| |
Reduced Levels of NAD in Skeletal Muscle and Increased Physiologic Frailty Are Associated With Viral Coinfection in Asymptomatic Middle-Aged Adults
|
| |
| |
Download the PDF here
Tran, Thanh PhDa,b; Pencina, Karol M. PhDa,b,c; Schultz, Michael B. PhDd; Li, Zhuoying PhDb; Ghattas, Catherine PhDb; Lau, Jackson PhDb; Sinclair, David A. PhD, AOd; Montano, Monty PhDa,b,c
Given the increased risk of functional decline and elevated inflammation in PLWH and skeletal muscle phenotypes that resemble nonhuman models for reduced levels of NAD in skeletal muscle, this study sought to investigate whether NAD levels in skeletal muscle of asymptomatic PLWH, in the context of prevalent HCV and CMV coinfection, differ from their uninfected counterparts. We also sought to determine whether potential differences could be related to other variables present in this asymptomatic cohort of preclinical middle-aged PLWH.
In conclusion, a cohort of middle-aged, asymptomatic PLWH compared with uninfected participants displayed reduced levels of total NAD, NAD+, and NADH in skeletal muscle that was in part explained by viral coinfection with HCV and/or CMV. A composite score for viral infection indicated associations with pathophysiologic frailty and circulating biomarkers for inflammation and immune activation. Collectively, the findings in this study support the presence of preclinical deficits that may help to explain previously observed inflammatory and bioenergetic derangements and support clinical follow-up studies to replete skeletal muscle NAD levels to improve physical function, quality of life, and overall healthspan in PLWH.
NAD+ is a key cofactor, both in cellular energy metabolism10 and in modulation of inflammatory signaling.11 In multiple species, NAD decline with age has been linked to deficits in mitochondrial function and metabolic capacity and decline in the activity of sirtuins, a class of NAD+-dependent enzymes that control inflammation, mitochondrial metabolism, and aging.
Abstract
Background:
People living with HIV (PLWH) are disproportionately burdened with multimorbidity and decline in physiologic function compared with their uninfected counterparts, but biological mechanisms that differentially contribute to the decline in muscle function in PLWH compared with uninfected people remain understudied.
Setting:
The study site was Brigham and Women's Hospital, Harvard Medical School, Boston, MA.
Methods:
We evaluated skeletal muscle tissue for levels of total nicotinamide adenine dinucleotide (NAD), NAD+, and nicotinamide adenine dinucleotide (NADH) in middle-aged asymptomatic PLWH, coinfected with hepatitis C virus and/or cytomegalovirus and compared them with uninfected control participants.
Results:
Of the 54 persons with muscle biopsy data, the mean age was 57 years with 33% women. Total NAD levels declined in skeletal muscle in association with HIV infection and was exacerbated by hepatitis C virus and cytomegalovirus coinfection, with lowest levels of total NAD, NAD+, and NADH among persons who were coinfected with all 3 viruses (P = 0.015, P = 0.014, and P = 0.076, respectively). Levels of total NAD, NAD+, and NADH in skeletal muscle were inversely associated with inflammation (P = 0.014, P = 0.013, and P = 0.055, respectively). Coinfections were also associated with measures of inflammation (CD4/CD8 ratio: P < 0.001 and sCD163: P < 0.001) and immune activation (CD38 and human leukocyte antigen-DR expression on CD8 T cells: P < 0.001). In addition, coinfection was associated with increased physiologic frailty based on the Veteran Aging Cohort Study 1.0 index assessment (P = 0.001).
In genetic studies using nonhuman model systems, mice lacking nicotinamide adenine dinucleotide (NAD+) in skeletal muscle because of ablation of the rate limiting NAD biosynthetic enzyme [nicotinamide phosphoribosyltransferase (NAMPT)] displayed similar features to that observed in the skeletal muscle of our cohort of PLWH (eg, internalized nuclei and reduced PGC-1α) with a dramatic decline in physical function as the mice aged.9
Conclusions:
Further research is warranted to determine the clinical relevance of preclinical deficits in NAD metabolites in skeletal muscle in association with viral coinfection and inflammation, as well as the observed association between viral coinfection and physiologic frailty.
INTRODUCTION
The success of effective combination antiretroviral therapy has dramatically increased life expectancy in people living with HIV (PLWH).1However, as PLWH age, they are prematurely burdened by multiple comorbid conditions, including a higher prevalence of functional limitations compared with their uninfected counterparts2 and chronically elevated biomarkers for inflammation [eg, C-reactive protein (CRP), interleukin-6 (IL-6), CD163, and CD14] and immune activation [eg, CD38 and human leukocyte antigen-DR [HL-DR]).3 PLWH also have a higher prevalence of viral coinfections, notably with hepatitis C virus (HCV) and cytomegalovirus (CMV) infection. HCV coinfection is common among PLWH in the United States with a prevalence of 20%-25%.4 Despite the recent success in sustained virologic repression with direct-acting agents, HCV continues to accelerate liver disease, adversely affecting mortality and quality of life and increasing the risk of physiologic frailty.5 CMV coinfection is nearly universal in PLWH and is associated with elevated inflammation, accelerated immune senescence, and also an increased risk of frailty.6
We recently reported that the skeletal muscle phenotype of a cohort of asymptomatic middle-aged PLWH exhibits elevated internalized nuclei, reduced nuclear PGC-1α, a master regulator of mitochondrial biogenesis, and subclinical deficits in physical function7-features that become increasingly common with advanced age. However, in the same cohort, skeletal muscle cross-sectional area, fiber-type distribution, and fiber size did not differ from uninfected participants of similar age and sex. Collectively, this is consistent with an asynchronous aging phenotype, with some but not all features of age occurring prematurely.8 In genetic studies using nonhuman model systems, mice lacking nicotinamide adenine dinucleotide (NAD+) in skeletal muscle because of ablation of the rate limiting NAD biosynthetic enzyme [nicotinamide phosphoribosyltransferase (NAMPT)] displayed similar features to that observed in the skeletal muscle of our cohort of PLWH (eg, internalized nuclei and reduced PGC-1α) with a dramatic decline in physical function as the mice aged.9
NAD+ is a key cofactor, both in cellular energy metabolism10 and in modulation of inflammatory signaling.11 In multiple species, NAD decline with age has been linked to deficits in mitochondrial function and metabolic capacity and decline in the activity of sirtuins, a class of NAD+-dependent enzymes that control inflammation, mitochondrial metabolism, and aging.12 Age-related decline of NAD is due in part to hydrolysis by an intrinsic NADase activity of the activation marker CD38.13 Notably, NAD deficits in skeletal muscle have been linked to reduced capillary density and physical endurance.14 In mice, restoring NAD levels in skeletal muscle15 reduces centrally located myonuclei, decreases inflammation, and increases mitochondrial biogenesis and physical activity.16 Thus, repletion of NAD is an attractive therapeutic modality for potentially reducing age-related inflammation (ie, inflammaging) and improving physical function.12
Given the increased risk of functional decline and elevated inflammation in PLWH and skeletal muscle phenotypes that resemble nonhuman models for reduced levels of NAD in skeletal muscle, this study sought to investigate whether NAD levels in skeletal muscle of asymptomatic PLWH, in the context of prevalent HCV and CMV coinfection, differ from their uninfected counterparts. We also sought to determine whether potential differences could be related to other variables present in this asymptomatic cohort of preclinical middle-aged PLWH.
DISCUSSION
A cornerstone of healthy aging is the maintenance of mobility and functional independence, and yet although PLWH have an increased life expectancy, they nevertheless experience premature loss in mobility and increased frailty risk.23 Physiologic mechanisms that underlay this functional decline in PLWH are poorly understood, but accumulating evidence points toward dysregulated inflammation and bioenergetics.24-27 Identifying targetable pathways contributing to these potential drivers of functional decline, especially before the onset of clinical symptoms, would provide an opportunity to improve health outcomes as PLWH age.
In our previous studies of asymptomatic middle-aged PLWH,7,17,18 we reported modest deficits in gait speed and stair climb power. Interestingly, we also observed increased internalized nuclei more typical of skeletal muscle in older persons28 and reduced levels of nuclear PGC-1α (a master regulator of mitochondrial biogenesis), suggesting compromised bioenergetics.7 Notably, in a follow-up study, physical activity measured with accelerometry revealed a significantly reduced activity profile in PLWH compared with uninfected participants.7,17 These data are consistent with independent studies, reporting that PLWH display reduced oxidative enzyme activity in skeletal muscle29 that may reduce aerobic capacity and exercise tolerance. Additional studies have also reported that PLWH also experience a disproportionate decline in grip strength and gait speed.26,27
NAD+ is a key cofactor in cellular energy metabolism.10 In mice, loss of NAD in skeletal muscle results in centrally located nuclei associated with myopathy and aging, and reduced activity of PGC-1α, resulting in a progressive decline in physical function and activity.9NAD also influences inflammatory signaling, in part, through NAD-dependent SIRT1 deacetylation of the p65 subunit of NF-κB, a heterodimeric transcription factor regulating multiple inflammatory genes.30 With aging, NAD levels gradually decline in multiple tissues,31 in part, because of age-related increases in NAD+-consuming enzymes, such as CD38.13 Notably, CD38 NADase activity has been reported to increase with HIV infection in vitro, thereby reducing levels of NAD in leukocytes.32 Deficits in NAD may be amenable to therapeutic intervention. For example, restoring NAD levels (eg, with nicotinamide riboside or nicotinamide mononucleotide15) in nonhuman models was shown to reverse the level of central nuclei, reduce inflammation in skeletal muscle, and increase mitochondrial biogenesis and physical activity.16
In this report, levels of NAD in skeletal muscle differed based on infection status, with reductions in total NAD, NAD+, and NADH strongly associated with viral coinfection (Fig. 2). In univariate analysis to identify predictors of NAD levels in skeletal muscle, the strongest predictors of total NAD, NAD+, and NADH were HCV and CMV coinfection and BMI (Table 2).
Multiple previous studies have observed functional impairment and frailty in PLWH compared with uninfected controls of similar age.23 Frailty has been defined as a loss in reserve capacity and increased vulnerability to stressors.33 Although frailty has most often been characterized as either a clinical syndrome34 or as an accumulation of deficits,35 there are currently as many as 29 different measures for frailty.36 Alternatively, the VACS score is based on standardized routinely collected clinical measures of multiorgan systems that as an index reflect physiological frailty.37 Physiologic frailty is an important subclinical landmark that may precede overt evidence of frailty.22,38Indeed, the VACS score predicts frailty-related outcomes (eg, hospitalizations, fractures, and falls) and is associated with measures of functional performance,39 inflammation,40 and more recently HCV infection.5 In this study, measurement of physiologic frailty using the VACS 1.0 index indicated that viral coinfection was associated with a higher score for physiologic frailty (Fig. 3).
Notably, inflammation and immune activation were associated with viral coinfection burden and inversely with NAD levels in skeletal muscle. A composite viral score reflecting burden of viral infections was associated with a reduced CD4/CD8 ratio (a general biomarker for inflammation), increased sCD163 (a monocyte biomarker of inflammation), increased HLA-DR+CD38+ (a biomarker for immune activation), and an increase in the VACS 1.0 index (a measure of physiologic frailty) (Fig. 3). Thus, the composite burden of asymptomatic infections affect drivers of aging (inflammation and immune activation) and warrant further study to determine a potential role for coinfection burden on biomarkers of aging, particularly in the context of asymptomatic or treated infections. In addition, levels of NAD in skeletal muscle were inversely associated with circulating inflammatory factors in blood, based on evaluation of a composite score for inflammation (IL-6, CRP, sCD163, and sCD14) (Table 2 and Fig. 2) and in sCD163 evaluated separately (data not shown). The mechanistic relationship between reduced NAD levels in skeletal muscle and elevated inflammation in blood remains unclear and requires further study.
HIV/HCV coinfection has been associated with a higher prevalence of clinically significant liver fibrosis.41 Although direct-acting agents have improved liver function in HIV/HCV coinfection,42 adverse patient-reported outcomes remain significant.43 Interestingly, HCV proteins are reported to inhibit the SIRT1-AMPK signaling pathway,44 and more recently, the HCV serine protease NS3/4A was shown to inhibit quinolinate phosphoribosyl transferase, a key enzyme in the de novo NAD synthesis pathway,45 suggesting a direct link between HCV infection and NAD. Notably, NAD treatment inhibited HCV replication in vitro and in vivo.45
The observed reduction in NAD levels in skeletal muscle of persons with viral coinfection calls into question the mechanism by which a primarily hepatotropic virus (ie, HCV) influences skeletal muscle NAD levels but does pose testable possibilities: (1) The de novo synthesis of NAD occurs primarily in the liver, with other tissues relying almost exclusively on circulating nicotinamide made by the liver.46 Therefore, subclinical liver function despite effective antiviral therapy (ie, direct-acting agents) may result in reduced bioavailable skeletal nicotinamide (and consequently reduced levels of skeletal NAD). Also, (2) the kynurenine-tryptophan pathway is disrupted by HIV infection and may be further exacerbated in HCV47 and/or CMV coinfection,48 compromising NAD levels in skeletal muscle tissue.
There are limitations to this substudy. First, the sample population is relatively small and will need to be validated in a larger cohort. Second, because there were no participants with HCV monoinfection, we cannot exclude potential differences in HCV monoinfection vs HIV/HCV coinfection. Third, our age range of 50-65 years in this study was not sufficient to identify potential age and NAD level associations in skeletal muscle. Finally, the sample size and asymptomatic health status for all infection in this population were insufficient to directly test NAD levels of traditional measures of geriatric syndrome such as frailty. These limitations restrict our ability to infer mechanism and underscore the need for a larger comprehensive mechanistic study but do point the way toward hypothesis-driven assessments.
CONCLUSIONS
In conclusion, a cohort of middle-aged, asymptomatic PLWH compared with uninfected participants displayed reduced levels of total NAD, NAD+, and NADH in skeletal muscle that was in part explained by viral coinfection with HCV and/or CMV. A composite score for viral infection indicated associations with pathophysiologic frailty and circulating biomarkers for inflammation and immune activation. Collectively, the findings in this study support the presence of preclinical deficits that may help to explain previously observed inflammatory and bioenergetic derangements and support clinical follow-up studies to replete skeletal muscle NAD levels to improve physical function, quality of life, and overall healthspan in PLWH.
|
|
| |
| |
|
|
|
