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Brault et al. J Transl Genet Genom. 2025;9:1-10 https://dx.doi.org/10.20517/jtgg.2024.83 Page 3

mismatch between energy supply and demand, the total pool of adenine nucleotides differs between
[21]
different cell types and between muscle fiber types [16,17] . Further, acceleration of adenine nucleotide
degradation by increased AMP deamination in skeletal muscle is sufficient to decrease ATP without
changing the ATP/ADP ratio [22,23] . Indeed, it is clear that these two measures, the ATP content and ATP/
ADP ratio, are regulated independently .
[24]
Measuring adenine nucleotides
Quantifying the energetic state in tissues or cells presents several challenges. First, ATP is rather labile and
can be quickly degraded during collection or processing [25-27] . Second, a single assay (e.g., simply measuring
ATP) is not sufficient, given that both ATP and ADP define the energetic state. Third, the measures of ATP
and ADP must be quantified in an absolute term (e.g., mmol/g or mM) and cannot be simply normalized to
arbitrary values, because arbitrary units would make the calculation of ATP/ADP meaningless.

ATP in muscle can be measured using a variety of assays, including magnetic resonance spectroscopy
[29]
(MRS) , fluorescent reporters , luciferase assays [30,31] , and high or ultra performance liquid
[28]
chromatography (HPLC or UPLC) [32,33] . These methods have been used for decades, but each has
limitations. MRS and fluorescent reporters are generally used in vivo, which avoids tissue collection
artifacts. However, they are not amenable to absolute quantification measures (only relative) and generally
cannot measure ADP directly in tissue , which makes validation across studies impossible and may even
[28]
be misleading. Luciferase assays can be used to quantify ATP directly and are generally performed on
extracts. However, ADP cannot be measured directly. UPLC assays are performed on tissue extracts, can
measure ATP and ADP (as well as NAD+, NADH, and adenine nucleotide degradation products such as
AMP and IMP) simultaneously, and are highly quantitative. Both luciferase and UPLC assays can only be
performed on tissue/cell extracts.

Tafazzin protein and interactions
TAFAZZIN protein contains mitochondrial localization and membrane anchoring domains, as well as a
unique hydrophilic domain that may serve as an exposed loop interacting with additional unidentified
[6]
proteins . Tafazzin can also sense mitochondrial membrane curvature and play a direct role in cristae
reorganization and stability [34,35] . However, Tafazzin is primarily known for the synthesis of mature
cardiolipin via promoting cardiolipin acyl chain remodeling, and is the characteristic lipid found in
mitochondrial inner membranes. Cardiolipin is associated with many of the complexes of oxidative
phosphorylation (OxPhos) and mitochondrial enzymes involved in ATP production, thereby stabilizing
OxPhos supercomplexes. Mitochondrial supercomplexes are assemblies of individual respiratory chain
[36]
complexes colocalized with cardiolipin found on the inner mitochondrial membrane , and increased
content of supercomplexes facilitates ATP synthesis [2,37-40] . Consistent with this, loss of cardiolipin in patients
or in models of BTHS leads to mitochondrial shape irregularities (e.g., swollen, collapsed cristae,
honeycomb-like formations, aggregates) [3,4,41-46] , decreased mitochondrial maximal oxygen consumption/
ATP generating capacity [8,9,47-57] , decreased mitochondrial efficiency as defined as phosphate-to-oxygen
ratio [45,56] , increased apoptosis, and either no change [48,51,58] or increase [48,52,53,57,59,60] in superoxide production.
Moreover, in addition to cardiolipin-deficient impairment of OxPhos, several BTHS models also exhibit
defects relating to the intermediary metabolism of fatty acids, carbohydrates, ketones, and amino acids .
[61]
Thus, as these alternative fuel substrates are less efficient, ATP production may also be indirectly
compromised via upstream metabolic disturbances.

In addition to direct effects on OxPhos enzymes, cardiolipin may also affect the energetic state by its ability
to bind with high affinity to the nucleotide metabolism enzymes nucleotide diphosphate kinase (NDPK-D
or nme23-H4 or NME4) and creatine kinase (CK). NME4 has a mitochondrial targeting sequence and is
[62]