Differential distribution and regulation of mouse cardiac Na+/K+-ATPase alpha1 and alpha2 subunits in T-tubule and surface sarcolemmal membranes.

OBJECTIVES: Two Na+/K+-ATPase (NKA) alpha-subunit isoforms, alpha1 and alpha2, are expressed in the adult mouse heart. The subcellular distribution of these isoforms in T-tubule and surface sarcolemmal (SSL) membranes and their regulation by cAMP-dependent protein kinase (PKA) is unclear. METHODS: We used formamide-induced detubulation of mouse ventricular myocytes to investigate differential functional distribution and regulation by PKA of alpha1 and alpha2 in T-tubule versus SSL membranes by measuring NKA current (I(pump)) and NKA-mediated Na+ efflux (-d[Na](i)/dt). RESULTS: I(pump) is composed of 88% alpha(1)-mediated I(pump) (Ialpha1) and 12% alpha2-mediated I(pump) (Ialpha2). alpha1 and alpha2 subunits demonstrate distinct ouabain affinities (105+/-6 and 0.3+/-0.1 micromol/L respectively) but similar affinity for intracellular Na+ (K(1/2)Na+ of 16.6+/-0.8 and 16.7+/-2.6 mmol/L respectively). Detubulation reduced (i) I(pump) density (1.42+/-0.1 to 1.20+/-0.04 pA/pF), (ii) cell capacitance (181+/-12 to 127+/-17 pF), and (iii) Ialpha2 contribution (12 to 6%). Total I(pump) density was approximately 60% higher in T-tubule (1.94 pA/pF, derived) vs. SSL membranes. Although T-tubule membranes represent only 30% of total surface area, they generate approximately 70% of Ialpha2 and approximately 37% of Ialpha1. Ialpha1 density was substantially higher than Ialpha2 in SSL (Ialpha1:Ialpha2 = 16:1) but this was markedly reduced in T-tubules (4:1). In addition to differential localisation, isoprenaline (ISO, 1 micromol/L) significantly increased alpha1-mediated NKA Na+ affinity (from 16.6+/-0.8 to 13.3+/-1.4 mmol/L) and caused a small increase in maximal NKA Na+ efflux rate. ISO had no effect on alpha2-mediated NKA activity. CONCLUSION: These data suggest that NKA alpha1 and alpha2 subunits are differentially localised and regulated by PKA in T-tubule and SSL membranes and may have distinct regulatory roles in cardiac excitation-contraction coupling.

FCCP is cardioprotective at concentrations that cause mitochondrial oxidation without detectable depolarisation.

OBJECTIVE: The role of mitochondria and in particular of mitochondrial uncoupling in the mechanism of cardioprotection is not defined. In the accompanying paper we have shown that pretreatment of isolated rat hearts with a low concentration (100 nM) of FCCP, prior to global ischaemia, is cardioprotective, while 300 nM FCCP exacerbates injury. Here we define the mitochondrial responses to increasing concentrations of FCCP and also to explore the equivalence of the cardioprotective doses of diazoxide. METHODS: Changes in mitochondrial respiration in response to FCCP and diazoxide were determined in isolated rat ventricular myocytes. In addition, mitochondrial state was monitored using confocal microscopy to record mitochondrial potential (TMRM) and redox state (NADH) during FCCP and diazoxide treatment. Myocytes were also voltage-clamped and whole cell currents recorded in response to 100 nM FCCP. RESULTS: FCCP (10-1000 nM) caused significant dose-dependent increase in oxygen consumption. Diazoxide (30 microM) failed to cause any measurable change in mitochondrial function. FCCP at 100 nM caused mitochondrial oxidation, but no change in mitochondrial membrane potential or (sarc)K(ATP) channel current, while at 300 nM, FCCP caused significant mitochondrial depolarisation. Diazoxide failed to induce any mitochondrial oxidation or depolarisation. CONCLUSIONS: Concentrations of FCCP that cause mitochondrial oxidation without depolarisation are cardioprotective. Higher FCCP concentrations dissipate mitochondrial membrane potential and exacerbate injury. This establishes the principle that mild mitochondrial uncoupling activates a protective mechanism. Diazoxide did not cause mitochondrial oxidation or mitochondrial depolarisation, suggesting it induces protection via another mechanism.