Epilepsy-causing KCNT1 variants increase KNa1.1 channel activity by disrupting the activation gate.

Gain-of-function pathogenic missense KCNT1 variants are associated with several developmental and epileptic encephalopathies (DEE). With few exceptions, patients are heterozygous and there is a paucity of mechanistic information about how pathogenic variants increase KNa1.1 channel activity and the behaviour of heterotetrameric channels comprising both wild-type (WT) and variant subunits. To better understand these, we selected a range of variants across the DEE spectrum, involving mutations in different protein domains and studied their functional properties. Whole-cell electrophysiology was used to characterise homomeric and heteromeric KNa1.1 channel assemblies carrying DEE-causing variants in the presence and absence of 10 mM intracellular sodium. Voltage-dependent activation of homomeric variant KNa1.1 assemblies were more hyperpolarised than WT KNa1.1 and, unlike WT KNa1.1, exhibited voltage-dependent activation in the absence of intracellular sodium. Heteromeric channels formed by co-expression of WT and variant KNa1.1 had activation kinetics intermediate of homomeric WT and variant KNa1.1 channels, with residual sodium-independent activity. In general, WT and variant KNa1.1 activation followed a single exponential, with time constants unaffected by voltage or sodium. Mutating the threonine in the KNa1.1 selectivity filter disrupted voltage-dependent activation, but sodium-dependence remained intact. Our findings suggest that KNa1.1 gating involves a sodium-dependent activation gate that modulates a voltage-dependent selectivity filter gate. Collectively, all DEE-associated KNa1.1 mutations lowered the energetic barrier for sodium-dependent activation, but some also had direct effects on selectivity filter gating. Destabilisation of the inactivated unliganded channel conformation can explain how DEE-causing amino acid substitutions in diverse regions of the channel structure all cause gain-of-function.

Impact of SGLT2 Inhibitors on Heart Failure: From Pathophysiology to Clinical Effects

Heart failure (HF) affects up to over 20% of patients with type 2 diabetes (T2DM), even more in the elderly. Although, in T2DM, both hyperglycemia and the proinflammatory status induced by insulin resistance are crucial in cardiac function impairment, SGLT2i cardioprotective mechanisms against HF are several. In particular, these beneficial effects seem attributable to the significant reduction of intracellular sodium levels, well-known to exert a cardioprotective role in the prevention of oxidative stress and consequent cardiomyocyte death. From a molecular perspective, patients’ exposure to gliflozins’ treatment mimics nutrient and oxygen deprivation, with consequent autophagy stimulation. This allows to maintain the cellular homeostasis through different degradative pathways. Thus, since their introduction in the clinical practice, the hypotheses on SGLT2i mechanisms of action have changed: from simple glycosuric drugs, with consequent glucose lowering, erythropoiesis enhancing and ketogenesis stimulating, to intracellular sodium-lowering molecules. This provides their consequent cardioprotective effect, which justifies its significant reduction in CV events, especially in populations at higher risk. Finally, the updated clinical evidence of SGLT2i benefits on HF was summarized. Thus, this review aimed to analyze the cardioprotective mechanisms of sodium glucose transporter 2 inhibitors (SGLT2i) in patients with HF, as well as their clinical impact on cardiovascular events.

EXPERIMENTAL SETUP FOR EVALUATING THE TRANSPORT FUNCTIONS OF LIVING CORNEAL ENDOTHELIAL CELLS

The paper summarizes the requirements and describes an experimental setup that allows us to study the dynamics of the concentration of intracellular sodium in the endothelium of the cornea.

Anti-VEGF therapy prevents Müller intracellular edema by decreasing VEGF-A in diabetic retinopathy

Background Although vascular endothelial growth factor A (VEGF-A) is known to play a key role in causing retinal edema, whether and how VEGF-A induces intracellular edema in the retina still remains unclear. Methods Sprague-Dawley rats were rendered diabetic with intraperitoneal injection of streptozotocin. Intravitreal injection of ranibizumab was performed 8 weeks after diabetes onset. rMC-1 cells (rat Müller cell line) were treated with glyoxal for 24 h with or without ranibizumab. The expression levels of inwardly rectifying K+ channel 4.1 (Kir4.1), aquaporin 4 (AQP4), Dystrophin 71 (Dp71), VEGF-A, glutamine synthetase (GS) and sodium-potassium-ATPase (Na+-K+-ATPase) were examined using Western blot. VEGF-A in the supernatant of the cell culture was detected with ELISA. The intracellular potassium and sodium levels were detected with specific indicators. Results Compared with normal control, protein expressions of Kir4.1 and AQP4 were down-regulated significantly in diabetic rat retinas, which were prevented by ranibizumab. The above changes were recapitulated in vitro. Similarly, the intracellular potassium level in glyoxal-treated rMC-1 cells was increased, while the intracellular sodium level and Na+-K+-ATPase protein level remained unchanged, compared with control. However, ranibizumab treatment decreased intracellular sodium, but not potassium. Conclusion Ranibizumab protected Müller cells from diabetic intracellular edema through the up-regulation of Kir4.1 and AQP4 by directly binding VEGF-A. It also caused a reduction in intracellular osmotic pressure.

Multinuclear MRI to disentangle intracellular sodium concentration and extracellular volume fraction in breast cancer

The purpose of this work was to develop a novel method to disentangle the intra- and extracellular components of the total sodium concentration (TSC) in breast cancer from a combination of proton ($$^{1}$$ 1 H) and sodium ($$^{23}\hbox {Na}$$ 23 Na ) magnetic resonance imaging (MRI) measurements. To do so, TSC is expressed as function of the intracellular sodium concentration ($$\hbox {C}_{\text {IC}}$$ C IC ), extracellular volume fraction (ECV) and the water fraction (WF) based on a three-compartment model of the tissue. TSC is measured from $$^{23}\hbox {Na}$$ 23 Na MRI, ECV is calculated from baseline and post-contrast $$^{1}$$ 1 H $$\hbox {T}_{{1}}$$ T 1 maps, while WF is measured with a $$^{1}$$ 1 H chemical shift technique. $$\hbox {C}_{\text {IC}}$$ C IC is then extrapolated from the model. Proof-of-concept was demonstrated in three healthy subjects and two patients with triple negative breast cancer. In both patients, TSC was two to threefold higher in the tumor than in normal tissue. This alteration mainly resulted from increased $$\hbox {C}_{\text {IC}}$$ C IC ($$\sim$$ ∼ 30 mM), which was $$\sim$$ ∼ 130% greater than in healthy conditions (10–15 mM) while the ECV was within the expected range of physiological values (0.2–0.25). Multinuclear MRI shows promise for disentangling $$\hbox {C}_{\text {IC}}$$ C IC and ECV by taking advantage of complementary $$^{1}$$ 1 H and $$^{23}\hbox {Na}$$ 23 Na measurements.

Abstract 15315: SGLT2 Inhibitor Ertugliflozin Decreases Elevated Intracellular Sodium, Improves Myocardial Energetics and Enhances Function in Metabolic Heart Disease

Background: Sodium-glucose co-transporter 2 (SGLT2) inhibitors are antidiabetic drugs of great interest in cardiology due to their improvement of heart failure outcomes independent of diabetes. As SGLT2 is not expressed in cardiomyocytes, the mechanism of such benefit remains unclear. Elevated myocardial intracellular sodium [Na + ] i has been found in heart failure and SGLT2 inhibition lowers [Na + ] i in isolated cardiomyocytes. Elevated [Na + ] i was shown to decrease mitochondrial calcium via mitochondrial Na/Ca exchanger (NCx MITO ), resulting in decreased mitochondrial ATP synthesis. We have previously shown that mice fed a diet high in fat and sugar (HFHS) develop metabolic heart disease (MHD) characterized by decreased mitochondrial ATP synthesis with decreased phosphocreatine (PCr), worsened diastolic function and contractile reserve. We hypothesize that the SGLT2 inhibitor ertugliflozin (ERTU) decreases the elevated [Na + ] i to improve energetics and contractile function in MHD. Methods and Results: Isolated hearts from mice after 6 months of HFHS vs. control diet (CD), +/- ERTU in the last month, were studied using 31 P and 23 Na NMR spectroscopy to measure PCr/ATP ratio and [Na + ] i , respectively. As expected, HFHS hearts showed lower PCr/ATP, diastolic dysfunction (↑LVEDP) and lack of contractile reserve (↓RPP) during high work protocol compared to CD hearts. Myocardial [Na + ] i was elevated more than 2-fold in HFHS compared to CD. One month of ERTU treatment decreased [Na + ] i and improved energetics and contractile function in HFHS to levels similar to or better than CD. Perfusion with CGP 37157, which inhibits NCx MITO , improved PCr/ATP in HFHS hearts. Conclusion: Lowering of myocardial [Na + ] i by ertugliflozin contributes to improved energetics and function in MHD. These results suggest targeting [Na + ] i as an effective strategy to improve cardiac dysfunction in MHD and other forms of heart disease associated with elevated myocardial [Na + ] i.

Ranibizumab prevents Müller cell edema by decreasing VEGF-A in diabetic retinopathy

Background: Diabetic macular edema (DME) is the most common cause of vision loss in patients with diabetic retinopathy. The efficacy of anti-VEGF therapy has been well demonstrated and become the standard of care in the management of DME. The present study is to explore the possible mechanism(s) of ranibizumab in protecting Müller cells from cellular edema in experimental diabetic retinopathy. Methods: Sprague-Dawley rats were rendered diabetes with intraperitoneal injection of streptozotocin. Intravitreal injection of ranibizumab was performed 8 weeks after diabetes onset. Four weeks later, the rats were killed and the retinas were harvested for examination. rMC-1 cells (rat Müller cell line) were treated with glyoxal for 24 hours, with or without ranibizumab. Cell viability was detected with CCK-8 assay. The expressions of inwardly rectifying K + channel 4.1 (Kir4.1), aquaporin 4 (AQP4), Dystrophin 71 (Dp71), vascular endothelial growth factor A (VEGF-A), glutamine synthetase (GS) and sodium-potassium-ATPase (Na + -K + -ATPase) were examined with Western blot. VEGF-A in the supernatant of cell culture was detected with ELISA. The intracellular potassium and sodium levels were detected with specific indicators. Results: Compared to the normal control, the protein expressions of Kir4.1, AQP4 and Dp71 were down-regulated significantly in diabetic rat retinas, which were prevented by ranibizumab. The above changes were recapitulated in vitro . As compared with the control, the intracellular potassium level in glyoxal-treated rMC-1 cells was increased, while the intracellular sodium level and Na + -K + -ATPase protein level remained unchanged. However, ranibizumab treatment increased Na + -K + -ATPase protein expression and decreased intracellular sodium, but not potassium level. Conclusion: Ranibizumab protected Müller cells from intracellular edema through up-regulation of Kir4.1, AQP4, and Dp71 by directly binding VEGF-A. It also increased the expression of Na + -K + -ATPase, contributing to reduction of the intracellular osmotic pressure.