Knockdown of the Cysteine Dioxygenase Gene in Damaged

Mitochondrial Cardiomyocytes May Be Protected by the Effective

Range of Taurine Level

Lifan Zhou

1,*

, Xinyi Zhou

, Yanxi Ren

, Yuwei Zha

and Jiarong Bei

School of bioscience, University of Nottingham Sutton Bonington Campus, Nottingham, NG7 2RD, U.K.

School of Biomedical Science, University of Bristol, Bristol, BS8 1TW, U.K.

Shenzhen Middle School, Shenzhen, 518001, China

Wuxi Big Bridge Academy, Wuxi, Jiangsu, 214115, China

Westover School, Middlebury, 06762, U.S.A.

Keywords: Taurine, CDO Gene, Cardiomyopathy Treatment.

Abstract: Cardiomyopathy is a disease caused by various reasons, including myocardial hypertrophy and autologous

gene defects. Taurine has been used in cardiomyopathy treatment for many years. It is a cytoprotective agent

that can protect the damaged mitochondria of the cardiomyocytes. Previous studies suggest that the high level

of taurine is primarily controlled by the cysteine dioxygenase (CDO) gene. This paper investigates the

optimum value of taurine concentration into CDO knockdown cardiomyocytes in vitro and vivo. In vitro,

taurine solution (ranges from 0μL to 1000μL) was added into the CDO knockdown cell culture medium daily

for every six groups. Then，we use ATP detection and Mitochondria membrane potential (MMP) detection

to investigate the effect of the taurine supplement. In vivo, 50 male mice are divided into ten groups fed with

taurine ranging from 1.0g to 3.0g as 0.2g increments. There is no result since all experiments are conducted

in virtual due to COVID-19. However, this paper is the first that provides the protocol to detect the right

taurine concentration in the case of CDO knockdown in cardiomyocytes. Thus, it may provide some ideas in

treating cardiomyopathy using a suitable amount of taurine in clinical.

1 INTRODUCTION

1.1 Background

Cardiomyopathy is characterized by a collection of

abnormal myocardial conditions including

myocardial mechanical and/or electrical dysfunction,

ventricular hypertrophy, and dilation, which can

eventually lead to cardiac death or progressive heart

failure (Zhuge, Ruiqi et al, 2017, Holmgren, D et al,

2003, Debray, François-Guillaume et al. 2007). And

this disease is often caused by genetic inheritance or

autoimmune disease (like lupus) (Cardiomyopathy

2017). A possible cause of cardiomyopathy is

mitochondria failure (Suzuki, Takeo, et al, 2002).

Cardiomyopathy can cause damage to the

mitochondria of myocardial cells, leading to a greater

possibility of heart failure (Chen, Yu-Han et al., 2019,

Pion et al., 1987, Moise, N S et al., 1991,

Marcinkiewicz, Janusz, Ewa Kontny. 2014).

Taurine (2-aminoethanesulfonic acid) is the most

abundant free amino acid in the human body. It plays

an important role in many important biological

processes, such as bile acid-binding, calcium

maintenance. Homeostasis, osmotic adjustment, and

membrane stability. In addition, the reduction of

apoptosis and its antioxidants. Activity seems to be

essential for cell protection taurine (Marcinkiewicz,

Janusz, Ewa Kontny, 2014, Lambert et al., 2015). In

1985, taurine (2-aminoethanesulphonic acid) was

first used in the treatment of congestive heart failure

in Japan (Azuma, et al., 1985, Azuma, et al., 1983),

and it is now established by numerous contemporary

works of literature that a decrease in the cellular level

of taurine considerably increases the possibility of

mitochondrial diseases, especially cardiomyopathy

(Suzuki, Takeo et al, 2002, Chen, Yu-Han et al., 2019,

Pion et al., 1987,

Marcinkiewicz, Janusz, Ewa

Kontny., 2014). Some studies have shown that taurine

as a cytoprotective agent can protect damaged

cardiomyocyte mitochondria (Marcinkiewicz,

944

Zhou, L., Zhou, X., Ren, Y., Zha, Y. and Bei, J.

Knockdown of the Cysteine Dioxygenase Gene in Damaged Mitochondrial Cardiomyocytes May Be Protected by the Effective Range of Taurine Level.

DOI: 10.5220/0011313400003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 944-950

ISBN: 978-989-758-595-1

Janusz, Ewa Kontny, 2014, Lambert et al., 2015). As

the most abundant free amino acid in excitable tissue,

taurine plays an essential role in several biological

functions including central nervous system

development and membrane stabilization. Studies

have also shown that mammals need to supplement

taurine by eating foods rich in taurine (Lambert, et al.,

2015).

In humans, taurine can be synthesized from other

sulfur-containing amino acids (Polakof, Sergio et al.

2018, Sampath, et al. 2020), one of which is cysteine.

It was long discovered that an enzyme, cysteine

dioxygenase (CDO), primarily controls the high level

of taurine in the human body (Wl, et al., 2019).

Regulating through the oxidation pathway of

cysteine, CDO expression level contributes to the

taurine biosynthesis in multiple human organs,

including the two major contributing organs: liver

and mammal glands (Ueki, Iori, Martha H Stipanuk.

2007). Through the CDO gene synthesis pathway, the

taurine content of taurine-containing plants can be

increased. Controlling the CDO gene can also help

control the content of taurine in the body. (Tevatia,

Rahul, et al. 2019)

Gene knockdown is considered better since it

achieves the same purpose and attains the same

results without directly regulating the genes, like

deletion in gene knockout or addition in gene knock-

in. It only affects the transcription and mostly

translation of a specific gene of interest, with high

accuracy and specificity. Plus, it will be far easier to

conduct knockdown than knockout.

Adenosine triphosphate (ATP) is formed by

connecting adenine, ribose, and 3 phosphate groups.

It releases more energy during hydrolysis and is the

most direct source of energy in the body. ATP release

and autocrine signals through purinergic receptors

promote T cell activation to form the immune synapse

formed by T cells and APC. (Ledderose, Carola et al.

2018) ATP can help human cells to carry out

immunity, and cardiomyopathy can reduce the ATP

produced by autogenous movement in the heart.

(Bloemink, Marieke et al., 2014, Ichihara, Sahoko, et

al., 2017)

1.2 Hypothesis

Therefore, this review will outline the important role

of taurine in mitochondrial cardiomyopathy. We

believe that increasing the content of taurine in the

body to a certain extent can help protect the damaged

mitochondria of cardiomyocytes. Change the original

CDO in the body to control the initial content of

taurine in the body. By changing the content of

taurine in the food used to help the experimenter to

supplement taurine, at the same time can detect the

content of ATP to select the most appropriate taurine

supplement. We believe that there should be a suitable

range for supplementing taurine content, which

should not be too high or too low.

2 EXPERIMENT DESIGN

2.1 Cardiomyocyte Cell Culture

Cardiomyocyte cell culture. Two groups of neonatal

cardiomyocytes are isolated from three-day-old

murine hearts, one from wild-type, the other from

mice with cardiomyopathy. The cells are resuspended

in DMEM supplemented with 10% fetal bovine

serum, 100units ml−1 penicillin, 100μg ml−1

streptomycin. After another 24 h with a regular

culture medium, 20μg ml−1 cytosine β-D-

arabinofuranoside will be added into the medium to

suppress non-interest cells. (Ladeira, et al, 2010)

2.2 CDO Knockdown in

Cardiomyocyte Cell Culture

Short interfering RNA (siRNA) Oligonucleotides.

In order to obtain CDO sequence siRNA

oligonucleotides, the experiment requires siRNA

manufacturer companies to design the required

complementary sequences, select potential target

sites, and then search with NCBI Blast to confirm the

specificity of each CDO exon expression. Since

there is no commercially available or known siRNA

that specifically downregulates the CDO gene in

murine cardiomyocytes, a positive control group

cannot be carried out. For the negative control group,

this work design non-targeting siRNAs that lack the

RNA sequences of interest in the targeting genome to

eliminate the possible experimental material

interference. (Han 2018) Aliquot the resuspended or

annealed siRNA into new tubes and store at −20 °C.

Single-Wall Carbon nanotubes (SWCNTs).

Ladeira et al. (Ladeira, et al, 2010) have already

validified that the covalent conjugation of siRNA to

SWCNTs for RNA interference and gene knockdown

is of high efficiency, especially in cell lines that are

poorly transfected, such as cardiomyocytes. SWCNT

is added into the cell medium with a concentration of

0.0250mg ml−1 for 48-h incubation of

cardiomyocytes. To guarantee the presence of

SWCNTs in the cells, the work use Raman

spectroscopy. The sample cell is excited by a He-Ne

laser (632.8 nm), and an oil objective lens with a

Knockdown of the Cysteine Dioxygenase Gene in Damaged Mitochondrial Cardiomyocytes May Be Protected by the Effective Range of

Taurine Level

945

magnification of 60 times and NA = 1.4 is used to

focus on the sample surface.

Transfection. Prepare a stable short SWCNT

(~200 nm length) aqueous solution using high-purity

short COOH-SWCNT dissolved in MilliQ water.

After centrifugation, 50nM CDO-specific siRNA was

added to 50μL of CNT aqueous solution, sonicated

for 30 minutes, and added to the cell culture medium.

For RNAifect preparation, wash the cells and provide

1 ml of fresh tissue culture medium. 50nM siRNA is

added to 3μL RNAifect, QIAGEN transfection

reagent, and then add 100μL tissue culture medium.

Following a 15-minute incubation at 37°C to allow

for complex formation, the mixture was reconstituted

with 900 mL of tissue culture media and subsequently

poured over the cells dropwise. The cells then are

plated into fibronectin-coated culture dishes at 37°C

in a 5% CO

incubator for two days. (Ladeira, et al,

2010)

Reverse Transcription Polymerase Chain

Reaction (RT-PCR) and Gel Electrophoresis. RT-

PCR is used for the evaluation of CDO knockdown

efficacy and the after-treatment reactions in the

negative control group. To purify the CDO mRNA of

interest, add 0.75mL of Trizol-LS® Reagent to

0.25ml of freshly isolated cardiomyocytes. To allow

the full dissociation of nucleoprotein complexes, mix

the cells multiple times with a pipette and then

incubate the lysates for 5 min at room temperature.

Then, centrifuge the lysates at 12,000 g for 10 min at

4°C, and discard the suspended liquid. To conduct

RT-PCR, using the High-Capacity cDNA Reverse

Transcription Kit, follow the instructions, and use

NCBI BLAST to create primers of interest. Then

place the tubes in a thermal-cycler and run RT-PCR

with 10 mins at 25°C, 2 hrs at 37°C, and 5 secs at

85°C. (Guan, Yang 2008) For the ethidium bromide-

stained gel analysis, 10μL each RT-PCR product is

loaded on a 7% polyacrylamide (19:1) gel and run for

3 hours at 120 V. The two groups of results are

compared and analyzed.

Western Blot. For a more detailed and

comprehensive investigation, the protein expression

of CDO is tested through Western Blot. Antibodies

are targeted towards CDO protein, and the protocol is

available online, See reference for more ( “

Antibodies, Proteins, Kits and Reagents for Life

Science.” Abcam).

In vivo siRNA delivery. Purchase C57BL/6 6–8-

week male mice from Beijing University. They are

kept in a 12:12 light/dark condition at 25°C on a chow

diet. Mice are anesthetized with pentobarbital sodium

(50mg/kg) by intraperitoneal injection. As shown in

Figure 1, 3mM of 20μL CDO-specific siRNAs are

administered intramyocardially into the left ventricle

using a 32G needle at roughly five regions around the

beginning section of the left anterior descending

coronary artery.

Figure 1: The injected locations are shown by the injectors.

Intramyocardial injection into the left ventricle (LV) at

roughly five places surrounding the beginning region of the

LAD coronary artery was performed. (The figure is adapted

from (Huang, Kun et al 2016)) .

2.3 Taurine Supplement into

Cardiomyocytes with CDO

Knockdown

a) In Vitro

Taurine Supplement. Inject taurine solution onto the

cell culture medium daily with 200μL as an increment

for each group, ranging from 0μL to 1000μL.

ATP Detection. The Detection Kit named

ab113849- Luminescent ATP Detection Assay Kit is

used to test the concentration of ATP. To extract ATP

in selected cardiomyocytes, PBS is first employed to

wash cells, and then add the cell lysis buffer on the

culture plate, which is then vibrated on the micro-

oscillator for 5 ~ 10min. To obtain the cell suspension

for further tests, first use a cell scraper to scrape the

cells on the culture plate, transfer the cell suspension

into a 1.5ml centrifuge tube, and fully vibrate on a

vortex oscillator for 30 seconds. Next, l dilute

luciferin with ATP assay buffer in the proportion of

1/20 and then add 1/10 volume of luciferase and

ddH

O to prepare the required volume of an assay

reagent. After all, the preparation adds the reagent

into the measuring tube and record the luminous unite

of firefly luciferase.

Mitochondria Membrane Potential (MMP)

Detection. The Detection Kit named ab112134-JC-10

Mitochondrial Membrane Potential Assay Kit is used

to test the membrane potential of cardiomyocytes’

mitochondria. For preparation, it is needed to place

cardiomyocytes overnight in a 90μL growth medium

at a 96-well plate. Then mix 50 µL of 100X JC-10 and

5 mL Assay Buffer A, treat cells by adding 10μL of

10X test compounds into PBS. Next incubate the cell

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

946

plate in a 37°C, 5% CO2 incubator for 4-6 hours to

induce apoptosis, and then add JC-10 dye-loading

solution into the cell plate. After the addition, another

30-min no-light incubation a 37°C, 5% CO2

incubation is needed. Then add the Assay Buffer B

into the dye loading plate, read the fluorescence

intensity, and monitor the fluorescence intensities at

= 490/525 nm (cut off at 515 nm) and 540/590

nm (cut off at 570 nm) for ratio analysis.

b) In Vivo

Experimental Period Estimation. In each of the

cell culture experiments, when the ATP and MMP

detection data from experimental groups deviate

significantly from the control group, this work will

make a note and regard it due to the ineffectiveness

of siRNAs. These statistics will be comprehensively

considered as an estimation of the efficient period of

our siRNA and be considered during in vivo

experiment conduction.

Taurine Supplement. Mice are chosen to do an

experiment on. Use 12 groups of male mice of the

same species, and each group has five mice. The first

group is all healthy mice without any treatment. The

second group is all mice with cardiomyopathy but

without any other treatment. The third group to the

twelfth group is mice with cardiomyopathy and with

CDO knockdown. These ten groups of mice are fed

with taurine, which is fed with 1.0g, 1.2g, 1.4g, 1.6g,

1.8g, 2.0g, 2.2g, 2.4g, 2.6g, 2.8g, and 3.0g.

Magnetic Resonance Imaging (MRI). The

mouse is under anesthesia and allowed to breathe

freely. First inhaled isoflurane into mice. Isoflurane

inhalation is currently the preferred method for mice

because anesthesia induction and wake-up are fast,

hemodynamic inhibition is minimal, and the depth of

anesthesia is easy to adjust. Then, the mice will

receive 1.0-2.0% isoflurane, 30%-50% oxygen, 50-

70% air. The location of the animals is also a critical

step, and they must be reproducible. Location affects

the quality of data and the degree to which motion

artifacts affect imaging. The mouse should be in the

prone position (fixed to an animal sled or other fixture

with tape or plastic pins). Then, an anesthesia cone is

provided through the nose, a breathing sensor is

usually connected to the abdomen, and the

temperature is measured through a fixed rectum.

After completing the previous step, the expected

gating synchronized with the ECG must be

performed. Finally, obtain cine cardiac imaging based

on multiphase gradient echocardiography.

High-Performance Liquid Chromatography.

After the mice are fed for 12 hours, blood samples

from their tail vein are collected, and each sample is

100μL. Add 5μL 1% heparin solution into each blood

sample and then centrifuge. Then, plasma samples

form. The pre-colum derivation is prepared, which

mixes 100μL blood sample with 50μL derivatization.

Add samples on y-reversed-phase column

chromatography, (125*3 mm, ODS Hypersil 3 m)

(VDS Optilab, Chromatographie Technik, GmbH),

the mobile phase is 27% methanol+73% 0.1 mol/L

Na2HPO4, 0.13 mmol/L EDTA water solution, speed

is 0.8 mL/min, detected volumetrically (ESA

Coulochem II; Bedford, Mass. USA) using three

electrodes, a guard (0.4 V), peroxidation (0.4 V) and

working (0.6 V) electrodes (analytical cell

ESA5011). The concentrations of taurine in mice’s

blood are tested out.

Computerized tomography (CT) and

anatomical observation. Mice euthanasia was

performed and left ventricular myocardial tissue was

extracted and utilized for detection.

3 RESULTS

3.1 Investigating CDO Knockdown in

Vitro

Expect experiment results as in Figure 2.

Figure 2: CDO has been knocked down using RT-PCR (a)

and Western Blot (b) (Mock trials).

3.2 Investigating Effect of Taurine

Levels in Vitro and in Vivo

a) ATP level in vitro using ATP Detection Assay

Kit.

From Figure 3, we expect a positive correlation

between the taurine supplement diet and ATP level.

Knockdown of the Cysteine Dioxygenase Gene in Damaged Mitochondrial Cardiomyocytes May Be Protected by the Effective Range of

Taurine Level

947

Figure 3: A positive correlation between taurine levels and

ATP levels is expected (Mock trials).

b) Mitochondrial Membrane Potential in vivo.

We expect a proper change in cell membrane

potential (Figure 4).

Figure 4: A proper change is expected in mock trails.

c) Taurine level in mice using MMR.

A typical mouse will contain 2.4 mg/g taurine or

(for better comparison with the other values: 100g

would contain 240mg) 4 ounces of a mouse would

equal over 2400 mg taurine (However, 2500mg is

minimum for a 10-lb cat). Compared with humans

and cats, the mouse exhibits a considerable

biosynthetic capacity for taurine.

d) Computerized tomography (CT) and

anatomical observation

Two symptoms of cardiomyopathy are thinner

myocardial wall and cardiac hypertrophy. CT is used

to supervise the change of these two symptoms of

each mouse (Figure 5). At last, dissect the hearts of

mice to see the condition of hearts (Figure 6). Since

it is a virtual experiment, there is no exact data. The

ideal situation is to find out mice of which taurine

level has a greater condition of the heart through

comparing these ill mice’s hearts with the healthy

ones. From this, the taurine range that can help cure

cardiomyopathy can be found out.

Figure 5: CT shows cardiomyopathy will cause thinner

myocardial wall and cardiac hypertrophy.

Figure 6: Thinner myocardial wall (a), cardiomyocyte

hypertrophy (c).

4 CONCLUSION

Due to the restricted conditions, the results are all

predicted. So, if all results meet the hypothesis, we

can propose that the protection of damaged

mitochondrial of cardiomyocytes with CDO

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

948

knockdown by a taurine supplement. However, there

will be still have some concerns about methods.

In the beginning of the experiment, this work

considered the knockdown CDO gene instead of

knockout it. This has also arisen a problem, which is

knockdown may have some uncertainty and

inaccuracy as we cannot be able to detect whether the

CDO gene was still knockdown during the entire

experiment. Also, further studies can be investigated

by using the knockout CDO gene method if this did

happen in a laboratory. Also, due to Han Haiyong

(Han 2018) mentioned in his paper that the efficient

effect of knockdown should be examined and to

determine the optimal time point for assessing

cellular effects of siRNA knockdown. Therefore, this

work will have some further studies on the monitor

the efficiency of knockdown and thus, used in mice

model. Moreover, in this report cannot ensure taurine

can be absorbed 100% by the human body whether

due to taurine consumption. Therefore, although

there were several repetitions of the experiment, we

cannot be 100% sure that the experiment will not

have the same absorption problems. This report also

considered the detection by high-pressure liquid

chromatography to detect the level of ATP in vitro,

but considering its stability is not as good as the kit,

we chose to use the kit after careful consideration. If

it is not possible to use the kit for an accurate surface

in real experiments, we can also choose to use high-

pressure liquid chromatography.

Apart from that, we are proud of an experiment is

the first to figure out that the range of taurine levels

that can affect cardiomyopathy under the condition of

CDO knockdown. However, more studies can be

taken to understand the underlying molecular

mechanisms. For example, test the mitochondrial

apoptosis pathway by Western blotting.

If the results of this experiment are true as

expected to prove that the content of taurine in the

body can have a positive and positive effect on

myocarditis, then this technology can be achieved by

consuming more taurine-rich foods. It helps patients

with cardiomyopathy relieve their symptoms, and at

the same time help patients achieve a greater quality

of life.

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