Phosphorylation and other conundrums of Na/Ca exchanger, NCX1.

The Na+/Ca2+ exchanger (NCX) is an important Ca2+ transport mechanism in virtually all cells in the body. There are three genes that control the expression of NCX in mammals. There are at least 16 alternatively spliced isoforms of NCX1 that target muscle and nerve and other tissues. Here we briefly discuss three remarkable regulatory issues or "conundrums" that involve the most prevalently expressed gene, NCX1. (1) How is NCX1 regulated by phosphorylation? We suggest that the macromolecular complex of NCX1 plays a critical role in the regulation of NCX. The role of the macromolecular complex and evidence supporting its existence and functional importance is presented. (2) Can there be transport block of a single "mode" of NCX1 transport by drugs or therapeutic agents? The simple answer is "no." A brief explanation is provided. (3) How can NCX1 knockout mice live? The answer is "by other compensatory regulatory mechanisms." These conundrums highlight important features in NCX1 and lay the foundation for new experiments to elucidate function and regulation of NCX1 and provide a context for investigations that seek to understand novel therapeutic agents.

Gender differences in Na/Ca exchanger current and beta-adrenergic responsiveness in heart failure in pig myocytes.

Clinical trials suggest females experience less heart failure (HF) progression, mortality, and arrhythmia frequency. HF increases Na/Ca exchanger (NCX) expression and activity contributing to both depressed contractility and ventricular arrhythmias, but whether gender modifies this effect is unknown. Left ventricular myocytes were isolated from control and from tachycardic pacing-induced failing swine hearts of both sexes. The Ni-sensitive NCX current (I(NCX)) was measured in voltage clamp after blocking other channels. In control myocytes there is no difference in basal I(NCX) and beta-adrenergic responsiveness between male and female animals. HF greatly increased I(NCX) and reduced beta-adrenergic responsiveness in males compared to females, an effect that was eliminated by PP1. Diuretic therapy (furosemide, 1 mg/kg/day) further enhanced I(NCX) and reduced beta-adrenergic responsiveness in females and eliminated the gender difference. Gender-specific differences in calcium handling may contribute to improved survival of females in HF.

Muscarinic modulation of the sodium-calcium exchanger in heart failure.

BACKGROUND: The Na-Ca exchanger (NCX) is a critical calcium efflux pathway in excitable cells, but little is known regarding its autonomic regulation. METHODS AND RESULTS: We investigated beta-adrenergic receptor and muscarinic receptor regulation of the cardiac NCX in control and heart failure (HF) conditions in atrially paced pigs. NCX current in myocytes from control swine hearts was significantly increased by isoproterenol, and this response was reversed by concurrent muscarinic receptor stimulation with the addition of carbachol, demonstrating "accentuated antagonism." Okadaic acid eliminated the inhibitory effect of carbachol on isoproterenol-stimulated NCX current, indicating that muscarinic receptor regulation operates via protein phosphatase-induced dephosphorylation. However, in myocytes from atrially paced tachycardia-induced HF pigs, the NCX current was significantly larger at baseline but less responsive to isoproterenol compared with controls, whereas carbachol failed to inhibit isoproterenol-stimulated NCX current, and 8-Br-cGMP did not restore muscarinic responsiveness. Protein phosphatase type 1 dialysis significantly reduced NCX current in failing but not control cells, consistent with NCX hyperphosphorylation in HF. Protein phosphatase type 1 levels associated with NCX were significantly depressed in HF pigs compared with control, and total phosphatase activity associated with NCX was significantly decreased. CONCLUSIONS: We conclude that the NCX is autonomically modulated, but HF reduces the level and activity of associated phosphatases; defective dephosphorylation then "locks" the exchanger in a highly active state.

Furosemide and the progression of left ventricular dysfunction in experimental heart failure.

OBJECTIVES: We tested the hypothesis that furosemide accelerates the progression of left ventricular systolic dysfunction in a tachycardia-induced porcine model of heart failure. BACKGROUND: Furosemide activates the renin-angiotensin-aldosterone system in patients with congestive heart failure (CHF). Such activation may contribute to CHF progression, but prospective data are lacking. METHODS: Thirty-two Yorkshire pigs were randomized to furosemide (1 mg/kg intramuscularly daily, mean 16.1 +/- 0.9 mg) or placebo. Thereafter, a pacing model of heart failure was utilized to produce systolic dysfunction in both sets of animals (fractional shortening <0.16 by echocardiogram). The goal was to determine if furosemide would accelerate the progression of left ventricular dysfunction in the "treated" group. After sacrifice, sodium-calcium exchanger currents and their responsiveness to isoproterenol were measured during voltage clamp. All investigators were blinded to treatment assignment. RESULTS: Furosemide shortened the time to left ventricular dysfunction (35.1 +/- 5.1 days in placebo versus 21.4 +/- 3.2 days for furosemide animals; p = 0.038, log-rank test). By day 14, aldosterone levels were significantly higher in furosemide animals (43.0 +/- 11.8 ng/dl vs. 17.6 +/- 4.5 ng/dl; p < 0.05). Serum sodium was reduced (133.0 +/- 0.9 mmol/l furosemide vs. 135.7 +/- 0.8 mmol/l placebo; p < 0.05), but no difference in norepinephrine, potassium, magnesium, creatinine, or urea nitrogen was present. Basal sodium-calcium exchanger currents were significantly increased and isoproterenol responsiveness depressed by furosemide. CONCLUSIONS: Tachycardic pigs given furosemide had significant acceleration of both contractile and metabolic features of CHF, including left ventricular systolic dysfunction, elevated serum aldosterone levels, and altered calcium handling in a controlled experimental model of heart failure.

Protein kinase A hyperphosphorylation increases basal current but decreases beta-adrenergic responsiveness of the sarcolemmal Na+-Ca2+ exchanger in failing pig myocytes.

The sodium-calcium exchanger (NCX) protein is the major cardiac calcium extrusion mechanism and is upregulated in heart failure (HF). NCX expression level and functional activity as regulated by beta-adrenergic receptor (beta-AR) stimulation in swine with and without tachycardia-induced heart failure were studied. The Ni2+-sensitive NCX current was measured in myocytes from HF and control animals in the basal state or in the presence of isoproterenol, forskolin, 8-Br-cAMP, okadaic acid, or protein phosphatase type 1. Western blot analysis revealed a significant increase in both the 120-kDa (29%) and 80-kDa (69%) fragments in HF (P<0.05 versus control). Despite this modest increase in protein, the basal peak outward NCX current was increased almost 5-fold in HF (P<0.05 versus control). Stimulation with isoproterenol, however, increased the control currents to a significantly greater extent than HF (500% increase in control versus 100% increase in HF, P<0.01); peak stimulated current was not different in HF and control. This reduction in responsiveness to beta-AR stimulation was refractory to forskolin, 8-Br-cAMP, or okadaic acid stimulation. In vitro protein kinase A back-phosphorylation revealed higher phosphorylation capacity of NCX protein in control versus HF, consistent with increased phosphorylation in vivo (hyperphosphorylation) in HF. Protein phosphatase type 1 exposure resulted in a significant reduction (73%) in peak basal current in HF (compared with no significant difference in controls), confirming that the increased basal NCX current in HF is predominantly attributable to hyperphosphorylation. NCX expression and activity are thus increased in HF, although beta-AR responsiveness is decreased because of NCX hyperphosphorylation.

Beta-adrenergic stimulation of pig myocytes with decreased cytosolic free magnesium prolongs the action potential and enhances triggered activity.

INTRODUCTION: Heart failure results in chronic beta-adrenergic stimulation, repolarization lability, and arrhythmias associated with early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs). Having described a significant reduction in intracellular free magnesium ([Mg2+]i) in experimental heart failure, we asked whether a reduction in [Mg2+]i would delay repolarization or facilitate EADs and/or DADs. METHODS AND RESULTS: Left ventricular myocytes were isolated from Yorkshire swine. Cytosolic free [Mg2+] was set at 0.12 mM (LoMg) or 1.2 mM (HiMg) through pipette dialysis. Action potentials (AP), Ca current (I(Ca)), and sodium/calcium exchange current (I(NCX)) were measured in the presence or absence of isoproterenol (2 microM) at 37 degrees C. Under basal conditions (0.1-Hz stimulation, 2 mM external [Ca2+]), reducing [Mg2+]i had no effect on AP duration and I(Ca) but did significantly enhance I(NCX). In contrast, during superfusion with isoproterenol, reduced [Mg2+]i caused a significant increase in AP duration at both 50% and 90% repolarization (APD50 and APD90) compared with HiMg (P < 0.05). LoMg cells manifested a high incidence of triggered activities, including spontaneous AP, EADs, and DADs (83.3% in LoMg, n = 12 vs 38.3% in HiMg, n = 13; P < 0.05). I(Ca) and I(NCX) were significantly increased in LoMg cells compared with HiMg cells (P < 0.05). CONCLUSION: Decreased cytosolic free magnesium prolongs AP duration and increases the incidence of triggered activity during beta-adrenergic stimulation. These effects may be due to increased I(Ca) and I(NCX) in the presence of reduced intracellular [Mg2+]. A magnesium-dependent increase in triggered activity coupled with delayed repolarization during beta-adrenergic stimulation could contribute to the arrhythmogenic substrate in heart failure.

Cytosolic free magnesium modulates Na/Ca exchange currents in pig myocytes.

OBJECTIVE: Cardiac Na/Ca exchanger (NCX) protein is up-regulated and intracellular free magnesium ([Mg(2+)](i)) is significantly reduced in experimental heart failure. We asked whether changes in [Mg(2+)](i) in a physiologically relevant range could alter the I(NCX). METHODS: The nickel-sensitive current was measured in voltage-clamped myocytes (Yorkshire pig; left ventricular) exposed to ramp pulses at 37 degrees C in Tyrode's solution containing ouabain, nifedipine and +/- Ni(2+) (5 mmol/l). The intracellular free [Ca(2+)] and [Mg(2+)] concentrations were set at 50 nmol/l and 1.25 mmol/l (HiMg) or 0.13 mmol/l (LoMg), respectively, through pipette dialysis. RESULTS: Reducing [Mg(2+)](i) resulted in a significant increase in both outward and inward Ni-sensitive current without a shift in the reversal potential. This effect was not due to the inadvertent reduction of intracellular free [ATP] secondary to binding of ATP to Mg(2+); reducing intracellular [ATP] in LoMg cells from 1.35 mmol/l to 0.18 mmol/l did not affect I(NCX). The intracellular free [Ca(2+)] was raised from 50 to 200 nmol/l, resulting in augmented inward and outward current due to calcium activation. HiMg attenuated both inward and outward currents significantly compared to LoMg, suggesting that [Mg(2+)](i) competes with [Ca(2+)](i) at the allosteric regulatory site. CONCLUSION: Cytosolic free magnesium modulates the I(NCX) over a physiologic range independent of [ATP](i). Reduced [Mg(2+)](i) in heart failure could contribute to altered calcium regulation of the NCX, contributing to the altered heart failure phenotype through enhanced NCX activity.