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.
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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
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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)
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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.
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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).
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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
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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).
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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.
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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).
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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)
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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
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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.
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