Andrew K Snabaitis

Kingston University, England, United Kingdom

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Publications (20)110.27 Total impact

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    ABSTRACT: The enzymatic activity of the type 2A protein phosphatase (PP2A) holoenzyme, a major serine/threonine phosphatase in the heart, is conferred by its catalytic subunit (PP2AC). PP2AC activity and subcellular localisation can be regulated by reversible carboxylmethylation of its C-terminal leucine309 (leu309) residue. Previous studies have shown that the stimulation of adenosine type 1 receptors (A1.Rs) induces PP2AC carboxylmethylation and altered subcellular distribution in adult rat ventricular myocytes (ARVM). In the current study, we show that the enzymatic components that regulate the carboxylmethylation status of PP2AC, leucine carboxylmethyltransferase-1 (LCMT-1) and phosphatase methylesterase-1 (PME-1) are abundantly expressed in, and almost entirely localised in the cytoplasm of ARVM. The stimulation of Gi-coupled A1.Rs with N(6)-cyclopentyladenosine (CPA), and of other Gi-coupled receptors such as muscarinic M2 receptors (stimulated with carbachol) and angiotensin II AT2 receptors (stimulated with CGP42112) in ARVM, induced PP2AC carboxylmethylation at leu309 in a concentration-dependent manner. Exposure of ARVM to 10 µM CPA increased the cellular association between PP2AC and its methyltransferase LCMT-1, but not its esterase PME-1. Stimulation of A1.Rs with 10 µM CPA increased the phosphorylation of protein kinase B at ser473, which was abolished by the PI3K inhibitor LY294002 (20 µM), thereby confirming that PI3K activity is upregulated in response to A1.R stimulation by CPA in ARVM. A1.R-induced PP2AC translocation to the particulate fraction was abrogated by adenoviral expression of the alpha subunit (Gαt1) coupled to the transducin G-protein coupled receptor. A similar inhibitory effect on A1.R-induced PP2AC translocation was also seen with LY294002 (20 µM). These data suggest that in ARVM, A1.R-induced PP2AC translocation to the particulate fraction occurs through a GiPCR-Gβγ-PI3K mediated intracellular signalling pathway, which may involve elevated PP2AC carboxylmethylation at leu309.
    PLoS ONE 01/2014; 9(1):e86234. DOI:10.1371/journal.pone.0086234 · 3.53 Impact Factor
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    ABSTRACT: Our aims in the present study were (1) to determine the effects of urotensin II (UT-II) on the sarcolemmal Na/H exchanger (NHE1) activity, and (2) to investigate possible kinase pathways for UT-II-mediated NHE1 stimulation. In single rat ventricular myocytes (n = 5-10/group) loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1, acid efflux rates (JH) were determined as an index of NHE1 activity by rate of recovery of intracellular pH (pHi) from NH4Cl-induced acidosis and the intrinsic buffering power. Phosphorylation of extracellular signal-regulated kinase (ERK), a key kinase of NHE1 activation, was determined by Western blot analysis. JH increased by 31%-71% relative to control in the presence of 100 nmol/L UT-II at pHi range of 6.6-7.0. Stimulation of NHE1 activity by UT-II was abolished by inhibitors of phospholipase C, protein kinase C, and ERK kinase; 2-nitro-4-carboxyphenil-N,N-diphenilcarbamate at 100 micromol/L, GF109203X at 300 nmol/L, and PD98059 at 50 micromol/L, respectively. Moreover, UT-II at 100 nmol/L produced a significant increase in cellular ERK1/2 phosphorylation, which was also inhibited by those inhibitors. Our study was the first to demonstrate that UT-II activates the cardiac sarcolemmal NHE1 and that the phenomenon may involve, at least in part, the phospholipase C-protein kinase C-ERK pathway.
    Journal of cardiovascular pharmacology 02/2010; 55(2):191-7. DOI:10.1097/FJC.0b013e3181cf0074 · 2.11 Impact Factor
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    ABSTRACT: The Na(+)/H(+) exchanger (NHE-1) plays a key role in pH(i) recovery from acidosis and is regulated by pH(i) and the ERK1/2-dependent phosphorylation pathway. Since acidosis increases the activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in cardiac muscle, we examined whether CaMKII activates the exchanger by using pharmacological tools and highly specific genetic approaches. Adult rat cardiomyocytes, loaded with the pH(i) indicator SNARF-1/AM were subjected to different protocols of intracellular acidosis. The rate of pH(i) recovery from the acid load (dpH(i)/dt)-an index of NHE-1 activity in HEPES buffer or in NaHCO(3) buffer in the presence of inhibition of anion transporters-was significantly decreased by the CaMKII inhibitors KN-93 or AIP. pH(i) recovery from acidosis was faster in CaMKII-overexpressing myocytes than in overexpressing beta-galactosidase myocytes (dpH(i)/dt: 0.195+/-0.04 vs. 0.045+/-0.010 min(-)(1), respectively, n=8) and slower in myocytes from transgenic mice with chronic cardiac CaMKII inhibition (AC3-I) than in controls (AC3-C). Inhibition of CaMKII and/or ERK1/2 indicated that stimulation of NHE-1 by CaMKII was independent of and additive to the ERK1/2 cascade. In vitro studies with fusion proteins containing wild-type or mutated (Ser/Ala) versions of the C-terminal domain of NHE-1 indicate that CaMKII phosphorylates NHE-1 at residues other than the canonical phosphorylation sites for the kinase (Ser648, Ser703, and Ser796). These results provide new mechanistic insights and unequivocally demonstrate a role of the already multifunctional CaMKII on the regulation of the NHE-1 activity. They also prove clinically important in multiple disorders which, like ischemia/reperfusion injury or hypertrophy, are associated with increased NHE-1 and CaMKII.
    Journal of Molecular and Cellular Cardiology 12/2009; 49(1):106-12. DOI:10.1016/j.yjmcc.2009.12.007 · 5.22 Impact Factor
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    Andrew K Snabaitis, Friederike Cuello, Metin Avkiran
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    ABSTRACT: Sarcolemmal Na(+)/H(+) exchanger (NHE) activity is mediated by NHE isoform 1 (NHE1), which is subject to regulation by protein kinases. Our objectives were to determine whether NHE1 is phosphorylated by protein kinase B (PKB), identify any pertinent phosphorylation site(s), and delineate the functional consequences of such phosphorylation. Active PKBalpha phosphorylated in vitro a glutathione S-transferase (GST)-NHE1 fusion protein comprising amino acids 516 to 815 of the NHE1 carboxyl-terminal regulatory domain. PKBalpha-mediated phosphorylation of GST-NHE1 fusion proteins containing overlapping segments of this region localized the targeted residues to the carboxyl-terminal 190 amino acids (625 to 815) of NHE1. Mass spectrometry and phosphorylation analysis of mutated (Ser-->Ala) GST-NHE1 fusion proteins revealed that PKBalpha-mediated phosphorylation of NHE1 occurred principally at Ser648. Far-Western assays demonstrated that PKBalpha-mediated Ser648 phosphorylation abrogated calcium-activated calmodulin (CaM) binding to the regulatory domain of NHE1. In adult rat ventricular myocytes, adenovirus-mediated expression of myristoylated PKBalpha (myr-PKBalpha) increased cellular PKB activity, as confirmed by increased glycogen synthase kinase 3beta phosphorylation. Heterologously expressed myr-PKBalpha was present in the sarcolemma, colocalized with NHE1 at the intercalated disc regions, increased NHE1 phosphorylation, and reduced NHE1 activity following intracellular acidosis. Conversely, pharmacological inhibition of endogenous PKB increased NHE1 activity following intracellular acidosis. Our data suggest that NHE1 is a novel PKB substrate and that its PKB-mediated phosphorylation at Ser648 inhibits sarcolemmal NHE activity during intracellular acidosis, most likely by interfering with CaM binding and reducing affinity for intracellular H(+).
    Circulation Research 09/2008; 103(8):881-90. DOI:10.1161/CIRCRESAHA.108.175877 · 11.09 Impact Factor
  • Journal of Molecular and Cellular Cardiology 06/2007; 42(6). DOI:10.1016/j.yjmcc.2007.03.131 · 5.22 Impact Factor
  • A. K. Snabaitis, F. Cuello, M. Avkiran
    Journal of Molecular and Cellular Cardiology 06/2007; 42(6). DOI:10.1016/j.yjmcc.2007.03.119 · 5.22 Impact Factor
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    ABSTRACT: Multiple stimuli of physiological and pathophysiological significance, including alpha1-adrenoceptor agonists, stimulate the cardiac sarcolemmal Na+/H+ exchanger isoform 1 (NHE1) through activation of the mitogen-activated or extracellular signal-regulated kinase (ERK) kinase (MEK) ERK-90-kDa ribosomal S6 kinase (RSK) signaling cascade. However, the individual contributions of ERK and RSK, which can each phosphorylate the NHE1 regulatory domain, to such stimulation are unknown. In the present study, we have used the novel RSK inhibitor fmk to determine the role of RSK as a direct regulator of NHE1 phosphorylation and activity in response to alpha1-adrenergic stimulation, in ventricular myocytes isolated from the adult rat heart. Initial experiments confirmed that pretreatment of myocytes with fmk before exposure to the alpha1-adrenoceptor agonist phenylephrine inhibited RSK C-terminal kinase activity and thereby RSK N-terminal kinase activation, without affecting MEK or ERK activation. Pretreatment of myocytes with fmk also inhibited phenylephrine-induced increases in NHE1 phosphorylation and the rate of NHE1-mediated H+ efflux under conditions of intracellular acidosis. These findings reveal, for the first time to our knowledge, that RSK is the principal regulator of NHE1 phosphorylation and activity after alpha1-adrenergic stimulation in adult myocardium.
    Molecular Pharmacology 04/2007; 71(3):799-806. DOI:10.1124/mol.106.029900 · 4.12 Impact Factor
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    ABSTRACT: G(q) protein-coupled receptor stimulation increases sarcolemmal Na(+)/H(+) exchanger (NHE1) activity in cardiac myocytes by an ERK/RSK-dependent mechanism, most likely via RSK-mediated phosphorylation of the NHE1 regulatory domain. Adenosine A(1) receptor stimulation inhibits this response through a G(i) protein-mediated pathway, but the distal inhibitory signaling mechanisms are unknown. In cultured adult rat ventricular myocytes (ARVM), the A(1) receptor agonist cyclopentyladenosine (CPA) inhibited the increase in NHE1 phosphorylation induced by the alpha(1)-adrenoreceptor agonist phenylephrine, without affecting activation of the ERK/RSK pathway. CPA also induced significant accumulation of the catalytic subunit of type 2A protein phosphatase (PP2A(c)) in the particulate fraction, which contained the cellular NHE1 complement; this effect was abolished by pretreatment with pertussis toxin to inactivate G(i) proteins. Confocal immunofluorescence microscopic imaging of CPA-treated ARVM revealed significant co-localization of PP2A(c) and NHE1, in intercalated disc regions. In an in vitro assay, purified PP2A(c) dephosphorylated a GST-NHE1 fusion protein containing aa 625-747 of the NHE1 regulatory domain, which had been pre-phosphorylated by recombinant RSK; such dephosphorylation was inhibited by the PP2A-selective phosphatase inhibitor endothall. In intact ARVM, the ability of CPA to attenuate the phenylephrine-induced increase in NHE1 phosphorylation and activity was lost in the presence of endothall. These studies reveal a novel role for the PP2A holoenzyme in adenosine A(1) receptor-mediated regulation of NHE1 activity in ARVM, the mechanism of which appears to involve G(i) protein-mediated translocation of PP2A(c) and NHE1 dephosphorylation.
    Journal of Biological Chemistry 08/2006; 281(29):20252-62. DOI:10.1074/jbc.M600268200 · 4.60 Impact Factor
  • Journal of Molecular and Cellular Cardiology 06/2006; 40(6):996-996. DOI:10.1016/j.yjmcc.2006.03.223 · 5.22 Impact Factor
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    ABSTRACT: Using adenoviruses encoding RGS2, RGS4 and Lsc (regulator of G protein signalling (RGS) domain of p115 RhoGEF), we investigated the contributions of G(q/11), Gi and G(12/13) proteins to G protein-coupled receptor (GPCR)-mediated activation of the extracellular signal-regulated kinase (ERK) pathway in adult rat ventricular myocytes (ARVM). Exposure to phenylephrine, endothelin-1 (ET-1) or thrombin induced significant activation of ERK1/2 and their downstream target 90 kDa ribosomal S6 kinase (p90RSK), which was abolished by overexpression of RGS4 (inhibits signalling via G(q/11) and Gi) or RGS2 (inhibits signalling via G(q/11)). Pertussis toxin (inhibits signalling via Gi) only partially attenuated the activation of ERK1/2 and p90(RSK) by phenylephrine and ET-1, but abolished such activation by thrombin. Overexpression of Lsc (inhibits signalling via G(12/13)) did not affect the responses to phenylephrine and ET-1, but suppressed the activation of ERK1/2 and p90RSK by thrombin. We conclude that full activation of the ERK pathway in ARVM by alpha1-adrenergic, ET-1 and thrombin receptors requires the activation of distinct families of heterotrimeric G proteins.
    Cellular Signalling 06/2005; 17(5):655-64. DOI:10.1016/j.cellsig.2004.10.008 · 4.47 Impact Factor
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    ABSTRACT: Aims: Inhibition of the Na+/H+-exchanger (NHE) preserves myocardial morphology and function in rat and mouse models of hypertrophy and failure. The mechanism(s) involved in such cardioprotective effects remain(s) unclear, but might involve blockade of increased protein kinase activity as observed in untreated hearts. Methods and results: We investigated the functional, morphological and biochemical consequences of NHE-inhibition with BIIB722 in rabbits with pacing-induced heart failure (HF). In sham rabbits treated with placebo (n = 9) or BIIB722 (30 mg/kg/day po, n = 9), LV end-diastolic diameter (LVEDD) and systolic fractional shortening (FS, %) remained unchanged. In HF rabbits (n = 9), LVEDD increased and FS decreased from 31.5 +/- 1.4 to 8.1 +/- 0.9 (p < 0.05) at 3 weeks of LV pacing (400 bpm). Apoptosis, fibrosis and myocyte cross-sectional area as well as p38MAPK phosphorylation and iNOS protein expression were significantly increased in HF compared to sham rabbits. The activity of the 90 kDa NHE-kinase was greater in HF than in sham rabbits. In HF rabbits receiving BIIB722 prior to (18.1 +/- 2.2, n = 9) or following 1 week (15.5 +/- 1.6, n = 7) of pacing, FS at 3 weeks was better preserved than in untreated HF rabbits (p < 0.05). Apoptosis, fibrosis, myocyte cross-sectional area, p38MAPK phosphorylation and iNOS protein expression were significantly reduced in HF rabbits receiving BIIB722. Conclusion: NHE-inhibition attenuates the functional, morphological and biochemical derangements of pacing-induced HF in rabbits.
    Cardiovascular Research 08/2004; 63(2):273-82. DOI:10.1016/j.cardiores.2004.04.014 · 5.81 Impact Factor
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    ABSTRACT: Activity of the Na+/H+ exchanger (NHE) isoform 1 (NHE1) is increased by intracellular acidosis through the interaction of intracellular H+ with an allosteric modifier site in the transport domain. Additional regulation is achieved via kinase-mediated modulation of the NHE1 regulatory domain. To determine if intracellular acidosis stimulates NHE1 activity solely by the allosteric mechanism, we subjected cultured neonatal rat ventricular myocytes (NRVM) with native NHE1 expression to intracellular acidosis (pHi approximately 6.6) for up to 6 min by transient exposure to NH4Cl and its washout in the presence of NHE inhibition (by zero [Na+]o or the NHE1 inhibitor cariporide) in HCO3- -free medium. After the desired duration of acidosis, NHE was reactivated (by reintroduction of [Na+]o or removal of cariporide), and the rate of recovery of pHi (dpHi/dt) was measured as the index of NHE activity. Regardless of the method used when intracellular acidosis was sustained for > or =3 min, subsequent NHE activity was significantly increased (>4-fold). Similar NHE stimulatory effects of sustained acidosis were observed in adult rat ventricular myocytes and COS-7 cells. Sustained (3 min) intracellular acidosis activated several NHE1 kinases in NRVM, in an in-gel kinase assay using as substrate a glutathione S-transferase fusion protein of the NHE1 regulatory domain. Detailed investigation of ERK and its downstream effector p90RSK, two putative NHE1 kinases, revealed time-dependent activation of both by intracellular acidosis in NRVM. Furthermore, inhibition of MEK1/2 by pretreatment of NRVM with two structurally distinct inhibitors, PD98059 (30 microM) or UO126 (3 microM), inhibited the activation of ERK and p90RSK and abolished the stimulation of NHE activity by sustained (3 min) intracellular acidosis. Our data show that not only the extent but also the duration of intracellular acidosis regulates NHE1 activity and suggest that the stimulatory effect of sustained intracellular acidosis occurs through a novel mechanism mediated by activation of the ERK pathway.
    Journal of Biological Chemistry 09/2003; 278(34):31676-84. DOI:10.1074/jbc.M304400200 · 4.60 Impact Factor
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    Andrew K Snabaitis, David J Hearse, Metin Avkiran
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    ABSTRACT: To characterise the effects of exogenous H(2)O(2) on sarcolemmal Na(+)/H(+) exchanger (NHE) activity and determine the roles of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase C (PKC) in observed effects. Sarcolemmal H(+) efflux rate (J(H)) was determined by microepifluorescence at a pH(i) of 6.70 in adult rat ventricular myocytes, after two consecutive acid pulses in HCO(3)(-)-free medium; before the second pulse, cells (n=7-10/group) were exposed to H(2)O(2) or vehicle and the change in J(H) (DeltaJ(H)) was used to quantify the change in NHE activity. ERK and p38 MAPK activities were determined by immunoblotting with phosphospecific antibodies. Relative to control, DeltaJ(H) was increased by a 10-min exposure to 100, but not 1 or 10 microM H(2)O(2) (1000 microM was not tolerated); 3 or 6 min exposure to 100 microM H(2)O(2) was without effect. ERK and p38 MAPK activities were both increased by 100 microM H(2)O(2) (peak at 6 min); the ERK kinase inhibitor PD98059 (10 microM), but not the p38 MAPK inhibitor SB203580 (1 microM), inhibited the H(2)O(2)-induced increase in DeltaJ(H). H(2)O(2)-induced ERK activation was inhibited not only by PD98059 (10 microM), but also by the non-selective tyrosine kinase inhibitor genistein (3-100 microM), the EGF receptor kinase inhibitor AG1478 (3-300 nM) and the Src family kinase inhibitor PP2 (0.1-10 microM). The PKC inhibitors GF109203X (0.3-10 microM) and chelerythrine (1-30 microM) were without effect on ERK activation, although the former abolished the H(2)O(2)-induced increase in DeltaJ(H). Our data demonstrate that, in adult rat ventricular myocytes, (i) hydrogen peroxide stimulates sarcolemmal NHE activity, (ii) this response requires activation of ERK and PKC, but not p38 MAPK, (iii) ERK activation occurs through tyrosine kinase-mediated, but PKC-independent, mechanisms
    Cardiovascular Research 03/2002; 53(2):470-80. DOI:10.1016/S0008-6363(01)00464-3 · 5.81 Impact Factor
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    A K Snabaitis, H Yokoyama, M Avkiran
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    ABSTRACT: Activation of the sarcolemmal Na(+)-H(+) exchanger (NHE) has been implicated as a mechanism of inotropic, arrhythmogenic, antiacidotic, and hypertrophic effects of alpha(1)-adrenoceptor (AR) stimulation. Although such regulation of sarcolemmal NHE activity has been shown to be selectively mediated through the alpha(1A)-AR subtype, distal signaling mechanisms remain poorly defined. We investigated the roles of various kinase pathways in alpha(1A)-AR-mediated stimulation of sarcolemmal NHE activity in adult rat ventricular myocytes. As an index of NHE activity, trans-sarcolemmal acid efflux rate (J(H)) was determined through microepifluorescence in single cells, during recovery from intracellular acidosis in bicarbonate-free conditions. Extracellular signal-regulated kinase (ERK), p38-mitogen-activated protein kinase (MAPK), and p90(rsk) activities were indexed on the basis of analysis of their phosphorylation status. In control cells, there was no change in J(H) in response to vehicle. Phenylephrine and A61603, an alpha(1A)-AR subtype-selective agonist, increased J(H), as well as cellular ERK and p90(rsk) activities. Neither agonist affected p38 activity, which was increased with sorbitol. The MAPK kinase inhibitor PD98059 abolished phenylephrine- and A61603-induced increases in J(H) and cellular ERK and p90(rsk) activities. In contrast, the PKC inhibitor GF109203X abolished phenylephrine- and A61603-induced increases in J(H) but failed to prevent the increases in ERK and p90(rsk) activities. Our findings suggest that alpha(1A)-AR-mediated stimulation of sarcolemmal NHE activity in rat ventricular myocytes requires activation of the ERK (but not the p38) pathway of the MAPK cascade and that the ERK-mediated effect may occur via p90(rsk). Activation of PKC is also required for alpha(1A)-AR-mediated NHE stimulation, but such regulation occurs through an ERK-independent pathway.
    Circulation Research 03/2000; 86(2):214-20. DOI:10.1161/01.RES.86.2.214 · 11.09 Impact Factor
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    AK Snabaitis, DJ Chambers
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    ABSTRACT: Polarized arrest, induced by tetrodotoxin (TTX) at an optimal concentration of 22 micromol/L, has been shown to reduce ionic imbalance and improve myocardial preservation compared with hyperkalemic (depolarized) arrest. Additional pharmacologic manipulation of ionic changes (involving inhibition of Na+ influx by the Na+/H+ exchanger [HOE694] and Na+/K+/2Cl- cotransporter [furosemide], and calcium desensitization [BDM]) may further improve long-term preservation. In this study, we (i) established optimal concentrations of each drug, (ii) determined additive effects of optimal concentrations of each drug and (iii) compared our optimal preservation solution to an established depolarizing cardioplegia (St Thomas' Hospital solution No 2: STH2) used during long-term hypothermic storage for clinical transplantation. The isolated working rat heart, perfused with Krebs Henseleit (KH) buffer was used; cardiac function was measured after 20 min aerobic working mode perfusion. The hearts (n=6/group) were arrested with a 2 ml infusion (for 30 sec) of the polarizing (control) solution (22 micromol/L TTX in KH) or control+drug and subjected to 5 hr or 8 hr of storage at 7.5 degrees C in the arresting solution. Postischemic function during reperfusion was measured (expressed as percentage of preischemic function). Dose-response studies established optimal concentrations of HOE694 (10 micromol/L), furosemide (1.0 micromol/L) and BDM (30 mmol/L) in the polarizing (control) solution. Sequential addition to the control solution (Group I) of optimal concentrations of HOE694 (Group II), furosemide (Group III), and BDM (Group IV) were compared with STH2 (Group V); postischemic recovery of aortic flow was 29+/-7%, 49+/-6%*, 56+/-2%*, 76+/-3%*, and 25+/-6%, respectively (*P<0.05 vs. I and V). Creatine kinase leakage was lowest, and myocardial ATP content was highest in Group IV. A polarizing preservation solution (KH+TTX) containing HOE694, furosemide, and BDM significantly enhanced long-term preservation compared with an optimized depolarizing solution (STH2) used clinically for long-term donor heart preservation.
    Transplantation 12/1999; 68(10):1444-53. DOI:10.1097/00007890-199911270-00003 · 3.78 Impact Factor
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    Andrew K. Snabaitis, David J. Chambers
    Transplantation 11/1999; 68. · 3.78 Impact Factor
  • M Avkiran, A K Snabaitis
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    ABSTRACT: The cardiac sarcolemmal Na(+)/H(+) exchanger (NHE) extrudes one H(+) in exchange for one Na(+) entering the myocyte, utilizing for its driving force the inwardly directed Na(+) gradient maintained by the Na(+), K(+)-ATPase. The exchanger is quiescent at physiological values of intracellular pH but becomes activated in response to intracellular acidosis. Recent evidence suggests that a variety of extracellular signals (e.g., adrenergic agonists, thrombin, endothelin, and oxidant stress) also modulate sarcolemmal NHE activity by altering its sensitivity to intracellular H(+). Because sarcolemmal NHE activity is believed to be an important determinant of the extent of myocardial injury during ischemia and reperfusion, regulation of exchanger activity by factors that are associated with ischemia is likely to be pathophysiological importance.
    Journal of Thrombosis and Thrombolysis 08/1999; 8(1):25-32. DOI:10.1023/A:1008938513337 · 2.04 Impact Factor
  • Andrew K. Snabaitis, Michael J. Shattock, David J. Chambers
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    ABSTRACT: We previously demonstrated improved myocardial preservation with polarized (tetrodotoxin-induced), compared with depolarized (hyperkalemia-induced), arrest and hypothermic storage. This study was undertaken to determine whether polarized arrest reduced ionic imbalance during ischemic storage and whether this was influenced by Na+/K +/2Cl- cotransport inhibition. We used the isolated crystalloid perfused working rat heart preparation (1) to measure extracellular K+ accumulation (using a K+-sensitive intramyocardial electrode) during ischemic (control), depolarized (K+ 16 mmol/L), and polarized (tetrodotoxin, 22 micromol/L) arrest and hypothermic (7.5 degrees C) storage (5 hours), (2) to determine dose-dependent (0.1, 1.0, 10 and 100 micromol/L) effects of the Na +/K+/2Cl- cotransport inhibitor, furosemide, on extracellular K+ accumulation during polarized arrest and 7.5 degrees C storage, and (3) to correlate extracellular K+ accumulation to postischemic recovery of cardiac function. Characteristic triphasic profiles of extracellular K+ accumulation were observed in control and depolarized arrested hearts; a significantly attenuated profile with polarized arrested hearts demonstrated reduced extracellular K+ accumulation, correlating with higher postischemic function (recovery of aortic flow was 54% +/-4% [P =.01] compared with 39% +/-3% and 32% +/-3% in depolarized and control hearts, respectively). Furosemide (0.1, 1.0, 10, and 100 micromol/L) modified extracellular K+ accumulation by -18%, -38%, -0.2%, and +9%, respectively, after 30 minutes and by -4%, -27%, +31%, and +42%, respectively, after 5 hours of polarized storage. Recovery of aortic flow was 53% +/-4% (polarized arrest alone), 56% +/-8%, 70% +/-2% (P =.04 vs control), 69% +/-4% (P =.04 vs control), and 65% +/-3% ( P =. 04 vs control), respectively. Polarized arrest was associated with a reduced ionic imbalance (demonstrated by reduced extracellular K+ accumulation) and improved recovery of cardiac function. Further attenuation of extracellular K + accumulation (by furosemide) resulted in additional recovery.
    Journal of Thoracic and Cardiovascular Surgery 08/1999; 118(1):123-34. DOI:10.1016/S0022-5223(99)70151-0 · 3.99 Impact Factor
  • A K Snabaitis, M J Shattock, D J Chambers
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    ABSTRACT: Hypothermic hyperkalemic cardioplegic solutions are currently used for donor heart preservation. Hyperkalemia-induced depolarization of the resting membrane potential (Em) may predispose the heart to Na+ and Ca2+ loading via voltage-dependent "window currents," thereby exacerbating injury and limiting the safe storage duration. Alternatively, maintaining the resting Em with a polarizing solution may reduce ionic movements and improve postischemic recovery; we investigated this concept with the reversible sodium channel blocker tetrodotoxin (TTX) to determine (1) whether polarized arrest was more efficacious than depolarized arrest during hypothermic long-term myocardial preservation and (2) whether TTX induces and maintains polarized arrest. The isolated crystalloid-perfused working rat heart preparation was used in this study. Preliminary studies determined an optimal TTX concentration of 22 micromol/L and an optimal storage temperature of 7.5 degrees C. To compare depolarized and polarized arrest, hearts were arrested with either Krebs-Henseleit (KH) buffer (control), KH buffer containing 16 mmol/L K+, or KH buffer containing 22 micromol/L TTX and then stored at 7.5 degrees C for 5 hours. Postischemic recovery of aortic flow was 13+/-4%, 38+/-2%, and 48+/-3%* (*P<.05 versus control and 16 mmol/L K+), respectively. When conventional 3 mol/L KCl-filled intracellular microelectrodes were used, Em gradually depolarized during control unprotected ischemia to approximately -55 mV before reperfusion, whereas arrest with 16 mmol/L K+ caused rapid depolarization to approximately -50 mV, where it remained throughout the 5-hour storage period. In contrast, in 22 micromol/L TTX-arrested hearts, Em remained more polarized, at approximately -70 mV, for the entire ischemic period. Blockade of cardiac sodium channels by TTX during ischemia maintained polarized arrest, which was more protective than depolarized arrest, possibly because of reduced ionic imbalance.
    Circulation 11/1997; 96(9):3148-56. DOI:10.1161/01.CIR.96.9.3148 · 14.95 Impact Factor
  • Andrew K. Snabaitis, David J. Chambers
    The Annals of Thoracic Surgery 09/1995; 60(3):864. DOI:10.1016/0003-4975(95)92712-U · 3.63 Impact Factor

Publication Stats

500 Citations
110.27 Total Impact Points


  • 2014
    • Kingston University
      England, United Kingdom
  • 2000–2009
    • King's College London
      • Cardiovascular Division
      Londinium, England, United Kingdom
  • 2007
    • University of California, San Francisco
      San Francisco, California, United States
  • 2006–2007
    • ICL
      Londinium, England, United Kingdom