Michael J. Shattock

Formation of 4-hydroxy-2-nonenal-modified proteins in ischemic rat heart

4-Hydroxy-2-nonenal (HNE) is a major lipid peroxidation product formed during oxidative stress. Because of its reactivity with nucleophilic compounds, particularly metabolites and proteins containing thiol groups, HNE is cytotoxic. The aim of this study was to assess the extent and time course for the formation of HNE-modified proteins during ischemia and ischemia plus reperfusion in isolated rat hearts. With an antibody to HNE-Cys/His/Lys and densitometry of Western blots, we quantified the amount of HNE-protein adduct in the heart. By taking biopsies from single hearts (n = 5) at various times (0, 5, 10, 15, 20, 35, and 40 min) after onset of zero-flow global ischemia, we showed a progressive, time-dependent increase (which peaked after 30 min) in HNE-mediated modification of a discrete number of proteins. In studies with individual hearts (n = 4/group), control aerobic perfusion (70 min) resulted in a very low level (296 arbitrary units) of HNE-protein adduct formation; by contrast, after 30-min ischemia HNE-adduct content increased by >50-fold (15,356 units, P < 0.05). In other studies (n = 4/group), administration of N-(2-mercaptopropionyl)glycine (MPG, 1 mM) to the heart for 5 min immediately before 30-min ischemia reduced HNE-protein adduct formation during ischemia by approximately 75%. In studies (n = 4/group) that included reperfusion of hearts after 5, 10, 15, or 30 min of ischemia, there was no further increase in the extent of HNE-protein adduct formation over that seen with ischemia alone. Similarly, in experiments with MPG, reperfusion did not significantly influence the tissue content of HNE-protein adduct. Western immunoblot results were confirmed in studies using in situ immunofluorescent localization of HNE-protein in cryosections. In conclusion, ischemia causes a major increase in HNE-protein adduct that would be expected to reflect a toxic sequence of events that might act to compromise tissue survival during ischemia and recovery on reperfusion.

Measurement of Na(+)-K+ pump current in isolated rabbit ventricular myocytes using the whole-cell voltage-clamp technique. Inhibition of the pump by oxidant stress.

Free radical-induced oxidant stress has been implicated in ischemia and reperfusion-induced injury in the heart. A number of studies have reported that oxidant stress reduces the activity of isolated Na+,K(+)-ATPase enzyme. We have studied the effects of oxidant stress on the Na(+)-K+ pump current recorded in isolated rabbit ventricular myocytes using the whole-cell voltage-clamp technique. Singlet oxygen and superoxide were generated by the photoactivation of rose bengal (50 nM). The compositions of Tyrodes and pipette solutions were designed to block channel currents and electrogenic Na(+)-Ca2+ exchange. Cells were dialyzed with a pipette solution containing 30 mM sodium via wide-tipped (1-2-M omega) electrodes, and outward Na(+)-K+ pump current was recorded during a voltage-ramp protocol. The validity of using such a ramp protocol was confirmed by comparison with steady-state Na(+)-K+ pump current measurements made at the end of 200-msec square-clamp steps. Active currents were abolished by potassium-free Tyrodes solution or ouabain (100 microM), and Na(+)-K+ pump current was defined as the Ko-sensitive fraction of recorded currents. The activation of Na(+)-K+ pump current by intracellular sodium and extracellular potassium revealed a concentration of potassium necessary for half-maximal activation of 18.7 mM for Nai and 1.88 mM for Ko. Oxidant stress inhibited Na(+)-K+ pump current at all voltages, such that after a 10-minute exposure to photoactivated rose bengal, Na(+)-K+ pump current measured at 0 mV was reduced by approximately 50%. The voltage dependence of Na(+)-K+ pump current was, however, not profoundly affected by oxidant stress. Passive membrane currents recorded in the absence of all major electrogenic ion channels, exchangers, or pumps were unaffected by oxidant stress. This observation suggests that, over the time course during which Na(+)-K+ pump inhibition and calcium overload occur, oxidant stress does not cause nonspecific membrane damage and changes in the passive resistance of the lipid bilayer. The inhibition of Na(+)-K+ pump activity by oxidant stress may contribute to ischemia/reperfusion injury and reperfusion-induced cellular calcium overload.

Ischemia-induced phosphorylation of phospholemman directly activates rat cardiac Na/K ATPase

Regulation of the Na/K ATPase by protein kinases is model‐specific. We have observed a profound activation of the sarcolemmal Na/K ATPase during cardiac ischemia, which is masked by an inhibitor of the enzyme in the cytosol. The aim of these studies was to characterize the pathways involved in this activation in the Langendorff‐perfused rat heart. Na/K ATPase activity was determined by measuring ouabain‐sensitive phosphate generation by cardiac homogenates at 37°C. In isolated sarcolemma, ischemia (30 min) caused a substantial activation of the Na/K ATPase compared with aerobic controls, which was abolished by perfusing the heart with staurosporine or H89. However, the α1 subunit of the Na/K ATPase was not phosphorylated during ischemia. The sarcolemmal protein phospholemman (PLM) was found associated with the Na/K ATPase α1 and β1 but not α2 subunits, and PLM increased its association with the catalytic subunit of PKA following ischemia. In vitro 14‐3‐3 binding assays indicated that PLM was phosphorylated following ischemia. These results indicate that the ischemia‐induced activation of the Na/K ATPase is indirect, through phosphorylation of PLM, which is an integral part of the Na/K ATPase enzyme complex in the heart. The role of PLM is analogous to phospholamban in regulating the sarcoplasmic reticulum calcium ATPase.

Mouse isolated perfused heart: Characteristics and cautions

1. Owing to the considerable potential for manipulating the murine genome and, as a consequence, the increasing availability of genetically modified models of cardiovascular diseases, the mouse is fast becoming a cornerstone of animal research. However, progress in the use of various murine preparations is hampered by the lack of facilities and skills for the adequate physiological assessment of genetically modified mice.

Cardiac ischemia causes inhibition of the Na/K ATPase by a labile cytosolic compound whose production is linked to oxidant stress

OBJECTIVE Intracellular Na rises rapidly during cardiac ischemia and this has been attributed to the combination of increased influx of Na via sodium-proton exchange and decreased activity of the Na/K ATPase. The aim of these studies was to investigate the effects of ischemia on Na/K ATPase function in Langendorff-perfused rat hearts. METHODS Na/K ATPase activity was determined by measuring ouabain-sensitive phosphate generation from ATP by cardiac homogenates. RESULTS Global ischemia (15 and 30 min) caused a substantial reduction in Na/K ATPase function despite high substrate availability in the assay. When sarcolemmal membranes were purified away from the cytosol a profound activation of the Na/K ATPase was revealed following ischemia, indicating that the inhibition was due to the cytosolic accumulation of an inhibitor of Na/K ATPase. The half-life of the inhibitor in cardiac homogenates was 10+/-3 min at room temperature. Perfusion with the antioxidant MPG (1 mmol/l) reduced the accumulation of this inhibitor, however MPG was without effect on Na/K ATPase function when added directly to the Na/K ATPase activity assay. While the inhibitor reduced the activity of cardiac and brain forms of the Na/K ATPase in bioassay experiments, no effect was observed on the renal and skeletal muscle forms of the enzyme. CONCLUSIONS An unstable cardiac and brain-specific inhibitor of the Na/K ATPase whose production is linked to oxidant stress, accumulates intracellularly during ischemia. Intracellular Na is a primary determinant of electro-mechanical recovery on reperfusion, so inhibition of the Na/K ATPase by this compound may be crucial in determining recovery from ischemia.

Membrane potential fluctuations and transient inward currents induced by reactive oxygen intermediates in isolated rabbit ventricular cells.

The cellular basis of reactive oxygen intermediate-induced arrhythmias was investigated in isolated rabbit ventricular cells using the whole-cell voltage- and current-clamp techniques. Singlet oxygen and superoxide were generated by the photoactivation of rose bengal. Single ventricular cells exposed to rose bengal (10-100 nM) exhibited spontaneous membrane potential fluctuations at plateau potentials and at the level of the resting membrane potential. The voltage fluctuations induced in the resting potential occasionally triggered repetitive action potential discharges. At the resting membrane potential, the magnitude and dominant frequency of the voltage fluctuations were 1-3 mV and 1.5 Hz, respectively. At plateau potentials, the amplitude of the voltage fluctuations was about 2-5 mV, and the dominant oscillatory frequency was 2.6 Hz. In voltage-clamp experiments, transient inward currents were induced on repolarization after a depolarizing clamp step. Oscillatory currents also occurred occasionally during clamp steps to positive potentials. The peak frequencies of transient inward currents recorded at -20 and -70 mV were approximately 3.7 and 2.3 Hz, respectively, indicating that these currents may underlie the arrhythmogenic membrane potential fluctuations observed in current-clamp experiments. The rose bengal-induced transient inward currents were shown to be dependent on the magnitude and duration of the preceding voltage step. Studies of the voltage dependence of transient inward currents showed that these currents remained inward even at positive potentials (+30 mV), and replacement of extracellular sodium with lithium decreased transient inward current to approximately 10% of its initial value. Thus, the major component of oxidant stress-induced inward current appears to be electrogenic Na-Ca exchange. This oscillatory transient inward current may be responsible for the arrhythmias induced in isolated hearts exposed to reactive oxygen intermediates, and since oxidant stress has been implicated in reperfusion injury, it is possible that similar oscillatory currents may underlie reperfusion-induced arrhythmias.

Na+/Ca2+ exchange and Na+/K+‐ATPase in the heart

This paper is the third in a series of reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation–contraction coupling and arrhythmias: Na+ channel and Na+ transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on cardiac Na+/Ca2+ exchange (NCX) and Na+/K+‐ATPase (NKA). While the relevance of Ca2+ homeostasis in cardiac function has been extensively investigated, the role of Na+ regulation in shaping heart function is often overlooked. Small changes in the cytoplasmic Na+ content have multiple effects on the heart by influencing intracellular Ca2+ and pH levels thereby modulating heart contractility. Therefore it is essential for heart cells to maintain Na+ homeostasis. Among the proteins that accomplish this task are the Na+/Ca2+ exchanger (NCX) and the Na+/K+ pump (NKA). By transporting three Na+ ions into the cytoplasm in exchange for one Ca2+ moved out, NCX is one of the main Na+ influx mechanisms in cardiomyocytes. Acting in the opposite direction, NKA moves Na+ ions from the cytoplasm to the extracellular space against their gradient by utilizing the energy released from ATP hydrolysis. A fine balance between these two processes controls the net amount of intracellular Na+ and aberrations in either of these two systems can have a large impact on cardiac contractility. Due to the relevant role of these two proteins in Na+ homeostasis, the emphasis of this review is on recent developments regarding the cardiac Na+/Ca2+ exchanger (NCX1) and Na+/K+ pump and the controversies that still persist in the field.

Nitric oxide regulates cardiac intracellular Na+ and Ca2 + by modulating Na/K ATPase via PKCε and phospholemman-dependent mechanism

In the heart, Na/K-ATPase regulates intracellular Na+ and Ca2 + (via NCX), thereby preventing Na+ and Ca2 + overload and arrhythmias. Here, we test the hypothesis that nitric oxide (NO) regulates cardiac intracellular Na+ and Ca2 + and investigate mechanisms and physiological consequences involved. Effects of both exogenous NO (via NO-donors) and endogenously synthesized NO (via field-stimulation of ventricular myocytes) were assessed in this study. Field stimulation of rat ventricular myocytes significantly increased endogenous NO (18 ± 2 μM), PKCε activation (82 ± 12%), phospholemman phosphorylation (at Ser-63 and Ser-68) and Na/K-ATPase activity (measured by DAF-FM dye, western-blotting and biochemical assay, respectively; p < 0.05, n = 6) and all were abolished by Ca2 +-chelation (EGTA 10 mM) or NOS inhibition l-NAME (1 mM). Exogenously added NO (spermine-NONO-ate) stimulated Na/K-ATPase (EC50 = 3.8 μM; n = 6/grp), via decrease in Km, in PLMWT but not PLMKO or PLM3SA myocytes (where phospholemman cannot be phosphorylated) as measured by whole-cell perforated-patch clamp. Field-stimulation with l-NAME or PKC-inhibitor (2 μM Bis) resulted in elevated intracellular Na+ (22 ± 1.5 and 24 ± 2 respectively, vs. 14 ± 0.6 mM in controls) in SBFI-AM-loaded rat myocytes. Arrhythmia incidence was significantly increased in rat hearts paced in the presence of l-NAME (and this was reversed by l-arginine), as well as in PLM3SA mouse hearts but not PLMWT and PLMKO. We provide physiological and biochemical evidence for a novel regulatory pathway whereby NO activates Na/K-ATPase via phospholemman phosphorylation and thereby limits Na+ and Ca2 + overload and arrhythmias. This article is part of a Special Issue entitled “Na+ Regulation in Cardiac Myocytes”.

Non‐steroidal anti‐inflammatory drugs (NSAIDs) inhibit vascular smooth muscle cell proliferation via differential effects on the cell cycle

Abnormal vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of both atherosclerosis and restenosis. Recent studies suggest that high‐dose salicylates, in addition to inhibiting cyclooxygenase activity, exert an antiproliferative effect on VSMC growth both in‐vitro and in‐vivo. However, whether all non‐steroidal anti‐inflammatory drugs (NSAIDs) exert similar antiproliferative effects on VSMCs, and do so via a common mechanism of action, remains to be shown. In this study, we demonstrate that the NSAIDs aspirin, sodium salicylate, diclofenac, ibuprofen, indometacin and sulindac induce a dose‐dependent inhibition of proliferation in rat A10 VSMCs in the absence of significant cytotoxicity. Flow cytometric analyses showed that exposure of A10 cells to diclofenac, indometacin, ibuprofen and sulindac, in the presence of the mitotic inhibitor, nocodazole, led to a significant G0/G1 arrest. In contrast, the salicylates failed to induce a significant G1 arrest since flow cytometry profiles were not significantly different from control cells. Cyclin A levels were elevated, and hyperphosphorylated p107 was present at significant levels, in salicylate‐treated A10 cells, consistent with a post‐G1/S block, whereas cyclin A levels were low, and hypophosphorylated p107 was the dominant form, in cells treated with other NSAIDs consistent with a G1 arrest. The ubiquitously expressed cyclin‐dependent kinase (CDK) inhibitors, p21 and p27, were increased in all NSAID‐treated cells. Our results suggest that diclofenac, indometacin, ibuprofen and sulindac inhibit VSMC proliferation by arresting the cell cycle in the G1 phase, whereas the growth inhibitory effect of salicylates probably affects the late S and/or G2/M phases. Irrespective of mechanism, our results suggest that NSAIDs might be of benefit in the treatment of certain vasculoproliferative disorders.

The inhibitory effect of phospholemman on the sodium pump requires its palmitoylation.

Background: Phospholemman regulates the plasmalemmal sodium pump in excitable tissues such as the heart. Results: Phospholemman is palmitoylated at two intracellular cysteines, and this reduces ion transport by the sodium pump. Conclusion: Phospholemman must be palmitoylated to inhibit the sodium pump. Significance: This is a potentially new way to regulate the sodium pump, an enzyme expressed in most eukaryotic cells. Phospholemman (PLM), the principal sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. We investigated post-translational modifications of PLM additional to phosphorylation in adult rat ventricular myocytes (ARVM). LC-MS/MS of tryptically digested PLM immunoprecipitated from ARVM identified cysteine 40 as palmitoylated in some peptides, but no information was obtained regarding the palmitoylation status of cysteine 42. PLM palmitoylation was confirmed by immunoprecipitating PLM from ARVM loaded with [3H]palmitic acid and immunoblotting following streptavidin affinity purification from ARVM lysates subjected to fatty acyl biotin exchange. Mutagenesis identified both Cys-40 and Cys-42 of PLM as palmitoylated. Phosphorylation of PLM at serine 68 by PKA in ARVM or transiently transfected HEK cells increased its palmitoylation, but PKA activation did not increase the palmitoylation of S68A PLM-YFP in HEK cells. Wild type and unpalmitoylatable PLM-YFP were all correctly targeted to the cell surface membrane, but the half-life of unpalmitoylatable PLM was reduced compared with wild type. In cells stably expressing inducible PLM, PLM expression inhibited the sodium pump, but PLM did not inhibit the sodium pump when palmitoylation was inhibited. Hence, palmitoylation of PLM controls its turnover, and palmitoylated PLM inhibits the sodium pump. Surprisingly, phosphorylation of PLM enhances its palmitoylation, probably through the enhanced mobility of the phosphorylated intracellular domain increasing the accessibility of cysteines for the palmitoylating enzyme, with interesting theoretical implications. All FXYD proteins have conserved intracellular cysteines, so FXYD protein palmitoylation may be a universal means to regulate the sodium pump.