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. 2025 Oct 22;57(1):52. doi: 10.1007/s00726-025-03485-y

Myocardial protection by acidic amino acids and natural dipeptides: potential for an underused resource

N King 1,✉
PMCID: PMC12545665  PMID: 41123699

Abstract

Stopping then restarting the blood flow to the heart can cause ischaemia reperfusion (IR) injury. This can happen during revascularisation following a myocardial infarction and during on pump cardiac surgery using cardioplegic arrest. Despite extensive studies to identify cardioprotective interventions, the myocardium continues to sustain significant injury. Therefore, there is a need to identify agents that can be used during IR. This review focuses on the potential cardioprotective role for acidic amino acids and natural dipeptides using evidence from experimental studies and clinical trials with particular emphasis on their membrane transport. Acidic amino acids are present at high concentration in the heart with a large tissue to plasma concentration gradient, where they are involved in protein synthesis and intermediary metabolism. During cardiac insults they are lost from heart cells but replenishment leads to cardioprotection through energy provision, protection against the production of reactive oxygen species production and improved calcium homeostasis. One important determinant of the intracellular concentration of acidic amino acids and natural dipeptides is membrane transport. The expression and activity of the acidic amino acids transporters EAAT1-3 and the dipeptide transporter, PEPT2 have been demonstrated in membrane vesicles and isolated cardiomyocytes. Improvements in our understanding of these different transport mechanisms should lead to the maximisation of acidic amino acid and natural dipeptide uptake during IR leading to improved cardioprotection.

Keywords: Myocardial protection, Membrane transport, Amino acids, Dipeptides

Introduction: Ischaemia/Reperfusion (IR) injury

Cardiovascular disease is an umbrella term covering all conditions affecting the heart and circulatory system, including coronary artery disease (CAD). The most serious consequence of CAD is thrombotic occlusion of a coronary vessel following the rupture of an atherosclerotic plaque leading to myocardial injury. Timely reperfusion is essential for rescuing the myocardium (Schirone et al. 2022); however, this introduces a new insult namely ischaemia/reperfusion (IR) injury. The pathophysiology of IR injury chiefly involves calcium overload, reactive oxygen species (ROS) production and mitochondrial dysfunction (Schirone et al. 2022). Further factors involved in IR injury include inflammation, apoptosis, and autophagy. (Schirone et al. 2022). This problem is also encountered during cardiac surgery with the fixing (ischaemia) and then removal (reperfusion) of the aortic cross clamp (Sabe et al. 2024). During this period the heart is stopped using cardioplegia, whilst oxygenated circulation is maintained using the heart-lung machine. In experiments using AC16 cardiomyocytes exposed to hydrogen peroxide only cardioplegic solutions containing high potassium were able to move the metabolome closest to the control non H2O2 exposed cells (Diao et al. 2023). Despite key advances in surgical technique allowing surgery on increasingly complex cases around 67% of CPB patients have post-operative complications, including atrial fibrillation, pulmonary dysfunction, or renal failure (Pahwa et al. 2021). Therefore, the search for the optimum means of myocardial protection continues.

Acidic amino acids in the heart

The dicarboxylic amino acids glutamate and aspartate are important for protein synthesis with glutamate also acting as a neurotransmitter. They are present at high concentrations in cardiomyocytes. For example, in the human heart the intracellular concentration of glutamate and aspartate is 6.2 ± 0.5 µmol/g wet weight and 1.32 ± 0.12 µmol/g wet weight. They are found at much lower concentration in human plasma with glutamate at 0.074 µmol/ml and aspartate at 0.011 ± 0.02 µmol/ml, yielding a tissue to plasma concentration ratio of 83 and 120 respectively (Suleiman et al. 1997). Both experimental models and human studies show that the concentration of these acidic amino acids significantly decreases during cardiac insults (Suleiman and Chapman 1993a, b; Suleiman et al. 1993, 1997). Importantly, this decrease was associated with an increase in the amino acid alanine, which has implications for metabolic stress (Suleiman et al. 1997; Venturini et al. 2009).

Glutamate and aspartate play an important role in intermediary energy metabolism. For instance, they are involved in the malate aspartate shuttle, which controls the distribution of reducing equivalents between the cardiomyocyte cytoplasm and mitochondria (Borst 2020). Glutamate and aspartate can be transaminated (and in the case of glutamate dehydrogenated) to form tricarboxylic acid (TCA) cycle intermediates, respectively α-ketoglutarate and oxaloacetate (Pisarenko 1996; Nguyen et al. 2022) followed by subsequent substrate level phosphorylation later in the cycle. Since energy metabolism is severely compromised in ischaemia (Schirone et al. 2022), it is this capacity of glutamate and aspartate to become a potential source of ATP that has led to their implication in myocardial protection.

Glutamate can also be formed from another amino acid, glutamine through the enzyme glutaminase and then further metabolised as described above (Pisarenko 1996). A recent report showed that the addition of 2.5mM glutamine prior to and after ischaemia improved the recovery of rat hearts, although the protection was associated with greater expression of heat shock protein 70 and O-linked β-N-acetylglucosamine and glutaminase activity was not measured (Kawakami et al. (2025). Unfortunately, there have been questions about glutamine’s stability (Jagušić et al. 2016; Dijkstra et al. 2023) leading to the successful use of glutamine containing dipeptides (Almashhadany et al. 2015; Lui et al. (2018).

Natural dipeptides in the heart

Natural dipeptides are formed of 2 amino acids joined by a peptide bond. For example, the dipeptide, carnosine contains β-alanine and histidine. Carnosine has several roles in the heart that could be advantageous during IR including quenching of reactive carbonyl species (Aldini et al. 2005), detoxifying aldehydes (Xie et al. 2013), intracellular pH buffering (Dolan et al. 2021) and modulation of energy metabolism (Yan et al. 2022). The latter was uncovered using omics technology. When rats were pretreated with an intraperitoneal injection of 250 mg/kg/day carnosine prior to isoprotenol induced myocardial infarction they had decreased histopathological changes, malondialdehyde, diene conjugate and protein carbonyl levels, but increased reduced glutathione (GSH) levels, superoxide dismutase and glutathione peroxidase activities compared to non-pretreated rats (Evran et al. 2014). Other dipeptides that offer cardioprotection include glycyl-L-glutamine (gly-gln, Almashhadany et al. 2015) and alanyl-L-glutamine (ala-gln, Hissa et al. 2011). These will be elaborated in the experimental studies section.

Incidentally, apart from glutamate synthesis, glutamine has been implicated in other metabolic pathways in the heart. A discussion of these is beyond the scope of this work, but for an excellent review see Shen et al. (2021).

In the following the evidence from experimental investigations and clinical trials for a role for acidic amino acids and dipeptides in myocardial protection is reviewed. This is succeeded by a discussion of the importance of membrane transport and finally a conclusion suggesting areas for future research.

Evidence from experimental studies: acidic amino acids

Table 1 summarises the different experimental studies that have investigated supplementation with acidic amino acids in various models of IR. The lowest glutamate concentration used was 0.01mM and the highest 100mM (Kimrose et al. (2018). In this study improved left ventricular pressure was observed for all concentrations < 100mM. These effects were not additive as the concentration increased. An improved recovery was observed in hypertrophic hearts accompanied by an increased intracellular concentration with aspartate (King et al., 2004). Glutamate and aspartate are typically used together at a concentration of 13mM of both (e.g. Zhang et al. 1997). None of the studies investigate molecular mechanisms.

Table 1.

Pre-clinical experimental studies investigating acidic amino acids

Study Species IR Model Concentration of Supplemented amino acid Results Additional information

Bittl and Shine

(1983)

 Rabbit 30 min low flow ischaemia or global no flow ischaemia 2mM glutamate Improved cardiac performance and increased αKetoglutarate level during ischaemia

Choong et al.

(1988)

 Rat 60 min low flow ischaemia 20 mM Glutamate

Glutamate during ischaemia only did improve functional recovery but did not improve energy metabolism.

Reperfusion with glutamate did improve pump recovery

Choong et al.

(1995)

 Rats 10 min global normothermic ischaemia then 4 h of cardioplegic arrest 10mM aspartate Improved functional recovery with 2 cardioplegia containing aspartate and better preservation of ATP

Ghomeshi et al.

(1997)

 Pig 30 min global normothermic ischaemia 13mM aspartate and glutamate in blood cardioplegia No difference in myocardial function, oxygen consumption and rate of energy decline Ionic supplementation improves recovery

Julia et al.

(1991)

 Immature dog 45 min global normothermic ischaemia 4 mL/Kg/hr aspartate and glutamate IV infusion

Preservation of tissue glutamate and aspartate.

Increased stroke work index

 Other compounds simultaneously infused

Kimose et al.

(2018)

 Rat 25 min normothermic global ischaemia initially then 87 min after cold cardioplegic arrest 0.01, 0.1, 1, 10, 20, 30 and 100mM glutamate in perfusate throughout Reperfusion LVDP increased with 0.1, 1, 10, 20, and 30mM. Not additive above 1mM. Glutamate increased in dose dependent manner with 10, 20, 30 and 100mM Glycogen increased with 100mM glutamate.

King et al.

(2004)

 Normal (WKY) and hypertensive Rat (SHR) 40 min global normothermic ischaemia for SHR and 70 min global normothermic ischaemia for WKY 0.5mM aspartate

Increase in tissue aspartate in SHR

Improved LVDP on reperfusion in SHR

 Also investigated EAAT3 expression which was increased in SHR

King et al.

(2006)

 SHR and WKY rat heart 20 min global normothermic ischaemia in working heart 0.5mM glutamate

Increase in tissue glutamate in SHR

Improved cardiac output on reperfusion in SHR

 Also investigated EAAT2 expression which was increased in SHR

Kofsky et al.

(1991)

 Immature dogs 60 min global normothermic ichaemia 13mM aspartate and glutamate in cardioplegia Preservation of heart rate, mean arterial pressure, cardiac output, stroke work index, and left atrial pressure vs. no ischaemia with parameters (except left atrial pressure) reduced in animals + ischaemia Used prolonged aortic clamping in the supplemented group

Løfgren et al.

(1995)

 Rat 40 min global no-flow ischaemia 20mM glutamate during reperfusion

Infarct size 51.4 ± 4.7% in control; 30.9 ± 3.6% in supplemented groups

LVDP depressed during reperfusion in supplemented groups

Protection with glutamate similar to Ischaemic preconditioning

Protection abrogated by transaminase inhibitor

Maiolino et al.

(Maiolino, et al., 2017)

 Cultured cardiomyocytes Exposure to hypoxia/reoxygenation 1mM Glutamate Promoted viability + mitochondrial function Effects abrogated by either NCX-1 or EAAT inhibitor.

Martinov et al.

(2014)

 Rats 30 min global ischaemia Glutamate transport inhibitor (LL-TBOA) Infarct area reduced Expression of EAAT1 and EAAT3.

Pisarenko et al.

(1985)

 Dog 2 min Low flow ischaemia at 60% of normal 3 mg/kg/min glutamate during reperfusion Less depression of cardiac performance. No change in oxygen consumption No effect of glutamate during normal coronary flow

Pisarenko et al.

(1989)

 Guinea pigs Low flow ischaemia for 25 min No additions. Reduction in amino acid pool on reperfusion

Pisarenko et al.

(1995)

 Rat 30 min of cardioplegic arrest (normothermic) or 5 h at 2 °C 20mM glutamate, 20mM aspartate or 20mM both All improved recovery but functional recovery best with aspartate or aspartate and glutamate.

Pisarenko et al.

(2006)

 Rat 40 min global normothermic ischaemia 21.5mM aspartate Recovery of function improved as was energy metabolism Arginine not as effective

Povlsen et al.

(2009)

 Obese type 2 diabetic Zucker fatty rats 40 min global ischaemia 15 or 30mM glutamate Supplementation (15mM) reduced infarct area in non diabetic only. 30mM reduced infarct size in both control + diabetic.

EAAT1 downregulated in diabetic. EAAT3 no difference.

Myocardial glutamate content increased in diabetic.

Pu et al.

(2008)

 Rabbit 30 min global ischaemia 10mL cardioplegia containing 850 mg aspartate Reduction in transmural repolarisation dispersion and occurrence of ventricular arrhythmias

Reed et al.

(1996)

 Rats Cardioplegia infused and pacing ceased. 13mM aspartate and glutamate in cardioplegia Little effect on energy metabolism

Rosenfeldt et al.

(1998)

 Rats Coronary ligation followed by 30 min global normothermic ischaemia 20 mM aspartate in cardioplegia

Greater power during reperfusion

Greater aortic flow and O2 consumption

Rosenkranz et al.

(1984)

 Dog 45 min global normothermic ischaemia 26mM glutamate in cardioplegia Better recovery of lactate and oxygen metabolism. Better recovery of postischaemic functional performance Compared cold and warm cardioplegia and used prolonged aortic clamping.

Singh et al.

(1989)

 Rat IV Isoproterenol 100 mg/kg aspartate or glutamate Reduced necrosis and reduced release of cardiac enzymes

Sivakumar et al.

(2008)

 Rats Subcutaneous injection of isoproterenol Aspartate or glutamate at 100 mg/Kg/day

Gred/GPx, TCA cycle enzymes and respiratory chain enzymes preserved with acidic amino acids

Same thing with ATP production

 All mitochondrial function

Sivakumar et al.

(2011)

 Rats Subcutaneous injection of isoproterenol Aspartate or glutamate (100 mg/Kg/day) Decreased cardiac marker enzymes, increased antioxidant enzymes, increased GSH and mitochondrial ATP, decreased lipid peroxides

Sun et al.

(2014)

 Rats 30 min ischaemia Glutamate transport inhibitor was used (dihydrokainate) at 1 mg/kg. Exacerbated reperfusion related arrhythmias and mitochondrial calcium overload and decreased SERCA2a expression Effects reduced with a noncompetitive NMDA receptor antagonist and by a glutamate release inhibitor

Weldner et al.

(1993)

 Immature rabbit 20 min global normothermic ischaemia followed by 90 min hypothermic ischaemia 20 mM glutamate in cardioplegia Better recovery of functional parameters and myocardial energy stores

Zhang et al.

(1997)

 Dogs 120 min cardioplegic arrest 13mM aspartate and glutamate Improved cardiac function Best recovery was when amino acid enrichment was accompanied by warm blood cardioplegia
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Loading isolated cardiomyocytes with 5.4mM KGlutamate led to increased ATP levels and protected against hypoxia and reperfusion (Williams et al. 2001). Subsequently it was shown that the glutamate loaded cardiomycoytes were also protected from reactive oxygen species generation leading to a novel hypothesis linking glutamate metabolism through glutamate dehydrogenase to the eventual detoxification of hydrogen peroxide by glutathione peroxidase (King et al. 2003). A proposed schema illustrating how acidic amino acids and dipeptides may protect the heart from IR is shown in Fig. 1. This shows dipeptides or glutamine as precursors of glutamate. The latter is then either transaminated and enters the TCA cycle as α-ketoglutarate or metabolised through glutamate dehydrogenase coupled eventually to glutathione peroxidase and the detoxification of hydrogen peroxide. Aspartate can also be transanimated to form oxaloacetate, another TCA cycle intermediate.

Fig. 1.

Fig. 1

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Cardioprotection by acidic amino acids and dipeptides. This shows 2 potential routes for the formation of TCA cycle intermediates from acidic amino acids in cardiomyocytes, which can then provide energy via substrate level phosphorylation. Also shown is glutamate metabolism via glutamate dehydrogenase leading to the detoxification of hydrogen peroxide and the entry points for glutamine and glutamine containing dipeptides. Numbers represent different enzymes. 1 = aspartate aminotransferase; 2 = alanine aminotransferase; 3 = glutaminase; 4 = cytosolic nonspecific dipedase; 5 = glutamate dehydrogenase; 6 = glutathione reductase; 7 = glutathione peroxidase. Gly-gln = glycyl-glutamine; ala-gln = alanyl-glutamine; GGSG = oxidised glutathione; GSH = reduced glutathione. Partly adapted from Williams et al. (2001)

Evidence from experimental studies: natural dipeptides

There are fewer experimental studies investigating the effect of natural dipeptides (Table 2). These can be divided according to the dipeptide used comprising carnosine, γglu-cys, glutamine containing dipeptides and one study investigating different combinations of leucine and tyrosine. The molecular mechanism underlying the protection brought about by 150 mg/kg N(2)-L-alanyl-L-Glutamine (ala-gln) injected intraperitoneally in rats 30 min before IR has been investigated. The ala-gln treated IR group showed increased protein expression of Bcl-2, JAK2, p-JAK2, STAT3 and p-STAT3 compared to the untreated IR group (Lui et al. 2019). This shows that activation of the JAK2/STAT3 pathway during IR in the presence of ala-gln leads to reduced apoptosis and the promotion cell survival (Lui at al. 2018). A proposed schema for this is shown in Fig. 2. This may be accompanied by increased inflammation and increased cytokine expression such as iL-6 and TNF-α (Yang et al. 2025). This maybe complicated by the interaction of the JAK/STAT pathway with other pathways such as p38 MAPK (Yang et al. 2025).

Table 2.

Studies in humans and clinical trials investigating acidic amino acids

Study Operation/Condition Supplemented amino acid Results Additional information

Bitzikas et al.

(2005)

 CABG 0.05 (units?) glutamate Myocardial glutamate levels increased. Also higher ATP. Better haemodynamic performance. Lower CKMB and Troponin T

Duman and Dogan

(2006)

 CABG Glutamate and aspartate to a total of 13 mM Better cardiac function at 1 h postoperatively but has disappeared after ≥ 6 h. No difference in marker enzyme release

Holme et al.

(2022)

GLUTAMICS II

 CABG in high risk patients 125 mM glutamate 20 min before releasing cross clamp and throughout first 150 min of reperfusion In diabetic patients less NT-proBNP released and AKI reduced.

Ji et al.

(2006)

 CABG 90 mL containing 158 mg potassium aspartate Lower troponin T release. Magnesium also included in the cardiopleia, which is more the focus of the paper

Lewis et al.

(2014)

 Aortic valve stenosis (AVS) or coronary artery disease None Left ventricle of female AVS patients high aspartate and glutamate

Pietersen et al.

(1998)

 Coronary artery disease 0.5 mg/kg glutamate Glutamate taken up by heart

Suleiman et al.

(1997)

 CABG None Cold crystalloid cardioplegia. Glutamate drops, aspartate no change. Glutamate and aspartate dropped during normothermic reperfusion.

Pisarenko et al.

(1989)

 Coronary artery disease None Glutamate secreted during pacing

Temizturk et al.

(2021)

 CABG 13 mM aspartate and glutamate in Del Nido Better post operative functional recovery and lower enzyme release Lower polymorphonuclear leucocytes in biopsy samples

Venturini et al.

(2009)

 Mitral valve disease None Dilated left ventricle had markedly higher glutamate compared to right ventricle. No differences in aspartate

Vidlund et al.

(2012)

GLUTAMICS

 CABG 125 mM glutamate infused prior to cardioplegic arrest and during reperfusion No difference in clinical endpoints Some patients had blood cardioplegia. Some had crystalloid cardioplegia.
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Fig. 2.

Fig. 2

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Schema illustrating the mechanism by which ala-gln confers protection. 1: ala-gln approaches (1) and binds to the JAK2 associated membrane receptor (2). This leads to the phosphorylation of JAK2 (3). Phosphorylated JAK2 stimulates the phosphorylation of STAT3 (4). Phosphorylated STAT3 dimerises (5) and translocates into the nucleus (6) where it stimulates gene expression leading to improved cell survival and increased inflammation (7). JAK2 – janus kinase 2; STAT3 – signal transducer and activator of transcription 3

Evidence from clinical trials: single and combined acidic amino acids

The role of glutamate in myocardial protection has been investigated in 2 randomised controlled clinical trials (RCTs), GLUTAMICS (Vidlund et al. 2012) and GLUTAMICS II (Holm et al. 2022). In GLUTAMICS patients undergoing coronary bypass grafting (CABG) were infused with either saline or 125mM glutamate prior to fixing the aortic clamp. Infusion was then continued after declamping the aorta for an additional 2 h. The only significant differences were that patients with left ventricular heart failure had a shorter ICU stay and time on the ventilator if they had received glutamate. This cohort also had lower lactate levels after.

protamine administration. Finally, there was a reduction in postoperative severe heart failure in the glutamate treated group compared to control, except in patients with diabetes (Vidlund et al. 2012).

GLUTAMICS II also enrolled patients undergoing CABG and infused with/without 125mM glutamate, but the patients were moderate to high risk and the primary endpoint was NT-proBNP release (Holm et al. 2022), which is a biomarker for heart failure (Weber and Hamm 2006). Both acute kidney injury and NT-proBNP release were significantly reduced in the glutamate infused group compared to control; however, this only occurred in patients without diabetes (Holm et al. 2022). This supported the findings in GLUTAMICS I (Vidlund et al. 2012) and suggests glutamate infusion could have a role in myocardial protection during cardiac surgery in patients without diabetes. The authors speculate that the reason for the difference between those with and without diabetes could be due to differences in mitochondrial EAAT1 expression (Vidlund et al. 2012). A similarly beneficial effect with respect to reperfusion injury has also been observed with cardioplegia enriched with aspartate (Ji et al. 2006).

Table 3 also contains clinical trials that have investigated adding aspartate and glutamate simultaneously. An early study showed that supplementation with a combined total of 13mM led to a significantly improved cardiac output in the first postoperative hour compared to control (Duman and Dogan 2006). These findings were supported by a relatively recent study where cardiac output and the stroke work index were significantly improved with 13mM glutamate/aspartate enriched Del Nido cardioplegia (Temiuzturk et al. (2021). This was accompanied by significantly reduced TnI and creatine kinase muscle brain band (CK-MB) release and lower plasma lactate concentration in comparison to non-supplemented controls. This suggests that in addition to improving functional parameters, cardioplegia enrichment with aspartate and glutamate may reduce membrane damage and improve metabolism. The underlying molecular mechanisms for this were not investigated.

Table 3.

Preclinical and experimental studies investigating natural dipeptides

Study Species IR Model Supplemented dipeptide Results Additional information

Almashhadany et al.

(2015)

 Rats 40 min global normothermic ischaemia 0.5, 2, 5mM gly-gln Improved functional recovery with 5mM in both young and middle-aged

Evran et al.

(2014)

 Rats Isoproterenol induced MI 250 mg/kg/day carnosine Decreased enzyme markers. Increased GSH, SOD and GPx

Hoshida et al.

(1994)

 Dog 90 min coronary occlusion 3 or 10 mg/kg γglu-cys Reduced infarct size and preserved GSH

Liu et al.

(2018)

 Rats Coronary ligation 150 mg/kg ala-gln

Damage markers reduced. Also increased protein for

Bcl-2, JAK2, p-JAK2, STAT3 and p-STAT3

Mitsui-Saitoh et al.

(2011)

 Guinea-pig 40 min global ischaemia 10 µM leu-tyr or tyr-leu No effect on post-ischaemic functional recovery Cyclo-leu-tyr did improve recovery

Nishinaka et al.

(1991)

 Dog 2 h LAD occlusion 10 mg/kg γglu-cys Reduced mitochondrial dysfunction and preserved GSH GSH before reperfusion had no effect on GSH depletion
Cultured H92C cells Simulated ischaemic conditions 0.5mM γglu-cys Reduced lactate dehydrogenase release

Wischmeyer et al.

(2003)

 rat 15 min global ischaemia 0.52 g/kg ala-gln Better functional recovery and metabolism Alanine no effect

Zhao et al.

(2020)

 Mice 30 min coronary occlusion 10 g/L carnosine in drinking water Smaller infarct area Similar effects with β-alanine feeding
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Evidence from clinical trials: natural dipeptides

The therapeutic use of ala-gln has been investigated in the setting of cardiac surgery (Table 4). For example, patients took 25 g ala-gln orally twice a day for 3 days prior to surgery and once on the morning of the surgery compared to a control group taking maltodextrin. Patients in the treated group had lower troponin I, CK-MB and myoglobin levels following surgery and a reduction in clinical complications (Sufit et al. 2012). This introduces the possibility of oral supplementation as an administration route.

Table 4.

Studies in humans and clinical trials investigating natural dipeptides

Study Operation/Condition Supplemented dipeptide Results Additional information
Hissa et al. (2011) CABG Ala-gln (250 mL of 20% solution) Reduction in blood glucose levels
Lomivorotov et al. (2011) CABG 0.4 g/kg ala-gln Lower cardiac damage markers. Better functional parameters
Sufit et al. (2012) Elective cardiac surgery Ala-gln (2 × 25 g) Reduction in cardiac damage markers. Reduction in pooled clinical complications
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The importance of membrane transport

Thirty-forty years age when the topic of myocardial protection by acidic amino acids was under investigation results were conflicting (Williams et al. 2001). Protection seemed to depend on whether the addition of exogenous acidic amino acids raised their intracellular concentration in cardiac tissue. When it did there was protection (Choong et al. 1988), when it did not there was none (Reed et al. 1996). Further support for the necessity of increasing the intracellular concentration for protection occurred when we developed a novel method for loading glutamate into isolated cardiomyocytes followed by greater resistance to oxidative or hypoxic stress (Williams et al. 2001; King et al. 2003). However, this relied on changes to membrane permeability and calcium concentration during enzyme digestion (Sollot et al. 1992) and was therefore untranslatable to open heart surgery. Another essential determinant of the intracellular concentration of any amino acid/dipeptide is membrane transport, an often-neglected topic in the heart. Could it be that poor understanding of the optimum conditions required to maximise acidic amino acid/dipeptides uptake into the heart during cardiac insults be hampering their protective potential?

Acidic amino acid transport

Classically, before the molecular identification and cloning of the responsible transporters, acidic amino acid uptake was investigated in brush border membrane vesicles (BBMV) isolated from either the kidney or small intestine (Weiss et al. 1978; Corcelli and Storelli 1983; Rajendran et al. 1987). This method was attractive as the vesicles lacked the metabolic machinery required to breakdown the amino acids enabling the transport to be followed using radiotracers. This resulted in the classification of acidic amino acid transporters into different transport systems (Kanai and Hediger 2003). The system responsible for the transport of acidic amino acids was called XAG−. We also adopted the traditional membrane vesicle approach when we carried out the first full characterisation of aspartate uptake in cardiac sarcolemmal vesicles (CSV) and isolated cardiomyocytes (King et al. 2001). We found that aspartate transport was dependent on an inward Na+ and outward K+ gradient, saturating with high affinity, electrogenic and over a time course produced the classic overshoot (King et al. 2001). The latter is important, because it enables the uptake of acidic amino acids against their large myocardial to plasma concentration gradient. This combined with the results from Dinkelborg et al. (1995) who found glutamate uptake into CSV to also be dependent on an inward H+ gradient is consistent with system XAG−. In an interesting investigation using isolated cardiomyocytes Dinkelborg et al. (1996) found that glutamate uptake was stimulated by anoxia.

A seminal moment occurred in the early 1990 s with the almost simultaneous molecular identification of 3 different genes encoding transporters that when expressed in different cell types showed acidic amino acid uptake consistent with system XAG− (Kanai and Hediger 2003) The human homologs were identified as the solute carrier 1 family comprising EAAT1 (SLC1A3), EAAT2 (SLC1A2), EAAT3 (SLC1A1), EAAT4 (SLC1A6) and EAAT5 (SLC1A7). Of these the only proteins detected in the heart are EAAT1 - EAAT3 (King et al. 2001, 2004, 2006; Martinov et al. 2014), although the mRNA for EAAT5 may also be present (Arriza et al. 1997).

In the hypertrophic heart the expression of EAAT2 and EAAT3 are increased accompanied by an increased rate of transport compared to the normal heart. This is associated with an increased tissue concentration when isolated and perfused hearts are supplemented with either 0.5 mM aspartate or glutamate leading to improved recovery from IR (King et al. 2004, 2006). Since then, it has been suggested that the protective effect of glutamate maybe linked with a functional interplay between EAAT1-3 and the cardiac sarcolemmal sodium calcium exchanger (NCX-1, Fig. 3, Maiolini et al. (Maiolino et al. 2017). In myoblasts expressing NCX-1 glutamate addition improved cell viability and mitochondrial function and normalised reverse mode NCX-1 activity (i.e. sodium exit in exchange for calcium entry). These effects were reversed in wild type myoblasts and in the presence of an NCX-1 inhibitor (DL-TBOA) or EAAT inhibitor (DL-TBOA). Too much calcium entry is detrimental (Schirone et al. 2022), therefore it is thought that the increase in NCX-1 activity only occurs to a new higher steady state level in the presence of continuing glutamate provision (Maiolini el al. 2017).

Fig. 3.

Fig. 3

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Schema illustrating how glutamate uptake via EAAT transporters is linked to improved calcium homeostasis and increased ATP synthesis. Glutamate entry via EAAT transporter(s) leads to increased SERCA-2 expression and Ca2+-ATPase activity in the sarcoplasmic reticulum, which improves calcium recapture following contraction. The sodium ions that accompany glutamate are exchanged with calcium ions leading to increased calcium dependent dehydrogenase activity and ATP synthesis in the mitochondria. These positive effects are abrogated in the presence of either EAAT inhibitors or NCX-1 inhibitors. DKA – dihydrokainate; DL-TBOA – DL-threo-β-benzyloxyaspartic acid; EAAT – excitatory amin acid transporter; NCX-1 – sarcolemmal sodium calcium exchanger 1; SN-6–2-[[4-[(4nitrophenyl)methoxy]phenyl]methyl]−4-thiazolidinecarboxylic acid ethyl ester

In rats subjected to coronary ligation and then release (IR) gabapentin use prevents glutamate secretion. In contrast the EAAT inhibitor dihydrokainate (DKA) had no effect suggesting that cardiac glutamate secretion is not mediated through the EAAT transporters. The presence of DKA increased the occurrence of ventricular arrhythmias accompanied decreased sarcoplasmic reticulum calcium ATPase activity and decreased SERCA-2 protein expression (Sun et al. 2013). Taken together with the results of Maiolini et al. (Maiolino, et al., 2017) this suggests a role for glutamate uptake in maintaining cardiac calcium homeostasis during IR (Fig. 3).

In a different investigation Martinov et al. (2014) confirmed the presence of EAAT1 and EAAT3 protein in the rat heart; however they found no improvement in functional parameters following IR when using the glutamate transport inhibitor, (2 S,3 S)−3-(3-(6-(6-(2-(2-(2-(2-(2-aminoethoxy)ethoxy)-ethoxy)ethoxy) acetamido)hexanamido)- hexanamido)−5-(4-(trifluoromethyl)benzamido)benzyloxy) aspartic acid (LL-BOA). Ventricular arrhythmias and calcium homeostasis were not measured. Another difference between these experiments and others using glutamate transport inhibitors is the absence of added exogenous glutamate. This is also consistent with the evidence suggesting myocardial protection occurs when the intracellular concentration of the acidic amino acid is increased in the presence of extra-myocardial glutamate.

Natural dipeptides transport

Dipeptide transport in the heart was first characterised in freshly isolated guinea-pig cardiomyocytes (Lin and King 2007). The key properties were dependence on an inward H+ gradient, saturation with a Km of 0.496mM and Vmax of 1470.5 ± 69.6 pmol/µL/min, and inhibition by a plethora of dipeptides and peptidomimetic drugs but not by the constituent single amino acids (Lin and King 2007). This was consistent with transport characteristics reported in the kidney cell line SKPT (Brandsch et al. 1995) and rat renal BBMV (Alghamdi et al. 2018; Ganapathy et al. 1995; Takahashi et al. 1998). Also consistent with these transport characteristics was the expression of PEPT2. This transporter is one member of The Proton-coupled Oligopeptide Transport family (Luo et al. 2023) of which only PEPT2 is expressed in heart Lin and King 2007; Alghamdi et al. 2021). In a further series of experiments the expression and activity of the PEPT2 transporter during ageing and in hypertension were investigated in CSV and heart ventricles isolated from Wistar, Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). With ageing, in CSV from normal Wistar rat hearts, the maximal rate of dipeptide uptake significantly increased at middle aged and then decreased in old age. This was accompanied by significantly greater PEPT2 protein expression in CSV from middle aged rats and higher SLC15A2 expression in middle aged hearts. This has implications for therapeutic peptidomimetic drug use in middle age. This may also be significant as we have shown that middle aged hearts are more susceptible to IR and were protected by gly-gln (Almashhadany et al. 2015) and the increased vulnerability of the hypertrophic heart to IR (King et al. 2004, 2006). In hypertrophic hearts (SHR) the maximal rate of dipeptide uptake was significantly lower compared to both Wistar and WKY. This correlated with a significantly lower PEPT2 protein expression but significantly higher SLC15A2 expression. This may reflect post translational modification in these hearts (Alghamdi et al. 2021) and is also relevant to the use of ACE inhibitors in hypertension.

Limitations and unanswered questions

There are limitations with the work described suggesting that more studies are required before clinical translatability can be considered. The studies reported in Tables 1, 2, 3 and 4 vary regarding concentration used and the timing of acidic amino acid/dipeptide addition. In GLUTAMICS II NT-proBNP and acute kidney injury were only reduced in diabetic patients (Holme et al. 2022), whilst in our studies only the hypertrophic heart showed improved recovery and greater EAAT expression (King et al. 2004, 2006). This suggests that protection maybe different for different patient groups. Cardiac surgery patients are usually taking a number of different drugs. Possible interactions between these drugs and acidic amino acids/natural dipeptides have not been investigated. Neither is it known how drugs interact with the EAATs/PEPT2. Consideration also should be made as to the cost of implementing strategies involving acidic amino acids/natural dipeptides, although these compounds are readily available through commercial sources. There is a need to address these important questions.

Future research directions

There is a need for clarification about the role of specific acidic amino acid transporters in myocardial protection. This could be achieved using knock-out mice. Assuming acidic amino acid secretion and uptake are mediated through different pathways more and confirmatory investigations of the effects of inhibiting acidic amino acid secretion whilst simultaneously providing exogenous acidic amino acids are required. There is also a need for greater consistency in relation to the concentration used and the type of cardioplegia used i.e. crystalloid vs. amino acid containing blood. More work is also required regarding natural dipeptides such as which if any glutamine containing dipeptide is most effective.

Conclusions

Improvements in our understanding of the mechanisms of membrane transport and possible means of protection have led to an increased knowledge as to how acidic amino acids and dipeptides could be utilised to protect the heart against IR injury. With respect to marrying together transport and protection the goal is to optimise conditions to maximise uptake and prevent secretion during IR to enable the protective effects to come to fruition. Once this has been achieved more large-scale clinical trials are warranted.

Acknowledgements

The author wishes to acknowledge Professor M - S Suleiman for critical reading of the manuscript.

Abbreviations

ACE

Angiotensin converting enzyme inhibitors

AKI

Acute kidney injury

Ala-gln

L-alanyl-L-glutamine

ATP

adenosine triphosphate

AVS

Aortic valve stenosis

BBMV

Brush border membrane vesicles

CABG

Coronary artery bypass grafting

CAD

Coronary artery disease

CDNP

Cytosol nonspecific dipeptidase

CKMB

Creatine kinase muscle brain band

CPB

Cardiopulmonary bypass

CSV

Cardiac sarcolemmal vesicles

Cys

Cysteine

DKA

Dihydrokainate

DL-TBOA

DL-threo-β-benzyloxyaspartic acid

EAAT

Excitatory amino acid transporter

Glu

Glutamate

GLUTAMICS

GLUTAMate for metabolics Intervention in Coronary Surgery

Gly-cys

[1-(2-hydroxyethyl)-1-methylbutyl]-(L)-cysteinylglycine

Gly-sar

Glycyl-sarcosine

GPx

Glutathione peroxidase

Gred

GLUTATHIONE reductase

GSH

Reduced glutathione

GSSG

Oxidised glutathione

HTK

Histidine-trypophan-ketoglutarate

ICU

Intensive care unit

IV

Intravenous

IR

Ischaemia reperfusion

JAK2

Janus kinase 2

LDH

Lactate dehydrogenase

LVDP

Left ventricular developed pressure

LL-TBOA

(2 S, 3 S)-3-(3-(6-(6-(2-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)acetamido)hexanamido)-5-(4-(trifluoromethyl)benzamido)benzyloxy)aspartic acid

NCX-1

Cardiac sarcolemmal sodium calcium exchanger

NT-proBNP

N-terminal pro B-type natriuretic peptide

PEPT

Peptide transporter

PHT

Peptide histidine transporter

RCT

Randomised controlled clinical trial

ROS

Reactive oxygen species

SN-6

2-[[4-[(4nitrophenyl)methoxy]phenyl]methyl]-4-thiazolidinecarboxylic acid ethyl ester

SERCA2a

Sarco/endoplasmic reticulum calcium ATPase

SOD

Superoxide dismutase

STAT3

Signal transducer and activator of transcription 3

SHR

Spontaneously hypertensive rat

SLC1

Solute carrier family

SOD

Superoxide dismutase

TCA

Tricarboxylic acid

TnI

Troponin I

WKY

Wistar Kyoto rat

XAG−

Amino acid transport system X-AG

Author contributions

This is solely the work of Nicola King.

Funding

No funding was received to assist with the preparation of this manuscript.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare no competing interests.

Ethical approval

Not applicable.

Consent to participate and consent for publication

Not applicable.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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