Glutamate-loading Stimulates Metabolic Flux and Improves Cell Recovery Following Chemical Hypoxia in Isolated Cardiomyocyte

Williams, Helen; King, Nicola; Griffiths, Elinor J.; Suleiman, M‐Saadeh

doi:10.1006/jmcc.2000.1474

Cited by 31 publications

(36 citation statements)

References 24 publications

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“…We used an established model of monitoring Ca 2+ transient in isolated adult rat cardiomyocytes. Rat cardiomyocytes were isolated using collagenase Type I (Worthington Biochemical Corporation, Lakewood, New Jersey, USA) and protease as described previously (Williams et al, 2001). Cells were loaded with Fura‐2‐AM (5 μM; Biotium, Cambridge, UK), and Ca 2+ transients were recorded at the excitation/emission of 415/510 nm using a photomultiplier as described elsewhere (Littlejohns et al, 2014).…”

Section: Methodsmentioning

confidence: 99%

Functional and cardioprotective effects of simultaneous and individual activation of protein kinase A and Epac

Khaliulin

Bond

James

et al. 2017

British J Pharmacology

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Background and PurposeMyocardial cAMP elevation confers cardioprotection against ischaemia/reperfusion (I/R) injury. cAMP activates two independent signalling pathways, PKA and Epac. This study investigated the cardiac effects of activating PKA and/or Epac and their involvement in cardioprotection against I/R.Experimental ApproachHearts from male rats were used either for determination of PKA and PKC activation or perfused in the Langendorff mode for either cardiomyocyte isolation or used to monitor functional activity at basal levels and after 30 min global ischaemia and 2 h reperfusion. Functional recovery and myocardial injury during reperfusion (LDH release and infarct size) were evaluated. Activation of PKA and/or Epac in perfused hearts was induced using cell permeable cAMP analogues in the presence or absence of inhibitors of PKA, Epac and PKC. H9C2 cells and cardiomyocytes were used to assess activation of Epac and effect on Ca2+ transients.Key ResultsSelective activation of either PKA or Epac was found to trigger a positive inotropic effect, which was considerably enhanced when both pathways were simultaneously activated. Only combined activation of PKA and Epac induced marked cardioprotection against I/R injury. This was accompanied by PKCε activation and repressed by inhibitors of PKA, Epac or PKC.Conclusion and ImplicationsSimultaneous activation of both PKA and Epac induces an additive inotropic effect and confers optimal and marked cardioprotection against I/R injury. The latter effect is mediated by PKCε activation. This work has introduced a new therapeutic approach and targets to protect the heart against cardiac insults.

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Section: Methodsmentioning

confidence: 99%

Functional and cardioprotective effects of simultaneous and individual activation of protein kinase A and Epac

Khaliulin

Bond

James

et al. 2017

British J Pharmacology

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“…These triggers alongside MPTP opening may signal the onset of necrosis or apoptosis. Whether glutamate affects MPTP opening in the heart is not currently known, although increased intracellular glutamate levels have been shown to improve calcium metabolism and protection of isolated ventricular cardiomyocytes against oxidative stress [16,35].…”

Section: Discussionmentioning

confidence: 99%

“…These glutamate-loaded cells have a greater NADH/NAD + ratio, higher ATP levels [35] and an enhanced capacity to remove intracellular reactive oxygen species during oxidative stress compared to control cells [16].…”

Section: Introductionmentioning

confidence: 98%

“…The isolation of cardiomyocytes in the presence of glutamate leads to an enhancement of their intracellular glutamate concentration [35]. These glutamate-loaded cells have a greater NADH/NAD + ratio, higher ATP levels [35] and an enhanced capacity to remove intracellular reactive oxygen species during oxidative stress compared to control cells [16].…”

Section: Introductionmentioning

confidence: 99%

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Expression and activity of the glutamate transporter EAAT2 in cardiac hypertrophy: implications for ischaemia reperfusion injury

King

Lin

McGivan

et al. 2006

Pflugers Arch - Eur J Physiol

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The expression and activity of the glutamate transporter, excitatory amino acid transporter 2 (EAAT2), in cardiac hypertrophy were investigated with respect to glutamate's potential as a cardioprotective agent. Sarcolemmal vesicles (SV) isolated from hypertrophic hearts of male spontaneously hypertensive rats (SHR) or normotrophic hearts from age-matched male Wistar Kyoto rats (WKY) were used to measure the relative level of EAAT2 expression by Western blotting and the initial rate of 0-0.3 mM L-[(14)C]glutamate uptake. The effects of 20-min global normothermic ischaemia +/-0.5 mM glutamate on cardiac function were measured in isolated working SHR/WKY hearts. In a separate series of hearts, glutamate, lactate and ATP levels were measured. Both the level of EAAT2 expression and the V (max) for sodium-dependent L-[(14)C]glutamate uptake were significantly greater in SHR SV compared to WKY SV. The reperfusion cardiac output (CO) of SHR hearts was significantly worse than that of the WKY hearts (24.3+/-2.2 ml/min vs 39.8+/-3.3 ml/min, n=7/9+/-SE, p<0.01). The addition of 0.5 mM L-glutamate improved the SHR reperfusion CO to 45.2+/-5 ml/min, (n=6+/-SE, p<0.01) but had no effect on WKYs (46.2+/-3.8 ml/min, n=6+/-SE). SHR with 0.5 mM L-glutamate had higher glutamate levels at the start of ischaemia, plus higher glutamate and ATP levels at the end of ischaemia compared to any other group. These results suggest that increased glutamate transporter expression and activity in the SHR hearts helped facilitate glutamate entry into the SHR cardiomyocytes leading to improved myocardial metabolism during ischaemia and better functional recovery on reperfusion.

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“…malate-aspartate shuttle [18], or indirectly, e.g. after transamination and subsequent feeding of the carbon backbone into the TCA cycle [14,26]. Other functions of amino acids in the heart include their requirement for protein synthesis [11], countering the production of reactive oxygen species [10,14], and effects on ionic, particularly calcium, homeostasis [14].…”

Section: Introductionmentioning

confidence: 99%

Demonstration of functional dipeptide transport with expression of PEPT2 in guinea pig cardiomyocytes

Lin

King

2006

Pflugers Arch - Eur J Physiol

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The transporters PEPT1 and PEPT2 accept a broad spectrum of substrates including small, naturally occurring peptides and peptidomimetic drugs. This study aimed to investigate for the first time whether these transporters are expressed and active in isolated cardiomyocytes. PEPT1/PEPT2 expression in rat kidney (positive control), guinea pig kidney and cardiomyocytes were investigated by reverse transcription polymerase chain reaction. L-Glycyl-L-[(14)C]sarcosine (Gly-sar) uptake was characterised using freshly isolated suspensions of adult male guinea pig cardiomyocytes. PEPT2-specific primers recognised mRNA of appropriate size and sequence in cardiomyocytes and kidney, whilst PEPT1 was expressed in the kidney only. The initial uptake (30 s) of 200 microM Gly-sar was dependent on extracellular pH with a maximum at pH 6.0 (237.8 +/- 12.2 pmol/microl) and a minimum at pH 8.0 (72.1 +/- 13.4 pmol/microl, n = 6 +/- SE, p < 0.01, T test). The K (m) and V (max) of Gly-sar uptake at pH 6.0 were 495.5 +/- 69.6 microM and 1470.5 +/- 69.6 pmol microl(-1) min(-1). The addition of 10 mM fosinopril, cefadroxil, carnosine, cyclacillin or a variety of L-amino acid containing dipeptides/tripeptides significantly reduced Gly-sar uptake. Gly-sar uptake was not affected by 10 mM D-ala-D-ala, glycine or sarcosine. These results support the presence of a functional dipeptide transporter in isolated cardiomyocytes, with accompanying expression of PEPT2.

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Glutamate-loading Stimulates Metabolic Flux and Improves Cell Recovery Following Chemical Hypoxia in Isolated Cardiomyocyte

Cited by 31 publications

References 24 publications

Functional and cardioprotective effects of simultaneous and individual activation of protein kinase A and Epac

Functional and cardioprotective effects of simultaneous and individual activation of protein kinase A and Epac

Expression and activity of the glutamate transporter EAAT2 in cardiac hypertrophy: implications for ischaemia reperfusion injury

Demonstration of functional dipeptide transport with expression of PEPT2 in guinea pig cardiomyocytes

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