Reduction In Pump Activity Research Articles

Abstract 12273: Crispr-Mutation of the Na + -k + Pump β1-Subunit Protects Against Cardiac Fibrosis and Heart Failure in Mice

Introduction: The Na+-K+ pump controls translocation of Na+ and K+ across the cell membrane which indirectly affects heart muscle contraction. Glutathionylation of the Na+-K+ pump’s β1 subunit (NKβ1) is a reversible post-translational oxidative modification, where a glutathione adduct forms a disulphide bond with the cysteine residue at site 45, leading to a reduction in pump activity. Using CRISPR/Cas9 technology, we created a transgenic mouse line (CRISPR-β1) that has a mutation in cysteine to serine at site 45 on NKβ1, causing insensitivity to β1-GSS. Hypothesis: We hypothesize that a mutation of the β1-GSS can inhibit transverse aortic constriction (TAC)-induced cardiac fibrosis. Methods and Results: TAC was induced in both the wild-type (WT) and CRISPR-NKβ1 mutated mice (CRISPR-β1) over a period of four weeks. Gene sequencing showed that cysteine 45 was replaced by serine, and immunoprecipitation of the free cysteine demonstrated that cysteine 45 was mutated in the β1 subunit of the CRISPR-β1 mice hearts. TAC significantly reduced cardiac function in the WT but not the CRISPR-β1 mice (ejection fraction: WT TAC: 36.24% ± 3.49%; CRISPR-β1 TAC: 57.5% ± 3.41%). Similarly, we found that the CRISPR-β1 mice were protected from cardiac hypertrophy, as evidenced by a significant reduction in cardiomyocytes size (WT TAC: 452.1 μm2 ± 30.74 μm2 ; CRISPR-β1 TAC: 271.1 μm 2 ± 11.0 μm 2 ). TAC significantly increased interstitial and perivascular fibrosis in the left ventricles of WT but not the CRISPR-β1 mice (interstitial: WT TAC: 18.4% ± 1.96%; CRISPR-β1 TAC: 5.2% ± 1.62%, perivascular: WT TAC: 42.6% ± 4.5%; CRISPR-β1 TAC: 22.01% ± 1.6%). Cardioprotective effects observed in the CRISPR-β1 mice were at least in part attributed to a significant reduction in the expression of ANP, BNP, Tgfb1, Fn1 and Ctgf genes. Conclusions: Our data indicate that CRISPR mutation of the Na+-K+ pump’s β1 subunit prevents the development of cardiac fibrosis and heart failure in a TAC-induced mouse model.

Intracellular Coupling via Limiting Calmodulin

Measurements of cellular Ca2+-calmodulin concentrations have suggested that competition for limiting calmodulin may couple calmodulin-dependent activities. Here we have directly tested this hypothesis. We have found that in endothelial cells the amount of calmodulin bound to nitric-oxide synthase and the catalytic activity of the enzyme both are increased approximately 3-fold upon changes in the phosphorylation status of the enzyme. Quantitative immunoblotting indicates that the synthase can bind up to 25% of the total cellular calmodulin. Consistent with this, simultaneous determinations of the free Ca2+ and Ca2+-calmodulin concentrations in these cells performed using indo-1 and a fluorescent calmodulin biosensor (Kd = 2 nm) indicate that increased binding of calmodulin to the synthase is associated with substantial reductions in the Ca2+-calmodulin concentrations produced and an increase in the [Ca2+]50 for formation of the calmodulin-biosensor complex. The physiological significance of these effects is confirmed by a corresponding 40% reduction in calmodulin-dependent plasma membrane Ca2+ pump activity. An identical reduction in pump activity is produced by expression of a high affinity (Kd = 0.3 nm) calmodulin biosensor, and treatment to increase calmodulin binding to the synthase then has no further effect. This suggests that the observed reduction in pump activity is due specifically to reduced calmodulin availability. Increases in synthase activity thus appear to be coupled to decreases in the activities of other calmodulin targets through reductions in the size of a limiting pool of available calmodulin. This exemplifies what is likely to be a ubiquitous mechanism for coupling among diverse calmodulin-dependent activities.

Short-term regulation of endothelial Na(+)-K(+)-pump activity by cGMP: a 133Cs magnetic resonance study.

The effect of nitric oxide radicals (NO) on the activity of porcine aortic endothelial Na(+)-K(+)-ATPase is reported. Measurements were made using an in vitro cell system and 133Cs magnetic resonance (NMR). It is shown that NO, through stimulation of guanylate cyclase, results in a reduction of pump activity. Similar observations were made using 8-Br-cGMP. Measurement of the cytosolic volume indicated no changes in volume during incubation with 8-Br-cGMP. Our measurements indicate a continuous regulation of endothelial Na(+)-K(+)-ATPase activity by endogenous NO. This regulation could be removed by L-NAME, resulting in a small increase in pump activity.

Regulation of the ASC system and Na+/K + pump activities in brown trout (Salmo trutta) hepatocytes

The present study investigates the regulation of Na+/K+ pump activity and alanine uptake in trout hepatocytes. Pump activity increased when cells were incubated in an amino-acid-free medium, while it was reduced in cells from fasted animals. Short-term exposure (3 h) to glucagon modified the activity of the pump in a complex seasonally dependent pattern: in experiments carried out in autumn and winter there was some inhibition, while in spring the pump was activated by this hormone. Pharmacological modification of levels of two intracellular signal transducers, namely cyclic AMP and Ca2+, always led to a reduction in pump activity. These experiments were conducted in May, when activation of the pump by glucagon exposure occurred. There is no apparent explanation for the mechanism by which this hormone modifies the activity of the pump. Glucagon also regulates the activity of system ASC (a Na+-dependent amino acid carrier with short-chain neutral amino acids as preferred substrates). This regulation also showed a seasonally dependent pattern, although the pattern was opposite to that found for the regulation of Na+/K+ pump activity.

Inhibition of Na/K ATPase Activity in Maternal and Neonatal Erythrocyte Ghosts in Pregnancy-Induced Hypertension

The specific activity of Na/K ATPase was determined to investigate the Sodium pump, in pregnancy-induced hypertension. A slight significant reduction in pump activity was observed in pregnancy induced hypertensive patients. A similar trend was shown in cord blood from pregnancy-hypertensive mothers, although not statistically significant. This reduction could explain the finding of higher intracellular Sodium levels in pregnancy-induced hypertension.

Hyperpolarization following long-lasting tetanic activation of hippocampal pyramidal cells

Long-lasting spike activation of CA3 hippocampal pyramidal neurons is shown to cause the development of a large and long-lasting (> 50 s) membrane hyperpolarization (PTH). Under normal conditions this PTH is mainly fiven by a relatively potential-independent process, presumably an electrogenic sodium pump. Following reduction in pump activity (cooling ouabain), the PTH remains but is mainly produced by a conductance process, presumably a K conductance increase resulting from a sodium-induced calcium release from intracellular stores.