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

1. The intestinal H(+)-coupled peptide transporter PepT1, displays a broad substrate specificity and accepts most charged and neutral di- and tripeptides. To study the proton-to-peptide stoichiometry and the dependence of the kinetic parameters on extracellular pH (pHo), rabbit PepT1 was expressed in Xenopus laevis oocytes and used for uptake studies of radiolabelled neutral and charged dipeptides, voltage-clamp analysis and intracellular pH measurements. 2. PepT1 did not display the substrate-gated anion conductances that have been found to be characteristic of members of the Na(+)- and H(+)-coupled high-affinity glutamate transporter family. In conjunction with previous data on the ion dependence of PepT1, it can therefore be concluded that peptide-evoked charge fluxes of PepT1 are entirely due to H+ movement. 3. Neutral, acidic and basic dipeptides induced intracellular acidification. The rate of acidification, the initial rates of the uptake of radiolabelled peptides and the associated charge fluxes gave proton-substrate coupling ratios of 1:1, 2:1 and 1:1 for neutral, acidic and basic dipeptides, respectively. 4. Maximal transport of the neutral and charged dipeptides Gly-Leu, Gly-Glu, Gly-Lys and Ala-Lys occurred at pHo 5.5, 5.2, 6.2 and 5.8, respectively. The Imax values were relatively pHo independent but the apparent affinity (Km(app) values for these peptides were shown to be highly pHo dependent. 5. Our data show that at physiological pH (pHo 5.5-6.0) PepT1 prefers neutral and acidic peptides. The shift in transport maximum for the acidic peptide Gly-Glu to a lower pH value suggests that acidic dipeptides are transported in the protonated form. The shift in the transport maxima of the basic dipeptides to higher pH values may involve titration of a side-chain on the transporter molecule (e.g. protonation of a histidine group). These considerations have led us to propose a model for coupled transport of neutral, acidic and basic dipeptides.

The intracellular concentrations of essential amino acids (EAA) in muscle are maintained relatively constant under a variety of conditions. However, the effect of a decrease in blood amino acid concentrations on intracellular concentrations is not clear. Similarly, the relation between intracellular and interstitial concentrations has not been determined in this circumstance. Thus the aim of this study was to determine the effect of hypoaminoacidemia on intracellular, interstitial, and plasma concentrations of EAA and the mechanisms responsible for the respective changes. Twelve normal pigs were investigated before and during 120 min of hemodialysis by use of stable-isotope tracer methodology, microdialysis technique, and muscle biopsies. During hemodialysis, there was a decrease in the interstitial fluid concentrations of phenylalanine, leucine, alanine, and lysine that corresponded to their decrease in plasma concentration. Nonetheless, the intracellular concentrations of these amino acids were maintained at the basal levels throughout the entire period due principally to a reduction in the rate of incorporation of amino acids into protein that was approximately equivalent to the decrease in uptake from the plasma. In conclusion, intracellular concentrations of amino acids are regulated to maintain relatively constant values, even when plasma and interstitial concentrations fall as a consequence of hemodialysis.

Abstracts

There are considerable data to support the general hypothesis that accumulation of [Na+]i during ischemia and early reperfusion leads, via Na+/Ca2+ exchange, to elevated [Ca2+]i, resulting in myocardial damage.1 2 3 4 5 6 7 8 9 10 Despite the strong support for the general aspects of this hypothesis, there is controversy regarding some details that have important implications for the design of therapeutic interventions. The relative importance of the increase in [Na+]i during ischemia versus the increase in [Na+]i during reperfusion in contributing to the rise in [Ca2+]i and resultant injury is debated. These issues are important because it has been suggested that inhibition of the Na+/H+ exchanger (NHE) during reperfusion alone would be beneficial. This would allow clinical intervention after an ischemic episode. It is also important to understand why an increase in [Na+]i is detrimental. It is commonly assumed that [Na+]i is detrimental because it leads to increased [Ca2+]i during reperfusion, either due to diminished Ca2+ efflux via Na+/Ca2+ exchange or due to increased Ca2+ influx due to reverse Na+/Ca2+ exchange. Recent data presented by Cross et al9 suggest that reverse Na+/Ca2+ exchange is involved in postischemic contractile dysfunction. However, an increase in [Na+]i could also be detrimental because of effects on K+ loss11 or energetics. An understanding of the mechanism responsible for the detrimental effects of Na+ accumulation is important for the design of therapeutic interventions. A study12 published in this issue of Circulation Research adds new insight into these important issues. Lazdunski et al …

The mechanism by which Clostridium perfringens enterotoxin (CPE) simultaneously inhibits RNA, DNA, and protein synthesis is unknown. In the current study the possible involvement of small molecule permeability alterations in CPE-induced inhibition of macromolecular synthesis was examined. Vero cells CPE-treated in minimal essential medium (MEM) completely ceased net precursor incorporation into RNA and protein within 15 minutes of CPE treatment. However, RNA and protein synthesis continued for at least 30 minutes in Vero cells CPE-treated in buffer (ICIB) approximating intracellular concentrations of most ions. Addition of intracellular concentrations of amino acids to ICIB (ICIB-AA) caused a further small but detectable increase in protein synthesis in CPE-treated cells. ICIB did not affect CPE-specific binding levels or rates. Similar small molecule permeability changes (i.e., 86Rb-release) were observed in cells CPE-treated in either ICIB or in Hanks' balanced salt solution. Collectively these findings suggest that CPE-treatment of cells in ICIB-AA ameliorates CPE-induced changes in intracellular concentrations of ions and amino acids and permits the continuation of RNA and protein synthesis. These results are consistent with and support the hypothesis that permeability alterations for small molecules are involved in the CPE-induced inhibition of precursor incorporation into macromolecules in Vero cells.

Concentration of free amino acids and dipeptides in porcine skeletal muscles with different oxidative patterns

The general anaesthetic propofol shows promise in protecting normal hearts against various cardiac insults, but little is known about its cardioprotective potential in hypertrophic hearts. This study tested the hypothesis that propofol at a clinically relevant dose would enhance functional recovery in hypertrophic hearts following ischaemia. Hypertrophic hearts from spontaneously hypertensive rats and hearts from their normotensive controls, Wistar Kyoto Rats, were equilibrated in the working mode prior to global normothermic ischaemia. Reperfusion commenced with 10min in Langendorff mode, followed by 30-min working reperfusion. Functional performance was measured throughout the working mode, whilst reperfusion damage was assessed from myocardial troponin I release during Langendorff reperfusion. Where used, 4μg/ml propofol was added 10min before ischaemia and was washed out 10min into working reperfusion. An additional protocol investigated recovery of hearts protected by normothermic hyperkalaemic cardioplegic arrest. Following 20-min ischaemia, reperfusion damage was significantly worse in hypertrophic hearts compared to normal hearts, whilst addition of propofol to hypertrophic hearts significantly improved the aortic flow (31 ± 5.8 vs. 11.6 ± 2.0ml/min, n = 6-7 ± SE, p < 0.05). Propofol also conferred significant protection following 30-min ischaemia where the recovery of cardiac output and stroke volume was similar to that for cardioplegia alone. Incubation with propofol improved the NADH/NAD(+) ratio in freshly isolated cardiomyocytes from hypertrophic hearts, suggesting possible improvements in metabolic flux. These findings suggest that propofol at the clinically relevant dose of 4μg/ml is as effective as cardioplegic arrest in protecting hypertrophic hearts against ischaemia-reperfusion.

1. To examine whether insulin resistance in uraemia extends to amino acid metabolism, the effect of physiological hyperinsulinaemia on plasma amino acid concentrations was studied in 17 chronically uraemic and 28 healthy subjects by using the euglycaemic insulin clamp technique. 2. In six uraemic and seven control subjects, splanchnic and leg exchange of amino acids was quantified with hepatic and femoral venous catheterization, and in five uraemic and eight control subjects intracellular free amino acid concentrations were determined in muscle tissue obtained by needle biopsy. 3. In response to hyperinsulinaemia the plasma concentrations of all amino acids except alanine decreased by 10-50% in both uraemic and control subjects. Splanchnic alanine uptake declined by 20% and leg amino acid release tended to decrease in both groups. 4. The intracellular levels of all measured amino acids except alanine fell significantly and to a similar extent in uraemic and control subjects. 5. These results indicate that insulin-mediated alterations in regional amino acid exchange and in plasma and intracellular amino acid concentrations are similar in uraemic and control subjects. 6. It is concluded that tissue insensitivity to insulin in uraemia does not extend to uptake or release of amino acids and that resistance to insulin can be selective in its effect on different metabolic functions.

Pediatric cardiac surgery with cardiopulmonary bypass (CPB) and cardioplegic arrest is associated with global myocardial ischemia-reperfusion (IR) injury, leukocyte activation, and a systemic inflammatory response that contributes to early postoperative morbidity and mortality. The mechanisms for acute myocardial dysfunction and end-organ injury after pediatric heart surgery are complex and incompletely understood. This study aimed to: (1) investigate the contributions of the toll-like receptor-4 (TLR-4) pro-inflammatory and the pro-survival hypoxia-inducible factor-1α/phosphatidylinositol 3-kinase (HIF-1α/PI3K) pathways to global myocardial IR injury and inflammation, and (2) evaluate the impact of nicorandil, a coronary vasodilator and anti-ischemic agent, on TLR-4 and HIF-1α/PI3K signaling and end-organ dysfunction in a neonatal piglet model of CPB and cardioplegic arrest.Piglets (4-5 weeks old, N = 12) were randomly assigned to IR or IR + nicorandil. After initiation of CPB with cardioplegic arrest, hearts were reperfused on partial CPB, followed by complete separation from CPB. Left ventricular (LV) systolic and diastolic pressures were continuously recorded, and serial blood and terminal tissue samples were collected to measure inflammation, oxidant stress (OS), and apoptosis.CPB with cardioplegic arrest transiently and significantly impaired both LV contractility (p = 0.01) and diastolic relaxation (p = 0.001) associated with a 39-fold increase in cardiac TLR-4 expression (p = 0.034), a 3-fold increase in IL-6 production (p = 0.01), and significant increases in plasma markers for end-organ injury, but without the expected upregulation in the HIF-1α/PI3K pro-survival pathway. Nicorandil pre-treatment significantly augmented LV systolic (p = 0.004) and diastolic (p = 0.002) functional recovery associated with attenuated upregulation of TLR-4 signaling pathways, augmented HIF-1α/PI3K pro-survival signaling, and reduced inflammation, OS, apoptosis, and end-organ injury.Our piglet model of CPB with cardioplegic arrest demonstrates that the TLR-4 pro-inflammatory signaling pathway plays an essential role in myocardial IR-induced cardiac dysfunction, inflammation, OS, apoptosis, and multi-organ injury. Nicorandil pre-treatment attenuates these effects. Nicorandil also upregulates the HIF-1α/PI3K pro-survival signaling pathway. These results suggest that nicorandil should be studied further to assess its potential therapeutic effects after CPB in children.

The proton transport properties of plasma membrane and tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue were examined and compared. Membrane vesicles isolated with 250 millimolar KCl in the homogenization media and recovered at low density following sucrose density gradient centrifugation displayed characteristics of proton transport (nitrate inhibition, no inhibition by orthovanadate, pH optimum of 7.75, pyrophosphate-driven proton transport) which were consistent with a tonoplast origin. When the KCl in the homogenization medium was replaced by 250 millimolar KI, sealed membrane vesicles were recovered at higher densities in sucrose gradients and displayed properties (orthovanadate sensitivity, no inhibition by nitrate, pH optimum of 6.5) consistent with a plasma membrane origin. A comparison of anion effects (potassium salts) upon DeltapH and DeltaPsi revealed a direct correspondence between the relative ability of anions to stimulate proton transport and reduce DeltaPsi. For tonoplast vesicles, the relative order for this effect was KI > KBr >/= KCl > KClO(3) > K(2)SO(4) while for plasma membrane vesicles, a different order KI > KNO(3) >/= KBr >/= KClO(3) > KCl > K(2)SO(4) was observed. Proton transport in plasma membrane and tonoplast vesicles was inhibited by fluoride; however, plasma membrane vesicles appeared to be more sensitive to this anion. In order to correlate anion effects in the two vesicle fractions with anion transport, the kinetics of anion stimulation of steady-state pH gradients established in the absence of monovalent ions was examined. Anions were added as potassium salts and the total potassium concentration (100 millimolar) was maintained through the addition of K(+)/Mes. For plasma membrane vesicles, chlorate and nitrate displayed saturation kinetics while chloride displayed stimulation of proton transport which followed a linear profile. For tonoplast vesicles, the kinetics of chloride stimulation of proton transport displayed a saturable component. The results of this study indicate differences in proton transport properties of these two vesicle types and provide information on conditions where proton transport in the two fractions can be optimized.

Myocardial protection with cardioplegia alone may be inadequate during complex aortic arch surgery, potentially resulting in postoperative myocardial insufficiency. We hypothesized that non-cardioplegic continuous myocardial perfusion (CMP) is feasible and safe to protect the heart while operating on the aortic arch, and improves cardiac outcome. Between April 2010 and April 2014, 144 patients (60% male, age: 60 ± 13 years) underwent complex aortic arch repair in our institution using prefabricated, branched aortic arch grafts. In 36 patients, the hearts were protected with a combination of cardioplegic cardiac arrest during cardiac procedures and subsequent non-cardioplegic CMP group during aortic arch repair. In 108 patients, myocardial protection was achieved by cardioplegic arrest (CA group) only. Preoperative risk factors were comparable in both groups. Acute aortic dissection was the indication for surgery in 42% (CMP) and 44% (CA) of patients; 22% (CMP) and 29% (CA) of patients underwent reoperations. Concomitant cardiac procedures were similar. CMP patients received a frozen elephant trunk more frequently (89 vs 66%, P = 0.0096). Cardiopulmonary bypass time (242 ± 50 vs 264 ± 68 min; P = 0.046), and cardiac ischaemic time (49 ± 32 vs 149 ± 56 min, P < 0.0001) were significantly lower in the CMP group. There were no conversions to CA in the CMP group. Aortic arch repair was not prolonged by CMP. Low cardiac output syndrome occurred less frequently in the CMP group (3 vs 22%, P = 0.0052). Thirty-day mortality was significantly lower in the CMP group (6 vs 21%, P = 0.040). There were no cardiac deaths in the CMP group (0 vs 9%, P = 0.067). Neurological outcome was comparable. Blood loss was higher in the CA group (P < 0.001). Routinely protecting the heart during complex aortic arch repair with non-cardioplegic CMP is a valuable new concept. The CMP technique is feasible and safe, does not prolong aortic arch repair, reduces myocardial damage and improves cardiac outcome. Further evaluation in a larger patient cohort is warranted to establish this novel technique.

&lt;b&gt;&lt;i&gt;Background&lt;/i&gt;&lt;/b&gt;. Myocardial ischemia, occurring as a consequence of imbalance between oxygen supply and demand, causes a rapid metabolic and structural&lt;br /&gt; impairment within the tissue. After a period of ischemia, sudden onset of reperfusion causes a transition to aerobic metabolism within living cells. Afterwards, emerging substrates initiate a chain of reactions leading to tissue injury. This situation is called “ischemia reperfusion injury”. Despite all technical advancements in anesthesia, myocardial protection and cardiac surgical techniques, we still face the clinical reflections of ischemia reperfusion (IR) injury.&lt;br /&gt; &lt;b&gt;&lt;i&gt;Materials and methods&lt;/i&gt;&lt;/b&gt;. The protective effect of cilostasole on IR injury in an animal model of experimental myocardial ischemia and reperfusion was investigated. In this regional myocardial ischemia model, male Wistar-Albino rats were used as subjects and they were allocated into three groups; ischemia (n=8), sham (n=8), and cilostazole (n=8). LAD was occluded for 45 minutes, and then reperfused for three hours. Rats received Cilostazole 20 mg/kg/d by gastric gavage once daily. During IR hemodynamic parameters were recorded. Serum analysis for CK-MB and Troponin T were analysed at 180th minute of ischemia. Ischemic zone was measured by dying with Evans Blue and infarct area was measured by dying with triphenyltetrazolium chloride.&lt;br /&gt; &lt;b&gt;&lt;i&gt;Results.&lt;/i&gt;&lt;/b&gt; Before the onset of LAD occlusion, as well as at 25th, 60th and 120th minutes of occlusion, all groups were similar in terms of blood pressure and pulse rate.&lt;br /&gt; The total area, affected area and necrotic area were calculated by using formulas; affected area ratio= affected area/total area X 100, necrotic area ratio = necrotic area/total affected area X 100, necrotic area and affected area ratio = necrotic area /affected area X 100.&amp;nbsp; Affected area and total area ratio was significantly higher in IR group, compared with cilostazole group (t=8.965; p&amp;lt;0.001). Similarly, necrotic area and total area ratio was higher in IR group, compared with cilostazole group (t=8.965; p&amp;lt;0.001). The necrotic area and affected area ratios were similar in IR and cilostazole groups (t=0.245; p=0.810). CK-MB level differences were not statistically significant between two groups (Z=0.382; p=0.721).&lt;br /&gt; Troponin levels were similar between IR and cilostazole groups and the difference was not statistically significant (Z=0.630; p=0.574). Pathological specimens of the heart were scanned for myocytolysis, PMNL and hemorrhage.&amp;nbsp; The difference between mean value of MDA enzyme levels were statistically significant (p&amp;lt;0.001) between all groups.&amp;nbsp; MDA enzyme levels, from higher to lower was IR, cilostazole and Sham group.&amp;nbsp; SOD levels (F=5.910; p=0.009) were significantly lower in Sham group when compared with IR group (p=0.008). The differences between Sham and cilostazole groups and IR and cilostazole gropus were not statistically significant (p=0.008). According to planimetric values and enzyme levels, cilostazole was found to be effective in reducing the ischemic zone, without effecting the necrotic zone in cardiac ischemia reperfusion damage. Therefore cilostazole has protective effects agains ischemia reperfusion damage.&lt;br /&gt; &lt;br /&gt; &lt;b&gt;Conclusion&lt;/b&gt;.&amp;nbsp;This study explored how cilostazol affects myocardial ischemia-reperfusion injury in rats, finding that cilostazol administration during reperfusion may protect against such injury. Through various analyses, we observed positive outcomes associated with cilostazol treatment, suggesting its potential in reducing myocardial damage. Further research is needed to understand the underlying mechanisms and optimize therapeutic strategies, but our findings highlight cilostazol's promise in improving clinical outcomes in cardiac interventions.

ObjectiveCold blood cardioplegia (CBC) has been advocated as an advancement in myocardial protection during cardiopulmonary bypass (CPB) and cardioplegic arrest (CA) leading to decreased postoperative morbidity. Bcl-2, Bad, and caspase...

Introduction: Nicorandil is a coronary vasodilator for treatment of ischemia-induced angina. Studies suggest nicorandil may offer myocardial protection against regional ischemia-reperfusion (IR) injury during coronary artery bypass grafting in adults. The impact of nicorandil on global IR-induced myocardial injury and left ventricular (LV) dysfunction after cardiopulmonary bypass (CPB) and cardioplegic arrest (CA) has not been investigated in children. Hypothesis: Nicorandil attenuates myocardial IR injury and LV dysfunction after CPB and CA in an immature animal model. Methods: Anesthetized piglets (4-5 weeks-old, 8.8±0.4 kg) were cannulated via the right atrium and ascending aorta for initiation of full-flow CPB (100 mL/kg/min). Following 30 mins of aortic cross-clamp (ACC) and CA, hearts were reperfused on full CPB for 60 mins, followed by partial CPB for 30 mins, and separation from CPB for 30 mins. Nicorandil (0.2 mg/kg IV bolus + 0.02 mg/kg/min IV continuous infusion) or saline (N=6 per group) was initiated 5 mins before ACC and continued throughout the protocol. Temperature, ECG, mean arterial pressure (MAP), and LV function (via high-fidelity Millar pressure catheter) were continuously recorded. Serial plasma samples were collected for measurement of oxidant stress, inflammation, and multi-organ injury (heart, lung, kidney, and brain). Results: Temperature, heart rate, and MAP were similar between nicorandil- and saline-treated groups. However, 120 mins after ACC removal, nicorandil treatment significantly improved LV contractility [LV +dP/dt: 2,962±678 mmHg/sec (nicorandil) vs. 1,278±332 mmHg/sec (saline), p=0.004] and LV relaxation [LV -dP/dt: -2,670±454 mmHg/sec (nicorandil) vs. -1,347±339 mmHg/s (saline), p=0.002]. Also, nicorandil significantly attenuated the IR-induced increase in circulating biomarkers for oxidant stress, inflammation, and multi-organ injury. Conclusions: In a piglet model of CPB with CA, nicorandil attenuates LV systolic and diastolic dysfunction and improves oxidant stress, inflammation, and multi-organ injury. These results suggest that nicorandil may prove beneficial to mitigate IR injury and accelerate LV functional recovery after open heart surgery in infants and children.

Cardioplegic arrest (CA) using cold blood cardioplegia (CBC) has been reported to reduce ischemia-reperfusion (IR)-induced myocardial injury via apoptosis. We studied key apoptotic mediators via the caspase-dependent and intrinsic pathways as well as poly(ADP)-ribosylating protein (PARP) activity in myocardial and peripheral tissues after CA and cardiopulmonary bypass (CBP). Right atrial (RA) and skeletal muscle(SM) was harvested from cardiac surgical patients with similar baseline characteristics (N =6) before and after CPB and CBC. Total and modified caspase-3, Bcl-2, Bad, apoptosis-inducing factor (AIF), and PARP were quantified by immunoblotting. Terminal caspase-3 activity was assessed and immunohistochemistry was performed for PARP and AIF. TUNEL staining was used for identification of apoptotic cells. Microarray gene expression analysis was performed using Affymetrix U95 GeneChip. In RA tissue, CA with CBC significantly increased phosphorylation of Bcl-2 (Ser70), Bad (Ser112) (2.63+/-0.4 and 1.77+/-0.3-fold respectively; P<0.05), and cleavage of the downstream caspase 3 (1.45+/-0.1-fold; P<0.05). There was no significant change in total protein levels. Also, there was an increase in mature AIF (57 kDa) levels (1.22+/-0.01-fold; P<0.05) and a trend toward nuclear translocation on histological staining. Caspase 3 activity was increased 1.5+/-0.14-fold (P<0.05). The number of apoptotic cells in atrial tissue increased after compared with before CPB/CA using TUNEL staining (1.55+/-0.66 versus 0.325+/-0.05%, respectively; P=0.03). In contrast, SM samples did not show any of the changes observed in RA tissue after CPB. Despite optimal current surgical myocardial protection, we found that CA with CBC induced both programmed cell death and survival signaling in myocardial tissue.