Even newer NRTIs (anti-HIV drugs) found to cause mitochondrial damage in muscle
[Lance Sherriff assisted in the transcription and compilation of this article.]
Mitochondria (the small organelles in cells that are critical for energy production) within the muscle fibers of antiretroviral-treated people living with HIV may be significantly damaged, according to a study presented last week in New York at the HIV and Aging Workshop by researchers from the Wellcome Centre for Microbial Research (WCMR) of Newcastle University.
The study, which looked at markers of mitochondrial function using a recently developed lab technique, found that people living with HIV who had taken the older nucleoside analog reverse transcriptase inhibitors (NRTIs), such as AZT, ddI, d4T and ddC, had significant mitochondrial deficiency compared to treatment-naive people living with HIV. This came as little surprise since the mitochondrial toxicity of those drugs is well established and has been associated with muscle weakness (myopathy), wasting and premature frailty in people who have taken those drugs.
However, what was not expected, was that the study also found significant mitochondrial defects in people who had only taken the newer NRTIs, such as abacavir, lamivudine, tenofovir and emtricitabine — the latter two being very widely used today. PCR performed on the specimens showed clear evidence that there were mutations in the mitochondrial DNA of treated individuals.
“Contemporary antiretroviral therapy may not be as mitochondrial clean as previously thought,” said Matthew Hunt, the PhD student at Newcastle University who presented the findings. However, the damage did not appear to be mediated by the age of the study participants.
Potential implications would be that clinicians need to be on the lookout for muscle weakness, muscle wasting and frailty in any patient who has been on prolonged treatment with the antiretroviral treatment regimens commonly being used today — regardless of their age — and consider regimens that spare or minimize exposure to NRTIs (even the new ones) — when and if possible — in any person already experiencing these complications. Such regimens do now exist. Meanwhile, researchers need to scale up work looking at the mechanisms involved in the damage caused by the contemporary NRTIs, as well as research interventions that may be useful.
[Note, as slides are not yet publicly available for these talks, references to some of the studies mentioned by speakers are not yet included. This will be corrected in later iterations of the article.]
Earlier at the meeting, Dr. Brendan Payne, the principle investigator at the WCMR (and Hunt’s instructor) provided more background into the role of mitochondrial dysfunction in aging and in aging-associated syndromes such as sarcopenia (muscle wasting) and frailty, which are much more common among people living with HIV over the age of 50 than in the similarly aged HIV-negative individuals.
There are many mitochondria within cells, and they perform a number of critical activities, but Payne focused on two that are key in the context of aging: adenosine triphosphate (ATP) synthesis (energy production) and maintaining reactive oxygen species (ROS) homeostasis.
ATP acts like a battery that stores energy used to power physiological processes such as muscle contraction. Mitochondria convert adenosine diphosphate (ADP) into ATP through a process known as cellular respiration. Without going into all the chemistry, this requires oxygen reduction reactions that drive electrons along the mitochondrial electron transport chain, a series of complexes within the inner membrane of the mitochondria. . [For those who are interested, there are some resources explaining the process in more detail online, such as this.]
Mitochondria have their own DNA (mtDNA) which contains the genes that encode for the proteins that make up these mitochondrial electron transport chains (one caveat, however, is that there are many more critical proteins that are not produced by mtDNA but are instead recruited into the mitochondria). mtDNA and mitochondria divide independently of the cell in which they reside, leading to many multiple mitochondria and multiple copies of mtDNA — with numbers ranging from a few hundred to tens of thousands of copies per cell. However, the mitochondria contain their own simple polymerases (enzymes that copy genetic material) which are more prone to errors (mutations) than the polymerases that copy our nuclear DNA.
When mtDNA mutations occur, it is typically only in some of these copies, which then co-exist in a mixed state with the wild-type (or unmutated) mtDNA. Over time, however, this mutant mtDNA may come to predominate in a cell. The process of going from a low level of mutation to high-level of mutation within the cell’s mtDNA is referred to as clonal expansion — and it typically happens when the cell enters a period of greater activity. However, if the mutant mtDNA cannot produce key mitochondrial proteins, ATP synthesis grinds to a halt and the cell becomes sick and dysfunctional.
But how does one measure the mitochondrial function in a person? According to Dr. Payne, the best way is to use phosphorus magnetic resonance spectroscopy (MRS) — which is like an MRI except that it captures the chemical signals in tissue. To look at what is happening in muscle, patients lie in the scanner and perform some exercise, such as moving their calf muscle. A coil sitting on their leg acquires the spectrum emitted and identifies the various metabolites of phosphorus, such as phosphocreatine, which becomes depleted as one exercises, and which is resynthesized as the muscle recovers (all part of cellular respiration).
How quickly phosphocreatine levels are replenished depends upon the quality of one’s mitochondrial function — with a slower recovery indicating a poorer function. Studies using phosphorus MRS have tracked the decline of mitochondrial function in individuals who have gone from a robust to pre-frail status — and these finding were supported by muscle biopsies from the same patients showing that mitochondrial protein and enzyme activities were also impaired as the they moved towards frailty. Similarly, there were lower levels mtDNA in the biopsies, which is a crude marker of mitochondrial biogenesis.
A few years ago, Payne and colleagues conducted a small study using phosphorus MRS in skeletal muscle of people living HIV that showed that a number had a slow recovery, suggesting impaired mitochondria. Another intriguing finding that also came out of this study was that the phosphorylation potential (the amount of ADP required to make a given amount of ATP — basically a measure of the resting/homeostatic health of mitochondria), was also diminished in the participants with HIV.
But Dr. Payne noted that MRS studies are very expensive, and muscle biopsy studies are invasive and painful — and so are not suitable for repeated measurement in large studies with very long follow-up periods. An alternative approach that was used in a large study at Johns Hopkins, involved measuring mtDNA levels in peripheral blood and tracking the clinical outcome of interest, which, in the case of this study, was mortality. They found that the lower the mtDNA copy level in blood, the worse the mortality. This finding was subsequently replicated in another cohort. However, it is not clear how the changes of mtDNA in blood are causally related to increased mortality.
Dr. Payne and the team of academics, clinicians and diagnostic scientists working at the WCMR have been working on developing a number of other laboratory techniques to better characterize mitochondrial deficits in tissue, particularly muscle, and how they directly affect clinical outcomes.
The NRTI study
The study that Hunt described used an automated immunofluorescence assay developed by WCMR that measures the contents of mitochondria in myofiber (muscle cell) biopsies. Specifically, the assay measured four markers simultaneously — the two most pertinent assess the quantity of mitochondrial electron transport chain complexes I and IV — each of which are, again, critical for ATP synthesis. A molecular analysis was then performed on fibers that were severely mitochondrially deficient to look for alterations in the mtDNA within the individual muscle fibers.
The study recruited a cohort of 37 people living with HIV (with a mean age of 48 years) who agreed to undergo muscle biopsies [and they should be called heroes for doing so]. 13 had never received antiretroviral therapy; 10 were taking contemporary NRTIs (tenofovir, abacavir, lamivudine, emtricitabine) and 14 others (who were also now taking contemporary NRTIs) who had previous exposure to older ‘historical’ NRTIs. A mean of 1229 myofibers were analyzed per participant. Clinical characteristics were also documented for a multivariate analysis.
Among the historical treated group, there were significant deficiencies in both complex I and complex IV (p=0.01 and p=0.004, respectively) compared to the treatment-naive individuals. Among the individuals only treated with the contemporary drugs, there were significant deficiencies in complex I (p=0.05) but not of complex IV. Similarly, the historically treated group had a significantly higher proportion of individual myofibers that were categorized as severely deficient in complex I (p<0.0001) and complex IV (p=0.04) compared to the treatment-naive individuals. Meanwhile, there was a strong trend towards the proportion of myofibers categorized as severely deficient in complex I (p=0.08) but not in complex IV among the participants taking the contemporary NRTIs.
In a multivariate analysis, looking at clinical characteristics, there was no association between CD4 cell counts [current — according to Hunt, the collection of nadir counts was incomplete] and mitochondrial deficits, nor [according to the study abstract] was there any association with the duration of HIV infection. With regard to duration of antiretroviral therapy, there was a correlation, but this could not be distinguished from the effect of the actual treatment group (individuals who were treated with historical NRTIs have been on treatment much longer than those who have only been on contemporary NRTIs). The same was true for age, as the older patients were more likely to have also taken historical NRTIs. So, in the multivariate analysis only historical and contemporary NRTI exposure were predictable of CI deficiency [which means that even younger individuals who have taken NRTIs may have accumulated mitochondrial deficits].
Molecular analysis was then performed to look at two sections (one in the minor and one in the major arc) of mtDNA in the severely deficient muscle fibers of those exposed to historical NRTIs or to only contemporary NRTIs. Both groups were found to have deletions in the muscle fiber’s mtDNA, however, in the historical group, the deletions were primarily in the major arc of the mtDNA, while in the contemporary group, there were deletions in both the major and minor arcs of the mtDNA. At present, it is unclear what this means, other than there may be more than one way for contemporary NRTIs to damage mtDNA.
The findings “merit further investigation into the molecular mechanisms behind these mitochondrial defects in the contemporary setting,” Hunt said.
Notably, during the discussion session, Scott LeTendre of the University of California, San Diego said that although the newer NRTIs do not inhibit mtDNA polymerase as potently as the old NRTIs, they still do somewhat. “They are also integrated into mtDNA and can then have an effect on mitochondrial replication,” he added.
Dr. Payne said that the time on the newer drugs will still probably prove to be a critical variable. “With relation to the newer NRTIs, which may possibly still have some mitochondrial toxicity, the other key factor is time. So, if you are on something for 10 years, that may be more than enough time,” he said.
Another issue was that it was not possible to tease out the individual mitochondrial toxicity of specific drugs in this limited cohort. This may be of particular importance for tenofovir which is being taken not only by people living with HIV, but as pre-exposure prophylaxis (PrEP) to prevent HIV acquisition.
Finally, the other missing piece is what are the clinical consequences of these mitochondrial deficits? Based upon the literature to date, over time, they may translate to muscle weakness and loss of resilience — including the early development of gerontological syndromes such as sarcopenia and frailty.
Hunt concluded by saying that “We also want to perform a multivariate analysis of mitochondrial dysfunction with physical frailty.”
Mitochondrial mechanisms in aging and potential interventions
Dr. Payne’s talk also focused on some other developments in mitochondrial research that might shed some light into how mtDNA mutations cause physiological aging, and what might be done to address it.
For instance, there is a new mitochondrial aging mouse model where the mitochondria have an unusually faulty polymerase that is prone to causing many mutations in the mtDNA. “The mouse,” he said, “has a very striking phenotype. It’s kind of hunched and thin, with thin hair . It also has a significantly reduced life expectancy — they tend to die just before a year of age.”
However, in aging humans such a high degree of mutations is not what is being seen. Instead, studies suggest that the burden of mutations associated with aging is much lower. What rather appears to be happening is that, over time, the dysfunctional mutant mitochondrial populations are expanding clonally and becoming the predominant mitochondria in the cell.
“What we are doing now in studies is to look at the molecular pathways – the signaling pathways – that might be driving this event,” he said.
Mitochondria are also a major source of reactive oxygen species (ROS), or free radicals (which are a bi-product of mitochondrial respiration). However, research into ROS in the last five years has moved away from the concept of ROS being a ‘bad actor’ that primarily causes damage to tissues. Dr. Payne said that their role as signaling molecules now appears to be more important. Such signaling roles are not always detrimental. For example, one sees an acute inflammatory response after exercise and similarly, there is a surge in ROS after a bout of exercise, but that is a beneficial signal that brings on an adaptive response. However, a better understanding of these signaling properties will be needed to channel them constructively.
For instance, if one can increase the number of functional mitochondria in cells, one should be able to increase the energy production and therefore the health of those cells and the individual. Most of the pathways converge on the molecule PGC-1alpha (peroxisome proliferator-activated receptor-gamma coactivator), which is the major regulator of mitochondrial biogenesis.
In human studies, exercise has been the most promising intervention for improving mitochondrial function. This has typically been endurance exercise, but there is now interest in high intensity exercise, which seems to be signaled through PGC-1alpha.
Payne cited a very recent study that has advanced the understanding of the exercise response. The study took sentry individuals who were given an exercise intervention that led to mitochondrial improvements, but there appeared to be some degree of a metabolic or signaling block in older individuals’ mitochondria. There were two molecular features of this block. One seemed to be a bottleneck in taking ADP and putting it through the cellular respiratory chain to produce ATP — and thus energy production was impaired. The second feature in the older adults was that there was a greatly increased amount of ROS production in response to the same degree of exercise challenge.
This imbalance of ADP and ATP parallels what was being shown in the MRS study that Payne described earlier.
As far as potential interventions to address these features, one that is currently receiving considerable attention involves nicotinamide adenine dinucleotide (NAD+), sirtuins (enzymes which mediate the anti-aging effects of caloric restriction in longevity studies in small animals) and PGC-1alpha.
One approach is to flood the system with extra NAD+, which opens the floodgates allowing more mitochondrial biogenesis. In mouse studies, they gave mice a compound called nicotinamide riboside, which increases the metabolite NAD+ and saw a modest but significant increase in lifespan. In addition, studies in aged mice show very little regeneration of muscle fiber after injury (exercise), but the mice that were given this supplement had active muscle regeneration. Dr. Payne believes much of this benefit had to have been due to rescuing mitochondrial function within stem cells.
Formulations of this particular supplement are available for sale online, Dr. Payne noted in a lighthearted manner. However, although a number of clinical studies have begun evaluating these supplements, they have yet to show clear benefit of supplementation in humans. As with any oral supplement, there may be physiological barriers that prevent it from being available at the right time and in the site where it is most needed (within the mitochondria). Nevertheless, research is ongoing.
Finally, in an exchange between Dr. Payne and Dr. LeTendre, there was a discussion of a factor that might drive some of the clonal expansion of defective mitochondria: stopping treatment.
Dr. LeTendre said that he had recently seen data suggesting that people who intermittently interrupt their therapy “develop mitochondrial mutations that then expand, so that overtime, there is a larger proportion of mitochondria, with mutations that do not seem to function as well.
“Absolutely,” Payne responded: “If you wanted to do a study like that, which would not be ethical, starting and then stopping antiretroviral therapy would be the most potent driver [of such clonal expansion],that would be exactly what our theoretical models suggest” he said.
[Notably, this is exactly what the ‘cure’ or ‘suppression while off-treatment’ studies are doing. To this writer, at least, this suggests that monitoring the mitochondria of people who go off treatment should be part of the safety analysis of all of these ‘cure’ studies.]
Hunt M, Zhu G, Greaves L, Payne B. Muscle mitochondrial function and contemporary anti-retroviral therapy. 9th International Workshop on HIV and Aging, September 13-14, 2018, New York. Abstract 22.
Payne B. Mitochondrial dysfunction in aging. 9th International Workshop on HIV and Aging, September 13-14, 2018, New York.
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