RESEARCH ARTICLE
Rat and mouse cardiomyocytes show subtle
differences in creatine kinase expression and
compartmentalization
Jelena Branovets, Ka¨rol Soodla, Marko Vendelin, Rikke Birkedal*
Laboratory of Systems Biology, Department of Cybernetics, Tallinn University of Technology, Tallinn, Estonia
* rikke@sysbio.ioc.ee
Abstract
Creatine kinase (CK) and adenylate kinase (AK) are energy transfer systems. Different stud-
ies on permeabilized cardiomyocytes suggest that ADP-channelling from mitochondrial CK
alone stimulates respiration to its maximum, VO2_max, in rat but not mouse cardiomyocytes.
Results are ambiguous on ADP-channelling from AK to mitochondria. This study was under-
taken to directly compare the CK and AK systems in rat and mouse hearts. In homogenates,
we assessed CK- and AK-activities, and the CK isoform distribution. In permeabilized cardi-
omyocytes, we assessed mitochondrial respiration stimulated by ADP from CK and AK,
VO2_CK and VO2_AK, respectively. The ADP-channelling from CK or AK to mitochondria was
assessed by adding PEP and PK to competitively inhibit the respiration rate. We found that
rat compared to mouse hearts had a lower aerobic capacity, higher VO2_CK/VO2_max, and dif-
ferent CK-isoform distribution. Although rat hearts had a larger fraction of mitochondrial CK,
less ADP was channeled from CK to the mitochondria. This suggests different intracellular
compartmentalization in rat and mouse cardiomyocytes. VO2_AK/VO2_max was similar in
mouse and rat cardiomyocytes, and AK did not channel ADP to the mitochondria. In the
absence of intracellular compartmentalization, the AK- and CK-activities in homogenate
should have been similar to the ADP-phosphorylation rates estimated from VO2_AK and
VO2_CK in permeabilized cardiomyocytes. Instead, we found that the ADP-phosphorylation
rates estimated from permeabilized cardiomyocytes were 2 and 9 times lower than the activ-
ities recorded in homogenate for CK and AK, respectively. Our results highlight the impor-
tance of energetic compartmentalization in cardiac metabolic regulation and signalling.
Introduction
Creatine kinase (CK) is thought to play a crucial role in storage and spatial transport of
energy-rich phosphates in tissues with high and fluctuating energy demands [1, 2]. In the
heart, there are three cytosolic CK isoforms (MM-, MB-, and BB-CK) and one mitochondrial
CK isoform (Mi-CK). Several studies on rat heart have reported that phosphotransfer via CK
is several times faster than ATP synthesis by oxidative phosphorylation in mitochondria [3–5],
and Mi-CK in the intermembrane space is coupled to the adenine nucleotide translocase
(ANT), which exchanges ATP for ADP, providing ADP for mitochondrial oxidative
PLOS ONE
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
1 / 25
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
OPEN ACCESS
Citation: Branovets J, Soodla K, Vendelin M,
Birkedal R (2023) Rat and mouse cardiomyocytes
show subtle differences in creatine kinase
expression and compartmentalization. PLoS ONE
18(11): e0294718. https://doi.org/10.1371/journal.
pone.0294718
Editor: Luis Eduardo M Quintas, Universidade
Federal do Rio de Janeiro, BRAZIL
Received: June 6, 2023
Accepted: November 6, 2023
Published: November 27, 2023
Peer Review History: PLOS recognizes the
benefits of transparency in the peer review
process; therefore, we enable the publication of
all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0294718
Copyright: © 2023 Branovets et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting information
files.
phosphorylation [6, 7]. For many years, the CK system was considered to have a pivotal role in
the regulation of cardiac energy metabolism [1, 2, 8–10].
Surprisingly, studies on genetically manipulated mouse models having modifications in
various components of the CK system like expression of different CK isoforms, creatine syn-
thesis or uptake have been equivocal [11]. Notably, the baseline cardiac function of mice with
compromised CK system is minimally disturbed [12–16] and compromised CK function does
not exacerbate heart failure [17, 18]. Thus, studies on mice have not corroborated the theory,
based mainly on rat experiments, that CK is crucial for energy transfer and regulation of oxida-
tive phosphorylation.
Our recent study on permeabilized cardiomyocytes from creatine-deficient AGAT [19] and
GAMT [20] mice also pointed to a difference between mouse and rat cardiomyocytes. We per-
formed an assay, where we recorded the respiration rate of permeabilized cardiomyocytes. In
addition to substrates for oxidative phosphorylation (glutamate and malate, GM) and creatine,
we added ATP to the solution to initiate endogenous ADP-generation by CK (creatine + ATP
! phosphocreatine + ADP + H+). Endogenous ADP then stimulated oxidative phosphoryla-
tion. We found that the rate of ADP-generation by CK sustained a respiration rate that was
~80% of the maximal respiration rate [21]. The channelling of ADP from CK to the mitochon-
dria was assessed by addition of phosphoenolpyruvate (PEP) and pyruvate kinase (PK) in
excess, which compete with the mitochondria for ADP. This is an experimental strategy to
assess how much of the CK is so closely associated with the mitochondria that ADP is chan-
nelled directly to the mitochondria without being released to the bulk phase, where it would be
consumed by PK. The addition of PEP and PK lowered the rate of oxidative phosphorylation
by ~75%, demonstrating that some ADP generated by CK is channelled to the mitochondria
and inaccessible to PK [21]. However, our results on mouse cardiomyocytes were in sharp
contrast to experiments on rat cardiomyocytes, where even in the presence of PEP and PK, the
rate of ADP-generation by CK sustained the respiration rate at its maximum [22, 23]. This dif-
ference raised the question whether rat and mouse hearts differ in terms of their CK activity,
either total and/or relative to the maximal respiration rate in the absence and presence of PEP
and PK.
Adenylate kinase (AK) is another well-known alternative phosphotransfer system in the
heart [9]. Its role in facilitating energy transfer and in the compartmentalization of adenine
nucleotides has also been investigated [24–29]. Although the contribution of AK-mediated
phosphoryl transfer to the total ATP turnover is only ~10%, some studies observed an
increased importance of AK under stress conditions [29, 30]. In our recent study on mouse
cardiomyocytes [21], we also assessed the respiration rate stimulated by endogenous ADP gen-
erated by AK. In the presence of substrates (GM) and ATP, the addition of AMP initiated the
endogenous ADP-generation by AK (AMP + ATP ! 2 ADP). The rate of ADP-generation by
AK sustained the respiration rate near its maximal rate, but the addition of PEP and PK abol-
ished the effect of adding AMP, suggesting that all ADP generated by AK was accessible to PK.
On the one hand, this is in agreement with the finding of a minimal AK activity in rat and
mouse heart mitochondria [31–33]. On the other hand, it contradicts results demonstrating a
stronger functional coupling between AK and mitochondrial respiration in rats [27]. Thus,
there is an inexplicable mismatch between different studies regarding the importance of mito-
chondrial AK in regulation of mitochondrial oxidative phosphorylation, and we speculated
whether species differences might be adding to the confusion.
The aim of the present study was to assess whether rat and mouse hearts are different in
terms of 1) the overall CK and AK activities in whole heart homogenates, and 2) how much
endogenous ADP generated by CK or AK stimulates oxidative phosphorylation in the absence
and presence of PEP and PK competing with the mitochondria for the consumption of ADP.
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
2 / 25
Funding: This work was supported by the Estonian
Research Council (www.etag.ee/en/), grant number
PRG1127. The funder had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
Abbreviations: AGAT, Arginine:glycine
amidinotransferase (EC 2.1.4.1); AK, Adenylate
kinase; AKADP_GMPS, Name of the protocol used to
record adenylate kinase-stimulated respiration with
glutamate, malate, pyruvate, and succinate as
substrates, followed by stimulation of maximal
respiration with 2mM ADP; AKf, Adenylate kinase
activity measured in the forward direction: ATP +
AMP ! ADP + ADP; AKPEP/PK_GMPS, Name of the
protocol used to record adenylate kinase-
stimulated respiration with glutamate, malate,
pyruvate, and succinate as substrates, followed by
inhibition of respiration with phosphoenolpyruvate
and pyruvate kinase; AKr, Adenylate kinase activity
measured in the reverse direction: ADP + ADP !
ATP + AMP; AMPK, AMP-activated protein kinase;
ANT, adenine nucleotide translocase; BB-CK,
Homodimeric brain isoform of creatine kinase; CK,
Creatine kinase; CKADP_GM, Name of the protocol
used to record creatine kinase-stimulated
respiration with glutamate and malate as
substrates, followed by stimulation of maximal
respiration with 2mM ADP; CKADP_GMPS, Name of
the protocol used to record creatine kinase-
stimulated respiration with glutamate, malate,
pyruvate, and succinate as substrates, followed by
stimulation of maximal respiration with 2mM ADP;
CKf, Creatine kinase activity measured in the
forward direction:ATP + creatine ! ADP +
phosphocreatine + H+; CKPEP/PK_GM, Name of the
protocol used to record creatine kinase-stimulated
respiration with glutamate and malate as
substrates, followed by inhibition of respiration
with phosphoenolpyruvate and pyruvate kinase;
CKPEP/PK_GMPS, Name of the protocol used to
record creatine kinase-stimulated respiration with
glutamate, malate, pyruvate, and succinate as
substrates, followed by inhibition of respiration
with phosphoenolpyruvate and pyruvate kinase;
CKr, Creatine kinase activity measured in the
reverse direction:ADP + phosphocreatine + H+ !
ATP + creatine; CM, Cardiomyocytes; CO,
Cytochrome oxidase; CS, Citrate synthase; Cyt aa3,
Cytochrome aa3; Cyt c, Cytochrome c; DTNB,
dithiobis(2-nitrobenzoic acid); FCCP, Carbonyl
cyanide 4-trifluoromethoxyphenylhydrazone;
GAMT, Guanidinoacetate N-methyltransferase (EC
2.1. 1.2); GM, Glutamate and malate; GMPS,
Glutamate, malate, pyruvate and succinate; MB-CK,
In whole heart homogenates, we recorded the activities of CK and AK in the direction of
both ADP- and ATP-generation. In addition, we recorded the activities of citrate synthase
(CS, a marker of mitochondrial density) and cytochrome oxidase (CO, a marker of oxidative
capacity, i.e. maximal O2 consumption rate per muscle mass) [34]. In permeabilized cardio-
myocytes, we recorded the respiration rate stimulated by endogenous ADP generated by either
CK or AK. In one set of experiments, we assessed how much CK or AK stimulated respiration
relative to its maximal rate by subsequent addition of ADP. In parallel, the ADP channelling
between kinases and mitochondria was assessed by subsequent additions of PEP and PK.
The maximal respiration rate is substrate-dependent [35, 36]. Most studies have used only
GM, which lead to NADH-linked electron flux only through complex I of the respiratory chain.
More substrates are required to obtain the maximal respiration rate. For example, a combina-
tion of glutamate, malate, pyruvate and succinate (GMPS) leads to NADH and FAD-linked elec-
tron flux through both complexes I and II of the respiratory chain [36, 37]. Here, the role of CK
was assessed with GM as in previous experiments [21–23], and with GMPS. Due to a limited
number of chambers in the respirometer, the role of AK was only assessed with GMPS. To the
best of our knowledge, this is the first time these recordings have been performed with GMPS.
Throughout this work, we compare whole heart homogenate with isolated cardiomyocytes.
However, whole heart homogenates are prepared using pieces of tissue, which in addition to
cardiomyocytes also contain extracellular matrix, endothelial cells, and fibroblasts. Cardio-
myocytes, endothelial cells and fibroblasts take up 70–80%, 3.2–5.3%, and 1.4–1.9% of the vol-
ume, respectively [38], and cardiomyocytes have a much larger mitochondrial volume (31–
40%) than the other cell types (5% for endothelial cells) [39–41]. Therefore, in order to com-
pare whole heart homogenate and isolated cardiomyocytes, we assumed that the CS activity in
non-cardiomyocytes was negligible and compared the data normalized to the CS activity.
Results
Animals and cell preparations
The morphological characteristics of the animals used in this study are given in Table 1.
Table 2 shows several characteristics of the cell suspensions. The viability of the cell
Table 1. Characteristics of the mice and rats used in the experiments.
n
BW, g
HW, mg
HW/BW, mg/g
Mice
15 (7)
27.3 ± 0.8
123.8 ± 2.3
4.7 ± 0.1
Rats
14 (7)
316.6 ± 8
920.4 ± 26
2.9 ± 0.1
Values are shown as mean ± SEM. BW, body weight; HW, absolute heart weight; HW/BW, relative heart weight. The
total number of animals is shown in column n. The absolute and relative heart weight (HW and HW/BW) are
reported for a smaller number of animals, indicated in parenthesis, because the hearts used for cardiomyocyte
isolation could not be weighed.
https://doi.org/10.1371/journal.pone.0294718.t001
Table 2. Characteristics of the isolated cardiomyocyte suspensions from mice and rats.
Viability %
Protein mg/ml
CS activity μmol/min/g protein
Cyt aa3 μmol/g protein
Mouse cardiomyocytes
75.0 ± 1.1
15.52 ± 0.80
806 ± 37
Rat cardiomyocytes
73.7 ± 1.9
20.80 ± 2.41
609 ± 9 ***
0.16 ± 0.01
Cardiomyocytes were isolated from 8 mice and 7 rats. Values are shown as mean ± SEM. Viability is the fraction of rod-shaped to the total number of cells. CS is the
citrate synthase activity, and cyt aa3 is the cytochrome aa3 content.
*** denotes p < 0.001, significant difference between species.
https://doi.org/10.1371/journal.pone.0294718.t002
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
3 / 25
Heterodimeric muscle-brain isoform of creatine
kinase; Mi-CK, Sarcomeric mitochondrial creatine
kinase; MM-CK, Homodimeric muscle isoform of
creatine kinase; P/O2 ratio, The phosphate to
oxygen ratio, describes how much ADP the
mitochondria phosphorylate to ATP per O2
consumed; PEP, Phosphoenolpyruvate; PK,
Pyruvate kinase; SERCA, sarcoendoplasmic
reticulum Ca2+-ATPase; TNB, thionitrobenzoate;
VADP_AK, Estimated rate of ADP-phosphorylation,
when mitochondria are stimulated by ADP
generated by adenylate kinase; VADP_CK, Estimated
rate of ADP-phosphorylation, when mitochondria
are stimulated by ADP generated by creatine
kinase; VADP_max, Estimated rate of ADP-
phosphorylation, when mitochondria are
stimulated by 2 mM exogenous ADP; VDAC,
Voltage dependent anion channel; VFCCP,
Respiration rate during maximal uncoupled
respiration rate; VO2_AK, Respiration rate stimulated
by adenylate kinase after addition of ATP and AMP;
VO2_CK, Respiration rate stimulated by creatine
kinase after addition of ATP with creatine in the
medium; VO2_max, Maximal coupled respiration rate
stimulated by 2 mM ADP; VO2_max_GM, Maximal
coupled respiration rate with glutamate and malate
as substrates; VO2_max_GMPS, Maximal coupled
respiration rate with glutamate, malate, pyruvate
and succinate as substrates.
suspensions did not differ between mice and rats. Although there was a tendency to a higher
protein content in rats than in mice, this was not statistically significant. CS activity, when nor-
malized to the protein content, was significantly higher in mouse than in rat cardiomyocytes.
The cytochrome aa3 (Cyt aa3) content was assessed only for cell suspensions from rat hearts
due to the large volume required for these measurements.
Enzyme activities and CK isoform distribution
The overall activities of CS, CK, AK and CO, measured in cardiac whole tissue homoge-
nates, are shown in Fig 1. In Fig 1A, the enzymatic activities normalized to the heart wet
weight are shown for comparison with the literature (see Discussion). In Fig 1B, the activi-
ties of CO, AK and CK normalized to the CS activity are shown for comparison with the res-
piration rates recorded in permeabilized cardiomyocytes (Figs 3 and 4). Since both CK- and
AK-catalysed phosphotransfer reactions are reversible, the activities of these enzymes were
Fig 1. The activities of citrate synthase (CS), adenylate kinase (AK), creatine kinase (CK) and cytochrome oxidase
(CO) in mouse and rat hearts. CS, AK and CK activities are represented as rates (μmolmin-1g ww-1) and CO activity as a
rate constant (min-1g ww-1). AKf and CKf, and AKr and CKr, are the forward (f) and reverse (r) reaction rates of AK and
CK, respectively. A: When normalized to the wet weight of the tissue, CS and AK activities were higher in mouse than in
rat heart. B: When normalized to the CS activity, the CK and CO activities (μmolmin-1IU CS-1) were higher in rat than in
mouse heart. The number of animals was n = 7 for mice and n = 7 for rats. * denotes p < 0.05, ** p < 0.01, *** p < 0.001,
**** p < 0.0001 significant difference between species.
https://doi.org/10.1371/journal.pone.0294718.g001
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
4 / 25
measured in both directions. In the forward direction, CK and AK generate ADP, i.e. CK
catalyses the reaction: creatine + ATP ! phosphocreatine + H+ + ADP, and AK catalyses
the reaction: ATP + AMP ! 2 ADP. In the reverse direction, CK catalyses the reaction:
phosphocreatine + H+ + ADP ! creatine + ATP, and AK catalyses the reaction: 2 ADP !
ATP + AMP.
When enzyme activities were normalized to the wet weight (Fig 1A), the CS activity was
~35% higher in mice than in rats (139.1 ± 4.7 vs 101.4 ± 1.6 μmolmin-1g ww-1 respectively;
p < 0.001). The AK activity was ~30% higher in mice than in rats in both directions
(p < 0.001). The CKr activity in the direction of ATP-production tended to be higher in rat
than in mouse heart (p = 0.0815), and the CKf activity measured in the direction of ADP-pro-
duction was 11% lower in mice than in rats (p < 0.05). The CO activity was similar for mice
and rats.
As the CS activity was different in mouse and rat hearts, normalizing hereto changed the
pattern (Fig 1B). The AK/CS activity (measured in both directions) was similar in mice and
rats, whereas the CK/CS activity was ~35% lower in mice than in rats in both directions (Fig
1B; p < 0.0001), and CO/CS activity was ~30% lower in mice than in rats (Fig 1B; p < 0.01).
The CK isoform distribution in the hearts of mice and rats was assessed by gel electrophore-
sis. A representative picture is shown in Fig 2. For each lane, the intensity of the four major
bands corresponding to Mi-, MM-, MB and BB-CK was quantified, and the fractional intensity
of each band was calculated. The averaged data are given in Table 3. Compared to mouse
hearts, rat hearts had a different CK isoform distribution with a smaller fraction of MM-CK,
and larger fractions of Mi-CK, MB-CK and BB-CK.
Stimulation of respiration by CK and AK
In permeabilized cardiomyocytes, the stimulation of respiration by CK or AK was assessed
relative to the maximal coupled respiration rate (VO2_max) (Fig 3). CK-stimulated
Fig 2. The CK isoform distribution in rat and mouse hearts. The CK isoform distribution in rat and mouse hearts was assessed by agarose gel
electrophoresis. A: Representative picture showing on the left the raw image, and, on the right the same image, in pseudocolour to highlight the bands.
B. The intensity profiles from the rat and mouse lanes shown in A.
https://doi.org/10.1371/journal.pone.0294718.g002
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
5 / 25
respiration rate was recorded with either substrates for complex I alone (GM; protocol
named CKADP_GM) or substrates for complexes I and II (GMPS; protocol named
CKADP_GMPS). AK-stimulated respiration was recorded only with substrates for complexes I
and II (GMPS; protocol named AKADP_GMPS). A representative experimental trace of
Table 3. Distribution of CK isoforms in the hearts of mice and rats.
Mi-CK, %
MM-CK, %
MB-CK, %
BB-CK, %
Mouse heart
31.6 ± 1.8
63.8 ± 1.6
3.7 ± 0.3
0.83 ± 0.10
Rat heart
37.1 ± 1.1*
50.5 ± 0.5 ***
11.1 ± 0.8 ***
1.20 ± 0.03*
The mitochondrial Mi-CK and the three cytosolic MM-, MB-, and BB-CK isoforms in cardiac homogenates from 4 mice and 4 rats were separated by electrophoresis,
and the fraction of each isoform is shown as mean ± SEM.
* denotes p < 0.05 and *** p < 0.001, significant difference between species.
https://doi.org/10.1371/journal.pone.0294718.t003
Fig 3. Stimulation of respiration by CK and AK assessed relative to the maximal respiration rate in permeabilized mouse and rat
cardiomyocytes. When normalized to the CS activity, CK stimulated the respiration rate (μmol O2min-1IU CS-1) more in rat than in mouse
cardiomyocytes. A: Representative example of a respirometer recording of CKADP_GMPS with permeabilized rat cardiomyocytes after
addition of: CM–cardiomyocytes; ATP– 2 mM ATP; ADP– 2 mM ADP; Cyt c– 8 μM cytochrome c; FCCP–FCCP was gradually increased,
the number behind shows the final concentration in μM. B: The averaged results of the experiments in the presence of GM, where
respiration was stimulated by CK. C and D: The averaged results of the experiments in the presence of GMPS, where respiration was
stimulated by AK (C) and CK (D). V0 was subtracted from the subsequent rates. In B and D, as creatine was already present in the
respiration chamber, ATP is the CK-stimulated respiration rate. In C, ATP is the rate stimulated by non-specific ATPases, and AMP is the
AK-stimulated respiration rate. ADP is the maximal respiration rate recorded with 2 mM ADP. The number of animals was n = 8 and
n = 5–7 for mice and rats, respectively. * denotes p < 0.05, ** p < 0.01, *** p < 0.001 significant difference between species.
https://doi.org/10.1371/journal.pone.0294718.g003
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
6 / 25
CKADP_GMPS is shown in Fig 3A. Due to the low solubility of creatine, it was present in the
medium before the start of the experiments at a concentration of 20 mM. After addition of
cardiomyocytes, and recording of the basal respiration rate (V0), the CK reaction was stimu-
lated by the addition of 2 mM ATP. Then, 2 mM ADP was added to stimulate the respiration
rate to VO2_max. Finally, cytochrome c (Cyt c) was added to test the intactness of the outer
mitochondrial membrane, and the ionophore carbonyl cyanide-p-trifluoromethoxyphenyl-
hydrazone (FCCP) was added in steps to determine the maximal electron flux capacity, i.e.
the maximal uncoupled respiration rate. For AK-stimulated respiration measurements,
AMP was added (instead of creatine) after addition of ATP. Fig 3B–3D show the averaged
results and the statistical analyses from the respiration measurements of CKADP_GM,
AKADP_GMPS, and CKADP_GMPS, respectively, normalized to the CS activity in cell suspen-
sions from mice and rats. Table 4 shows the respiration rates stimulated by CK (VO2_CK) and
AK (VO2_AK), and the maximal coupled respiration rate (VO2_max) normalized to the CS
activity of the cell suspension. In addition, it shows VO2_CK and VO2_AK relative to VO2_max.
Note that in Fig 3 and Table 4, all respiration rates recorded after V0 had V0 subtracted
before analysis. The same notation was used throughout the study.
Mitochondrial respiration has a lower phosphate to oxygen (P/O2) ratio with GMPS than
with GM, i.e. with GMPS as substrates, fewer ADP molecules are phosphorylated to ATP for a
given O2 consumption. This is because complex I substrates translocate 10 H+/O2, whereas
complex II substrates translocate 6 H+/O2 across the inner mitochondrial membrane [42]. As
the proton gradient across the inner mitochondrial membrane is the driving force for oxida-
tive phosphorylation by the F1F0 ATPase, the higher H+/O2 with GM than with GMPS II leads
to the theoretical P/O2 ratios of 6 and 4 for GM and GMPS, respectively [43]. To determine
whether differences in respiration rates were associated with differences in ADP-phosphoryla-
tion rates, we multiplied the respiration rates in Table 4 with these P/O2 ratios to calculate the
rates of ADP-phosphorylation, when respiration was stimulated by CK (VADP_CK), AK (VAD-
P_AK), or 2 mM ADP (VADP_max). These values are shown in Table 5.
Table 4. Respiration rates of permeabilized mouse and rat cardiomyocytes stimulated by CK, AK, or 2 mM ADP.
VO2_CK
VO2_AK
VO2_max
VO2_CK /VO2_max
VO2_AK/VO2_max
nmol O2 /min/IU CS
%
Mouse
GM
55 ± 2 ##
61 ± 3
91 ± 2
GMPS
93 ± 4 ####
89 ± 9 ####
124 ± 4
75 ± 1
79 ± 2
Rat
GM
77 ± 4
72 ± 4
107 ± 4
GMPS
113 ± 4 ##
79 ± 7 ###
128 ± 7
89 ± 3
71 ± 3
Substrate
****
****
***
Species
***
***
*
The respiration of permeabilized cardiomyocytes was recorded in the presence of either GM or GMPS as substrates. Under these conditions, the respiration rate is
limited by the availability of ADP. The respiration was stimulated by endogenous ADP generated by CK (VO2_CK), or AK (VO2_AK). The maximal respiration rate
(VO2_max), was recorded in the presence of 2 mM exogenous ADP. In addition, the fractional stimulation of respiration by CK and AK relative to VO2_max was
determined (VO2_CK /VO2_max and VO2_AK/VO2_max, respectively). Values from 8 mice and 5–7 rats are shown as mean ± SEM.
## denotes p < 0.01,
### p < 0.001,
#### p < 0.0001, significantly different from VO2_max.
* denotes p < 0.05,
*** p < 0.001,
**** p < 0.0001, significant effect of substrate or species. We found no interaction between substrates and species.
https://doi.org/10.1371/journal.pone.0294718.t004
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
7 / 25
The CK-stimulated respiration rates, VO2_CK, were species dependent and significantly
higher in rat than in mouse cardiomyocytes in both CKADP_GM and CKADP_GMPS (Fig 3B and
3D, p < 0.01 and p < 0.001, respectively). However, after addition of ADP, Cyt c and FCCP,
there was no significant difference in respiration rates between mice and rats (Fig 3B and 3D).
As a result, the CK-stimulated respiration rate relative to the maximal respiration rate,
VO2_CK/VO2_max, was higher in rats than in mice (Table 4; p < 0.001).
The CK-stimulated respiration rates were also substrate dependent (Table 4, compare Fig
3B and 3D), but the estimated rates of ADP-phosphorylation (VADP_CK) were only affected by
species and not substrate (Table 5). This suggests that the rate with which CK generated ADP
to stimulate respiration was not affected by substrates but resulted in different VO2_CK rates
because of the substrate dependent P/O2 ratios.
The respiration rate stimulated by AK, VO2_AK, did not differ between mouse and rat cardi-
omyocytes (AKADP_GMPS; Fig 3C). However, when assessed relative to the maximal respira-
tion, VO2_AK/VO2_max was slightly higher in mouse than in rat cardiomyocytes (Table 4;
p < 0.05).
When VO2_max was normalized to the CS activity of the cell suspension, there was no dif-
ference between mouse and rat cardiomyocytes (Fig 3 and Table 4). However, the CS activity
was higher in mouse than in rat cardiomyocytes (Table 2). When VO2_max was normalized to
the protein content of the cell suspension (see S1 Table), it was significantly higher in mouse
than in rat cardiomyocytes. Thus, VO2_max correlated with the CS activity of the cell
suspension.
VO2_max was significantly higher than VO2_CK except in rat cardiomyocytes with only com-
plex I substrates (GM). VO2_max was also significantly higher than VO2_AK in both mouse and
rat cardiomyocytes (Table 4).
VO2_max was significantly affected by the substrates. In the presence of GM, VO2_max was
lower than in the presence of GMPS (Table 4). VADP_max was also significantly affected by the
substrates (Table 5). Thus, in the presence of 2 mM ADP, the rate of ADP-phosphorylation
was lower with GM alone than with GMPS.
The quality of the permeabilized cardiomyocytes was assessed through the coupling effi-
ciency of respiration and the Cyt c test. The coupling efficiency of respiration, calculated as
1-V0/VO2_max, indicates the proportion of oxygen used for ATP synthesis. In permeabilized
cardiomyocytes, a high coupling efficiency indicates that they are 1) adequately permeabilized
Table 5. Estimated rates of ADP-phosphorylation by mitochondria stimulated by CK, AK or 2 mM ADP.
VADP_CK
VADP_AK
VADP_max
nmol ADP/min/IU CS
Mouse
GM
332 ± 13
367 ± 19
GMPS
373 ± 14
354 ± 41
498 ± 18
Rat
GM
461 ± 23
433 ± 27
GMPS
451 ± 17
315 ± 26
512 ± 28
Substrate
****
Species
***
We used the data from Table 4 to estimate the rates of ADP-phosphorylation by mitochondria, when respiration was stimulated by endogenous ADP from CK
(VADP_CK) or AK (VADP_AK), or 2 mM exogenous ADP (VADP_max). The respiration rates were multiplied by a P/O2 ratio of 6 or 4, for GM and GMPS, respectively, as
explained in the main text. Values from 8 mice and 5–7 rats are shown as mean ± SEM.
*** denotes p < 0.001,
**** p < 0.0001, significant effect of substrate or species. We found no interaction between substrates and species.
https://doi.org/10.1371/journal.pone.0294718.t005
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
8 / 25
and respond to ADP addition, and 2) not damaged, as this would cause an elevated V0.
According to Chance and Williams, tightly coupled mitochondria show a substrate-dependent
4- to 10-fold increase in respiration rate upon addition of ADP [44]. This corresponds to a
coupling efficiency of 0.75–0.90. In the present experiment, the coupling efficiency was similar
in mouse and rat cardiomyocytes but substrate dependent and lower with GMPS than with
GM (81.6 ± 0.5% and 89.7 ± 0.7%, respectively; p < 0.0001; data were pooled for rats and
mice).
The addition of Cyt c is commonly used to test the intactness of the outer mitochondrial
membrane. If the outer mitochondrial membrane is damaged, electron transfer will be com-
promised as Cyt c leaks out of the mitochondria. The associated decline in respiration rate will
be rescued upon addition of Cyt c. In our experiments, the addition of Cyt c did not signifi-
cantly affect the respiration rate in any of the measurements (Fig 3), indicating that in the car-
diomyocytes in the present study, the outer mitochondrial membrane was intact.
The addition of FCCP, an uncoupler of respiration, did not increase the respiration rate
with GM only. But with GMPS, FCCP increased the respiration rate by ~40% (Fig 3). The
phosphorylation control ratio, calculated as VO2_max/VFCCP, was 106 ± 2.1 and 58.3 ± 0.6%
with GM and GMPS, respectively (pooled data from rats and mice).
Channelling of ADP from CK or AK to the mitochondria
The ADP channelling between CK or AK and the mitochondria was assessed in parallel
experiments, where the kinase stimulated respiration was subsequently inhibited by addition
of PEP and PK. Using the same substrate-kinase combinations as before, CK-stimulated res-
piration rate was recorded with either GM (CKPEP/PK_GM) or GMPS (CKPEP/PK_GMPS), and
AK-stimulated respiration was recorded only with GMPS (AKPEP/PK_GMPS). Fig 4A shows a
representative experimental trace from a recording of CKPEP/PK_GMPS. After addition of cardi-
omyocytes (CM) to the respiration chamber, addition of ATP to the solution (already con-
taining 20 mM creatine) stimulated CK to generate endogenous ADP. This ADP distributed
in the solution and stimulated respiration. Then, endogenous PK was stimulated by addition
of PEP and competed with mitochondrial respiration for some of the ADP. Subsequently,
exogenous PK in excess was added to the chamber. Thus, PK converted all accessible ADP in
the solution to ATP, leaving only ADP that was directly channelled from CK to mitochondria
to stimulate respiration. For AK-stimulated respiration measurements, AMP was added
(instead of creatine) after addition of ATP. Fig 4B–4D, shows the averaged results and the sta-
tistical analysis from the respiration measurements of CKPEP/PK_GM, AKPEP/PK_GMPS, and
CKPEP/PK_GMPS, respectively, normalized to the CS activity in cell suspensions from mouse
and rat hearts.
CK-stimulated respiration was significantly higher in rats than in mice irrespective of the
substrates (Fig 4B; p < 0.0001; Fig 4D; p < 0.01), in agreement with the results in Figs 1B and
3. In CKPEP/PK_GM, the addition of PEP and PK lowered respiration rate more in rat than in
mouse cardiomyocytes (by 75 ± 1% and 58 ± 1%, respectively; p < 0.001), and the respiration
rate in the presence of PEP and PK was lower in rat than in mouse (Fig 4B). In CKPEP/PK_GMPS,
the addition of PEP and PK also lowered respiration rate more in rat than in mouse cardio-
myocytes (by 62 ± 1% and 48 ± 1%, respectively; p < 0.001), as the initially higher respiration
rate in rat cardiomyocytes was lowered to the same level as in mice in the presence of PEP and
PK (Fig 4D).
In AKPEP/PK_GMPS, there was no difference between mice and rats, and the respiration rate
was lowered to the same level as before addition of AMP (Fig 4C; compare rates at ATP and
PK).
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
9 / 25
Discussion
This is the first study to show that there are differences between rat and mouse hearts in terms
of both aerobic capacity, CK isoform distribution, and intracellular compartmentalization.
When comparing the measurements in homogenate and permeabilized cardiomyocytes, we
were surprised to find that CK and, to an even greater extent, AK activities assessed in heart
homogenates were much higher than the ADP-phosphorylation rates estimated from the CK-
and AK-stimulated respiration rates of permeabilized cardiomyocytes. This is a consequence
of intracellular compartmentalization and demonstrates that results from whole-heart homog-
enates cannot be directly extrapolated to the situation in permeabilized cardiomyocytes.
Rat hearts have a lower oxidative capacity than mouse hearts
The CS activities reported in the present study were close to activities reported in other studies
[35, 45–48]. The CS activity was higher in mouse than in rat in both whole heart homogenates
and cell suspensions (Fig 1A and Table 2). Furthermore, when normalized to protein content,
VO2_max was higher in mouse than in rat cardiomyocytes (S1 Table). Thus, all three measure-
ments indicate that rat hearts have a lower oxidative capacity than mouse hearts.
Fig 4. Channeling of ADP from creatine kinase (CK) and adenylate kinase (AK) to mitochondria in
permeabilized mouse and rat cardiomyocytes. As in Fig 3, when normalized to the CS activity, CK stimulated the
respiration rate (μmol O2min-1IU CS-1) more in rat than in mouse cardiomyocytes. However, this species difference
was reversed (B) or lost (D) after addition of PEP and PK to compete with the mitochondria for endogenous ADP
from CK. A: Representative example of a respirometer recording of CKPEP/PK_GMPS with permeabilized rat
cardiomyocytes after addition of: CM–cardiomyocytes; ATP– 2 mM ATP; PEP– 5 mM PEP; PK– 20 U/ml PK. B: The
averaged results of the experiments in the presence of GM, where respiration was stimulated by CK. C and D: The
averaged results of the respiration experiments in the presence of GMPS, where respiration was stimulated by AK (C)
and CK (D). V0 was subtracted from the subsequent rates. For further explanation, see the legend of Fig 3 and the main
text. The number of animals was n = 7–8 and n = 5–7 for mice and rats, respectively. * denotes p < 0.05, ** p < 0.01,
*** p < 0.001, **** p < 0.0001 significant difference between species.
https://doi.org/10.1371/journal.pone.0294718.g004
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
10 / 25
In skeletal muscle, the oxidative capacity correlates with the CO activity [34]. However, in
the present study, the CO activity was similar in mouse and rat hearts (Fig 1A), and VO2_max
correlated with the CS activity (Fig 3). This suggests that, in heart muscle, the CS activity is a
better marker of oxidative capacity.
As the CS activity correlated with the mitochondrial oxidative capacity, and one of our
aims was to assess the rate of ADP-generation by AK and CK relative to the mitochondrial oxi-
dative capacity, we normalized the respiration data to the CS activity. As described in the
Introduction, we assumed that CS is mainly from cardiomyocytes, and that the CS in other cell
types is negligible. Furthermore, we assumed that this distribution of CS is similar in rat and
mouse heart, so that normalizing to CS allowed us to compare the reaction rates in whole
heart homogenates and isolated cardiomyocytes across the species.
Rat hearts have more Mi-CK and B-CK, and mouse hearts have more
M-CK
The CK isoform distribution was also different in mouse and rat hearts (Fig 2 and Table 3).
Mouse hearts had a larger fraction of MM-CK, whereas rat hearts had larger fractions of Mi-,
MB- and BB-CK. The large fraction of MB-CK in rat heart is consistent with results in the liter-
ature [14, 17, 49]. We are uncertain whether rat heart benefits functionally from having a
greater expression of B-CK. B-CK has a higher creatine affinity than M-CK [50], but the total
creatine content seems to be similar in rat and mouse ventricles (~80 nmol/mg protein) [49,
51]. MM-CK and BB-CK are both mainly soluble, but a fraction associates to cellular struc-
tures, and they seem to do so differently. Their N-terminal regions differ, and due to four con-
served lysine residues, MM-CK binds to the M-band, whereas BB-CK does not [52, 53].
Instead, B-CK binds to the I-band of the myofibrils [52, 54, 55]. MM-CK is also found at the I-
band of the myofibrils, but here, it is bound loosely and through phosphofructokinase of the
glycolytic pathway [56]. Both MM-CK and BB-CK are known to associate with membranes. In
muscle tissue, MM-CK associates near the sarcoendoplasmic reticulum Ca2+-ATPase
(SERCA) and the Na+/K+-ATPase [57, 58]. More recent evidence suggests that BB-CK also
locates near membrane structures, in some cases in a manner that is regulated through phos-
phorylation by AMP-activated protein kinase (AMPK), suggesting that this association may be
weaker and transient depending on the state of the cell [59]. We were unable to find informa-
tion regarding the heterodimeric MB-CK. At present, we speculate whether the differences in
CK isoform composition in rat and mouse heart relate to the different binding properties of
M- and B-CK, but this warrants further studies.
Rat hearts have a higher CK activity and larger fraction of Mi-CK, but less
ADP from CK is channelled to the mitochondria
Traditionally, the reverse CK activity is recorded in homogenates. When taking into account
temperature differences, the CK activities in rat and mouse hearts (Fig 1A) were close to and a
little higher, respectively, than reported in other studies [45, 46, 48, 60]. When normalized to
the wet weight, CKr tended to be higher and CKf activity was slightly, but significantly higher
in rat than in mouse heart (Fig 1A).
When normalized to the CS activity, the CK activities were clearly higher in rat than in
mouse heart (Fig 1B). This difference was also reflected in the experiments on permeabilized
cardiomyocytes, where stimulation of CK led to higher VO2_CK in rat than in mouse cardio-
myocytes (Fig 3B and 3D and Table 4). As expected, VO2_max was substrate dependent with
VO2_max_GM being lower than VO2_max_GMPS (compare Fig 3B and 3D; Table 4). This has also
been shown before [35, 36]. As a result, VO2_CK/VO2_max was also substrate dependent
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
11 / 25
(Table 4; effect of substrate), and consistently higher in rat than in mouse heart (Table 4; effect
of species).
Although rat cardiomyocytes had a higher VO2_CK/VO2_max, the subsequent addition of
PEP and PK to competitively inhibit the flux of ADP from CK to mitochondria lowered the
respiration rate more in rat than in mouse cardiomyocytes. In the presence of PEP and PK, the
respiration rate was similar (CKPEP/PK_GMPS) or slightly lower (CKPEP/PK_GM) in rats than in
mice (Fig 4B and 4D). This suggests that in mouse hearts, a larger fraction of ADP generated
by CK was channelled to the mitochondria. However, the fraction of Mi-CK was lower in
mouse than in rat hearts (Fig 2 and Table 3). It may seem contradictory that mouse cardio-
myocytes have less Mi-CK and greater ADP channelling to the mitochondria. However, this
can be explained by differences in the intracellular compartmentalization. The greater
channelling of ADP from CK to mitochondria in mouse heart could be due to a tighter cou-
pling of cytosolic CK as well as Mi-CK to the respiration.
As to Mi-CK, in isolated mitochondria, approximately half of the ADP generated by Mi-CK
is translocated by the ANT into the matrix, while the rest diffuses out through the voltage
dependent anion channel (VDAC) in the outer mitochondrial membrane [6, 61]. In vivo and
in permeabilized cardiomyocytes, the mitochondrial outer membrane permeability is more
restricted [62]. This, in turn, is expected to increase the channeling between Mi-CK and ANT.
Thus, it is possible that there is more direct transfer between Mi-CK and ANT or the outer
mitochondrial membrane is less permeable to ADP in mouse cardiomyocytes, so more ADP
from Mi-CK cycles within the mitochondria and is inaccessible to PK.
As to cytosolic CK, it was shown in Mi-CK knockout mice that cytosolic CK can also be
coupled to respiration [63] possibly due to intracellular diffusion barriers in the cytosol, which
can group CK and mitochondria [39, 64–67]. The extent of channelling we observe between
cytosolic CK and the mitochondria depends on the interplay between cytosolic CK, PK, and
diffusion barriers. It is possible that in mouse cardiomyocytes, a larger fraction of cytosolic CK
is on the mitochondrial side of the diffusion barriers, or the diffusion barriers are less perme-
able, so more ADP cycles between CK and the mitochondria and is inaccessible to PK.
If the cytosolic diffusion barriers are less permeable to ADP, then they are presumably also
less permeable to the diffusion of proteins. This is a relevant point for the present experiments
on permeabilized cardiomyocytes, which do not necessarily reflect the situation in vivo,
because some cytosolic CK and PK may diffuse out of the cells, and also some exogenous PK
diffuses into the cell. The diffusion of CK out of the cardiomyocytes does not on its own inhibit
respiration, because CK continues to generate ADP in the solution. However, it may lead to an
underestimation of the overall coupling between all CK isoforms and respiration. Thus, if the
cytosolic diffusion barriers are less permeable in mouse cardiomyocytes, it is also possible that
the species differences in the PEP-PK assay are caused in part by the differences in CK and PK
diffusion between solution and cardiomyocytes. Clearly, further studies are needed to pinpoint
the exact mechanism behind the different outcomes of the PEP-PK assay. Nevertheless, our
results suggest that compartmentalization and/or energy transfer is different in rat and mouse
cardiomyocytes.
The lowering of respiration rate by PEP and PK in mouse cardiomyocytes is in agreement
with our previous finding [21]. With the present study, we extend this finding to rat cardio-
myocytes. However, our results contradict the findings from another group, who found on rat
cardiomyocytes that with GM as substrates CK-stimulated respiration rate was similar to
VO2_max even in the presence of PEP and PK [22, 23]. We speculated whether this difference
between studies could be because the cardiomyocytes in the other study had damaged mito-
chondria. If the mitochondria are damaged, VO2_max will be very low and VO2_CK/VO2_max will
be high. In order to compare our data with those of others, we also normalized our maximal
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
12 / 25
respiration rate in rat cardiomyocytes to the Cyt aa3 content (S1 Table, VO2_max/cyt aa3). In the
present study, VO2_max_GM was ~35% higher than reported by others (273 ± 19 versus 178 ± 34
nmol O2min-1nmol cyt aa3
-1) [68]. However, in rat cardiomyocytes in the present study, the
addition of PEP and PK lowered the respiration rate by 75% (Fig 4B). As the difference in
VO2_max between studies was smaller than the lowering of respiration rate by PEP and PK, the
higher VO2_max in the present study can only partially explain the difference between the stud-
ies. In the present study, the cell viability was acceptable (Table 2), the addition of Cyt c had no
effect on respiration rate (Fig 3B–3D), and the coupling efficiency of respiration was high.
Therefore, we are confident that our preparation was sound.
Taken together, our study confirms that intracellular compartmentalization leads to a local
pool of phosphates circulating between mitochondria and CK and suggests that this is more
predominant in mouse than in rat cardiomyocytes. It must be noted that these experiments
were performed on isolated, non-contracting cardiomyocytes, and it is uncertain how defor-
mation of the cells during contraction affects the compartmentalization.
The rate of ADP-generation by CK is lower than the maximal rate of ADP-
consumption by the mitochondria
As noted above, rat hearts had a higher VO2_CK/VO2_max than mouse hearts. According to our
data, mitochondrial and cytosolic CK stimulated respiration rate to 90% of the maximal rate
with GMPS in rat (Fig 3D and Table 4). This is far from the finding on isolated rat hearts that
the CK reaction rate is 10 times higher than the maximal respiration rate [4, 5], but it is in
agreement with a previous study [69]. This may explain why the CK shuttle is bypassed under
extreme workloads [69], and why studies on transgenic mice with disturbances in the CK sys-
tem have been equivocal regarding the importance of the CK system [11].
Rat and mouse hearts have similar VO2_AK/VO2_max
In homogenates, the AK activity normalized to the wet weight was ~30% higher in mouse than
in rat hearts, measured in both directions (Fig 1A). In mouse hearts, the AK activity was ~1.5–
2 times higher than reported previously [21, 45, 47], whereas in rat hearts, it was only slightly
higher than in another study [46]. In contrast to our previous study on creatine-deficient mice,
where AK had the highest activity of the kinases [21], the present results showed that the AK
activity was lower than the CK activity in both mouse and rat hearts, when measured in the
direction of ATP-production. The AK activities were similar in the forward and reverse direc-
tions, which is compatible with another study [70], showing that the AKf/AKr ratio is ~1.3.
When the AK activity was normalized to the CS activity in homogenates, it was similar in
mouse and rat heart (Fig 1B). In agreement with this, there was no difference between rat and
mouse in the respiration experiments (Fig 3C). However, relative to the maximal respiration
rate, VO2_AK/VO2_max was slightly higher in mouse than in rat hearts (Table 4), but below 80%.
In our previous experiments, AK stimulated respiration to the maximum, but they were per-
formed with only GM as substrates [21]. In the present study, AK-stimulated respiration mea-
surements were performed only with GMPS, and as VO2_max is higher with GMPS than with
GM, it was not surprising, that VO2_AK/VO2_max was lower than recorded with GM, as was also
the case for CK (Fig 3B and 3D, Table 4).
No channelling of ADP from AK to the mitochondria
When the flux of ADP from AK to the mitochondria was inhibited by the subsequent addition
of PEP and PK, the respiration rate was lowered to the same level as before the addition of
AMP in both rat and mouse cardiomyocytes (Fig 4C). This was similar to our previous results
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
13 / 25
on mouse cardiomyocytes [21], and in agreement with the low expression of AK2 in the
mouse heart [32], but in contrast to another study on isolated rat heart mitochondria [27]. We
hypothesize that methodological differences could cause this discrepancy between studies. The
present study suggests that the majority of AK is cytosolic in both rat and mouse hearts.
AK and CK activities in homogenate are higher than estimated VADP_AK
and VADP_CK in permeabilized cardiomyocytes
We estimated the rates of ADP-generation by CK and AK in permeabilized cardiomyocytes
(VADP_CK and VADP_AK, Table 5) by multiplying the respiration rates with the P/O2 ratios (4
for GMPS and 6 for GM). This represents their forward reaction rates, and we had expected
that they would be similar to the forward reaction rates recorded in homogenate (CKf and
AKf, Fig 1B). Surprisingly, we found that the rates estimated from permeabilized cardiomyo-
cytes were much lower than in homogenate. CKf was ~2 times higher and AKf was ~9 times
higher than VADP_CK and VADP_AK, respectively. This difference is highlighted in Fig 5. Our
finding suggests that in permeabilized cardiomyocytes with the intracellular structures left
intact, local substrate and product concentrations in the vicinity of an enzyme can be very dif-
ferent from the concentrations in solution. One factor is that diffusion of substrates into the
permeabilized cardiomyocytes is restricted so the substrate concentrations could be smaller
Fig 5. Intracellular compartmentalization shapes energy transfer in cardiomyocytes. In permeabilized
cardiomyocytes (left panel; data from Table 5, units converted to μmol ADPmin-1IU CS-1), diffusion of substrates to
the center of the cell is restricted. Thus, kinases in the center of the cell may be exposed to smaller concentrations of
substrates than are present in the surrounding solution. More importantly, as diffusion out of the cell is restricted, the
products accumulate near the kinase and inhibit the reaction rate. In contrast, when recording the kinase activity in
homogenate (right panel; data from Fig 1), the kinases are in solution, where diffusion is much faster. Thus, there is no
build-up of products, and the reaction takes place without inhibition.
https://doi.org/10.1371/journal.pone.0294718.g005
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
14 / 25
than in solution. More importantly, the diffusion of products out of the permeabilized cardio-
myocytes is restricted, so the products accumulate near the kinases, inhibiting the reaction
rate. We have illustrated this in Fig 5.
The restriction of diffusion in cardiomyocytes has been studied for several decades. In car-
diomyocytes, intracellular membranes such as the transverse tubules, the sarcoplasmic reticu-
lum, and the mitochondria, together with protein dense parts of the sarcomeres constitute
barriers that form modules [64, 65]. These modules influence energy transfer within the cell
[39]. Local environments are also known to play a crucial role in cAMP signalling [71], excita-
tion-contraction coupling [72] and mitochondrial calcium uptake [73, 74]. In the present
study, we were surprised to see that compartmentalization had such a large effect on the AK
activity. This, in turn, is likely to affect not only ATPase function, but also energetic signalling
through, for example, AMPK, which is a cellular energy sensor implicated in both acute signal-
ling and regulation of gene expression [75]. Our results highlight the challenge for computa-
tional models, which should take into account the intracellular heterogeneity of substrate and
product concentrations and the spatial limitations of their diffusion inside the cell [62, 64, 65].
Conclusions
In the present study, we found species differences between mouse and rat hearts. Rat hearts
had a lower oxidative capacity than mouse hearts. As a result, CK/CS and VO2_CK/VO2_max
were higher in rat than in mouse, and the distribution of CK isoforms was different. In rat
heart, although VO2_CK/VO2_max and the fraction of Mi-CK was higher than in mouse heart,
less ADP was channelled from CK to the mitochondria. This suggests differences in the
compartmentalization of mouse and rat cardiomyocytes.
An interesting finding of this study was that AK/CS activity in whole tissue homogenates
was several times higher than the VADP_AK estimated from the respiration rate in isolated per-
meabilized cardiomyocytes. This difference is a consequence of intracellular compartmentali-
zation. Our results highlight how intracellular structural organization shapes energetic
compartmentalization, which plays a pivotal role in energy homeostasis, signalling, and regula-
tion of cardiac metabolism.
Materials and methods
All experiments and animal procedures complied with directive 2010/63/EU of the European
Parliament for the protection of animals used for scientific purposes and were approved by the
Project Authorisation Committee for Animal Experiments in the Estonian Ministry of Rural
Affairs. All methods are reported in accordance with ARRIVE guidelines.
Animals
The animals used in this study were 7–10 months for mice and 10–12 months for rats. Due to
the low quality of cardiomyocytes isolated from male rat hearts, only females were used in this
study. Sprague-Dawley rats were a gift from the Laboratory of Neurobiology at Tallinn Univer-
sity of Technology. C57BL/6J Ola Hsd mice were originally from Envigo RMS B.V. (The Neth-
erlands). The animals were kept in the animal facility of Tallinn University of Technology at
an ambient temperature of 22–22.8˚C and a 12:12 hours light:dark cycle. They had free access
to water and food (V1534-000 Rat/mouse maintenance from Ssniff Spezialdia¨ten GmbH,
Germany).
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
15 / 25
Isolation of cardiomyocytes
Cardiomyocytes from mouse [15] and rat [76] hearts were isolated using a slightly modified
version of a method described previously. The mice were anesthetized with a mixture of keta-
mine/dexmedetomidine (150 mg/kg and 0.5mg/kg, respectively) and received an injection of
250U of heparin to prevent blood coagulation. When the toe-pinch reflex was absent, the ani-
mal was euthanized by cervical dislocation. The rats were anesthetized with 2% isoflurane
using Open-Drop system (or Drop Jar method) (https://animal.research.uiowa.edu/iacuc-
guidelines-anesthesia) [77]. Briefly, the rats were placed in a closed container of known volume
(~5l) with tightly fitting lid, a gauze pad soaked with appropriate volume (~0.5 ml) of isoflur-
ane was placed in the bottom of the container and the animals were left to inhale isoflurane
vapours. When they lost the righting reflex and breathing had slowed but was regular, 2500U
of heparin was injected intraperitoneally and they were allowed to sleep a little bit more.
Under deep isoflurane anaesthesia, the rats were euthanized by decapitation. The hearts of
both rat and mouse were excised and immediately placed in ice-cold wash solution consisting
of the following (mM): 117 NaCl, 5.7 KCl, 1.5 KH2PO4, 4.4 NaHCO3, 1.7 MgCl2, 21 HEPES,
20 taurine, 11.7 glucose, and 10 2,3-butanedione monoxime (pH was adjusted to 7.4 with
NaOH). It was cannulated via the aorta on a Langendorff perfusion system. The heart was first
perfused with wash solution at 38.5˚C at a constant pressure of 80 cm H2O. When the heart
was washed free of blood, the perfusion was switched to a constant flow with digestion solution
containing 0.37–0.435 mg/ml Liberase DL (Roche) and 1.36 mg/ml of dispase II (Roche). The
pressure was observed for 10–15 minutes or ~30 min (mouse and rat heart, respectively) until
the pressure had decreased to 40–50% of the initial. When the heart was soft, the perfusion was
stopped. The ventricles were cut into smaller pieces, transferred to a beaker with digestion
solution and incubated further at 38.5˚C with gentle shaking until the tissue started falling
apart. Cells were harvested with a Pasteur pipette several times and filtered through a 100μm
cell strainer (EASYstrainerTM Cell Strainer, Greiner Bio-One) into a vial with sedimentation
solution consisting of wash solution (without 2,3-butanedione monoxime) containing addi-
tional 2mM pyruvate, 10 μM leupeptin, 2 μM soybean trypsin inhibitor, and 3 mgmL-1 BSA.
The viable cells were separated by sedimentation or by centrifugation for 2 min at 300 rpm/
12g. During the first washes, extracellular Ca2+ was gradually increased to 2 mM to ensure
Ca2+ tolerance of the cells. Then, extracellular Ca2+ was washed out again by washing the cells
three times with 5–8 ml of sedimentation solution. The isolated cells were stored in this solu-
tion at room temperature until use within 3 hours.
To assess the quality of the cell preparation, the yield of the cell suspension was measured
with a 1000 or 5000 μl pipette (Eppendorf), and a 1:10 dilution of the cells was counted in a
chamber to estimate the total number of cells as well as the viability (the percentage of live cells
relative to the total number of cells).
Respiration measurements
For the respiration experiments, we used protocols similar to those described previously [21,
35]. In brief, we used a Strathkelvin RC 650 Respirometer equipped with six 1302 O2-elec-
trodes connected via a 929 Oxygen System interface (all from Strathkelvin Instruments Lim-
ited, UK). The respirometer was thermostatted to 25˚C (Julabo F12-ED, JULABO
Labortechnik GmbH). The respiration measurements in cardiomyocytes were performed in 2
ml of respiration solution consisting of 110 mM sucrose, 60 mM K-lactobionic acid, 3 mM
KH2PO4, 3 mM MgCl2, 20 mM HEPES, 20 mM taurine, 0.5 mM EGTA, 0.5 mM dithiothreitol
(DTT) (pH was adjusted to 7.1 with KOH). 5 mgmL-1 BSA and 25 μgmL-1 saponin were
added just before use. Saponin was present in the respiration chamber throughout the
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
16 / 25
measurements. It interacts with cholesterol to form pores in the sarcolemma, which contains
90% of the cellular cholesterol [78]. At this concentration, saponin does not damage the mito-
chondrial membranes, which have a very low cholesterol content [79]. CK experiments were
performed in the presence of 2.5 mM glutamate and 2 mM malate only (CKGM) and in the
presence of 2.5 mM glutamate, 2 mM malate, 5 mM pyruvate and 15 mM succinate
(CKGMPS). AK experiments were carried out only in the presence of 2.5 mM glutamate, 2
mM malate, 5 mM pyruvate and 15 mM succinate (AKGMPS).
We used CK and AK protocols similar to those described in [21]. First, 5 ul of cell suspen-
sion, for CKGMPS and AKGMPS, and 10 ul for CKGM were added to the respiration chambers.
Cells were allowed at least 5 min to permeabilize before the steady-state basal respiration rate,
Vo, was recorded. After that, we stimulated AK by adding 2 mM ATP and 1 mM AMP, or CK
by adding 2 mM ATP while 20 mM creatine was already present in the respirometer chamber
before the cells were added.
In the chambers, where we recorded CKPEP/PK_GM, CKPEP/PK_GMPS, and AKPEP/PK_GMPS,
this was followed by the addition of 5 mM PEP and 20 U/ml exogenous PK.
In the parallel chambers, where we recorded CKADP_GM, CKADP_GMPS, and AKADP_GMPS,
this was followed by the addition of 2 mM ADP, 10 μM Cyt c and stepwise titration of FCCP
in 2.5 μM steps until the maximum uncoupled respiration rate, VFCCP, was reached.
As a reference, respiration rate measured at 2 mM ADP was taken as the maximal coupled
respiration rate, VO2_max. This measurement was used to estimate AK and CK stimulated res-
piration rates (VO2_AK and VO2_CK, respectively) relative to VO2_max (VO2_CK/VO2_max and
VO2_AK/VO2_max), and to calculate the coupling efficiency of respiration as follows: (VO2_max−-
V0)/VO2_max [37]. This allowed us to determine the oxidative phosphorylation control ratio,
defined as: VO2_max/VFCCP [37].
Homogenization
Cardiac homogenates were prepared as in Barsunova et al. [80]. The mice and rats were anes-
thetized and killed as described above for the isolation of cardiomyocytes. The heart was
quickly removed from animals and immediately transferred to a glass beaker with ice-cold iso-
lation solution. The heart was trimmed of any obvious fat and connective tissue, gently blotted
to remove excess fluids, weighed, cut into several pieces if needed (for rat heart), and then
stored in cryovials at -80˚C until further experiments. All subsequent homogenization proce-
dures were carried out on ice. The heart tissue was minced with scissors into small pieces,
transferred to a glass homogenizer, and ice-cold homogenization buffer was added to a con-
centration of 50 mg tissue/ml buffer. The buffer consisted of 5 mM HEPES, 1mM EGTA, 0.1%
Triton X-100, 1 mM DL-Dithiothreitol, and 1 tablet of cOmplete Mini Protease inhibitors per
10 ml buffer (Roche, Merck) (pH 8.7). Next, the heart tissue was ground with a pestle attached
to a drill until the solution was homogenous. The homogenized samples were incubated on ice
for one hour before use. Fresh, non-diluted homogenates were use to measure CO activity.
The remaining homogenates were kept at -80˚C until activities of CS, CK and AK were
measured.
Enzyme activities
Enzyme activities were recorded using Evolution 600 spectrophotometer (Thermo Fisher Sci-
entific) equipped with a Peltier water-cooled cell changer (SPE 8 W, Thermo Fisher Scientific)
to maintain temperature at 25˚C.
CO and CS activities were determined as described earlier [80]. CO activity was deter-
mined by measuring the decrease in absorbance, caused by oxidation of Cyt c by cytochrome
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
17 / 25
oxidase, at 550 nm [81]. The reaction took place in 1 ml 13 mM sodium phosphate buffer
(pH 7.4) containing 0.4 mg/ml Cyt c, which had been reduced with Na-dithionite. After
recording the initial absorbance for 10–20 seconds, the reaction was initiated by the addition
of 10 μl of undiluted homogenate. The reaction of Cyt c oxidation represents a first-order
reaction with respect to reduced Cyt c and is observed as a logarithmical decline in absorp-
tion (as a function of time). The rate constant, obtained by fitting to the equation δ(Absorp-
tion) / δ(time) = k (Absorption), was normalized to the tissue wet weight, min−1g−1. The
IOCBIO Kinetics software for fitting is open source and available at https://iocbio.gitlab.io/
kinetics.
CS activity was recorded using a coupled enzyme assay in a total volume of 1 mL CS buffer
containing the following (in mM): 100 TrisHCl buffer (pH 8.1), 0.1 5,5’ -dithiobis(2-nitroben-
zoic acid) (DTNB), and 0.3 acetyl-CoA. The assay was started by the addition of 10 μL or 15–
20 μL of diluted (1:10) cell suspension or heart homogenate, respectively. The change in absor-
bance was recorded for 2 min at 412 nm before (for reference) and after addition of 0.5 mM
oxaloacetate. The enzyme activity was calculated using the extinction coefficient for thionitro-
benzoate (TNB), which is 14150 M−1 cm−1 at 25˚C [82].
The activities of CK and AK in the reverse direction (ATP-production) were measured in a
coupled enzyme assay in a total volume of 1ml respiration buffer (without BSA and saponin)
(see composition in Respiration measurements section) containing the following: 10 mM glu-
cose, 0.6 mM NADP, 2 mM ADP, 5 Uml-1 hexokinase and 5 Uml-1 glucose-6-phosphate
dehydrogenase. The reaction was initiated by the addition of 3–5 ul of diluted (1:10) heart
homogenate. The increase in absorbance was measured for 3 min at 340 nm before (AK activ-
ity) and after addition of 10 mM creatine phosphate (CK activity + AK activity). In the second
run, 50 uM P1,P5-Di(adenosine-5’)pentaphosphate was added to the buffer to inhibit AK. The
absorbance was measured for 3 min at 340 nm before (to verify AK inhibition) and after addi-
tion of 10 mM creatine phosphate (CK activity).
The activities of CK and AK in the forward direction (ATP-consumption) were measured
in a total volume of 1ml respiration buffer containing the following: 5 mM PEP, 2 mM ATP,
30 mM NADH, 5 U/ml PK, 2.5 U/ml lactate dehydrogenase. First, the absorbance was mea-
sured for 3 min at 340 nm with buffer to check whether the response is stable (to record the
absorbance shift). Then 10 ul of diluted (1:10) heart homogenate was added (non-specific
ATPase activity). Finally, the decrease in absorbance was measured after addition of 20 mM
creatine (CK activity) or 1 mM AMP (AK activity).
The activities of CK and AK in both directions were measured with conditions similar to
those used in respiration measurements and were calculated using the extinction coefficient
for NADH/NADPH (ε340 = 6.220 mM−1 cm−1).
All enzyme activity measurements were performed in triplicate (in quadruplicate for CO)
and the results averaged. Data was analysed using IOCBIO Kinetics [83].
Determination of CK isoforms
The rat and mouse homogenates were also used to determine the CK isoform distribution as
previously described [84]. The CK isoforms were separated by native agarose gel electrophore-
sis on a 1% agarose gel of approximately 1 mm thickness. The gel was transferred to a ceramic
plate, which in turn was placed upon a cooling pad maintained at 15˚C by a thermostat (Julabo
F12-ED, JULABO Labortechnik GmbH). A small strip of filter paper was used to mark the
row of loading spots about 1/3 from the anode. Drops of 1 μl homogenate (corresponding to
tissue extract from 50 μg heart wet weight) were put on each loading spot with rat and mouse
samples put one after another. Electrophoresis buffer was added to the anodic and cathodic
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
18 / 25
chamber, and wicks consisting of 4 layers of filter paper were used to connect the gel to the
buffer in the two chambers. The gel was run at 250 V for 120 min.
The gel was transferred to the imaging chamber of an ImageQuant 400 (GE Healthcare Life
Sciences). A filter paper the size of the gel was soaked in 5 ml visualization buffer and put on
top of the gel. As the solution from the filter paper entered the gel, CK in the gel catalyzed the
first of a chain of reactions: phosphocreatine + ADP + H+ ! creatine + ATP. ATP from this
reaction was used by hexokinase in the reaction: ATP + glucose ! ADP + glucose-6-phos-
phate. Finally, glucose-6-phosphate was used by glucose-6-phosphate-dehydrogenase in the
reaction: glucose-6-phosphate + NADP+ ! 6-phospho-D-glucono-1,5-lactone + H+
+ NADPH. The increase in NADPH from the assay coupled to CK was followed by transillu-
mination with UV-light and image capture with a SYBR Green filter. After ~20 min, the filter
paper was carefully removed to image the NADPH signal in the gel. The gel pictures were ana-
lyzed using ImageJ software. Each lane was marked with a rectangle, and the area of each
intensity profile peak corresponding to Mi-CK, MM-CK, MB-CK and BB-CK was noted. The
relative intensity of each isoform was calculated.
The gel electrophoresis buffer consisted of (in mM): Tris 60, Tricine 60, EGTA 1, dithiotrei-
tol 1, Triton X-100 0.1%, pH 8.6.
The visualization buffer consisted of (in mM): N-Acetyl-L-cysteine 120, phosphocreatine
120, glucose 70, MgAcetate 50, MES 22, ADP 9, β-Nicotinamide adenine dinucleotide phos-
phate 9, P1P5-Di(adenosine-50) pentaphosphate pentasodium salt 0.2, pH 7.4. Immediately
before use, hexokinase and glucose-6-phosphate dehydrogenase were both added to the visual-
ization buffer to a final concentration of 5 IU/ml.
Determination of Cyt aa3 content
The Cyt aa3 content in isolated rat cardiomyocytes was determined using a spectroscopic
method independent of myoglobin contamination. This assay relies on the selective reduction
of mitochondrial cytochromes by the action of sodium cyanide [85]. The rat cardiomyocytes
were solubilized with 5% TritonX-100 in 0.1M potassium phosphate buffer (pH 7.5). The first
graph (oxidized cytochromes) was obtained by scanning from 500 to 650 nm using an Evolu-
tion 600 spectrophotometer (Thermo Fisher Scientific Inc.) equipped with a Peltier water
cooled cell changer (SPE 8 W; Thermo Fisher Scientific Inc.) to maintain temperature at 25˚C.
The second graph (reduced cytochromes) was obtained the same way after reduction with 2
mM sodium cyanide in the presence of 15 mM ascorbic acid. The differential absorbance
(reduced versus oxidized cytochromes) at 605 and 630 nm was used for quantification of respi-
ratory chain Cyt aa3 content (cytochrome c oxidase), using the extinction coefficient
ε605 = 18.6 mM-1 cm-1 [86].
Normalization
For better comparison of our results to the findings of earlier studies, the respiration rates
from permeabilized cardiomyocytes were normalized to protein content, Cyt aa3 content (rat
cardiomyocytes only), and CS activity. Enzyme activities in homogenates were normalized to
wet weight and CS activity. The protein content in cardiomyocytes was measured spectropho-
tometrically in a BioSpec-nano (Shimadzu Scientific Instruments Inc., Columbia, MD) as pre-
viously described [21].
Statistics
The values are shown as mean ± standard error of the mean (SEM). Statistical analysis of most
data was performed using unpaired Student’s t-test using R. However, the difference between
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
19 / 25
VO2_CK and VO2_max, and VO2_AK and VO2_max was assessed using a paired t-test. Furthermore,
for the comparison of VO2 rates and VADP rates (Tables 4 and 5, respectively), the effect of spe-
cies and substrate was analysed by a mixed type ANOVA. p < 0.05 was considered statistically
significant.
The raw data are given in the S2 Table.
Supporting information
S1 Table. Maximal respiration rate, VO2_max, and maximal ADP-phosphorylation rate,
VADP_max, normalized to the protein content of the cell suspensions, and VO2_max, normal-
ized to the cytochrome aa3 content. The maximal respiration rate, VO2_max, was recorded in
the presence of either GM or GMPS, and 2 mM ADP (see representative recording in Fig 3A).
The rate was normalized to the protein content and, for rat cardiomyocytes only, the cyto-
chrome aa3 content. For statistical purposes, only the results from CK recordings are given.
The corresponding maximal ADP-phosphorylation rate, VADP_max, was calculated assuming
P/O2 ratios of 6 and 4 for GM and GMPS, respectively (see main text). Values from 8 mice and
7 rats are shown as mean ± SEM. * denotes p < 0.05, ** p < 0.01, *** p < 0.001, ****
p < 0.0001, significant effect of substrate, species, or interaction between substrates and spe-
cies.
(DOCX)
S2 Table. Raw data from the experiments.
(ODS)
S1 Raw images.
(PDF)
Author Contributions
Conceptualization: Rikke Birkedal.
Formal analysis: Jelena Branovets, Marko Vendelin, Rikke Birkedal.
Funding acquisition: Marko Vendelin, Rikke Birkedal.
Investigation: Jelena Branovets, Ka¨rol Soodla, Rikke Birkedal.
Methodology: Jelena Branovets, Rikke Birkedal.
Supervision: Rikke Birkedal.
Visualization: Jelena Branovets, Marko Vendelin.
Writing – original draft: Jelena Branovets, Rikke Birkedal.
Writing – review & editing: Jelena Branovets, Marko Vendelin, Rikke Birkedal.
References
1.
Bessman SP, Carpenter CL. The creatine-creatine phosphate energy shuttle. AnnuRevBiochem. 1985;
54:831–62. https://doi.org/10.1146/annurev.bi.54.070185.004151 PMID: 3896131
2.
Wallimann T, Wyss M, Brdiczka D, Nicolay K, Eppenberger HM. Intracellular compartmentation, struc-
ture and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands:
the “phosphocreatine circuit” for cellular energy homeostasis. BiochemJ. 1992 Jan; 281 (Pt 1):21–40.
https://doi.org/10.1042/bj2810021 PMID: 1731757
3.
Ingwall JS. ATP and the Heart: An Overview. In: Ingwall JS, editor. ATP and the Heart. Boston, MA:
Springer US; 2002. p. 3–6. (Basic Science for the Cardiologist). https://doi.org/10.1007/978-1-4615-
1093-2_1
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
20 / 25
4.
Bittl JA, Ingwall JS. Reaction rates of creatine kinase and ATP synthesis in the isolated rat heart. A 31P
NMR magnetization transfer study. J Biol Chem. 1985 Mar; 260(6):3512–7. PMID: 3972835
5.
Katz LA, Swain JA, Portman MA, Balaban RS. Relation between phosphate metabolites and oxygen
consumption of heart in vivo. Am J Physiol. 1989; 256(1 Pt 2):H265–74. https://doi.org/10.1152/
ajpheart.1989.256.1.H265 PMID: 2912189
6.
Vendelin M, Lemba M, Saks VA. Analysis of Functional Coupling: Mitochondrial Creatine Kinase and
Adenine Nucleotide Translocase. Biophys J. 2004 Jul; 87(1):696–713. https://doi.org/10.1529/biophysj.
103.036210 PMID: 15240503
7.
Gellerich FN, Schlame M, Bohnensack R, Kunz W. Dynamic compartmentation of adenine nucleotides
in the mitochondrial intermembrane space of rat-heart mitochondria. Biochimica et Biophysica Acta
(BBA)—Bioenergetics. 1987 Feb 11; 890(2):117–26. https://doi.org/10.1016/0005-2728(87)90012-0
PMID: 3801462
8.
Saks VA, Khuchua ZA, Vasilyeva EV, Belikova OY, Kuznetsov AV. Metabolic compartmentation and
substrate channelling in muscle cells. Role of coupled creatine kinases in in vivo regulation of cellular
respiration–a synthesis. MolCell Biochem. 1994 Apr; 133–134:155–92. https://doi.org/10.1007/
BF01267954 PMID: 7808453
9.
Dzeja PP, Terzic A. Phosphotransfer networks and cellular energetics. JExpBiol. 2003 Jun; 206(Pt
12):2039–47. https://doi.org/10.1242/jeb.00426 PMID: 12756286
10.
Joubert F, Mazet JL, Mateo P, Hoerter JA. 31P {NMR} detection of subcellular creatine kinase fluxes in
the perfused rat heart: contractility modifies energy transfer pathways. J Biol Chem. 2002 May 24; 277
(21):18469–76. https://doi.org/10.1074/jbc.M200792200 PMID: 11886866
11.
Lygate CA, Neubauer S. The Myocardial Creatine Kinase System in the Normal, Ischaemic and Failing
Heart. In: Lopaschuk GD, Dhalla NS, editors. Cardiac Energy Metabolism in Health and Disease. New
York, NY: Springer; 2014. p. 155–68. (Advances in Biochemistry in Health and Disease). https://doi.
org/10.1007/978-1-4939-1227-8_10
12.
Hove M ten Lygate CA, Fischer A Schneider JE, Sang AE, Hulbert K, et al. Reduced inotropic reserve
and increased susceptibility to cardiac ischemia/reperfusion injury in phosphocreatine-deficient guanidi-
noacetate-N-methyltransferase-knockout mice. Circulation. 2005 May; 111(19):2477–85. https://doi.
org/10.1161/01.CIR.0000165147.99592.01 PMID: 15883212
13.
Nahrendorf M, Streif JU, Hiller KH, Hu K, Nordbeck P, Ritter O, et al. Multimodal functional cardiac MRI
in creatine kinase-deficient mice reveals subtle abnormalities in myocardial perfusion and mechanics.
Am J Physiol Heart Circ Physiol. 2006 Jun 1; 290(6):H2516–21. https://doi.org/10.1152/ajpheart.
01038.2005 PMID: 16415075
14.
Faller KME, Atzler D, McAndrew DJ, Zervou S, Whittington HJ, Simon JN, et al. Impaired cardiac con-
tractile function in arginine:glycine amidinotransferase knockout mice devoid of creatine is rescued by
homoarginine but not creatine. Cardiovasc Res. 2018 Mar 1; 114(3):417–30. https://doi.org/10.1093/
cvr/cvx242 PMID: 29236952
15.
Branovets J, Sepp M, Kotlyarova S, Jepihhina N, Sokolova N, Aksentijevic D, et al. Unchanged mito-
chondrial organization and compartmentation of high-energy phosphates in creatine deficient GAMT-/-
mouse heart. Am J Physiol Heart Circ Physiol. 2013 Jun 21; Available from: http://ajpheart.physiology.
org/content/early/2013/06/17/ajpheart.00919.2012
16.
Aksentijević D, Zervou S, Eykyn TR, McAndrew DJ, Wallis J, Schneider JE, et al. Age-Dependent
Decline in Cardiac Function in Guanidinoacetate-N-Methyltransferase Knockout Mice. Front Physiol.
2020 Jan 21; 10. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6985570/ PMID:
32038270
17.
Lygate CA, Aksentijevic D, Dawson D, Hove M ten, Phillips D, de Bono JP, et al. Living Without Creatine
Unchanged Exercise Capacity and Response to Chronic Myocardial Infarction in Creatine-Deficient
Mice. Circulation Research. 2013 Mar 15; 112(6):945–55. https://doi.org/10.1161/CIRCRESAHA.112.
300725 PMID: 23325497
18.
Nahrendorf M, Spindler M, Hu K, Bauer L, Ritter O, Nordbeck P, et al. Creatine kinase knockout mice
show left ventricular hypertrophy and dilatation, but unaltered remodeling post-myocardial infarction.
Cardiovascular Research. 2005 Feb 1; 65(2):419–27. https://doi.org/10.1016/j.cardiores.2004.10.006
PMID: 15639481
19.
Choe C un, Nabuurs C, Stockebrand MC, Neu A, Nunes P, Morellini F, et al. l-arginine:glycine amidino-
transferase deficiency protects from metabolic syndrome. Human Molecular Genetics. 2013 Jan 1; 22
(1):110–23. https://doi.org/10.1093/hmg/dds407 PMID: 23026748
20.
Schmidt A, Marescau B, Boehm EA, Renema WKJ, Peco R, Das A, et al. Severely altered guanidino
compound levels, disturbed body weight homeostasis and impaired fertility in a mouse model of guanidi-
noacetate N-methyltransferase (GAMT) deficiency. Hum Mol Genet. 2004 May 1; 13(9):905–21.
https://doi.org/10.1093/hmg/ddh112 PMID: 15028668
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
21 / 25
21.
Branovets J, Karro N, Barsunova K, Laasmaa M, Lygate CA, Vendelin M, et al. Cardiac expression and
location of hexokinase changes in a mouse model of pure creatine deficiency. American Journal of
Physiology-Heart and Circulatory Physiology. 2020 Dec 18; 320(2):H613–29. https://doi.org/10.1152/
ajpheart.00188.2020 PMID: 33337958
22.
Guzun R, Timohhina N, Tepp K, Monge C, Kaambre T, Sikk P, et al. Regulation of respiration controlled
by mitochondrial creatine kinase in permeabilized cardiac cells in situ: Importance of system level prop-
erties. Biochimica et Biophysica Acta (BBA)—Bioenergetics. 2009 Sep 1; 1787(9):1089–105.
23.
Tepp K, Shevchuk I, Chekulayev V, Timohhina N, Kuznetsov AV, Guzun R, et al. High efficiency of
energy flux controls within mitochondrial interactosome in cardiac intracellular energetic units. Biochi-
mica et Biophysica Acta (BBA)—Bioenergetics. 2011 Dec; 1807(12):1549–61. https://doi.org/10.1016/j.
bbabio.2011.08.005 PMID: 21872567
24.
Laterveer FD, Nicolay K, Gellerich FN. Experimental evidence for dynamic compartmentation of ADP at
the mitochondrial periphery: coupling of mitochondrial adenylate kinase and mitochondrial hexokinase
with oxidative phosphorylation under conditions mimicking the intracellular colloid osmotic pressure.
Mol Cell Biochem. 1997 Sep; 174(1–2):43–51. PMID: 9309664
25.
Dzeja PP, Zeleznikar RJ, Goldberg ND. Adenylate kinase: kinetic behavior in intact cells indicates it is
integral to multiple cellular processes. Mol Cell Biochem. 1998 Jul; 184(1–2):169–82. PMID: 9746320
26.
Zeleznikar RJ, Goldberg ND. Adenylate Kinase-catalyzed Phosphoryl Transfer Couples ATP Utilization
with Its Generation by Glycolysis in Intact Muscle. J Biol Chem. 1995 Mar 31; 270(13):7311–9. https://
doi.org/10.1074/jbc.270.13.7311 PMID: 7706272
27.
Gellerich FN. The role of adenylate kinase in dynamic compartmentation of adenine nucleotides in the
mitochondrial intermembrane space. FEBS Lett. 1992 Feb 3; 297(1–2):55–8. https://doi.org/10.1016/
0014-5793(92)80326-c PMID: 1551437
28.
Dzeja P, Terzic A. Adenylate Kinase and AMP Signaling Networks: Metabolic Monitoring, Signal Com-
munication and Body Energy Sensing. Int J Mol Sci. 2009 Apr 17; 10(4):1729–72. https://doi.org/10.
3390/ijms10041729 PMID: 19468337
29.
Aksentijević D, Lygate CA, Makinen K, Zervou S, Sebag-Montefiore L, Medway D, et al. High-
energy phosphotransfer in the failing mouse heart: role of adenylate kinase and glycolytic
enzymes. Eur J Heart Fail. 2010 Dec; 12(12):1282–9. https://doi.org/10.1093/eurjhf/hfq174 PMID:
20940173
30.
Dzeja PP, Vitkevicius KT, Redfield MM, Burnett JC, Terzic A. Adenylate kinase-catalyzed phospho-
transfer in the myocardium: Increased contribution in heart failure. Circ Res. 1999 May 28; 84
(10):1137–43. https://doi.org/10.1161/01.res.84.10.1137 PMID: 10347088
31.
Jacobus WE, Lehninger AL. Creatine kinase of rat heart mitochondria. Coupling of creatine phosphory-
lation to electron transport. J Biol Chem. 1973; 248(13):4803–10. PMID: 4718746
32.
Noma T. Dynamics of nucleotide metabolism as a supporter of life phenomena. The Journal of Medical
Investigation. 2005; 52(3,4):127–36. https://doi.org/10.2152/jmi.52.127 PMID: 16167529
33.
Pucar D, Bast P, Gumina RJ, Lim L, Drahl C, Juranic N, et al. Adenylate kinase AK1 knockout
heart: energetics and functional performance under ischemia-reperfusion. Am J Physiol Heart Circ
Physiol. 2002 Aug 1; 283(2):H776–782. https://doi.org/10.1152/ajpheart.00116.2002 PMID:
12124227
34.
Larsen S, Nielsen J, Hansen CN, Nielsen LB, Wibrand F, Stride N, et al. Biomarkers of mitochondrial
content in skeletal muscle of healthy young human subjects. J Physiol. 2012 Jul 15; 590(Pt 14):3349–
60. https://doi.org/10.1113/jphysiol.2012.230185 PMID: 22586215
35.
Karro N, Laasmaa M, Vendelin M, Birkedal R. Respiration of permeabilized cardiomyocytes from mice:
no sex differences, but substrate-dependent changes in the apparent ADP-affinity. Sci Rep. 2019 Aug
29; 9(1):12592. https://doi.org/10.1038/s41598-019-48964-x PMID: 31467353
36.
Lemieux H, Blier PU, Gnaiger E. Remodeling pathway control of mitochondrial respiratory capacity
by temperature in mouse heart: electron flow through the Q-junction in permeabilized fibers. Scien-
tific Reports. 2017 Jun 6; 7(1):2840. https://doi.org/10.1038/s41598-017-02789-8 PMID:
28588260
37.
Gnaiger E. Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 4th
ed. Innsbruck, Austria: Steiger Druck GmbH, Axams, Austria; 2014. 81 p. www.bioblast.at/index.php/
Gnaiger_2014_MitoPathways
38.
Zhou P, Pu WT. Recounting cardiac cellular composition. Circ Res. 2016 Feb 5; 118(3):368–70. https://
doi.org/10.1161/CIRCRESAHA.116.308139 PMID: 26846633
39.
Birkedal R, Laasmaa M, Branovets J, Vendelin M. Ontogeny of cardiomyocytes: ultrastructure optimiza-
tion to meet the demand for tight communication in excitation–contraction coupling and energy transfer.
Philosophical Transactions of the Royal Society B. 2022 Nov 21; https://royalsocietypublishing.org/doi/
10.1098/rstb.2021.0321 PMID: 36189816
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
22 / 25
40.
Blouin A, Bolender RP, Weibel ER. Distribution of organelles and membranes between hepatocytes
and nonhepatocytes in the rat liver parenchyma. A stereological study. J Cell Biol. 1977 Feb; 72
(2):441–55. https://doi.org/10.1083/jcb.72.2.441 PMID: 833203
41.
Davidson SM. Endothelial mitochondria and heart disease. Cardiovascular Research. 2010 Oct 1; 88
(1):58–66. https://doi.org/10.1093/cvr/cvq195 PMID: 20558442
42.
Brand MD, Chien LF, Ainscow EK, Rolfe DF, Porter RK. The causes and functions of mitochondrial pro-
ton leak. BiochimBiophysActa. 1994 Aug; 1187(2):132–9. https://doi.org/10.1016/0005-2728(94)
90099-x PMID: 8075107
43.
Nicholls DG, Ferguson, S. J. Bioenergetics, 4th edition. 4th ed. Elsevier; 2013. 434 p.
44.
CHANCE B, WILLIAMS GR. Respiratory enzymes in oxidative phosphorylation. I. Kinetics of oxygen
utilization. JBiolChem. 1955 Nov; 217(1):383–93. PMID: 13271402
45.
Momken I, Lechêne P, Koulmann N, Fortin D, Mateo P, Doan BT, et al. Impaired voluntary running
capacity of creatine kinase-deficient mice. J Physiol (Lond). 2005 Jun 15; 565(3):951–64. https://doi.
org/10.1113/jphysiol.2005.086397 PMID: 15831533
46.
De Sousa E, Veksler V, Minajeva A, Kaasik A, Mateo P, Mayoux E, et al. Subcellular creatine kinase
alterations. Implications in heart failure. CircRes. 1999 Jul; 85(1):68–76. https://doi.org/10.1161/01.res.
85.1.68 PMID: 10400912
47.
Kaasik A, Veksler V, Boehm E, Novotova M, Minajeva A, Ventura-Clapier R. Energetic crosstalk
between organelles: architectural integration of energy production and utilization. Circ Res. 2001; 89
(2):153–9. https://doi.org/10.1161/hh1401.093440 PMID: 11463722
48.
Veksler VI, Kuznetsov AV, Anflous K, Mateo P, van Deursen J, Wieringa B, et al. Muscle Creatine
Kinase-deficient Mice II. CARDIAC AND SKELETAL MUSCLES EXHIBIT TISSUE-SPECIFIC
ADAPTATION OF THE MITOCHONDRIAL FUNCTION. J Biol Chem. 1995 Aug 25; 270
(34):19921–9.
49.
Boehm E, Chan S, Monfared M, Wallimann T, Clarke K, Neubauer S. Creatine transporter activity and
content in the rat heart supplemented by and depleted of creatine. American Journal of Physiology-
Endocrinology and Metabolism. 2003 Feb 1; 284(2):E399–406. https://doi.org/10.1152/ajpendo.00259.
2002 PMID: 12531746
50.
Matsushima K, Uda K, Ishida K, Kokufuta C, Iwasaki N, Suzuki T. Comparison of kinetic constants of
creatine kinase isoforms. International Journal of Biological Macromolecules. 2006 Mar 30; 38(2):83–8.
https://doi.org/10.1016/j.ijbiomac.2005.12.023 PMID: 16451808
51.
Wallis J, Lygate CA, Fischer A, ten Hove M, Schneider JE, Sebag-Montefiore L, et al. Supranormal
myocardial creatine and phosphocreatine concentrations lead to cardiac hypertrophy and heart failure:
insights from creatine transporter-overexpressing transgenic mice. Circulation. 2005 Nov 15; 112
(20):3131–9. https://doi.org/10.1161/CIRCULATIONAHA.105.572990 PMID: 16286605
52.
Stolz M, Wallimann T. Myofibrillar interaction of cytosolic creatine kinase (CK) isoenzymes: allocation of
N-terminal binding epitope in MM-CK and BB-CK. JCell Sci. 1998 May; 111 (Pt 9):1207–16. https://doi.
org/10.1242/jcs.111.9.1207 PMID: 9547297
53.
Hornemann T, Stolz M, Wallimann T. Isoenzyme-specific interaction of muscle-type creatine kinase
with the sarcomeric M-line is mediated by NH(2)-terminal lysine charge-clamps. JCell Biol. 2000 Jun;
149(6):1225–34. https://doi.org/10.1083/jcb.149.6.1225 PMID: 10851020
54.
Wallimann T, Turner DC, Eppenberger HM. Localization of creatine kinase isoenzymes in myofibrils. I.
Chicken skeletal muscle. J Cell Biol. 1977 Nov; 75(2 Pt 1):297–317. https://doi.org/10.1083/jcb.75.2.
297 PMID: 264112
55.
Wallimann T, Kuhn HJ, Pelloni G, Turner DC, Eppenberger HM. Localization of creatine kinase isoen-
zymes in myofibrils. II. Chicken heart muscle. J Cell Biol. 1977 Nov; 75(2 Pt 1):318–25. https://doi.org/
10.1083/jcb.75.2.318 PMID: 264113
56.
Kraft T, Hornemann T, Stolz M, Nier V, Wallimann T. Coupling of creatine kinase to glycolytic enzymes
at the sarcomeric I-band of skeletal muscle: a biochemical study in situ. J Muscle Res Cell Motil. 2000;
21(7):691–703. https://doi.org/10.1023/a:1005623002979 PMID: 11227796
57.
Grosse R, Spitzer E, Kupriyanov VV, Saks VA, Repke KR. Coordinate interplay between (Na+ + K
+)-ATPase and creatine phosphokinase optimizes (Na+/K+)-antiport across the membrane of vesicles
formed from the plasma membrane of cardiac muscle cell. BiochimBiophysActa. 1980 Dec; 603
(1):142–56.
58.
Rossi AM, Eppenberger HM, Volpe P, Cotrufo R, Wallimann T. Muscle-type MM creatine kinase is spe-
cifically bound to sarcoplasmic reticulum and can support Ca2+ uptake and regulate local ATP/ADP
ratios. J Biol Chem. 1990 Mar 25; 265(9):5258–66. PMID: 2318892
59.
Schlattner U, Klaus A, Ramirez Rios S, Guzun R, Kay L, Tokarska-Schlattner M. Cellular compartmen-
tation of energy metabolism: creatine kinase microcompartments and recruitment of B-type creatine
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
23 / 25
kinase to specific subcellular sites. Amino Acids. 2016 Aug 1; 48(8):1751–74. https://doi.org/10.1007/
s00726-016-2267-3 PMID: 27318991
60.
Ventura-Clapier R, Kuznetsov A, Veksler V, Boehm E, Anflous K. Functional coupling of creatine
kinases in muscles: species and tissue specificity. Mol Cell Biochem. 1998; 184(1–2):231–47. PMID:
9746324
61.
Gellerich F, Saks VA. Control of heart mitochondrial oxygen consumption by creatine kinase: the impor-
tance of enzyme localization. Biochem Biophys Res Commun. 1982 Apr; 105(4):1473–81. https://doi.
org/10.1016/0006-291x(82)90954-8 PMID: 7103968
62.
Simson P, Jepihhina N, Laasmaa M, Peterson P, Birkedal R, Vendelin M. Restricted ADP movement in
cardiomyocytes: Cytosolic diffusion obstacles are complemented with a small number of open mito-
chondrial voltage-dependent anion channels. Journal of Molecular and Cellular Cardiology. 2016 Aug;
97:197–203. https://doi.org/10.1016/j.yjmcc.2016.04.012 PMID: 27261153
63.
Boehm E. Maintained Coupling of Oxidative Phosphorylation to Creatine Kinase Activity in Sarcomeric
Mitochondrial Creatine Kinase-deficient Mice. Journal of Molecular and Cellular Cardiology. 1998 May;
30(5):901–12. https://doi.org/10.1006/jmcc.1998.0692 PMID: 9618231
64.
Ramay HR, Vendelin M. Diffusion Restrictions Surrounding Mitochondria: A Mathematical Model of
Heart Muscle Fibers. Biophysical Journal. 2009 Jul 22; 97(2):443–52. https://doi.org/10.1016/j.bpj.
2009.04.062 PMID: 19619458
65.
Illaste A, Laasmaa M, Peterson P, Vendelin M. Analysis of Molecular Movement Reveals Latticelike
Obstructions to Diffusion in Heart Muscle Cells. Biophysical Journal. 2012 Feb 22; 102(4):739–48.
https://doi.org/10.1016/j.bpj.2012.01.012 PMID: 22385844
66.
Birkedal R, Laasmaa M, Vendelin M. The location of energetic compartments affects energetic commu-
nication in cardiomyocytes. Front Physiol. 2014; 5: 376. https://doi.org/10.3389/fphys.2014.00376
PMID: 25324784
67.
Jepihhina N, Beraud N, Sepp M, Birkedal R, Vendelin M. Permeabilized Rat Cardiomyocyte Response
Demonstrates Intracellular Origin of Diffusion Obstacles. Biophysical Journal. 2011 Nov 2; 101
(9):2112–21. https://doi.org/10.1016/j.bpj.2011.09.025 PMID: 22067148
68.
Timohhina N, Guzun R, Tepp K, Monge C, Varikmaa M, Vija H, et al. Direct measurement of energy
fluxes from mitochondria into cytoplasm in permeabilized cardiac cells in situ: some evidence for Mito-
chondrial Interactosome. J Bioenerg Biomembr. 2009 Jun; 41(3):259–75. https://doi.org/10.1007/
s10863-009-9224-8 PMID: 19597977
69.
Vendelin M, Hoerter JA, Mateo P, Soboll S, Gillet B, Mazet JL. Modulation of Energy Transfer Pathways
between Mitochondria and Myofibrils by Changes in Performance of Perfused Heart. J Biol Chem. 2010
Nov 26; 285(48):37240–50. https://doi.org/10.1074/jbc.M110.147116 PMID: 20847056
70.
Noda L. ADENOSINE TRIPHOSPHATE-ADENOSINE MONOPHOSPHATE TRANSPHOSPHORY-
LASE: III. KINETIC STUDIES. Journal of Biological Chemistry. 1958 May 1; 232(1):237–50.
71.
Mika D, Leroy J, Vandecasteele G, Fischmeister R. PDEs create local domains of cAMP signaling.
Journal of Molecular and Cellular Cardiology. 2012 Feb; 52(2):323–9. https://doi.org/10.1016/j.yjmcc.
2011.08.016 PMID: 21888909
72.
Weber CR, Piacentino V, Ginsburg KS, Houser SR, Bers DM. Na-Ca(2+) exchange current and sub-
membrane [Ca(2+)] during the cardiac action potential. CircRes. 2002 Feb; 90(2):182–9.
73.
Franzini-Armstrong C. ER-mitochondria communication. How privileged? Physiology (Bethesda). 2007
Aug; 22: 261–8.
74.
Williams GSB, Boyman L, Chikando AC, Khairallah RJ, Lederer WJ. Mitochondrial calcium uptake.
PNAS. 2013 Jun 25; 110(26):10479–86. https://doi.org/10.1073/pnas.1300410110 PMID:
23759742
75.
Hardie DG. AMP-activated protein kinase: a key regulator of energy balance with many roles in
human disease. J Intern Med. 2014 Dec; 276(6):543–59. https://doi.org/10.1111/joim.12268 PMID:
24824502
76.
Sepp M, Vendelin M, Vija H, Birkedal R. ADP Compartmentation Analysis Reveals Coupling between
Pyruvate Kinase and ATPases in Heart Muscle. Biophysical Journal. 2010 Jun; 98: 2785–93. https://
doi.org/10.1016/j.bpj.2010.03.025 PMID: 20550890
77.
Itah R, Gitelman I, Davis C. A replacement for methoxyflurane (Metofane) in open-circuit anaesthesia.
Lab Anim. 2004 Jul; 38(3):280–5. https://doi.org/10.1258/002367704323133664 PMID: 15207039
78.
Lange Y. Disposition of intracellular cholesterol in human fibroblasts. Journal of Lipid Research. 1991
Feb 1; 32(2):329–39. PMID: 2066666
79.
Saks VA, Veksler VI, Kuznetsov AV, Kay L, Sikk P, Tiivel T, et al. Permeabilized cell and skinned fiber
techniques in studies of mitochondrial function in vivo. Mol Cell Biochem. 1998 Jul; 184(1–2):81–100.
PMID: 9746314
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
24 / 25
80.
Barsunova K, Vendelin M, Birkedal R. Marker enzyme activities in hindleg from creatine-deficient AGAT
and GAMT KO mice–differences between models, muscles, and sexes. Sci Rep. 2020 May 14; 10
(1):1–9.
81.
Christensen M, Hartmund T, Gesser H. Creatine kinase, energy-rich phosphates and energy metabo-
lism in heart muscle of different vertebrates. J Comp Physiol [B]. 1994; 164(2):118–23. https://doi.org/
10.1007/BF00301652 PMID: 8056878
82.
Eyer P, Worek F, Kiderlen D, Sinko G, Stuglin A, Simeon-Rudolf V, et al. Molar absorption coefficients
for the reduced Ellman reagent: reassessment. Analytical Biochemistry. 2003 Jan 15; 312(2):224–7.
https://doi.org/10.1016/s0003-2697(02)00506-7 PMID: 12531209
83.
Vendelin M, Laasmaa M, Kalda M, Branovets J, Karro N, Barsunova K, et al. IOCBIO Kinetics: An
open-source software solution for analysis of data traces. PLOS Computational Biology. 2020 Dec 22;
16(12):e1008475. https://doi.org/10.1371/journal.pcbi.1008475 PMID: 33351800
84.
Karro N, Sepp M, Jugai S, Laasmaa M, Vendelin M, Birkedal R. Metabolic compartmentation in rainbow
trout cardiomyocytes: coupling of hexokinase but not creatine kinase to mitochondrial respiration. J
Comp Physiol B, Biochem Syst Environ Physiol. 2016 Aug 13;
85.
Balaban RS, Mootha VK, Arai A. Spectroscopic Determination of Cytochrome c Oxidase Content in Tis-
sues Containing Myoglobin or Hemoglobin. Analytical Biochemistry. 1996 Jun 1; 237(2):274–8. https://
doi.org/10.1006/abio.1996.0239 PMID: 8660576
86.
Liao GL, Palmer G. The reduced minus oxidized difference spectra of cytochromes a and a3. Biochi-
mica et Biophysica Acta (BBA)—Bioenergetics. 1996 Jun 13; 1274(3):109–11. https://doi.org/10.1016/
0005-2728(96)00014-x PMID: 8664303
PLOS ONE
Creatine kinase compartmentalization in rat and mouse cardiomyocytes
PLOS ONE | https://doi.org/10.1371/journal.pone.0294718
November 27, 2023
25 / 25

Rat and mouse cardiomyocytes show subtle differences in creatine kinase expression and compartmentalization.
Branovets, Jelena,Soodla, Kärol,Vendelin, Marko,Birkedal, Rikke