In the current research, we have shown that ddI and ddI-containing regimens are associated with the greatest degree of mtDNA suppression in HSMCs, most notably in myoblasts. The patterns of mtRNA decrease were different from those of mtDNA, particularly for cells treated with NRTI combinations. These findings suggest that evaluating markers beyond mtDNA can provide additional information on the potential adverse effects of these drugs on mitochondrial function.
To our knowledge, this is the first study that has investigated the impact of NRTIs and NRTI combinations on mitochondria in human skeletal muscle myoblasts, a cell type likely to be susceptible to mitochondrial damage in vivo. Myoblasts can differentiate into myotubes (32
) and are more abundant in the skeletal muscle of infants and children than in adults (8
). Moreover, age is known to alter the potential of myoblasts to differentiate into myotubes (22
) and to affect myoblast metabolism and proliferation (3
). These differences are of particular interest, because our data indicate that the decline in mtDNA resulting from ddI exposure was greater for myoblasts than myotubes. Thus, it is likely that children are more vulnerable than adults to the mitochondrial toxicity of ddI which could negatively impact growth and development. Recent in vivo data also demonstrated that mitochondrial damage at birth in monkey offspring exposed perinatally to AZT-ddI was severe and there was no improvement during the first year of life, with significant reduction of mtDNA in muscle compared to levels for other NRTI regimens (6
). Of note, ddI is the only purine analog that is commonly used in developing countries, in contrast to other NRTIs such as AZT and d4T, which are pyrimidine analogs. This is of particular concern in developing countries, where ddI is widely used as part of the first-line treatment of HIV-infected children (28
The decrease in mtDNA abundance is determined by either a decrease in replication or an increase in degradation of mtDNA. Important factors for the replication of mtDNA include the nuclear genes encoding the mtDNA-specific replication and transcription factors such as POLG and Tfam. In our current study, the mRNA expression for POLG
did not demonstrate any significant changes in HSMCs treated with ddI. It is possible that a longer observation beyond the 5 days used for our study might have altered the mRNA expression of POLG
. It is also possible that the decline in mtDNA was partially due to other pathways, including reactive oxygen species production, uncoupling proteins, and depletion of deoxyribonucleotide triphosphate pools in mitochondria (29
). In contrast, the degradation of mitochondria by autophagy, specifically called mitophagy (19
), plays a central role in the degradation of whole mitochondria and their contents (15
). Therefore, the effects of each NRTI on mitophagy may, in part, determine the degree of mtDNA and mtRNA degradation.
Interestingly, NRTIs were found to induce different patterns of decline for mtDNA and mtRNA levels in HSMCs. ddI and ddI-containing regimens showed a rapid decline in mtDNA levels but slower and smaller declines in mtRNA. Moreover, NRTI combinations seemed to lower the mtRNA levels in HSMCs to a significantly greater extent than single NRTIs, demonstrating the cumulative negative impact of NRTI combinations on mtRNA levels, a finding not observed in mtDNA levels. Recent data obtained using HepG2 cells also showed similar negative effects of NRTI combinations on mitochondria (34
). These findings, combined with our current data, suggest the importance of evaluating NRTI combinations and not relying on single-drug studies if the impact of antiretrovirals on mitochondria is to be assessed in in vitro models (14
), because patients take NRTI combinations as a part of HAART.
The decline in mtRNA levels for MTCYB
with ddI and ddI-containing regimens was more pronounced than the decline in MTCO3
mtRNA levels, suggesting a differential impact of ddI on mitochondrial oxygen phosphorylation (OXPHOS) complexes. Importantly, MTCYB
encodes a subunit of respiratory complex III, and MTCO3
encodes subunits of respiratory complex IV; both transcriptions are initiated by the same heavy-strand promoter (PH
). Therefore, the difference may be explained by the instability of each mtRNA due to the different lengths of poly(A) tails at the 3′ ends of mtRNA in MTCYB
). Our current data, combined with those of a previous study using lymphoblast lines (10
), suggest the importance of investigating additional markers beyond mtDNA, such as mtRNA levels, for the evaluation of mitochondrial toxicity. Further investigations are needed, however, to fully understand the long-term consequences of these mtDNA and mtRNA changes.
In agreement with our previous in vivo data (30
), mtDNA significantly increased in myoblasts treated with AZT-containing regimens. Similar findings are also observed in other in vitro models, including human hepatoblastoma (HepG2) cells (5
) and HSMCs (2
). The mechanisms of increase in mtDNA by AZT are still unknown; however, these results strongly suggest that AZT upregulates genes encoding mtDNA. Because AZT causes significant mitochondrial damage in different in vitro models (14
), the increase in mtDNA may reflect a compensatory response to mitochondrial dysfunction resulting from different causes, including mtDNA POLG (23
), oxidative stress (16
), and increases in mtDNA and mitochondrial mass by oxidative stress (18
There are limitations of this study. First, we do not know the actual concentrations of NRTIs in HSMCs, although the drug levels in culture were designed to approximate those used clinically. Even so, the intracellular drug concentrations used for this study may not reflect the concentrations in vivo. One study using an in vivo rat model demonstrated that the intracellular concentration of ddI in rat hepatocytes was almost half the extracellular concentration, whereas the intracellular and extracellular concentrations of AZT were similar (27
). Evaluating the assays using concentrations higher than 1× Cmax
may be more informative for ddI. Second, although we demonstrated changes in mtDNA and mtRNA abundance, the actual functions of mitochondria were not evaluated in the current study. These include lactate production, mitochondrial membrane potential, and other relevant mitochondrial parameters. Finally, as noted earlier, the duration of treatment of 2 to 5 days may not reflect the drug effect on mitochondria in patients receiving long-term antiretroviral therapy.
In conclusion, we have shown that ddI and ddI-containing regimens in clinically relevant concentrations have a significant impact on mtDNA and mtRNA levels in HSMCs, most notably myoblasts. The results are consistent with our previously published in vivo data demonstrating that mtDNA levels in PBMCs were significantly affected by ddI. These data suggest that the use of ddI in children should include provider awareness of the potential of mitochondrial toxicity, especially in developing countries, where ddI has been used widely as a first-line antiretroviral therapy.