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mtDNA Damage & Heart Disease/End Organ Disease: Role of Pyrimidine Depletion in the Mitochondrial Cardiotoxicity of Nucleoside Analogue Reverse Transcriptase Inhibitors - pdf attached
 
 
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JAIDS Journal of Acquired Immune Deficiency Syndromes: 15 December 2010
 
Balcarek, Kerstin MD*; Venhoff, Nils MD*; Deveaud, Catherine PhD; Beauvoit, Bertrand PhD; Bonnet, Jacques MD; Kirschner, Janbernd MD; Venhoff, Ana C PhD*; Lebrecht, Dirk PhD*; Walker, Ulrich A MD* From the *Department of Rheumatology and Clinical Immunology, Medizinische Universitatsklinik, Freiburg, Germany; Centre of Chronic Immunodeficiency (CCI), Freiburg, Germany; INSERM U828, Pessac, France; Department of Neuropediatrics and Muscle Disorders, Medizinische Universitatsklinik, Freiburg, Germany; and Department of Rheumatology, Basel University, Basel, Switzerland. Parts of the data have been presented at the following meetings: 10th International Workshop on Adverse Drug Reactions and Lipodystrophy in HIV, November 6-8, 2008, London, United Kingdom; 9th International Congress on Drug Therapy in HIV Infection, November 9-13, 2008, Glasgow, United Kingdom.
 
From Jules: studies have NOT FOUND any benefit with regards to lipoatrophy through the use of uridine supplemtation although the makers of this product promised benefit and Walker the author of this paper was part of that research effort. What's interesting here is the finding that nukes AZT & ddC induced cardiomyopathy......mtDNA damage can lead to organ damage: "mitochondrial toxicity, which may affect liver,1 adipose tissue,2 skeletal muscle,3 and other organs. Animal models suggest that thymidine analogue NRTI also cause a mitochondrial cardiomyopathy4,5 although in clinical practice overt myocardial failure is only occasionally observed,6-8 NRTI may, however, cause more subtle cardiac abnormalities. Zidovudine for example has been associated with left ventricular hypertrophy in adults,9 and fetal and perinatal exposure is associated with reduced left ventricular mass, septal wall thickness and increased left ventricular afterload.10"
 
"Both NRTI induced a cardiomyopathy with mitochondrial enlargement, a disrupted cristal architecture on electron microscopy and diminished myocardial mtDNA copy numbers.....Our findings demonstrate that zidovudine and zalcitabine induce a cardiomyopathy in which mtDNA-encoded respiratory chain deficiency plays a significant role."
 
Abstract
 
Objective: Long-term antiretroviral treatment with nucleoside analogue reverse transcriptase inhibitors (NRTI) may result in a cardiomyopathy due to mitochondrial DNA (mtDNA) depletion. An intact mitochondrial function is required for the synthesis of intramyocardial pyrimidine nucleotides, which in turn are building blocks of mtDNA. We investigated if NRTI-related cardiomyopathy can be prevented with pyrimidine precursors.
 
Methods: Mice were fed with zidovudine or zalcitabine with or without simultaneous Mitocnol, a dietary supplement with high uridine bioavailability. Myocardia were examined after 9 weeks.
 
Results: Both NRTI induced a cardiomyopathy with mitochondrial enlargement, a disrupted cristal architecture on electron microscopy and diminished myocardial mtDNA copy numbers. The myocardial mtDNA-encoded cytochrome c-oxidase I subunit was impaired more profoundly than the nucleus-encoded cytochrome c-oxidase IV subunit. The myocardial formation of reactive oxygen species and mtDNA mutations was enhanced in zidovudine and zalcitabine treated animals. Mitocnol attenuated or normalized all myocardial pathology when given with both NRTI, but by itself had no intrinsic effects and no apparent adverse effects.
 
Conclusions: Zidovudine and zalcitabine induce a mitochondrial cardiomyopathy, which is antagonized with uridine supplementation, implicating pyrimidine pool depletion in its pathogenesis. Pyrimidine pool replenishment may be exploited clinically because uridine is well tolerated.
 
INTRODUCTION
 
Nucleoside analogue reverse transcriptase inhibitors (NRTI) are widely used in the antiretroviral treatment of HIV-infected individuals. A major limitation of their long-term use is their mitochondrial toxicity, which may affect liver,1 adipose tissue,2 skeletal muscle,3 and other organs. Animal models suggest that thymidine analogue NRTI also cause a mitochondrial cardiomyopathy4,5 although in clinical practice overt myocardial failure is only occasionally observed,6-8 NRTI may, however, cause more subtle cardiac abnormalities. Zidovudine for example has been associated with left ventricular hypertrophy in adults,9 and fetal and perinatal exposure is associated with reduced left ventricular mass, septal wall thickness and increased left ventricular afterload.10
 
The currently prevailing hypothesis for this form of myocardial damage is that triphosphorylated NRTI interfere with the metabolism of cardiac mitochondria by inhibiting polymerase gamma, the enzyme which replicates mitochondrial DNA (mtDNA).11 Zalcitabine for example, is a relatively potent NRTI inhibitor of polymerase gamma in clinically relevant concentrations, whereas zidovudine in contrast is only a relatively weak inhibitor.11 Recent investigations suggest that zidovudine may predominantly cause mtDNA depletion by a second mechanism, namely by interfering with thymidine kinase 2 (TK2), a mitochondrial enzyme which is the major thymidine kinase in nonreplicating tissues such as the heart.12,13 Through inhibition of TK2, zidovudine may limit the supply of thymidine phosphates as mtDNA building blocks, also resulting in mtDNA depletion.12,13 It has also been shown that zidovudine induces oxidative stress,11,14 an effect which may be secondary to respiratory chain failure and by itself also impairs mtDNA replication.15
 
Last but not least, severe mtDNA depletion and consequent respiratory chain failure can also induce myocardial pyrimidine depletion because a normal electron flux through the respiratory chain is essential for the activity of dihydroorotate dehydrogenase, an enzyme which is indispensable for pyrimidine de novo synthesis16 and critical for cell survival.
 
As it has been recently shown in vitro that pyrimidine loss sensitizes and contributes to the mitochondrial toxicity of thymidine analogues,17 we aimed to explore the role of pyrimidine depletion in the pathogenesis of the NRTI-induced mitochondrial cardiomyopathy and to study if uridine, a nucleoside which replenishes pyrimidines via the salvage pathway, may be therapeutically employed to antagonize the onset of zidovudine cardiomyopathy. As a source of exogenous uridine, we used Mitocnol, a dietary supplement with a high uridine bioavailability.18
 
DISCUSSION
 
The present study analyzed the effects of uridine supplementation on NRTI-mediated mitochondrial toxicity in cardiac muscle. Our findings demonstrate that zidovudine and zalcitabine induce a cardiomyopathy in which mtDNA-encoded respiratory chain deficiency plays a significant role. The reduction of COX subunit I expression suggests that quantitative and qualitative mtDNA lesions are important if not pivotal in this form of respiratory dysfunction and does not support mitochondrial toxicity secondary to other mechanisms such as increased ROS formation.
 
Our results also exhibit that the marked mitochondrial cardiotoxicity can be antagonized by oral uridine supplementation and support a central role of intracellular pyrimidine pools in its pathogenesis. As discussed above, mtDNA depletion by zidovudine may result from the inhibition of mitochondrial TK2, with consecutive depletion of normal deoxypyrimidine pools as mtDNA building blocks.12,13 This mechanism operates predominantly in postmitotic tissues such as the cardiac muscle in which thymidine kinase 1 activity as an alternate pathway of thymidine phosphorylation is down-regulated.12,27
 
Thymidine triphosphate concentrations are critical and rate limiting for mitochondrial DNA replication28 and reduced pyrimidine pools promote mutagenesis by reducing replication fidelity.28 Thus, the diminished thymidine triphosphate pools resulting from the inhibition of TK2 can explain the reduced wild-type mtDNA content and the increased prevalence of mtDNA deletions in zidovudine-treated myocardia.
 
The murine "common" mtDNA deletion26 was also present in the hearts of mice treated with zalcitabine, a polymerase gamma inhibitor not known to antagonize TK2 activity. MtDNA mutations with zalcitabine may arise from ROS which are produced by the defective respiratory chain.29,30 Mutagenic pyrimidine nucleotide depletion may also arise from defective de novo synthesis by dihydroorotate dehydrogenase secondary to the dysfunctional oxidative phosphorylation.28 Both mechanisms can explain our observation that ROS formation as assessed by myocardial MDA content was fully normalized with uridine supplementation.
 
Most importantly our study demonstrates that uridine supplementation attenuates all aspects of NRTI-related cardiomyopathy. The exact mechanism of the beneficial effect of uridine is not fully delineated, but it is likely that uridine itself or its metabolites disinhibit mtDNA replication by competing with zidovudine and zalcitabine as pyrimidine nucleotides either at TK2 or at polymerase gamma. Alternatively, uridine may compete with pyrimidine NRTI at other steps of intracellular NRTI transport or phosphorylation.31,32 Uridine may in addition correct an intracellular pyrimidine deficit which in itself is sufficient to induce apoptosis.17,33
 
The dose of the uridine supplement used in this study deserves some consideration. The dosage of Mitocnol administered to mice in our study corresponded on a per body weight basis to a human dose of 24 g/70 kg, and on a per body surface area basis to 13 g/m2 of Mitocnol.34 Mitocnol consists of 0.6 g of uridine plus 5.4 g of triacetyl uridine, a uridine precursor with several-fold higher uridine bioavailability over conventional uridine.35 Mitocnol constitutes the active ingredient of a commercially available dietary supplement named NucleomaxX. NucleomaxX is delivered in sachets (36 g), each of which contain 6 g of Mitocnol plus other substances to camouflage the bitter taste of uridine. Continuous administration of 3 sachets of NucleomaxX per day to men yields mean uridine plasma levels of 163 µM about 1 hour after administration. These uridine plasma levels are in a similar order of magnitude as those found to be protective against the mitochondrial toxicity of pyrimidine nucleoside analogues in vitro.32 This was the rationale for the dose administered to the mice in this study. In vitro studies have also indicated that it took considerably longer for mitochondrial toxicity to develop in the absence of uridine (weeks), than it took for uridine to abrogate established mitochondrial toxicity (days).32 This relatively quick therapeutic effect of uridine relative to the more prolonged development of mitochondrial toxicities in these in vitro studies may allow for intermittent dosing to "reset the mitochondrial clock", an approach which has been tested in clinical trials of NucleomaxX for NRTI-related hepatotoxicity and lipoatrophy.36,37 A number of studies in indications not related to myocardial disease or HIV infection have demonstrated the safety and good tolerability of uridine and triacetyl uridine.18,32 High doses of uridine were also shown to not compete with NRTI at HIV reverse transcriptase.31,36,38-40 Thus our work may not only open a new avenue for the therapy of NRTI-related cardiotoxicity in HIV patients and potentially also for the cardiomyopathies associated with inherited mtDNA mutations.41-43
 
RESULTS
 
Macroscopic and Microscopic Pathology

 
Daily fluid consumption, body weight, and heart weight were unaffected by zidovudine or zalcitabine treatment (data not shown). On post mortem examination, no clinical signs of cardiomyopathy were detected.
 
The histopathological cardiomyopathy score was increased after treatment with zidovudine (540% of untreated control values; P < 0.001) and with zalcitabine (313% of control values; P < 0.001; Table 1). Cotreatment with Mitocnol significantly attenuated the zidovudine-induced and zalcitabine-induced histopathological lesions but did not have an intrinsic effect (Fig. 1).
 
On ultrastructural investigations, the mitochondria in the Mitocnol-only hearts did not show abnormalities (Fig. 2). The myofibrillar lattice of the cardiomyocytes of animals treated with zidovudine or zalcitabine in the absence of Mitocnol was in disarray, the cardiomyoctes contained enlarged mitochondria in which the cristal architecture was disrupted. Cotreatment with Mitocnol normalized the myocardial ultrastructure.
 
Myocellular Lipids
 
Neither zidovudine nor zalcitabine augmented myocellular lipid deposition compared with untreated controls as evidenced by oil red O staining (data not shown) and biochemical quantification (Table 1). Zalcitabine, however, induced a slight, but quantitatively significant decrease of cardiac lipids. Mitocnol treatment per se did not alter myocardial lipids.
 
MtDNA Copy Number
 
In mice treated with zidovudine alone, the mean mtDNA copy numbers were diminished by 13% compared with control values (P = 0.02). Zalcitabine caused a similar mtDNA depletion (14%; P = 0.01). Whereas Mitocnol did not have an intrinsic effect on mtDNA copy numbers, this pyrimidine precursor fully normalized mtDNA copy numbers in zidovudine-treated and zalcitabine-treated animals. Among all animals, mtDNA copy numbers were inversely correlated with the cardiomyopathy score (r = -0.67; P < 0.001; Fig. 3A).
 
MtDNA-Encoded Respiratory Chain Subunits
 
The mean COX I/COX IV ratio was substantially reduced in the zidovudine group (57% of control values; P < 0.001) and to a lesser extent in the zalcitabine group (82%; P = 0.005) (Table 1). The COX I protein expression relative to COX IV was not altered in mice treated with Mitocnol alone. Mitocnol however attenuated the zidovudine-induced downregulation of COX I expression relative to COX IV (P = 0.03). Furthermore, Mitocnol normalized the COX I expression in mice cotreated with zalcitabine. Among all mice, the COX I/COX IV ratio was inversely correlated with the cardiomyopathy score (r = -0.51; P < 0.001; Fig. 3B).
 
MDA Levels and Lipid Peroxidation
 
MDA levels are an indirect indicator of lipid peroxidation due to reactive oxygen species (ROS). Compared with controls, MDA levels were increased by 70% (P < 0.001) in hearts exposed to zidovudine, and by 106% (P < 0.001) in hearts exposed to zalcitabine (Table 1). Uridine supplementation normalized cellular MDA formation in both zidovudine-treated and zalcitabine-treated animals (both P < 0.05).
 
The cardiac MDA content was positively correlated with the cardiomyopathy score (r = 0.68; P < 0.001; Fig. 3C); mtDNA copy numbers were inversely correlated with MDA levels (r = -0.70; P < 0.001; Fig. 3D).
 
MtDNA Deletions
 
A 445 bp PCR product, corresponding to the "common" mtDNA 4974 bp deletion in mice,26 was identified in all zidovudine and zalcitabine-treated animals. In contrast, 70% of the myocardia of the control animals and 80% of Mitocnol-only myocardia did not harbor detectable amounts of the deletion. In all cases in which the "common" mtDNA deletion was detectable in the control or Mitocnol-only myocardia, the deletion quantity was lower than the lowest deletion quantity in the zidovudine or zalcitabine hearts.
 
On average, the quantity of the mtDNA mutation was increased by 433% in the zidovudine group compared with controls (P < 0.001), and by 454% in the zalcitabine group (P < 0.001; Table 1; Fig. 4). With Mitocnol cotreatment, the frequency of the mtDNA deletion relative to controls was 305% in the zidovudine group (P = 0.001) and 313% in the zalcitabine group (P = 0.002). Thus cotreatment with Mitocnol diminished the presence of the mtDNA common deletion significantly compared with NRTI without Mitocnol (both P < 0.001).
 
Among mice in which the "common" mtDNA deletion was detectable, its intensity was positively correlated with the myocardial MDA content (r = 0.60; P < 0.001; Fig. 3E). The intensity of the "common" mtDNA deletion was inversely correlated with the COX I/COX IV ratio (r = -0.52; P < 0.001) and positively correlated with the cardiomyopathy score (r = 0.70; P < 0.001).
 
 
 
 
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