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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