The L-lactate was quantified using an enzymatic kit (Lactate Assay Kit, MAK064, Sigma-Aldrich), according to the manufacturer's recommendations. In this assay, lactate concentration is measured by enzymatic analysis, which produces a colorimetric (570 nm) product in proportion to the lactate present Lactate standards for colorimetric detection dilute 10 μl of 100 nmole/μl lactate standard with 990 μl lactic acid assay buffer to generate 1 nmole/μl standard solution. And add 0, 2, 4, 6, 8, 10ul 1nmole / μl lactate standards to 96 well plates to create 0 (blank), 2, 4, 6, 8, 10nmole / well standards. Add Lactate Assay Buffer to each well to make a volume of 50 μl. The assays require 50 μl of sample for each reaction (well). Tissue or cells are homogenized with 4 volumes of the Lactate Assay Buffer. To remove insoluble material, samples centrifuged at 13,000 rpm for 10 min. And to remove lactate dehydrogenase, samples should be deproteinized with a 10 kDa MWCO spin filter Soluble fractions can be analyzed directly. Directly by adding in duplicate to 96 well plate. Bring samples to final volume of 50 μl /well with Lactate Assay Buffer.
Add 50 μl of master reaction mix (Lactate Assay Buffer 46 μl, Lactate Enzyme Mix 2 μl, Lactate Probe 2 μl) required for each reaction (well). Use a shaker or pipette and protect the plate from light and incubate the reaction for 30 min at RT. Measure absorbance at 570 nm.
１０ G. Western blotting assay
Protein extracts from cultured HL-1 cardiomyocytes. Preparation HL-1 cell pellet and lysed in RIPA buffer containing proteinase inhibitor cocktail. The cell lysates were further homogenized by short sonication, centrifuged at 13,000 rpm for 10 min at 4°C. and the resulting supernatant collected.
DC ™ Protein Assay (BIO-RAD) was performed for protein concentration quantification.
Samples containing the same amount of protein were run under conditions of 80 V 20 min, 120 V 1 h using 10-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the protein was then Transferred to vinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% BSA for 1h and incubated with primary , Actin (Santa Cruz [sc1616] 1:2,000), anti-Tubulin (Santa Cruz [sc32293] 1:2,000), anti-Lamin A (Abcam [ab26300] 1:5,000), (anti-LDHA (Abcam [ab9002] 1:10,000), anti-LDHB (Abcam [ab85319] 1:5,000),anti VDAC (Abcam [ab140817] 1:5,000) and anti-MG-H1(AGE) (cell biolabs [STA-011] 1:1,000) for 2 h at RT or overnight at 4 ℃. After washing with TBS containing Tween-20 (TBST), the membranes were incubated with peroxidase-conjugated secondary mouse, rabbit and sheep antibodies (1:5,000) for 1 h. Protein bands incubated with enhanced chemiluminescence reagents (ECL) were visualized using Developer. To ensure equal protein loading, Anti-Tubulin, Actin and lamin A were used as a control for protein level quantification.
H. Quantification of methylglyoxal by HPLC
0.1 g of cell harvest or tissue homogenized with 1 ml of 10 mM ortho-phenylenediamine (o-PD) in 0.45 N perchloric acid (PCA). After Bead homogenization and sonication, incubated for 24 h in RT on orbital shaker in dark. PCA treatment allowed protein to precipitate proteins from the samples and to inhibit metabolic reactions. Samples were centrifuged at 12,000 rpm for 10 min at 4°C to remove precipitated protein. Methyl glyoxal (MGO) was derivatized with a final concentration of 10 mM o-PD to form stable 2-methyl quinoxaline (2-MQ). In 10 minutes of PCA treatment, after removing the precipitated protein, o-PD was added to the supernatant. On the other hand, for 3 h or 24 h of PCA treatment, o-PD was added to the sample with PCA for 3 h or 24 h and incubated for 3 h or 24 h at RT (Chaplen et al., 1996).
Before performing HPLC system, the sample was further centrifuged at 13000 rpm for 10 min at 4℃, supernatant is used as a sample. 20 μl of samples were quantified on Agilent 1200 HPLC system
１１
via ZORBAX eclipse plus c18 4.6 x 150mm, 5-micron column, by plotting the concentration of standard 2-methylquinoxaline (2-MQ) derivative as a function of peak area detected at 315 nm.
Acetonitrile 20% was used for mobile phase. Each samples was run for 30 min with a flow rate of 1 ml/min. 5-Methylquinoxaline (5-MQ) was use as internal standard (Dhar et al., 2009).
I. Methylglyoxal Determination by ELISA
The methylglyoxal was quantified using Competitive ELISA (Cell biolabs, STA-811, OxiSelect™
Methylglyoxal (MG) Competitive ELISA Kit), according to the manufacturer's recommendations.
The OxiSelect™ Methylglyoxal (MG) ELISA Kit is an enzyme immunoassay developed for rapid detect and quantify of MG-H1 (methylglyoxal-hydroimidazolone) protein adducts. The amount of MG adducts in a protein sample is calculated by comparing the absorbance with the absorbance of a known MG-BSA standard curve. Just before use, prepare MG-conjugate coated ELISA plate. 100 μl of 500 ng/ml of MG-conjugate to each well to tested and incubate overnight at 4 ℃. Remove the MG-conjugate coating solution and wash twice with 1 x PBS. Then, 200 μl of Assay diluent is added to each well and blocked for 1 hour at RT in an orbital shaker. Transfer the plate to 4 ℃ and remove the assay diluents immediately before use. Add 50μl of samples and standards to the wells of the MG-conjugate coated plate. Add 50μl of diluted anti-MG antibody to each well and incubate at RT for 1 h on an orbital shaker. After incubation, anti-MG antibody is added followed by HRP conjugated secondary antibody. Add 100 μl of substrate solution to each well. Incubate for 20 min at RT on an orbital shaker. To stop the reaction, 100 μl of stop solution is added to each well to stop the enzyme reaction. Results should be read immediately.
Read the absorbance of each well using 450 nm on a microplate reader. Protein adducts in protein samples are quantified by comparison with a predetermined MG-BSA standard curve.
J. Mitochondria isolation
Mitochondria isolation was performed using the Mitochondria Isolation Kit for Cultured Cells (cat.89874, Thermo Scientific). Add protease inhibitors to Reagent A and Reagent C immediately before use. Add 800 μl of mitochondrial isolation reagent A, vortex for 5 s at medium speed and incubate the tube for exactly 2 min on ice. Add 10 μl mitochondrial isolation reagent B and vortex at maximum speed for 5 s. Incubate the tube for 5 min on ice and vortex every minute at full speed.
Add 800μL Mitochondrial isolation reagent C. Invert the tube several times to mix (do not vortex).
１２
Centrifuge tubes for 10 min at 700 x g at 4°C. Transfer the supernatant to a new 2.0 mL tube and centrifuge at 12,000 x g for 15 min at 4°C. The supernatant containing the cytosol fraction is transferred to a new tube. The isolated mitochondria are contained in pellets. Add 500 μl Mitochondrial separation reagent C to the pellet and centrifuge for 5 min at 12,000 x g. Remove the supernatant. Store mitochondrial pellets on ice before further experimental treatment.
K. Complex activity Ⅰ enzyme activity assay
Mitochondria OXPHOS complex Ⅰ (NADH dehydrogenase) enzyme activity assay was measured using Complex I Enzyme Activity Assay Kit (Abcam, ab109721). Antibodies captured on precoated microplates immobilize complex I, whose activity has been determined upon oxidation of NADH to NAD+ and simultaneous reduction of provided dyes to increase absorbance at OD 450 nm. The prepared samples are put in a microplate wells and incubated at RT for 3 h. Assay solution is prepared by mixing 1 x dilution buffer, 20 x NADH, 100 x Dye. Wash plate wells with wash buffer and add 200 μl of Assay solution to each well. Read absorbance of each well on a microplate reader using 450 nm in kinetic mode at RT for up to 30 min.
L. Statistical analysis
All data were calculated as the average of at least three independent samples. Results were reported as mean ± SEM. Data was analyzed using Student's t-test and one-way ANOVA. Statistical significance was determined if P value ≤ 0.05.
１３ III. RESULTS
A. LDHB plays a major role in D-lactate metabolism
Five LDH isozymes are formed by combining LDHA (subunit A) and LDHB (subunit B) in different proportions. The LDHA subunit prefers to convert pyruvate to lactate and the LDHB subunit prefers to convert lactate to pyruvate. LDH1 has a large LDHB subunits ratio and LDH5 has a large LDHA subunits ratio. LDH activity was confirmed when L-lactate and D-lactate were added to isoenzyme pattern gel (Fig.4A). D-lactate was also confirmed to have L-LDH activity and compared to L-lactate, the activity of LDH1 was greater than that of LDH5 in D-lactate (Fig.4B).
We thought that LDH1 plays an important role in L-lactate and D-lactate metabolism.
１４ Figure 4. LDH enzyme activity in extract
(A) Native-PAGE and LDH activity staining, (B) analyzed activity percentage bar graph.
(*p<0.05,**p<0.01,***p<0.001 by Student’s t-test)
１５ B. LDH enzyme activity to D-lactate
In order to confirm the D-lactate specific activity of LDH1, the enzyme activities of LDH1 and LDH5 were measured under D-lactate 20 mM and NAD 5 mM conditions. The activity of LDH5 was only 37.8% of LDH1 (LDH1-3.86 D-lactate (mM) / mg / min, LDH5- 1.46 D-lactate (mM) / mg / min) (Fig.5). LDH 1 was found to be important in the D-form lactate because it consists only of LDHB subunits.
１６ Figure 5. LDH enzyme activity in pure enzyme
Human LDH1 and LDH5 activity to D-lactate (*p<0.05,**p<0.01,***p<0.001 by Student’s t-test)
１７
C. Loss of LDHB in cardiomyocytes increased D-lactate
I performed knockdown LDHB in cardiomyocyte cell line HL-1 with siRNA, and knockdown efficiency was verified by Western blotting (Fig. 6A) and LDH isoenzyme staining (Fig. 6B). In addition, D-lactate level was increased by 200% in the LDHB knockdown HL-1 cells (Fig. 6C).
１８
Figure 6. LDHB knockdown HL-1cells increased D-lactate level
(A) Western blot, LDHB and LDHA was normalized with lamin A, (B) LDH isoenzyme assay with L-lactate, (C) D-lactate measurement, (D) Mitochondria complex1 activity assay.
(*p<0.05, **p<0.01, ***p<0.001 by Student’s t-test)
１９
D. Loss of LDHB in cardiomyocytes increased Methylglyoxal
The most generally used method for measurement of methylglyoxal involves the derivatization of methylglyoxal with 1,2-diaminobenzene derivatives, such as o-phenylenediamine (o-pd), subsequent quantify of the resulting quinoxaline with high performance liquid chromatography (HPLC). 2-MQ derivatized with methylglyoxal by o-phenylenediamine (o-pd). Methylglyoxal was determined by quantification of 2MQ via HPLC analysis (Agilent 1200 HPLC system via ZORBAX eclipse plus c18 4.6 x 150mm, 5-micron column). I analyzed MG-derivatives to identify peaks in chromatograms of samples at the same retention time (Fig.7A; retention time: 2MQ 8.1 min, 5MQ 15.5 min) to analyze the internal standard 5MQ and 2MQ. In the LDHB KD HL-1 cells, methylglyoxal level was increased by 35% (Fig.7B). D-lactate was not metabolized, and D-lactate was increased, confirmed that methylglyoxal was increase.
２０
Figure 7. LDHB Knockdown increased Methylglyoxal in HL-1 cell
(A) HPLC results (B) Methylglyoxal measurement analyzed percent bar graph by HPLC (*p<0.05,**p<0.01,***p<0.001 by Student’s t-test)
２１ E. Loss of LDHB in cardiomyocytes increased AGEs
The interaction of methylglyoxal with arginine leads to the formation of the specific AGEs, methylglyoxal-derived hydroimidazolone (MG-H1) and tetra hydro pyrimidine (THP) (Vistoli et al., 2013). methylglyoxal-derived hydroimidazolone (MG-H1) is the major product of MG-specific glycation and about 90% of all adducts. Methylglyoxal levels were measured indirectly with MG-H1.
In the LDHB KD HL-1 cells, AGEs (MG-H1: hydroimidazolone) level was increased by 150%
(Fig.8A,8B). The methylglyoxal accumulation was again measured using MG-H1 ELISA kit (Fig.8C).
In other words, AGEs accumulated in LDHB knockdown cells. The experiment was performed triple and calculated as the average value
２２
Figure 8. LDHB Knockdown increased MG-H1(AGEs) in HL-1 cell
(A) Western blot analysis for AGEs, (B) MG-H1 was normalized with Actin, (C) MG-H1 ELISA.
(*p<0.05,**p<0.01,***p<0.001 by Student’s t-test)
２３
F. Loss of LDHB in cardiomyocytes decreased complex activity
In addition, I found that mitochondrial complex activity was reduced in LDHB KD cells (Fig.9). It can be seen that LDHB deficiency induces mitochondrial defects.
２４
Figure 9. LDHB Knockdown reduced Complex Ⅰ activity in HL-1 cell
Mitochondria complexⅠactivity assay (*p<0.05, **p<0.01, ***p<0.001 by Student’s t-test)
２５ G. Generation of LDHB heart specific KO mouse
Previously our lab generated LDHB gene knockout mouse by Cre/loxP system (Fig. 10A).
Conditional knockout is referred to as tissue specific knockout. Conditional mouse has floxed alleles with loxP sites on either side of the target gene to be removed. To knock out tissue-specific knockdown, Cre recombinase is expressed using a promoter of a gene that expresses only in the desired tissue. When Cre recombinase is expressed, target genes between the two loxP sites are removed. LDHB genes with loxP sites on both sides can be recombinated and inactivated by Cre recombination enzyme that recognizes two loxP sites. Recombination occurs only in cells that express Cre recombinase. Thus, LDHB remains active in all cells and tissues that do not express Cre recombinase. I recombined Cre recombination enzymes only in heart specific tissue. Through Western blot (Fig.10C) and isoenzyme patterning analysis (Fig.10D), I confirmed successful generation of Heart specific LDHB knockout mouse (Saihali, 2014; Song Mi, 2017).
２６
Figure 10. Generation of heart specific knockout mouse
(A) LDHB Knockout mouse model by Cre/loxP system, (B) analysis of control and targeted ES cells, (C) protein level of LDHB expression in LDHB knock out mouse heart tissue, (D) LDH isozyme pattern in LDHB knock out mouse heart tissue
２７
H. LDHB knockout mouse heart increased methylglyoxal
Methylglyoxal increased in LDHB knockdown HL-1 cell experiments, and the same experiment was performed in LDHB knockout mouse. As expected, in the LDHB knockout heart, Methylglyoxal level was increased by 24.4% (Fig.11).
２８
Figure11. Loss of LDHB increase methylglyoxal in knockout mouse
Methylglyoxal level was determined with HPLC and normalized with tissue wet weight (*p<0.05,**p<0.01,***p<0.001 by Student’s t-test)
２９ I. LDHB knockout mouse heart increased AGEs
In knockout of LDHB hearts, measurements were performed using the MG-H1 ELISA kit. As expected, Methylglyoxal level was increased by 45% (Fig.12A). Furthermore, AGEs accumulated in LDHB knockout heart (Fig.12B), and interestingly, Western blot data showed mitochondria was more sensitive to compare with whole lysate (Fig.12C). More AGEs were found in mitochondria than whole cells.
３０
Figure 12. Loss of LDHB increase AGEs in knockout mouse
(A) Monoclonal anti-AGEs (MG-H1) antibody was used for ELISA, (B),(C) Western blot for AGEs in isolated mitochondria and whole cells (*p<0.05,**p<0.01,***p<0.001 by Student’s t-test)
３１ J. LDHB deficiency cause mitochondrial dysfunction
Mitochondrial function is not only important for energy metabolism, but also plays an important role in the aging process. Mitochondrial function depends on the oxford complex of the mitochondrial lining. In addition, the essential subunits of the complex are encoded by mitochondrial DNA, so mitochondrial DNA homeostasis is important for the aging process.
Methylglyoxal, on the other hand, can damage not only proteins, but also lipids and DNA. The increased methylglyoxal levels in the LDHB knockout heart were thought to damage mitochondria more than WT. Indeed, LDHB knockout heart decreased mitochondrial DNA levels (Fig.13A, 13B), mitochondrial mRNA levels (Fig.13C) and tRNA levels also decreased. (Fig.13D) Also, LDHB knockout heart reduced mitochondrial complex activity and Mitochondrial transcription factor A (TFAM) (Fig.13E). TEM imaging showed that most of the mitochondria morphology were unhealthy in the LDHB knockout heart (Fig.13F).
３２
Figure 13. Loss of LDHB causes mitochondrial dysfunction
(A) Southern blot, mitochondrial DNA was normalized with 18s rRNA, (B) % mt DNA analysis, (C) northern blot, mitochondrial mRNA levels, (D) and mitochondrial tRNA levels, (E), western blot, mitochondrial protein levels, (F) TEM image (*p<0.05,**p<0.01,***p<0.001 by Student’s t-test)
３３ K. Loss of LDHB induces cardiomyopathy
Heart specific LDHB knockout cause cardiomyopathy. Survival was reduced in mouse at 30 weeks of age (Fig.14).
40-week-old knockout mouse significantly increased the heart / weight ratio indicating myocardial hypertrophy, but 20-weeks- knockout mouse slightly increased but were not significant (Fig.15A).
In addition, 40-week-old KO mouse had myofibrosis (Fig.15B).
３４ Figure 14. LDHB knockout mouse Survival rat
３５ Figure 15. Loss of LDHB induces cardiomyopathy
(A) Heart weight (g)/ body weight (g) ratio, (B) Trichrome staining (*p<0.05,**p<0.01,***p<0.001 by Student’s t-test)
３６
Ⅳ. DISCUSSION
The mechanism of D-lactate, the detoxification product of methylglyoxal at present, is not yet known. It is hypothesized that accumulation of D-lactate will increase methylglyoxal due to feedback inhibition. Increasing methylglyoxal will result in the accumulation of AGEs, disruption of cell function and various diseases. So, I think that the metabolism of D-lactate is important for methylglyoxal detoxification. I think LDH, which converts lactate to pyruvate, will play an important role in D-lactate metabolism.
Lactate dehydrogenase (LDH) activity was confirmed when L-lactate and D-lactate were added to the isoenzyme pattern gel (Fig. 4A). This result shows that L-lactate dehydrogenase also metabolizes D-lactate. In addition, the activity of LDHB was higher than that of LDHA in D-lactate compared to L-lactate (Fig. 4B). This means that isoenzyme with LDHB subunits play a greater role in D-lactate metabolism. I found that LDH plays a role in D-lactate metabolism and that LDHB plays an important role.
To demonstrate whether LDHB is involved in D-lactate metabolism, experiments were performed with LDHB removal from HL-1 cells. siRNA LDHB knockdown in HL-1 cells showed an increase in D-lactate compared to the control (Figure 6C). I demonstrate a decrease in D-lactate metabolism in the absence of LDHB. As hypothesized, experiments were conducted to confirm that an increase in D-lactate caused an increase in methylglyoxal. Quantitative results of Methylglyoxal in LDHB knockdown Cells, methylglyoxal level increased 20% compared to control (Fig. 7). As expected, AGE increases as methylglyoxal increases, confirming the accumulation of AGEs in LDHB knockdown cells (Fig.8). It was measured by whether AGEs that interfere with cell function will destroy the mitochondria of my interest. In the absence of LDHB, mitochondrial complex activity was reduced (Fig.9). Further experimentation is needed to measure the change in glyoxalase Ⅰ, Ⅱ levels in the LDHB deficiency of the glyoxalase pathway, the detoxification pathway of methylglyoxal.
Based on the previous results, experiments were performed with the mouse removed from LDHB to determine if the actual disease was induced. Methylglyoxal was quantified in LDHB knockout mouse cardiac extract as in cell experiments (Fig.11), and AGEs were also increased by 40% (Fig.12A). In addition, mitochondria were isolated from LDHB knockout mouse heart tissues and confirmed the
３７
accumulation of AGEs (Fig.12B). I thought that LDHB deficiency damaged mitochondria. So, I confirmed that it causes mitochondrial dysfunction in LDHB KO mouse.
The function of mitochondria depends on the electron transport complex of the inner membrane.
Mitochondrial DNA levels were decreased in LDHB knockout mouse (Fig.13A, 13B) and RNA levels were also decreased (Fig.13C, 13D). Similarly, mitochondrial transcription factor A (TFAM) protein and mitochondrial complex subunit protein levels were also reduced in LDHB knockout mouse (Fig.13E). In addition, electron microscopy confirmed that the morphology of mitochondria was not normal in the LDHB knockout mouse heart (Fig.13F). LDHB knockout mouse had a reduced survival rate after 30 weeks (Fig.14), and 40-week-old LDHB knockout mouse had cardiac hypertrophy (Fig.15A) and myofibrosis (Fig.15B). This confirmed that LDHB deficiency caused cardiomyopathy.
Since LDHB is present in many hearts, I used only cardiac specific mouse. Further experimentation with the whole body LDHB knockout mouse is considered necessary.
In summary, LDH uses D-lactate as a substrate as well as L-lactate. LDHB plays an important role in D-lactate metabolism. LDHB loss increases D-lactate and methylglyoxal. It also causes mitochondrial defects and induces cardiomyopathy.
３８
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