Cardiospecific Overexpression of ATPGD1 (Carnosine Synthase) Increases Histidine Dipeptide Levels and Prevents Myocardial Ischemia Reperfusion Injury.

BACKGROUND Myocardial ischemia reperfusion (I/R) injury is associated with complex pathophysiological changes characterized by pH imbalance, the accumulation of lipid peroxidation products acrolein and 4-hydroxy trans-2-nonenal, and the depletion of ATP levels. Cardioprotective interventions, designed to address individual mediators of I/R injury, have shown limited efficacy. The recently identified enzyme ATPGD1 (Carnosine Synthase), which synthesizes histidyl dipeptides such as carnosine, has the potential to counteract multiple effectors of I/R injury by buffering intracellular pH and quenching lipid peroxidation products and may protect against I/R injury. METHODS AND RESULTS We report here that β-alanine and carnosine feeding enhanced myocardial carnosine levels and protected the heart against I/R injury. Cardiospecific overexpression of ATPGD1 increased myocardial histidyl dipeptides levels and protected the heart from I/R injury. Isolated cardiac myocytes from ATPGD1-transgenic hearts were protected against hypoxia reoxygenation injury. The overexpression of ATPGD1 prevented the accumulation of acrolein and 4-hydroxy trans-2-nonenal-protein adducts in ischemic hearts and delayed acrolein or 4-hydroxy trans-2-nonenal-induced hypercontracture in isolated cardiac myocytes. Changes in the levels of ATP, high-energy phosphates, intracellular pH, and glycolysis during low-flow ischemia in the wild-type mice hearts were attenuated in the ATPGD1-transgenic hearts. Two natural dipeptide analogs (anserine and balenine) that can either quench aldehydes or buffer intracellular pH, but not both, failed to protect against I/R injury. CONCLUSIONS Either exogenous administration or enhanced endogenous formation of histidyl dipeptides prevents I/R injury by attenuating changes in intracellular pH and preventing the accumulation of lipid peroxidation derived aldehydes.

area at risk

carnosine synthase

ischemia reperfusion

low‐flow ischemia

reactive oxygen species

Clinical Perspective

What Is New?

This research demonstrates that enhancing myocardial production of histidyl dipeptides by cardiospecific carnosine synthase overexpression or feeding carnosine and β‐alanine imparts protection from ischemia reperfusion injury.

Decrease in intracellular pH and the accumulation of reactive aldehydes are 2 deleterious characteristics of ischemia reperfusion injury.

Histidyl dipeptides by their dual ability to buffer intracellular pH and scavenging reactive aldehydes impart protection from ischemia reperfusion injury.

What Are the Clinical Implications?

Histidyl dipeptides are food components found in red meat that are increased by exercise.

Therefore, our findings lay the foundation to test the translational feasibility of these dipeptides as a prophylactic measure to prevent ischemic heart diseases.

Myocardial ischemia leads to a complex series of pathological changes that involve multiple deleterious events such as a switch to anaerobic glucose metabolism and intracellular acidification and increased generation of reactive oxygen species (ROS), which initiate the formation of toxic lipid peroxidation products. If uninterrupted, these changes lead to extensive tissue injury and sustained contractile dysfunction. Therefore, the most effective intervention to rescue ischemic myocardium is timely reperfusion using thrombolytic therapy or by percutaneous coronary intervention. However, reperfusion by itself initiates a second cascade of complex pathological events, collectively defined as “reperfusion injury,” characterized by rapid restoration of intracellular pH ([pH]i), release of cardiac enzymes, mitochondrial permeability transition pore opening, and eventually cell death.1 In the past, most therapeutic interventions to limit ischemia reperfusion (I/R) injury were designed to target a single effector (eg, antioxidants, pH buffers, or metabolic substrates),which showed only limited efficacy because preventing one aspect of I/R did not necessarily attenuate injury attributed to other contributing processes.

Multifunctional histidyl dipeptides, such as carnosine (β‐alanine‐histidine), have been evolutionarily conserved for at least 530 million years and are present in the muscles of families as distantly related as fish, amphibians, birds, and mammals.2 Although the specific roles of these peptides in muscle physiology is still unclear, it is thought that by the virtue of their dissociation constant (pKa) value, which is close to physiological pH (7.01), some of these peptides may buffer [pH]i.3 This property may be particularly useful under conditions of hypoxia, when high amounts of protons are generated by anaerobic glycolysis. However, in addition to buffering, these peptides can also quench ROS as well as reactive aldehydes such as acrolein, and 4‐hydroxy‐trans‐2‐nonenal (HNE),4, 5, 6 produced during the oxidation of membrane lipids. It has also been proposed that histidyl dipeptides act as a neuromodulators or a neurotransmitters.7 In most vertebrates, histidyl dipeptides are present in high concentrations in skeletal muscle (1–10 mmol/L) and heart (0.1–1 mmol/L) tissues2 in which they are locally synthesized by the enzyme ATPGD1 (carnosine synthase) from their precursors β‐alanine and histidine.8 Although the chemical properties of histidyl dipeptides are well suited to limit the effects of tissue hypoxia and I/R injury, it is unclear whether increasing the endogenous synthesis of these peptides by ATPGD1 overexpression protects against tissue injury and whether the complex I/R pathophysiology is affected by augmenting histidyl dipeptide levels.

During myocardial ischemia, the most prompt and marked change that occurs within the seconds after the onset of ischemia is cellular acidosis.9, 10 This decrease in [pH]i causes a rapid breakdown of high‐energy phosphates, increases inorganic phosphates, and downregulates contraction near the ischemic zone.9, 11 In response to ischemic acidosis, H+ ions are extruded by the Na+‐H+ exchanger to normalize [pH]i during reperfusion, but this results in intracellular accumulation of Na+, which in turn is exchanged for Ca2+ overload and the opening of the mitochondrial permeability pore.13 Several Na+‐H+ exchanger inhibitors in animal models had either resulted in modest recovery or had significant detrimental effects on I/R injury, but the clinical trials involving Na+‐H+ exchanger inhibitors were largely negative.14, 15 In addition to acidosis, I/R generates toxic lipid peroxidation products, which accumulate in the heart even after reperfusion.16, 17 These products, particularly HNE and acrolein, can lead to coronary vasodilation18 and decrease systolic pressure19 and arrhythmias.20, 21 They can also deplete ATP levels, alter Na+ and K+ conductances, and induce cell death.20 The contribution of these aldehydes to I/R injury is supported by extensive evidence showing that the deletion of enzymes that detoxify these aldehydes, including glutathione‐S‐transferase P,22 aldose reductase,23, 24 and aldehyde dehydrogenase 2, increases I/R injury.25, 26 Therefore, a combinatorial strategy that could reduce acidification and prevent the accumulation of reactive aldehydes might be well suited to ameliorate concurrently several adverse factors of I/R injury.

Previous work from our laboratory and others have shown that the perfusion of isolated murine hearts by carnosine offers modest protection against I/R injury,27, 28 and intravenous injection of carnosine in rats imparts cerebroprotection.29 Nevertheless, from these studies it is not clear whether these dipeptides act at intracellular sites or if these effects are systemic. Importantly, it is not known whether the cardioprotective effects of carnosine are related to pH buffering and/or aldehyde quenching and how it affects glucose use in the heart. In addition, in humans, histidyl dipeptides are rapidly hydrolyzed by the carnosine‐specific peptidase carnosinase present in the serum, which limits its bioavailability.30 Recent clinical trials in heart failure patients showed that carnosine feeding did not improve cardiac function, reflecting a limitation to carnosine ingestion in this setting.31 Hence, we increased the myocardial histidyl dipeptide pool by the cardiospecific overexpression of ATPGD1. Our results showed that ATPGD1 overexpression enhances the myocardial pool of histidyl dipeptides and diminished myocardial infarct size. The results of mechanistic studies showed that both aldehyde quenching and [pH]i buffering are essential actions for optimal cardio protection by histidyl dipeptides.

METHODS

Adult male C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All animal protocols were reviewed and approved by the University of Louisville Institutional Animal Use and Care Committee. Detailed methodology is described in Data S1. ATPGD1‐transgenic (Tg) mice were generated using the α–myosin heavy chain promoter and subjected to in vivo coronary ligation and reperfusion to induce I/R injury. Nuclear magnetic resonance (NMR) was used to quantify changes in pH in isolated perfused hearts, and metabolites were measured by liquid chromatography–mass spectrometry or gas chromatography–tandem mass spectrometry (see Data S1, and raw data will be provided on request). All of the data and methods used in the analysis and material used to conduct research will be made available at request.

Statistical Analysis

The levels of carnosine and infarct size in carnosine and β‐alanine–treated and untreated mice hearts, infarct size of C57BL/6, wild type littermates (WT mice) and ATPGD1‐Tg mice hearts subjected to in vivo ischemia reperfusion, and postischemic functional recovery following global ischemia in WT and ATPGD1‐Tg–isolated mice hearts were analyzed by using unpaired t test. High‐energy phosphates measured by 31P NMR and 13C lactate formation generated during low‐flow ischemia (LFI) and reperfusion in WT and ATPGD1‐Tg mice hearts were analyzed using repeated analysis of variance. Synthesis of carnosine in hearts isolated from WT and ATPGD1‐Tg mice were analyzed by using the Mann‐Whitney nonparametric test. Cytotoxicity of isolated cardiomyocytes isolated from WT and ATPGD1‐Tg mice hearts that were subjected to normoxia and hypoxia/reperfusion and generation of aldehyde protein adducts after ischemia in the WT and ATPGD1‐Tg mice hearts were analyzed by using 2‐way analysis of variance followed by bonferroni test. To estimate the survival time of isolated cardiomyocytes between different treatments, 4 different models of estimating survival were evaluated as mentioned previously.32 Fits of exponential, Weibull, linear exponential, and γ distribution to the data were estimated using a regression method for survival distribution fitting.33 The best fits were obtained for the Weibull distribution. This distribution has been used to determine the survival in continuous carcinogenesis and isolated cardiomyocytes.22, 32, 34 Survival data for cardiac myocytes were modeled using the Weibull survival distribution. Curve fitting was performed using the Proc NLIN procedure in SAS version 9.4 software (SAS Institute, Inc., Cary, NC). Two Weibull distributions were compared as described previously.35 The number of observations “n” refers to the number of different mice hearts, and ~100 cells were counted from each mouse heart. Statistical significance was accepted at P<0.05. Group data are mean±SD and mean±SEM.

RESULTS

Carnosine and β‐Alanine Feeding Protect the Heart From I/R Injury

To increase intracellular levels of carnosine, we placed adult male C57BL/6 mice on drinking water containing β‐alanine (20 mg/mL). β‐alanine is the nonproteinogenic rate‐limiting amino acid that is used for carnosine synthesis. It is widely used as a food supplement to increase carnosine levels in skeletal muscle.36, 37 As shown in Figure 1A, after 7 days on β‐alanine–containing water, carnosine levels were ≈7‐fold higher in the hearts of treated than untreated mice (0.31±0.05 nmoles/mg protein versus 2.26±0.08 nmoles/mg protein). There was no change in anserine levels (0.0754±0.008 nmoles/mg protein versus 0.068±0.001 nmoles/mg protein; Figure S1E). To evaluate the effect of elevated myocardial carnosine levels, these mice were subjected to coronary ligation for 30 minutes followed by 24 hours of reperfusion. Heart tissues were perfused with Evans blue dye and stained with 2,3,5‐triphenyl tetrazolium chloride to characterize the area at risk (AAR) and infarct size. Figure 1B shows the representative 2,3,5‐triphenyl tetrazolium chloride micrographs of the heart tissues. Quantitative analysis of Evans blue dye and 2,3,5‐triphenyl tetrazolium chloride staining, indicated that the AAR/left ventricle ratio was similar between the untreated and β‐alanine–treated mice (Figure 1C). However, the infarct area as a ratio of AAR was significantly lower in the β‐alanine–treated than in the untreated mice (Figure 1D). To examine whether carnosine itself was as effective as its precursor, we placed the WT mice on drinking water ±10 mg/mL carnosine for 7 days, which led to a 5‐fold increase in myocardial carnosine levels (0.15±0.04 nmoles/mg protein versus 0.71±0.19 nmoles/mg protein; Figure 1E). Again, there was no change in anserine levels (Figure S1F). Similar to β‐alanine, carnosine feeding protected the heart from I/R damage, as reflected by a lower infarct area/AAR ratio in the hearts of the carnosine‐fed mice subjected to coronary ligation and perfusion (Figure 1H). Taken together, these results suggest that increasing myocardial levels of carnosine via the delivery of carnosine or its precursor has an infarct‐sparing effect. In addition, these findings strengthened the rationale for testing the role of ATPGD1, which synthesizes these dipeptides.8

Figure 1

β‐alanine and carnosine feeding protect against ischemia reperfusion injury.

A. Wild‐type C57BL/6 mice were provided drinking water without (n=4) or with β‐alanine (20 mg/mL; n=3) for 7 days. Myocardial carnosine levels were measured by ultra‐performance liquid chromatography/tandem mass spectrometry. Inset is a representative chromatogram of carnosine and internal standard (IS) tyrosine histidine. B, Hearts of untreated and β‐alanine–fed mice subjected to 30 minutes of ischemia followed by 24 hours of reperfusion. Representative cross‐sections of 2,3,5 triphenyltetrazolium chloride staining shows the infarct zone depicted by a white area, the red area is the area at risk (AAR), and the blue region is the remote zone. (Scale bar=1 mm.) C and D, The relative ratios of the AAR to the left ventricle (LV) area and the infarct area (IF) to the AAR were compared between the untreated (n=8) and β‐alanine–fed (n=9) fed mice. E, Mice provided drinking water with or without carnosine (10 mg/mL) for 7 days; n=3 mice in each group. F, Representative cross‐sections of the untreated and carnosine‐treated mice subjected to 30 minutes of ischemia followed by 24 hours of reperfusion. G and H, Relative ratio of AAR to the LV area and IF to the AAR were compared between the untreated (n=9) and carnosine‐treated mice groups (n=11). *P<0.05 vs the untreated mice group.

Cardiospecific Overexpression of ATPGD1 Protects Against I/R Injury

The enzyme ATPGD1 ligates β‐alanine and histidine to form carnosine, which could be further methylated by carnosine N‐methyltransferase to form anserine.2, 8, 38 To examine whether the cardiospecific ATPGD1 overexpression increases myocardial levels of histidyl dipeptides, we generated the Tg mice overexpressing the mouse ATPGD1 gene under the control of α–myosin heavy chain promoter on a C57/BL6 background. Western blot analysis confirmed that the ATPGD1 expression in the heart was increased ≈20‐ to 25‐fold compared with the littermate non‐Tg control (WT) mice (Figure 2A). Liquid chromatography–mass spectrometry measurements showed that the carnosine and anserine levels were increased 30‐ to 40‐fold and 10‐ to 12‐fold, respectively, in the ATPGD1‐Tg compared with the WT hearts (carnosine, WT: 0.31±0.04 nmoles/mg protein versus ATPGD1Tg: 14.57±0.39 nmoles/mg protein; anserine, WT: 0.075±0.008 nmoles/mg protein versus ATPGD1Tg: 0.791±0.137 nmoles/mg protein; Figure 2B, Figure S1A and S1B). The levels of both of these dipeptides in gastrocnemius skeletal muscle remained unchanged, attesting to the fidelity of the α–myosin heavy chain promoter (Figure S1C and S1D). Echocardiographic analysis showed that at baseline morphometric data and function were similar between the ATPGD1‐Tg and WT mice hearts (Table S1). To determine the effect of ATPGD1 overexpression on I/R injury, we subjected the WT and ATPGD1‐Tg mice hearts to coronary ligation and reperfusion. Quantitative analysis of the 2,3,5‐triphenyl tetrazolium chloride staining showed that the AAR was similar between the WT and ATPGD1‐Tg mice hearts; however, the infarct size decreased significantly by ATPGD1 overexpression (Figure 2C through 2E). Taken together, these findings suggest that increasing the endogenous production of histidyl dipeptides within the heart protects from I/R injury.

Figure 2

Cardiospecific overexpression of ATPGD1 (carnosine synthase) increases histidyl dipeptide levels and protects against ischemia reperfusion injury.

A, Representative Western blot of ATPGD1 in wild‐type (WT) and ATPGD1‐transgenic (Tg) hearts (i); lower panel represents the band intensity normalized to tubulin between the 2 groups (ii); n=4 in each group. B, Fold changes in carnosine and anserine levels in WT and ATPGD1‐Tg hearts, n=4 in each group. C, Representative images of 2,3,5‐triphenyl tetrazolium chloride–stained WT and ATPGD1‐Tg hearts after 30 minutes of ischemia followed by 24 hours of reperfusion. D and E, Quantification of the ratio of area of risk (AAR) and left ventricle (LV) and the ratio of infarct size (IF) and AAR in the WT (n=6) and ATPGD1‐Tg (n=7) hearts. Results are mean±SEM. *P<0.05 vs WT mice hearts.

ATPGD1 Overexpression Improves Postischemic Function and Protects Myocytes From Hypoxia Reoxygenation Injury

Although our results showed that carnosine protects against I/R in vivo, this could be attributed to direct effects of the dipeptide in cardiomyocytes or neurohormonal and inflammatory responses triggered by coronary occlusion and reperfusion. Hence, to exclude neurohormonal and inflammatory responses, we perfused isolated WT and ATPGD1‐Tg mice hearts in the Langendorff mode for 10 to 12 minutes and then subjected them to 30 minutes of global ischemia followed by 45 minutes of reperfusion. At baseline, the left ventricular developed pressures were comparable between the WT and ATPGD1‐Tg hearts; however, during reperfusion, the ATPGD1‐Tg hearts had improved systolic functional recovery, as demonstrated by higher left ventricular developed pressures compared with the WT mice hearts (Figure 3A). Similarly, the release of the myocardial enzymes lactate dehydrogenase and creatine kinase was less in the ATPGD1‐Tg than in the WT hearts, indicating the cardiospecific protection attributed to ATPGD1 overexpression (Figure 3B and 3C).

Figure 3

ATPGD1 (carnosine synthase) overexpression prevents contractile dysfunction and cardiomyocytes death.

A, Changes in the developed pressure of hearts isolated from the wild‐type (WT) and ATPGD1‐transgenic (Tg) mice during 10 to 12 minutes of perfusion followed by 30 minutes of ischemia and 45 minutes of reperfusion. Data are mean±SEM, n=4 mice hearts in each group; *P<0.05 vs WT heart. B and C, Levels of lactate dehydrogenase (LDH) and creatine kinase (CK) released in the perfusate collected at different intervals of reperfusion. D, LDH release in the WT and ATPGD1‐Tg isolated cardiomyocytes under normoxia and hypoxia/reoxygenation (H/R) conditions. Results are mean±SEM, n=6 in each group. *P<0.05 vs WT normoxia, # P<0.05 vs ATPGD1‐Tg normoxia, and ^P<0.05 vs WT H/R‐treated cardiomyocytes. LVDP indicates left ventricular developed pressures.

To localize further the site of action of ATPGD1, we isolated the cardiac myocytes from the WT and ATPGD1‐Tg mice hearts and subjected them to either normoxia (5 hours) or hypoxia (3 hours) followed by 2 hours of reoxygenation. The WT cardiac myocytes subjected to hypoxia and reoxygenation showed greater lactate dehydrogenase release than normoxic cardiac myocytes; however, the lactate dehydrogenase release in myocytes from the ATPGD1‐Tg hearts subjected to hypoxia and reoxygenation was similar to that from normoxic WT cells, and significantly lower than the WT myocytes subjected to hypoxia and reoxygenation (Figure 3D). Collectively, these data indicate that increased intracellular levels of carnosine protect myocytes from the deleterious effects of hypoxia and reoxygenation.

ATPGD1 Overexpression Prevents Aldehyde Accumulation in Ischemic Hearts and Protects Aldehyde Toxicity

Given our findings that ATPGD1‐Tg mice hearts are protected from I/R injury, we next determined the mechanism by which ATPGD1 overexpression affects the heart during ischemia. For this, we subjected the WT and ATPGD1‐Tg mice hearts to 40 minutes of coronary ligation in vivo and analyzed the ischemic zone of the ligated hearts and the anterior zone of the sham‐operated hearts for protein‐HNE and protein‐acrolein adducts by Western blotting. Our results showed that the protein‐HNE adducts were more abundant in the ischemic zone of the WT ischemic hearts than in the anterior zone of the sham‐operated WT and ATPGD1 hearts. Importantly, the accumulation of the protein‐HNE adducts was mitigated in the ischemic zone of the ATPGD1‐Tg mice hearts (Figure 4A through 4C). Moreover, less protein‐acrolein adducts accumulated in the ischemic zone of ATPGD1‐Tg ischemic hearts (Figure 4D through 4F). These observations are consistent with the notion that elevated levels of carnosine decreases the accumulation of lipid peroxidation–derived aldehydes in the ischemic hearts.

Figure 4

ATPGD1 (carnosine synthase) attenuates aldehyde accumulation and toxicity.

Representative Western blots of the wild‐type (WT) and ATPGD1‐transgenic (Tg) mice hearts after 30 minutes of sham and coronary ligations. The anterior zone (AZ) from sham‐operated and the ischemic zone (IZ) from coronary‐ligated mice hearts were developed with (A) anti–4‐hydroxy‐trans‐2‐nonenal (HNE) and (D) anti‐acrolein antibodies and normalized with Amido‐black (AB). Relative band intensities of HNE (B and C) and acrolein‐modified (E and F) protein bands. Data are mean±SEM. ^P<0.05 vs WT sham AZ, *P<0.05 vs ATPGD1‐Tg AZ, # P<0.05 vs WT IZ; n=4 in each group. G, Representative images of adult cardiomyocytes isolated from the WT and ATPGD1‐Tg hearts at 0 and 60 minutes of superfusion with HNE (50–60 μmol/L). (Scale bar=100 μmol/L.) H, Survival plots of cardiomyocytes isolated from WT (n=7) and ATPGD1‐Tg (n=5) hearts superfused with HNE, 100 cells from each heart. Data are shown as discrete points, and the curves are best fits of a Weibull survival function. I, Graph showing the mean lifetime of WT and ATPGD1‐Tg cardiomyocytes in the presence of HNE are presented as mean±SEM. *P<0.05 vs WT HNE superfused cardiomyocytes. J, Representative images of myocytes isolated from WT and ATPGD1‐Tg hearts at 0 and 60 minutes of superfusion with acrolein (5 μmol/L). (Scale bar=100 μmol/L.) K, Survival plots of cardiomyocytes isolated from WT (n=7) and ATPGD1‐Tg (n=6) hearts superfused with HNE, 100 cells from each heart. Data are shown as discrete points and the curves are best fits of the Weibull survival function. L, Graph showing the mean lifetime of WT and ATPGD‐Tg cardiomyocytes. *P<0.05 vs WT acrolein‐treated myocytes.

Given that aldehyde protein‐adduct accumulation was diminished in the ATPGD1‐Tg ischemic hearts, we next tested whether ATPGD1 overexpression would protect against aldehyde toxicity. Previous work has shown that these aldehydes induce myocyte hypercontracture.20, 21 Therefore, we perfused myocytes isolated from the WT and ATPGD1‐Tg hearts with HNE for 60 minutes, continuously monitored their shape under a light microscope, and calculated cell survival using the Weibull distribution function. These analyses showed that although HNE perfusion caused hypercontracture in both WT and ATPGD1‐Tg cardiac myocytes, the mean lifetime of WT myocytes was 55±2 minutes, whereas the ATPGD‐1‐Tg survived with a mean lifetime of 72±6 minutes (Figure 4G through 4I). The ATPGD1‐Tg cardiac myocytes were also resistant to acrolein‐induced hypercontracture. The mean lifetime of WT cardiomyocytes superfused with acrolein was 55±2 minutes, which was less than that of ATPGD1‐Tg myocytes (72±5 minutes; Figure 4J through 4L). Collectively, these results suggest that high intracellular levels of carnosine protect against the toxicity of a structurally diverse range of aldehydes generated by lipid peroxidation.

ATPGD1 Overexpression Prevents Ischemic Changes in [pH]i and High‐Energy Phosphate Content

Given that ATPGD1 overexpression enhanced myocardial histidyl dipeptide levels, which can buffer pH,2 we next tested whether it could also preserve change in [pH]i during myocardial I/R. For this, we used 31P NMR spectroscopy to measure the changes in myocardial ATP, phosphocreatine (PCr), and [pH]i during I/R. Hearts isolated from the WT and ATPGD1‐Tg mice were subjected to 20 minutes of perfusion, followed by 20 minutes of LFI (10% of baseline) and 50 minutes of reperfusion. Figure 5A and 5D shows the representative 31P NMR spectra at baseline, LFI, and reperfusion of the WT and ATPGD1‐Tg isolated hearts, respectively. From the left to right, the peaks present inorganic phosphate; PCr; and γ, α, and β phosphates of ATP; and the area under the curve of respective resonances were used to calculate the amount of each metabolite. The baseline spectra of PCr was similar between the WT and ATPGD1‐Tg hearts, which was reduced to ~50% of baseline in both the WT and ATPGD1‐Tg hearts during LFI; however, the decrease was slightly less pronounced in the ATPGD1‐Tg hearts. During reperfusion, the PCr recovery in the WT hearts was nearly 100%; however, in the ATPGD1‐Tg hearts, the PCr recovery exceeded the baseline levels and remained higher than in WT hearts throughout the reperfusion (Figure 5B). Basal ATP levels were similar between the WT and the ATPGD1‐Tg hearts, and LFI resulted in a 40% to 50% decrease in ATP levels in the WT hearts, which did not recover during reperfusion. In contrast, the ATP levels in the ATPGD1‐Tg hearts decreased to only 30% to 40% of the baseline during LFI and remained higher than in the WT hearts (Figure 5C). The inorganic phosphate increased substantially in both the WT and ATPGD1‐Tg hearts during LFI and reached preischemic levels during reperfusion in both groups (Figure 5E). These results suggest that increasing the abundance of carnosine in the heart prevents the loss of high‐energy phosphates during ischemia and promotes their restoration during reperfusion.

Figure 5

ATPGD1 (carnosine synthase) overexpression enhances buffering capacity during ischemia reperfusion injury.

Representative 31P NMR spectra from wild‐type (WT) (A) and ATPGD1‐transgenic (Tg) (D) hearts during baseline, low‐flow ischemia (LFI), and reperfusion. Pi, PCr, γ, α, β denotes inorganic phosphate, phosphocreatine, and resonances of ATP, respectively. Changes in PCr (B), ATP (, Pi (E), and pH (F) during baseline, LFI, and reperfusion. Data are mean±SEM; n=6 mice in each group. *P<0.05 vs WT, # P<0.05 vs WT baseline, and ^P<0.05 vs ATPGD‐Tg baseline.

At baseline, [pH]i was similar between the WT and ATPGD1‐Tg hearts (7.13±0.04 versus 7.14±0.01), but in WT hearts, [pH]i fell rapidly by 0.34±0.02 pH units after 10 minutes and 0.45±0.02 pH units after 20 minutes of LFI. In contrast, there was less acidosis in the ATPGD1‐Tg hearts. In these hearts, the mean fall of [pH]i was 0.23±0.02 units after 10 minutes and 0.32±0.03 units after 20 minutes of LFI. During the first 10 minutes of reperfusion, the [pH]i in the WT hearts reached 6.92±0.01 and thus had recovered 0.21±0.01 pH units. However, in the ATPGD1‐Tg hearts, the [pH]i reached 7.03±0.01, suggesting a greater recovery of [pH]i compared with the WT hearts. The rates of [pH]i recovery during 20 to 50 minutes of reperfusion was delayed in the WT hearts and did not reach preischemic levels, whereas the [pH]i in the ATPGD1‐Tg hearts after 30 to 40 minutes of reperfusion was similar to baseline (7.08±0.01 versus 7.13±0.01; Figure 5F). Taken together, these results provide direct evidence that ATPGD1 overexpression augments intracellular buffering and mitigates acidosis during I/R.

Both Buffering and Aldehyde Quenching Are Essential for Cardiac Protection by Histidyl Dipeptides

Because ATPGD1 overexpression seems to protect against carbonyl stress and [pH]i imbalance, we next examined which of these properties might be essential for cardioprotection. To test the relative contribution of aldehyde quenching and buffering, we synthesized the natural carnosine analog anserine, which is methylated at the Nπ residue of histidine and has aldehyde‐binding capacity similar to carnosine (Figure S2).39 However, in comparison with carnosine, anserine has a lower buffering capacity (pKa 7.15).40 In addition, we also synthesized balenine, which is methylated at the Nτ residue of histidine, and therefore has a higher buffering (pKa 6.93), yet lower aldehyde binding, capacity than does carnosine.21, 40 Thus, unlike carnosine, balenine does not protect cardiomyocytes against aldehyde‐induced hypercontracture.21 To determine how the differential biochemical properties of these dipeptides influences I/R injury, we perfused isolated C57/BL6 mice hearts with either anserine or balenine (1 mmol/L) and subjected these hearts to I/R as previously. The results of these experiments showed that neither balenine nor anserine was able to improve the recovery of postischemic contractile function (Figure 6A and 6B). The release of cardiac enzymes (creatine kinase and lactate dehydrogenase) in balenine‐perfused and anserine‐perfused hearts was similar in magnitude to their release in untreated WT hearts (Figure 6C through 6F). From these results, we infer that the loss of either the buffering capacity or aldehyde binding abolishes the cardioprotective effects of histidyl dipeptides, and therefore both of these properties are essential for the cardioprotective effects of carnosine.

Figure 6

Histidyl dipeptide analogs balenine and anserine were unable to restore the contractile function.

Changes in the developed pressure of C57BL/6 mice hearts perfused with (A) balenine and (B) anserine followed by 30 minutes of ischemia and 45 minutes of reperfusion. Data are mean±SEM; n=4 mice hearts in each group. *P<0.5 vs wild‐type heart. Levels of creatine kinase (CK) () and lactate dehydrogenase (LDH) (E and F) released in the perfusate collected at different intervals of reperfusion. LVDP indicates left ventricular developed pressures.

Anaerobic glycolysis is increased in carnosine synthase‐transgenic (Tg) ischemic hearts.

A, Wild‐type (WT) and ATPGD1‐Tg isolated hearts were perfused for 10 minutes with 13C D‐glucose in the Langendorff mode, followed by 20 minutes of low flow ischemia. Levels of (A) 13C D‐glucose and (B) 13C‐lactate in perfusates collected at different time intervals. Data are mean±SEM; n=5 mice in each group. *P<0.05 vs WT heart. ATPGD1 indicates carnosine synthase.

ATPGD1 Overexpression Preserves Intermediary Metabolism During Ischemia

Extensive evidence suggests that elevated glucose levels could be beneficial to improve recovery from I/R injury.41, 42, 43 Our results so far suggested that ATPGD1 overexpression prevents intracellular acidification during ischemia. Although it has been proposed that carnosine promotes glycolysis by buffering pH,2 direct evidence for such a proglycolytic effect is lacking. To examine whether ATPGD1 overexpression affects glycolysis, we perfused isolated WT and ATPGD1‐Tg hearts with13C glucose for 10 minutes followed by 20 minutes of LFI. Perfusates from these hearts were collected at different times and analyzed for13C glucose and13C lactate by liquid chromatography–mass spectrometry. We found that the13C glucose levels measured at different time points of LFI were similar between the WT and ATPGD1‐Tg hearts. However,13C lactate formation during 1, 2, and 10 minutes of LFI was higher in the perfusates of the ATPGD1‐Tg rather than the WT hearts (Figure 6B and 7A). Taken together, these results suggest that despite the similar levels of glucose uptake, ATPGD1 overexpression increases glucose use under ischemic conditions, presumably by increasing the rates of glycolysis by buffering [pH]i.

DISCUSSION

The major findings of this study are that cardiac myocyte–restricted overexpression of ATPGD1 enhances myocardial production of carnosine and thereby protects the heart from I/R injury. In addition, augmenting myocardial carnosine levels by β‐alanine or carnosine supplementation also imparts cardioprotection. Our results show that ATPGD1 overexpression decreased the formation of protein‐aldehyde adducts in the ischemic heart and diminished the toxicity of lipid peroxidation products. We also found that the overexpression of ATPGD1 attenuated I/R‐induced changes in [pH]i and intermediary metabolism that accompany myocardial I/R. The protective effects of carnosine were not duplicated by the structurally related histidyl dipeptides anserine and balenine, suggesting that the multifunctional chemistry of carnosine is essential for cardioprotection. Taken together, these findings for the first time reveal a direct cardioprotective role of ATPGD1 and uncover new aspects of cardioprotection against myocardial ischemic injury targeted by ATPGD1‐synthesized dipeptides. Given that ATPGD1 synthesizes multifaceted dipeptides, which are readily hydrolyzed in human serum diminishing its bioavailability, these results suggest that modulating the endogenous production via ATPGD1 overexpression may be a key strategy to prevent and attenuate myocardial I/R injury and related pathological conditions in humans.

Carnosine, anserine, and balenine are evolutionarily conserved dipeptides that are present in a high abundance in energetically active tissues such as the striated muscles and neurons.2 Among these dipeptides, carnosine is widely present in the humans, whereas anserine and balenine are mostly found at relatively higher levels in birds and whales, respectively.40 In this study, we found that the mouse heart contains 300 to 350 nmoles/g protein of carnosine and 75 to 80 nmoles/g protein of anserine, which are consistent with the expected distribution of these peptides in mammalian hearts. Nevertheless, despite their near ubiquitous distribution, the physiological role of these peptides is understudied, and it is unclear what specific function they perform in different tissues. Most previous studies on histidyl dipeptides has focused on their potential ability to increase exercise capacity, and β‐alanine is frequently ingested by professional athletes as a performance‐enhancing supplement. The skeletal muscle levels of carnosine are increased by high‐intensity exercise, suggesting that it plays an important role in skeletal muscle physiology.36 Although the specific mechanisms by which carnosine increases exercise capacity remain unknown. It is widely believed that it can enhance exercise performance by buffering [pH]i and thereby preserve glycolysis during hypoxia36, 44, 45 however, direct evidence regarding the proglycolytic effects is lacking.

In addition to buffering pH, carnosine can also react with a variety of electrophiles, such as the reactive aldehydes generated by lipid peroxidation. Indeed, in our previous work we found that carnosine reacts directly with HNE and acrolein and that the resultant conjugates are metabolized and excreted in mouse and human urine.27 Therefore, carnosine may be an endogenous antioxidant that removes reactive aldehyde species, acting in parallel to glutathione‐linked detoxification. This notion is supported by our recent observation that an increase in carnosine levels attributed to β‐alanine supplementation results in increased scavenging of lipid peroxidation products in human skeletal muscle, generated during high‐intensity exercise.36 Carnosine treatment has also been reported to protect the brain from ischemic damage,29 and topical administration of carnosine accelerates wound healing in db/db mice.46 Carnosine perfusion in isolated hearts has also been shown to marginally improve functional recovery of isolated ischemic rat and mice hearts.27, 28, 47 Nonetheless, the mechanisms underlying the cardioprotective effects of carnosine remain unknown, and it is unclear whether carnosine protects by targeting extracellular sites or intracellular pathology and which cell types in the heart are targeted. Therefore, we generated ATPGD1‐Tg mice to selectively increase carnosine levels within cardiomyocytes, and we undertook a comprehensive evaluation of the mechanisms of carnosine‐mediated cardioprotection.

We found that the cardiospecific overexpression of ATPGD1 reduced the infarct size both in vivo and ex vivo and that cardiac myocytes overexpressing ATPDGD1 were resistant to hypoxia reoxygenation injury. Although cardiospecific ATPGD1 overexpression enhanced the myocardial levels of histidyl dipeptides 40‐ to 50‐fold and mice fed with β‐alanine and carnosine enhanced the myocardial carnosine levels 4‐ to 7‐fold, both treatments had similar infarct‐sparing effects, suggesting that physiological dosing of β‐alanine and carnosine could be cardioprotective. Together these observations provide unequivocal evidence that carnosine protects the heart by targeting sites and events within cardiac myocytes, without directly affecting extracellular events, neurohormonal changes, or even inflammatory responses that accompany myocardial I/R.

Previous work has shown that myocardial ischemia is associated with acidification and a decrease in ATP levels attributed to the progressive failure of glucose and fatty acid metabolism. Myocardial ischemia also creates conditions that promote ROS generation, which exacerbates tissue injury and induces cell death. However, the relationship between acidification and loss of ATP is unclear, and the contribution of these changes to the ROS generation has not been clarified. Moreover, it is unclear to which extent ROS themselves or the secondary products they generate mediate tissue injury. Although free radicals and ROS are highly reactive, they are short lived and therefore much of their injury has been attributed to their secondary, more stable, products, such as the aldehydes generated by lipid peroxidation.48, 49 Several studies have shown that lipid peroxidation–derived aldehydes accumulate in cardiac tissue within 5 to 10 minutes of ischemia and that these aldehydes propagate the I/R injury.16, 22, 50 Of the several reactive aldehydes generated during I/R, HNE and acrolein are the most potent and toxic species that promote ischemic injury.51, 52, 53 Given the extensive evidence linking reactive aldehydes to tissue injury, we investigated whether increasing myocyte levels of carnosine protects the heart by detoxifying lipid peroxidation products. We found that ATPGD1 overexpression decreased the accumulation of protein‐HNE and protein‐acrolein adducts in the heart and that myocytes isolated from the ATPGD1‐Tg heart were resistant to HNE and acrolein‐induced hypercontracture. When taken together, these observations support the notion that the cardioprotective effects of high carnosine levels may be related to a greater antioxidant capacity of myocytes, specifically their greater ability to detoxify the toxic products of lipid peroxidation.

In addition to preventing oxidative injury, carnosine could also protect the heart by buffering changes in [pH]i. Because of its histidyl group, carnosine is an efficient buffer with a pKa value near [pH]i.40 Previous studies with racehorses and greyhound dogs as well as elite human sprinters have shown that high levels of histidyl dipeptides can increase the intracellular buffering capacity of skeletal muscle.54, 55 Moreover, it has been recently suggested that the buffering potential of the heart, augmented by exercise, might be associated with an increase in the cardiac pool of histidyl dipeptides.56, 57 Changes in [pH]i are an important aspect of I/R injury. Extensive data have shown that acidification during I/R promotes cardiac injury by affecting cardiac metabolism and mechanics.58, 59, 60 Because carnosine has been shown to act as a mobile buffer that regulates the Ca2+ and H+ signaling in isolated cardiac myocytes,61 it seems that intracellular buffering may be important aspect of its cardioprotective properties. Indeed, we found that although ATPGD1 overexpression did not affect basal [pH]i, it prevented the steep fall in pH and decreased the perpetuation of acidotic pH during reperfusion. These results showing that ATPGD1 overexpression diminishes the decline in [pH]i provides direct evidence that [pH]i buffering by histidyl dipeptides may be an essential feature of the cardioprotective effects of carnosine. Nevertheless, pH buffering does not seem to be the sole mechanism of protection because perfusion with balenine, which is as efficient a buffer as carnosine, did not ameliorate I/R injury, suggesting that simply buffering [pH]i during I/R is not sufficient for limiting I/R and that properties of carnosine, other than buffering [pH]i, may be pertinent to its cardioprotective effects. Nonetheless, the lack of protection by anserine, which has a lower buffering capacity than carnosine, suggests that pH buffering by carnosine is important. Because anserine has a lower buffering capacity, we suggest that both aldehyde quenching and buffering intracellular pH are important components of carnosine‐mediated protection. Therefore, in comparison with previous interventions with buffers and antioxidants, carnosine has a more superior protective profile. Based on this evidence, it appears that buffering and aldehyde quenching properties of carnosine, optimized by natural selection, may be ideally suited for the protection of ischemic tissue. This property of carnosine seems to be in contrast to anserine and balenine, which are mostly abundant in avian and whale hearts40 suggesting that greater abundance of carnosine may be acquired by land mammals to optimize both the buffering and aldehyde‐quenching properties of histidyl dipeptides.

Our findings further suggest that the mechanism linking the cardioprotective effects of carnosine may relate to the prevention of acidification and quenching of reactive aldehydes generated during I/R injury. These changes are likely to prevent metabolic derangements in ischemic tissue. Indeed, previous work has shown that interventions that promote glucose supply and use protect the heart during ischemia and improve recovery during reperfusion.43, 62, 63, 64 Clinical studies with glucose‐insulin‐potassium therapy, which augments glucose metabolism during acute myocardial ischemia, improves the outcome in patients receiving thrombolytic therapy.65, 66 In addition, the protective effects of buffering [pH]i may also be related to the preservation of energy metabolism. For instance, it has been reported that during ischemia, delaying [pH]i decline by proton‐buffering agents preserves glycolysis and prevents myocardial injury.67, 68 Our experiments with ATPGD1 hearts provide a direct link between the two phenomena. We found that although glucose uptake in the WT and ATPGD1‐Tg mice hearts was similar, lactate formation was increased, suggesting that increasing the buffering capacity during ischemia facilitates anaerobic glycolysis. That ATPGD1 hearts also showed less severe depletion of high‐energy phosphates further reinforces the view that myocardial energetics could be preserved during ischemia by buffering [pH]i. Whether the salutary effects of ATPGD1 expression on glycolysis are attributed to buffering, aldehyde quenching, or direct effects on metabolic pathways requires further investigation. Nevertheless, our current data provide strong evidence that carnosine prevents oxidative stress, acidification, and metabolic derangements that are key features of myocardial I/R injury and therefore have high clinical relevance. Because carnosine is an endogenous dipeptide and its myocardial levels could be modulated by exogenous supplementation or by modulation of the endogenous production, it could be used therapeutically to promote recovery from myocardial injury or prophylactically to prevent myocardial injury in susceptible individuals.

Sources of Funding

This work was supported by the National Institutes of Health: R01HL122581‐01 (Baba), R01HL55477, GM127607 (Bhatnagar).

Disclosures

None.

Data S1 Table S1 Figures S1 and S2 References 21, 27, 36 and 69–79

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