (PGJ2)] do not enhance Na+ transport in cultured renal collecting duct principal mpkCCDc14 cells, as assessed by short-circuit current measurements. However, the PPARgamma antagonist 2-chloro-5-nitro-N-4-pyridinyl-benzamide (T0070907), and to a lesser extent 2-chloro-5-nitrobenzanilide (GW9662), were found to decrease Na+ reabsorption across mpkCCDc14 cell layers. Furthermore, pretreatment of monolayers with T0070907 diminished the insulin-stimulated sodium transport. PPARgamma agonist PGJ2 did not enhance insulin-stimulated Na+ flux via ENaC. We also show that PPARgamma enhances ENaC activity when all three subunits are reconstituted in Chinese hamster ovary (CHO) cells. GW9662 inhibits ENaC activity when ENaC subunits are coexpressed in CHO cells with PPARgamma. In contrast, rosiglitazone has no effect on ENaC activity. We conclude that PPARgamma activity is important for maintaining basal and insulin-dependent transepithelial Na+ transport and ENaC activity.

Muscarinic M1 modulation of acid-sensing ion channels.

Acid-sensing ion channels (ASICs) are ligand-gated cation channels that are highly expressed in nervous system. Little is known about the regulation of these channels. Therefore, we tested whether muscarinic M1 receptors can modulate ASICs. The muscarinic agonist oxotremorine methiodide applied to the bath solution strongly inhibited the whole-cell current in Chinese hamster ovary cells heterologously expressing ASIC1a and M1 receptors. Maximal current was inhibited 30% during muscarinic receptor stimulation. These effects were fast, fully reversible and subunit specific. The acid-sensing current in population of isolated rat hippocampus CA1 and striatum interneurons, thought to be carried primarily by ASIC1a, was similarly inhibited by oxotremorine methiodide. Thus, the current study identifies ASIC1a as a novel target for muscarinic signaling.

Intrinsic voltage dependence of the epithelial Na+ channel is masked by a conserved transmembrane domain tryptophan.

Tryptophan residues critical to function are frequently located at the lipid-water interface of transmembrane domains. All members of the epithelial Na+ channel (ENaC)/Degenerin (Deg) channel superfamily contain an absolutely conserved Trp at the base of their first transmembrane domain. Here, we test the importance of this conserved Trp to ENaC/Deg function. Targeted substitution of this Trp in mouse ENaC and rat ASIC subunits decrease channel activity. Differential substitution with distinct amino acids in alpha-mENaC shows that it is loss of this critical Trp rather than introduction of residues having novel properties that changes channel activity. Surprisingly, Trp substitution unmasks voltage sensitivity. Mutant ENaC has increased steady-state activity at hyperpolarizing compared with depolarizing potentials associated with transient activation and deactivation times, respectively. The times of activation and deactivation change 1 ms/mV in a linear manner with rising and decreasing slopes, respectively. Increases in macroscopic currents at hyperpolarizing potentials results from a voltage-dependent increase in open probability. Voltage sensitivity is not influenced by divalent cations; however, it is Na+-dependent with a 63-mV decrease in voltage required to reach half-maximal activity per log increase in [Na+]. Mutant channels are particularly sensitive to intracellular [Na+] for removing this sodium abolishes voltage dependence. We conclude that the conserved Trp at the base of TM1 in ENaC/Deg channels protects against voltage by masking an inhibitory allosteric or pore block mechanism, which decreases activity in response to intracellular Na+.

Regulation of ENaC expression at the cell surface by Rab11.

The epithelial Na(+) channel (ENaC) is an essential channel responsible for Na(+) reabsorption. Coexpression of Rab11a and Rab3a small G proteins with ENaC results in a significant increase in channel activity. In contrast, coexpression of Rab5, Rab27a, and Arf-1 had no effect or slightly decreased ENaC activity. Inhibition of MEK with PD98059, Rho-kinase with Y27632 or PI3-kinase with LY294002 had no effect on ENaC activity in Rab11a-transfected CHO cells. Fluorescence imaging methods demonstrate that Rab11a colocalized with ENaC. Rab11a increases ENaC activity in an additive manner with dominant-negative dynamin, which is a GTPase responsible for endocytosis. Brefeldin A, an inhibitor of intracellular protein translocation, blocked the stimulatory action of Rab11a on ENaC activity. We conclude that ENaC channels, present on the apical plasma membrane, are being exchanged with channels from the intracellular pool in a Rab11-dependent manner.