Expression of Ldh-c (Sperm-Specific Lactate Dehydrogenase Gene) in Skeletal Muscle of Plateau Pika, Ochotona curzoniae, and its Effect on Anaerobic Glycolysis

Yang Wang1, Zhide Cheng2, Mengjie Tang3, Haixia Zhou1, Xiaolu Yuan4, Muhammad Aqeel Ashraf5, Shuting Mao1 and Jing Wang1,*

1Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital, Zhengzhou University, Jianshe East 1 Road, Zhengzhou 450000, Henan, China.

2Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Wuhan, Hubei, China.

3Hunan Provincial Cancer Hospital, The Affiliated Tumor Hospital of Xiangya Medical School of Central South University, Changsha, Hunan, China.

4Deptt. Pathology and Pathophysiology, School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei, China

5Faculty of Science and Natural Resources, University Malaysia, Sabah 88400 Kota Kinabalu Sabah, Malaysia

 

Yang Wang and Zhide Cheng contributed equally to this study.

ABSTRACT

The plateau pika (Ochotona curzoniae) has a strong adaptability to hypoxic plateau environment. We found that the sperm-specific lactate dehydrogenase (LDH-C4) gene Ldh-c is expressed in plateau pika skeletal muscles. In order to shed light on the effect of LDH-C4 on the anaerobic glycolysis in plateau pika skeletal muscle, 20 pikas were randomly divided into two groups the inhibitor group (experimental) and the control group, reach of 10 pikas. The pikas of experimental group were injected with 1 mL of 1 mol/L N-isopropyl oxamate, a specific LDH-C4 inhibitor, in biceps femoris muscle of hind legs, each leg with 500 μL. The pikas of control group were injected with the same volume of normal saline. The mRNA and protein expression levels of Ldh-c in plateau pika skeletal muscles were determined by real-time PCR and Western blot. The LDH activities, lactate contents and ATP levels in skeletal muscle were compared between the experimental group and the control group. The results showed that 1) the expression levels of Ldh-c mRNA and protein were 0.804±0.059 and 0.979±0.176, respectively; 2) 30 min after administration of 1 mL of 1 mol/L N-isopropyl oxamate injected in biceps femoris muscle, the concentration of N-isopropyl oxamate in blood was 0.08 mmol/L; 3) in skeletal muscles of the inhibitor group and the control group, the LDH activities were 6.30±0.50 U/mg and 10.01±0.59 U/mg, the contents of LD were 0.28±0.039 mmol/g and 0.84±0.16 mmol/g, and the ATP level were 8.15±1.03 nmol/mg and 12.06±1.23 nmol/mg (P<0.01); 5) the inhibition rates of N-isopropyl oxamate to LDH, LD and ATP were 37.12%, 66.27%, and 32.42%, respectively. The results suggest that Ldh-c expresses in skeletal muscles of plateau pika, and the pika skeletal muscle may get at least 32.42% ATP for its activities by LDH-C4 catalyzed anaerobic glycolysis, which reduces the dependence on oxygen and enhances the adaptation to the hypoxic environments.

Article Information

Received 14 August 2016

Revised 24 December 2016

Accepted 06 January 2017

Available online 02 May 2017

Authors’ Contribution

YW, ZC and JW conceived and designed the study. YW, ZC, MT, HZ and XY contributed in experimental work, acquisition of data analyzed and drafting of the manuscript. ZC, MT and SM statistically evaluated the data. JW, YW and MAA wrote the article.

Key words

Sperm-specific lactate dehydrogenase, Anaerobic glycolysis, Skeletal muscle, Ochotona curzoniae Plateau pika, Ldh-c, N-isopropyl oxamate, LDH activity.

DOI: http://dx.doi.org/10.17582/journal.pjz/2017.49.3.905.913

* Corresponding author: yangwangmd@163.com

0030-9923/2017/0003-0905 $ 9.00/0

Copyright 2017 Zoological Society of Pakistan

INTRODUCTION

 

The Qinghai-Tibet Plateau is the highest plateau on earth at an average of over 4,000 m above sea level, which possesses a unique harsh environment of climate and geography, such as hypoxia, cold, and strong ultraviolet radiation. Hypoxia is the most obvious climate characters on the plateau, which expectedly have profound effects on animal survival. Over long years evolution, many plateau-native animals have developed their own unique mechanisms to the adaptation in the plateau environment.

Plateau pika (Ochotona curzoniae), a dominant small mammal in the alpine meadow ecosystems across the Qinghai-Tibetan plateau, China (Smith and Foggin, 1999; Lai and Smith, 2003). Pikas play an important role as a keystone species in maintaining ecosystem functions for providing food for predators and underground nests for small birds, and in promoting nutrient recycling within alpine ecosystems (Yang et al., 2007). Within perpetual evolution, the pika underwent a series of adaptative changes to the harsh environment. First, the pika obtained oxygen effectively by larger pulmonary alveoli superficial and higher capillary density (Wang et al., 2008a), thin walled pulmonary arterioles and blunted hypoxic pulmonary vasoconstriction (Ge et al., 1998), an increase in erythrocyte count (Wang et al., 2008b), reduction in the mean corpuscular volume (Ye et al., 1994), changes in hemoglobin (Hb) (He et al., 1994) and 2,3-diphosphoglycerate concentrations (Ge et al., 1998), and an increase in the oxygen affinity to Hb (He et al., 1994). Moreover, pika has a strong cardiac pumping function by having a larger heart and smaller right-to-left ventricular plus septum weights (Qi et al., 2008); Thirdly, pika has a high ratio of oxygen utilization by increasing capillary and mitochondrial densities (Wei et al., 2006), and concentration of myoglobin in tissues (Wang et al., 2008; Qi et al., 2008). In addition to these physiological mechanisms, pika reduces dependence on oxygen by increasing anaerobic glycolysis in skeletal muscle (Zhu et al., 2009) and gluconeogenesis in liver (Sun et al., 2013). The molecular basis of these adaptations in the pika have occurred because of a series of genetic evolutionary changes, including HIF-1α (Li et al., 2009; Zhao et al., 2004), hemoglobin (Yang et al., 2007), vascular endothelial growth factor (VEGF) (Li et al., 2013; Zheng et al., 2011) testis-specific lactate dehydrogenase (LDH-C4) (Wang et al., 2013), pyruvate carboxylase (Sun et al., 2013), myoglobin (Qi et al., 2008), cytochromec oxidase (Luo et al., 2008), neuron nitric oxide synthase (nNOS) (Pichon et al., 2009), and leptin (Yang et al., 2006, 2008).

The lactate dehydrogenase (LDH) family enzymes catalyze the inter-conversion of pyruvate to lactate with the concomitant oxidation/reduction of NADH to NAD+ (Everse and Kaplan, 1973). Different forms of LDH are the product of three different genes: Ldh-a, Ldh-b, and Ldh-c which encode A, B and C subunits, respectively (Li, 1989; Li et al., 1989). LDH consists of A and B subunits that assemble into homo- or hetero- tetramers that are distributed in the body in various combinations reflecting the metabolic requirements of different tissues and are consistent with the catalytic properties of the isozymes (Cahn et al., 1962; Fine et al., 1963). However, the homotetramer LDH-C4 was previously only detected in testis and spermatozoa and not in any other tissues or cells (Goldberg, 1964, 1975, 1984; Coonrod et al., 2006). In later studies, it was found that Ldh-c is expressed not only in testis and sperm, but also in different types of human cancers (Gupta, 1999, 2012; Koslowski et al., 2002). LDH-C4 is an iso-, allo-, and an auto-antigen. Animals, after immunization its LDH-C4 show enhanced fertility in homologous species and reduced fertility in heterologous species (Gupta and Syal, 1997; Gupta, 1999). In our previous study, we identified that Ldh-c is expressed not only in testis and sperm, but also in somatic tissues of plateau pika (Wang et al., 2013). We also found that pika LDH-C4 has an affinity for pyruvate that is 90-fold higher than that for lactate in our prevoious study, and it was beneficial to catalyze the conversion of pyruvate to lactate even at high concentration of lactate in our previous study (Wang et al., 2016).

 

Skeletal muscle fibers can be characterized by their metabolic processes, namely oxidative and glycolytic muscles, oxidative fibers primarily use oxidative phosphorylation to generate adenosine triphosphate (ATP) (Lieber and Friden, 2002). Also, oxidative fibers contain a large number of myoglobin, an oxygen-binding protein that can store oxygen and speed its delivery to mitochondria within the muscle cell. In contrast, glycolytic muscle has the less levels of myoglobin and relies on glycolysis to supply muscle energy (Lieber and Friden, 2002).

Previous study has shown that N-isopropyl oxamate was an effective and selective inhibitor due to the close chemical structure existing between N-isopropyl oxamic acid and the best substrate for HADH-isozyme II, the α-keto isocaproate (Elizondo et al., 2003). N-isopropyl oxamate has similar chemical and molecular stucture as pyruvate, as shown in Figure 2, which contributes to its affinity with LDH-C4.

 

 

In order to shed light on the expression of Ldh-c and its function on tolerance performance of plateau pika, in the current study, we investigated the expression level of Ldh-c in pika’s skeletal muscle. N-isopropyl was used to testify the biochemical index of plateau pika skeletal muscle after LDH-C4 was inhibited. The molecular mechanism was elucidated by measuring the LDH activity, lactate content and ATP level in pika’s skeletal muscle.

 

MATERIALS AND METHODS

Synthesis of LDH-C4 inhibitor N-isopropyl oxamate

Ethyl N-isopropyl oxamate was shaken with 50 mL NaOH for half an hour and extracted in ether. The aqueous phase was separated and acidified with HCl. Ether extraction and evaporation gave a crude product which, on mixing with light petroleum, soon became crystalline. The crystals were purified by recrystallization from chloroform (5.3 g, 81%): mp 113-114°C, 1H-NMR (CDCl3) δ 1.25 (d, J=6.5 Hz, 6H), 4.07(m, 1H), 7.25 (broad s, 1H), 9.4 (broad s, 1H), IR (KBr) 3294, 2980, 1770, 1677, 1558, 1360 cm-1.

Animal procedure

Plateau pikas were live-trapped at Laji Mountain in Qinghai Province, China, at an altitude of 3,850 m above sea level. The environmental temperature was 10-20°C outside. The average body weight of plateau pikas was 198±9 g. All adult animals, used in this study, were in good health and were randomly divided into 2 groups (sample size was 10 for each group) and treated as follows: i) Control Group: plateau pikas injected with 0.5 mL normal saline in each bilateral biceps femoris of hind legs; ii). Experimental Group: plateau pikas injected with 0.5 mL N-isopropyl oxamate in each bilateral biceps femoris of hind legs. All animals were sacrificed 30 min after injection. After experiment, all animals were anesthetized with sodium pentobarbital (5%) and then sacrificed by cervical dislocation immediately before dissection. Skeletal muscle was rapidly removed and frozen in liquid nitrogen for long-term storage. All procedures involved in the handling and care of animals were in accordance with the China Practice for the Care and Use of Laboratory Animals and were approved by the China Zoological Society (permit number: GB 14923-2010).

RNA extraction and quantification of Ldh-c mRNA level by real-time PCR

Total RNA was isolated using TRIzol reagent (Invitrogen Corp, USA). RNA concentration and purity were assessed by UV spectrophotometry (1.8 <A260/A280<2.0). RNA integrity was checked using electrophoresis. Reverse transcription reaction was carried out starting from 4 μg of total RNA using the First Strand cDNA Synthesis kit (Thermo Scientific, USA). To make standard curves, 1 μL of first-strand cDNA was amplified with Premix Ex Taq Version Kit (TaKaRa BIO Inc., Japan), and quantification of PCR products was checked for plotting standard curves. The initial product concentration was set at 1 and standard curves were generated using a ten-fold serial dilution ranging from 1 to 10−7.

Real-time PCR was performed using the SYBR Premix Ex TaqTM II (TaKaRa BIO Inc., Japan) protocol on BIO-RAD Connect real-time PCR detection system with cycling conditions of 95°C for 3 min, followed by 40 cycles of 95°C for 30 s and 60°C for 30 s. β-actin was used as an internal control. The PCR primers for Ldh-c and β-actin were designed as follows: Ldh-c: forward, 5’-TATCGAGAATCTGATCGCAGAAGAC-3’ and reverse, 5’-GGGCAAGTTCATCAGCCAAATCC-3’, the amplicon length was 130 bp. β-actin: forward, 5’-CTCTTCCAGCCCTCCTTCTT-3’ and reverse 5’-AGGTCCTTACGGATCTCCAC-3’, the amplicon length was 98 bp. The Ldh-c mRNA level was normalized with β-actin mRNA to compensate for variations in initial RNA amounts. Normalization was carried out by dividing the logarithmic value of Ldh-c by the logarithmic value of β-actin.

Western blot analysis

Total cellular proteins were extracted by RIPA lysis buffer containing protein inhibitors, BCA protein assay kit (Pierce Biotechnology, USA) was used to assess protein concentration. Proteins were separated by SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane (0.22 μm). After blocking the non-specific binding sites for 2 h with 5% non-fat milk, the membranes were incubated with a rabbit monoclonal antibody against LDHC (SIGMA-ALDRICH, USA, at 1:4,000 dilution) or GAPDH (Genetex, USA, at 1:5,000 dilution) at 4°C overnight. The membranes were then washed with TBST (Tris-Buffered Saline with Tween-20) for six times at room temperature for 10 min. After washing, the target protein was probed with the horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (Santa Cruz, USA, at 1:6,000 dilution) at 37˚C for 2 h. After ten times washing, the bound antibody was detected by chemiluminescence with the ECL Detection Reagent (Pierce Biotechnology, USA) (Zhang et al., 2015; Jin et al., 2016).

Assay of LDH activities, LD contents and ATP levels

The samples of skeletal muscle were homogenized on ice as a 1:9 (W/V) dilution in 0.9% physiological saline. The homogenate was centrifuged at 15,000 r/min at 4°C for 10 min, and the supernatant was collected. The total protein concentration, LDH activity and content of lactic acid (LD) were determined using commercially available assay kits according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, China). The amount of ATP was measured by the luciferin-luciferase method (John, 1970) following the protocol of ATP detection kit (Beyotime, China). The luminescence from a 100 μL sample was assayed in a luminometer (Promega, GloMax 20/20, USA) together with 100 μL ATP detection buffer from the ATP detection kit. Standard curves were also generated and the protein concentration of each sample was determined using the BCA Protein assay (Pierce, USA).

Data analysis

All values were expressed as mean ± standard deviation (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA) and Duncan’s test using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). A value of p<0.05 was considered statistically significant.

 

RESULTS

mRNA and protein level of Ldh-c expression

Figure 3 shows the mRNA and protein levels of Ldh-c, examined by qRT-PCR and Western blot assays, in skeletal muscle of plateau pika. The relative expression levels of Ldh-c mRNA and protein were 0.80±0.06 and 0.98±0.18, respectively.

 

 

N-isopropyl oxamate as LDH-C4 inhibitor

N-isopropyl oxamate is more specific inhibitor for LDH-C4 than N-propyl oxamate at concentrations up to 0.1 mmol/L (Wang et al., 2015). When pikas were injected with 1 mL of 1 mol/L N-isopropyl oxamate in hind legs for 30 min, HPLC analysis showed the inhibitor concentration was 0.08 mmol/L in the blood (Wang et al., 2015). While at this concentration, LDH-C4 was inhibited by 70%, the LDH-A4 and LDH-B4 were only inhibited by about 5% (Wang et al., 2015). Therefore, N-isopropyl oxamate was an optimal inhibitor to study the function of LDH-C4 in glycolysis of plateau pikas.

 

LDH activities, LD contents and ATP levels in the skeletal muscle

Figure 4 shows LDH activities 10.01±0.59 U/mg, and 6.35±0.51 U/mg in skeletal muscle of control and inhibitor groups, respectively.

 

LD contents in skeletal muscle were 0.85±0.17 mmol/ g, and 0.29±0.05 mmol/g, in control group and inhibitor group, respectively. ATP levels in skeletal muscle were 12.06±1.23 nmol/mg and 8.15±1.03 nmol/mg in control group and inhibitor group, respectively. The inhibition rates of LDH, LD and ATP in the skeletal muscle were 37%, 66%, and 32%, respectively (** p<0.01).

 

DISCUSSION

 

Existing reports show that the plateau pika has high microvascular density myoglobin content, large surface area and mitochondria number in skeletal muscle (Zhu et al., 2009). LDH isozymes LDH-A4, LDH-A3B1, LDH-A2B2 (Zhu et al., 2009) in skeletal muscle suggest that pika skeletal muscle mainly relies on the aerobic respiration.

In our previous study we found that LDH-C4 had a lower Km for pyruvate (~0.052 mmol/L) and a higher Km for lactate (~4.934 mmol/L) compared with LDH-A4 and LDH-B4 (Wang et al., 2016). This finding implies that LDH-C4 has an affinity for pyruvate that is 90-fold higher than that for lactate and suggests that pyruvate turnover to lactate may be high even at large contents of endogenous or extracellular lactate in vivo. This finding is also supported by the finding that addition of excess lactate did not affect ATP generation in spermatozoa (Hereng et al., 2011).

Comparative results showing the Ki value of N-isopropyl oxamate on murine LDH-A4, LDH-B4 and LDH-C4 of 0.01 mmol/L, 0.40 mmol/L and 0.80 mmol/L (Wong et al., 1997) suggest that N-isopropyl oxamate has strong inhibitory effect on LDH-C4 and weak effects on LDH-A4 and LDH-B4.

In present study, the results showed that Ldh-c expresses in skeletal muscle of plateau pika. The injection of 1 mol/L N-isopropyl oxamate in the biceps femoris of plateau pika for 30 min resulted in blood concentration of N-isopropyl oxamate of 0.08 mmol/L. At this concentration, N-isopropyl oxamate inhibited 70% LDH-C4 activity without affecting the enzymatic activity of recombinant pika LDH-A4 and LDH-B4 (Wang et al., 2015). After experiment, LDH activity and LD and ATP content in biceps femoris of treated pikas decreased significantly compared to untreated animals, and inhibition rates of LDH, LD, and ATP by N-isopropyl oxamate were 37%, 66% and 32%, respectively.

The extraordinary function of pika Ldh-c was also due to its amino composition. We had compared the amino sequence comparison of pika LDHA, LDHB and LDHC with other mammalian species. As shown in Figure 5, Pika (Ochotona curzoniae) Ldh-a (HQ704676) has 95.20%, 96.10%, 94.60% and 94.30% amino homologous respectively, to that of Rattus norvegicus, Mus musculus, Bos taurus and Homo sapiens (Fig. 5A); Ldh-b (HQ704677) has 98.80%, 97.30%, 97.05% and 96.50%, respectively (Fig. 5B); Ldh-c (HQ704678) had 87.70%, 74.70%, 75.30% and 87.30%, respectively (Fig. 5C). These results show that pika LDHC amino homologous to the LDHC of other species is significant lower than LDHA and LDHB, suggesting that the kinetic properties of LDH-C4 are also related to its amino acid composition. In addition, Pika LDHA and LDHB have 77% and 72% amino homology with LDHC (Fig. 5D). LDHC shares more in common with LDHA than LDHB. This theory is proved by that Ldh-c. arose from a second independent gene duplication event, by duplication of the Ldh-a during mammalian evolution (Li et al., 1983, 2002; Millan et al., 1987). In addition, based on the amino acid constitution of pika LDHC subunit, we found that the pika LDH-C4 contains 26 and 20 more residues of isoleucine than LDH-A4 and LDH-B4. Isoleucine residues in pika LDH-C4 form part of the hydrophobic region present only at the active site of pika LDH-C4 (Wang et al., 2015). It seems that this hydrophobic region allows the enzyme to discriminate between N-isopropyl oxamate α-keto and α-hydroxy acids with different side chains, facilitating, through hydrophobic interactions, the proper binding of those substrates as N-isopropyl oxamate with nonpolar side chains and rejecting those with polar side chains.

 

CONCLUSIONS

 

Collectively, our results suggest that decreased ATP in skeletal muscle of plateau pika in hypoxic environment is due to inhibition LDH-C4 activity. Pika have reduced oxygen dependence and enhanced adaptation to hypoxic environments due to increased anaerobic glycolysis by LDH-C4 in skeletal muscle since this is the role of LDH-A4 in most species on plain land environment.

 

ACKNOWLEDGEMENTS

 

This work was supported by the Youth Foundation of First Affiliated Hospital of Zhengzhou University, the National Natural Science Foundation of China (No.31260512), and Natural Science Foundation of Qinghai Province, China (No. 2014-ZJ-714).

 

Statement of conflict of interest

Authors have declared no conflict of interest.

 

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