1 An impaired metabolism of nucleotides underpins a novel mechanism of cardiac remodeling leading to Huntington’s disease related cardiomyopathy. Marta Toczek 1 , Daniel Zielonka 2 , Paulina Zukowska 1 , Jerzy T. Marcinkowski 2 , Ewa Slominska 1 , Mark Isalan 3 , Ryszard T. Smolenski 1** , Michal Mielcarek 3* 1 Department of Biochemistry, Medical University of Gdansk, 1 Debinki Str, 80-210, Gdansk, Poland. 2 Department of Social Medicine, Poznan University of Medical Sciences, 6 Rokietnicka Str, 60-806, Poznan, Poland. 3 Department of Life Sciences, Imperial College London, Exhibition Road, SW7 2AZ, London, UK. * Corresponding author: Michal Mielcarek, Department of Life Sciences, Imperial College London, Exhibition road, SW7 2AZ, London, UK, tel. +44 207 59 46482, fax: +44 2075942290,e-mail: [email protected]; [email protected]** Co-corresponding author: Ryszard Smolenski, Department of Biochemistry, Medical University of Gdansk, 1 Debinki Str, 80-210, Gdansk, Poland, tel:+48 58 349 14 63, fax: +48 58 349 14 65, e-mail: [email protected]
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An impaired metabolism of nucleotides underpins a novel mechanism of cardiac remodeling leading to Huntington’s disease related cardiomyopathy.
Marta Toczek1, Daniel Zielonka2, Paulina Zukowska1, Jerzy T. Marcinkowski2, Ewa Slominska1, Mark Isalan3, Ryszard T. Smolenski 1**, Michal Mielcarek3*
1 Department of Biochemistry, Medical University of Gdansk, 1 Debinki Str, 80-210, Gdansk, Poland.
2 Department of Social Medicine, Poznan University of Medical Sciences, 6 Rokietnicka Str, 60-806, Poznan, Poland.
3 Department of Life Sciences, Imperial College London, Exhibition Road, SW7 2AZ, London, UK.
* Corresponding author: Michal Mielcarek, Department of Life Sciences, Imperial College London, Exhibition road, SW7 2AZ, London, UK, tel. +44 207 59 46482, fax: +44 2075942290,e-mail: [email protected]; [email protected]
** Co-corresponding author: Ryszard Smolenski, Department of Biochemistry, Medical University of Gdansk, 1 Debinki Str, 80-210, Gdansk, Poland, tel:+48 58 349 14 63, fax: +48
(Adenosine monophosphate deaminase 3) transcripts remained unchanged in both HD mouse
models (Fig. 7B).
We were also interested in validating the transcriptional profiles of genes engaged in
guanine, inosine and hypoxanthine degradation pathways, such as Gda (Guanine deaminase),
Pnp (Purine nucleoside phosphorylase) and Xdh (Xanthine dehydrogenase) [32, 33]. We
found that all transcripts that are involved in these pathways were unaltered in the hearts of
HD mouse models (Fig. 7C).
Finally, to further examine the source of the energy shift in the HD hearts, we studied the
transcriptional profile of key players involved in energy metabolism. Hk2 (Hexokinase 2)
catalyses the phosphorylation of glucose, the rate-limiting first step of glycolysis and is
known as a molecule involved in energy metabolism and cellular protection [34]. Hk2
transcripts were significantly down-regulated in HD hearts (Fig. 7D). There were no changes
in the expression of Ppargc1a (Peroxisome proliferator-activated receptor gamma coactivator
1-alpha) and Ppara (Peroxisome proliferator-activated receptor alpha) mRNAs, while Prkaa1
(AMP- activated protein kinase) transcripts were significantly up-regulated in the hearts of
HD mouse models (Fig.7D). Taken together, these data indicate a transcriptional remodelling
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of genes involved in de novo purine biosynthesis, in the purine nucleotide salvage pathway
and in catabolism of nucleotides.
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4. DISCUSSION
Although HD has been described primarily as a neurological disorder, there is solid
evidence emphasizing the contribution of peripheral pathology to disease progression,
including skeletal muscle atrophy [35] and heart failure [7]. In fact, recent clinical data
confirmed the presence of contractile heart dysfunction in HD pre-symptomatic patients [8],
that might lead to HD-related cardiomyopathy or even a cardiac sudden death. On the other
hand, there are examples from studies based on HD animal models that have affirmed cardiac
pathological events such as brady- and tachyarrhythmias, variations in heart rates and cardiac
remodelling [5] and these are in line with the clinical data [8]. However, there is still an
urgent need for more insights into the molecular pathologies that might lead to heart
malfunction in HD patients.
In pre-clinical settings, the R6/2 mouse model - that displays impairment in cardiac
functions - had significant alterations in mitochondrial structure, including the loss of
mitochondrial elongated shapes and diffused mitochondrial densities [36, 37]. These
morphological changes may lead directly to cardiac energy metabolism imbalances. Indeed, a
recent study suggested that cardiac Fas-dependent and mitochondria-dependent apoptotic
pathways were activated in R6/2 hearts [38]. Mitochondrial structure disarrangement may
thus result in an energy status imbalance in cardiomyocytes.
This type of energy imbalance is not only restricted to the heart but also underpins a
decline in an energy metabolism and decreased oxidation in skeletal muscles [14], and is a
feature similar to muscle wasting syndrome in cancer cachexia [39]. HD subjects also showed
a deficit in mitochondrial oxidative metabolism in skeletal muscles, as judged by a significant
decrease in phosphocreatine recovery after exercise [40]. Additionally, in vitro muscle cell
cultures exhibited abnormalities in mitochondrial membrane potential and cytochrome 3
release [41].
Following on from these findings, we found a reduced cardiac ATP level, combined with a
lower ATP/ADP ratio in two HD mouse models of varying severity (R6/2, HdhQ150).
Interestingly, a reduced ATP level has already been positively associated with the severity of
human heart failure [42] and a decreased ATP pool was also observed in heart tissue obtained
from patients with a dilated and restricted cardiomyopathy [43]. A reduced single
mitochondrion ATP flux may limit sarcomere contraction, leading to a compensatory
proliferation of the cardiac mitochondria, while the myocardium may continue to contract
inefficiently and dyssynchronously due to its adaptation, as previously anticipated [5, 44].
Notably, the cardiac guanine-to-adenine nucleotides ratio was amplified, likely as a
compensatory mechanism to fewer adenine nucleotides. This was in agreement with a
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previous finding of a higher concentration of guanine nucleotides in human hearts with
dilated and hypertrophic cardiomyopathy [45]. It is well known that guanine nucleotides are
an essential component of G-protein signalling, with links to the adenylate cyclase
machinery, and so changes in their concentration may impair regulatory mechanisms in the
heart [46].
It is also well established that the major source of ATP regeneration is the creatine kinase
system [47]. Depletion of the phosphocreatine (PCr) concentration is a typical feature of heart
failure and has been described in many different animal models of cardiomyopathy [48, 49].
A reduced PCr concentration, and PCr/Cr ratio, is other cardiac energy metabolism
parameters that we found to be deregulated in HD settings. A decreased PCr/Cr ratio directly
translates into lower values of phosphorylation potential, leading to a decreased cardiac
muscle contraction and an impaired heart rate as previously described in HD mouse models
[5].
To examine the steady-state internal redox status in HD hearts, we assessed the levels of
oxidized and reduced forms of nicotinamide adenine dinucleotides – NAD+ and NADH. We
demonstrated a reduced NADH level and NADH/NAD+ ratio in the mouse models. This may
be an indication of redox imbalance, and could suggest an inherent dysfunction of
mitochondria, or be indicative of alterations in a mitochondrial function upstream of
oxidative phosphorylation [50]. In particular, this could indicate a disrupted efficiency of the
Krebs cycle. Further studies should be done to identify whether this is caused by enzyme
inhibition or down-regulation, or whether there is insufficient supply of acetyl-CoA or
anaplerotic substrates.
The cardiac energy metabolism and steady state internal redox imbalance, found in both
symptomatic HD mouse models, may activate energy regenerating pathways like AMP-
activated protein kinase (AMPK) phosphorylation. AMPK is a heterotrimeric complex kinase
comprising a catalytic subunit (α-subunit) and two regulatory subunits (β- and γ-subunits).
Both α- and β-subunits form two isoforms, α1, α2 and β1, β2, while the γ-subunit is present in
three γ1, γ2, and γ3 isoforms [22]. The α-subunit contains the catalytic domain of the
serine/threonine protein kinase (Thr172) whose phosphorylation is crucial for AMPK
activation [51]. We found that AMPK was hyperphosphorylated in HD mouse model hearts.
It is known that AMPK plays an important role in ATP regeneration by the cellular uptake of
glucose, β-oxidation of fatty acids and regulation of mitochondrial biogenesis [52]. It is likely
that an enhanced AMPK activity results in a cardiac substrate preference shift in symptomatic
HD animals, as was demonstrated by decreased 13C glutamate enrichment and a reduced ratio
of [13C glutamate] to [13C alanine] in HD mouse model hearts.
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Similarly, a reduced glucose oxidative metabolism was observed both in skeletal muscle in
HD mouse models [14] and in the cardiac mitochondria obtained from patients with a dilated
cardiomyopathy [12]. A decreased glucose metabolism could be the cause or result of lower
transcript levels of Hk2 (Hexokinase 2; an enzyme that phosphorylates glucose). Hexokinase
activity is the initiating step for virtually all glucose utilization pathways observed in HD
mouse model hearts. Moreover, there are some data suggesting that a reduction in hexokinase
2 levels might result in a decreased cardiac function and altered remodelling of the heart, in
ischemia-reperfusion, by increasing cell death, fibrosis and reducing angiogenesis in
cardiomyocytes [53].
No significant changes in transcripts of molecules involved in the metabolism of fatty
acids such as Ppargc1a (Peroxisome proliferator-activated receptor gamma coactivator 1-
alpha) and Ppara (Peroxisome proliferator-activated receptor alpha) were identified. Thus,
one may conclude that the myocardium failure in HD could be caused by alterations in
specific energy substrate metabolism [54]. In particular, a failing heart typically shifts
metabolism from carbohydrate oxidation towards metabolism of fatty acids, which may cause
a decline in contractile function and intensify the progression of pump failure [53]. Thus,
such observed shifts determine reductions in myocardial oxygen consumption efficiency and
we observed this phenomenon in HD hearts. Although the protective AMPK pathway was not
properly activated, as judged on the lack of ATP regeneration, it is likely that mutant HTT
can exert loss or gain of function effects on mitochondrial enzymes. Alternatively, in HD
there could be a lack of trophic signals from the CNS [5, 55] that may lead to impairment of
proteins involved in energy metabolism.
Despite the fact that there is no global transcriptional deregulation in hearts, in either HD
mouse model [5], we found a significant down-regulation of genes involved in purine de
novo biosynthesis, such as Adsl, Adlssl1, Gart, and in reconversion of adenine nucleotides,
like Gmps and Akt1. Interestingly, it is well established that an induction of genes involved in
de novo purine nucleotide synthesis is observed in cardiac hypertrophy in rats [56]. Our
findings are in line with a previous study showing a substantial acceleration of purine
synthesis and turnover in HTT−/− murine embryonic stem cells, likely due to increased purine
biosynthesis [57]. It is possible that reduced purine synthesis and re-conversion of adenine
nucleotides led to the changes in catabolism of nucleotides that we observed in HD mouse
models. Indeed, we found a number of adenosine catabolites such as inosine, hypoxanthine,
xanthine, uridine or uric acid at increased levels in the sera of the HD murine models. More
importantly, uridine and hypoxanthine levels were also found to be increased in the plasma of
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symptomatic HD patients. Hence, for the first time, our study identified biomarkers that
might be linked to HD progression both in pre-clinical and clinical settings.
It is likely that the catabolites that we we observed in sera were released by the affected
heart and/or skeletal muscle tissues. In fact we showed that these catabolites were
accumulated within the heart mass in the R6/2 mouse model [58]. In addition, these findings
are supported by the observation of the up-regulation of genes involved in the adenosine
degradation pathway, such as Ada or Dpp4. Adenosine is known to be a protective agent in
ischemic heart failure [59] and administration of adenosine metabolism inhibitors results in
an improvement of cardiac function [60]. Moreover, it is well known that polymorphisms
within the Ada gene can predispose to chronic heart failure [61]. Altered transcripts of genes
responsible for ATP degradation, such as Entpd2 (Ectonucleoside triphosphate
diphosphohydrolase 2) and Nme3 (Nucleoside diphosphate kinase 3), were observed in the
HdhQ150 mouse model and could interfere with regulation of the ATP pool. Entpd2 has been
identified as a key enzyme with a role in regulating nucleotide-mediated signaling, and in
controlling the rate, amount and timing of nucleotide degradation [62]. It is well-established
that nucleoside diphosphate kinase possesses a histidine kinase activity for G-beta proteins
that could potentially contribute to receptor-independent regulation of cAMP synthesis and
the contractility of cardiomyocytes [63]. Nonetheless, no changes were observed in the
transcript levels of genes involved in nucleotide catabolite pathways, such as guanine, inosine
or hypoxanthine degradation. Therefore down-regulation of synthesis of adenine nucleotides,
and changes in their catabolism, may intensify the deterioration in energy equilibrium.
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5. CONCLUSIONS
In summary, HD mouse models develop a notable decrease in cardiac energy metabolism,
concomitant with AMPK hyperphosphorylation. This may contribute to a shift in cardiac
substrate preference that is accompanied by decreased oxidation and lack of ATP
regeneration, leading to the accumulation of nucleotide catabolites in sera, both in HD mice
and in HD clinical samples (Fig. 8). In addition, cardiac transcriptional dysregulation of
genes occurs, involving processes such as purine biosynthesis and catabolism of adenine
nucleotides, which may in turn activate the development of pathological features leading to
HD-related cardiomyopathy One may conclude that the energy imbalance and altered
metabolism of nucleotides is likely to be a contributor to the apparent contractile heart
dysfunction that has been previously reported in HD mouse models [5].
Our data strongly suggest that there is value in embarking on the development of new
therapies to improve energy metabolism in HD affected hearts. Likely candidates would
include inhibitors of mitochondrial permeability transition pore (mPTP) opening and
compounds in clinical trials, such as CoQ10 and creatine [64]. Other candidates would
include substances that provide anaplerotic substrates for the Krebs cycle, such as branched-
chain amino acids (BCAA) - whose concentrations have been found to be decreased in HD
patients' plasma; reviewed in [65].
Alternatively, PPAR activators are already widely-used in pre-clinical trials [64]. These
small molecule drugs promote the expression of genes that enhance energy production and
optimize the quality control of proteins and organelles. Moreover, Dickey and colleagues
recently tested the PPARδ agonist KD3010 in HD N171-82Q transgenic mice. They observed
improved motor function, decreased neurodegeneration and longer survival in HD mice [66].
Based on these promising results it would be of interest to evaluate how PPAR activation
might improve the energy metabolism of other tissues affected in HD.
Finally, a different therapeutic strategy may include testing molecules to improve the
metabolism of nucleotides directly. Our previous studies identified that a combined treatment
of cardiomyocytes with adenosine metabolism inhibitors and substrates for nucleotide
synthesis resulted in improvements in cardiac mechanical function, in the energy status and
of the adenine nucleotide pool [67, 68]. Ultimately, this therapeutic avenue that may well be
amenable to small molecule therapeutics and may even be widely applicable to other
neurodegenerative diseases.
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6. ACKNOWLEDGEMENT
This work was supported by the National Science Centre of Poland (2011/01/B/NZ4/03719)
and (2015/17/N/NZ4/028410); Foundation for Polish Science (TEAM/2011-8/7), and
funding from European Research Council grant H2020 - ERC-2014-PoC 641232 -
Fingers4Cure. These funders had no role in study design, data collection and analysis,
decision to publish or preparation of the manuscript.
7. CONFLICTS OF INTEREST STATEMENT
None declared.
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LEGENDS TO FIGURES Figure 1. Adenine nucleotides, phosphocreatine and creatine levels in symptomatic HD
mouse model hearts. (A) ATP and ADP (B) levels in wild-type, R6/2 and HdhQ150 mice.
(C) AMP level. (D) The ATP/ADP ratio. (E) Phosphocreatine and creatine levels. (F) The
PCr/Cr ratio. Data presented as mean ± SEM; n=5; * p<0.05, ** p<0.01.
Figure 2. Total levels of adenine and guanine nucleotides, and NAD+ or NADH levels in
HD mouse model hearts. (A) The total concentration of adenine nucleotides in wild-type,
R6/2 and HdhQ150 mouse hearts. (B) The total concentration of guanine nucleotides. (C)
Guanine/adenine nucleotides ratio. (D) NAD+ and NADH levels (E). F. NADH/NAD+ ratio.
Data presented as mean ± SEM; n=5; * p<0.05.
Figure 3. Intensive increases of adenine nucleotide catabolites and uridine concentration
in the sera of HD mouse models. (A) Inosine, hypoxanthine, xanthine and uric acid levels
in wild-type, R6/2 and HdhQ150 mouse serum. (B) Uridine concentration. Data presented as
mean ± SEM; n=5; ** p<0.01, *** p<0.001.
Figure 4. Concentrations of uric acid, hypoxanthine and uridine in the plasma of HD
patients. (A) Uric acid level. (B) Hypoxanthine concentration. (C) Linear regression of
mutant CAG repeat length and hypoxanthine concentration. (D) Linear regression of HD
disease burden and hypoxanthine concentration. (E) Uridine concentration. (F) Linear
regression of uridine levels and mutant CAG repeat length. (G) Linear regression of uridine
concentration and HD disease burden. Data presented as mean ± SEM; n=5; * p<0.05; ***
p<0.001.
Figure 5. Changes in the cardiac substrate preference and AMP-regulated protein
kinase phosphorylation level in HD mouse models. (A) 13C glutamate / 13C glucose ratio in
the blood of wild-type, R6/2 and HdhQ150 mice. (B) 13C alanine / 13C glucose ratios in the
blood of HD mouse models. (C) 13C glutamate / 13C alanine ratio in the hearts of HD mouse
models (D). The AMPK phosphorylation level in the hearts of HD mouse models. Data
presented as mean ± SEM; n=5; * p<0.05; *** p<0.001.
Figure 6. Transcriptional remodeling of genes involved in synthesis of nucleotides. (A)
Transcripts of genes involved in de novo purine biosynthesis (Adsl (Adenylosuccinate lyase),
An impaired metabolism of nucleotides underpins a novel mechanism of cardiac remodeling leading to Huntington’s disease related cardiomyopathy. Marta Toczek, Daniel Zielonka, Paulina Zukowska, Jerzy T Marcinkowski, Ewa Slominska, Mark Isalan, Ryszard T. Smolenski, Michal Mielcarek
ATP [µM]
ADP [µM]
NAD+ [µM]
WT 7.2 ± 5.1 2.8 ± 0.13 4.7 ± 1.4
R6/2 8.5 ± 4.9 4.5 ± 0.11 4.1 ± 1.4
WT 8.6 ± 3.6 6.1 ± 3.1 3.9 ± 1.5
HdhQ150 8.5 ± 3.3 6.6 ± 1.9 2.6 ± 1.5
Supplementary Table 1. Adenine nucleotides (ATP, ADP) and NAD+ levels in sera from
mice. Results presented as mean ± SD, n=5.
37
HD
patients Control
Male/ female
4/1 3/2
Age [y] 59 ± 11 44 ± 8 Body mass
[kg] 67 ± 10 68 ± 12
BMI 23 ± 2 23 ± 3 Mutant CAG repeat size
43 ± 4 -
Age of onset [y]
46 ± 10 -
Disease duration [y]
12 ± 4 -
Disease burden
440 ± 152 -
Motor score
54 ± 24 -
Intensity of chorea
17 ± 8 -
Supplementary Table 2. Characteristics of the study population.
One of the HD patients has been diagnosed with ischemic heart conditions. All other HD patients and healthy controls were cardiologically normal. Results presented as mean ± SD,
n=5.
38
ATP [µM]
ADP [µM]
NAD+ [µM]
HD patients
1.71 ± 1.15 * 1.27 ± 0.46 1.51 ± 0.46
Control 3.22 ± 0.71 1.24 ± 0.75 2.66 ± 1.21
Supplementary Table 3. Adenine nucleotides (ATP, ADP) and NAD+ levels in HD patients and control plasma. Results presented as mean ± SD, n=5, * p<0.05.
39
Supplementary Figure 1. Correlation between hypoxanthine concentration in HD
patients’ plasma and: A. Disease duration. B. Motor score. C. Intensity of chorea. n=5.
40
Supplementary Figure 2. Correlation of uridine concentration in HD patients’ plasma and: A. Disease duration. B. Motor score. C. Intensity of chorea. n=5.
41
Metabolite concentration in serum/plasma
R6/2 HD mouse model vs. control mouse
HdhQ150 HD mouse model vs. control mouse
HD patients vs. healthy control
ATP No changes No changes
Hypoxanthine
Uric acid
No changes
Uridine
Supplementary Table 3. Summary of metabolites' concentrations in HD mouse models serum and HD patients' plasma.